1

MC LC
Chng1: M U 1. i tng, nhim v v ni dung ca vi sinh vt hc cng nghip 2. Lc s pht trin ca vi sinh vt hc cng nghip 3. V tr v yu cu mn hc Cu hi n tp Chng2: C S KHOA HC CA VI SINH VT HC CNG NGHIP 1. c im cu to v hot ng sng ca vi sinh vt 2. C s ha sinh ca vi sinh vt hc cng nghip 3. C s di truyn vi sinh vt Cu hi n tp Chng 3: S PHN LOI SN PHM 1. Phn loi theo tnh cht thng mi 2. Phn loi theo sinh l trao i cht Cu hi n tp Chng 4: CC PHNG PHP V K THUT LN MEN 1. Qu trnh ln men 2. Vi sinh vt 3. C cht dinh dng 4. Nhu cu v oxy v s thng kh trong qu trnh ln men 5. Kh trng 6. Phng php nui 7. Ni ln men Cu hi n tp Chng 5: S THU NHN SINH KHI T BO 1. Tiu chun v chng 2. Mi quan h vi sinh trng 3. Cht lng sn phm 4. Ging khi ng 5. Protein n bo (SCP) Cu hi n tp Chng 6: CC SN PHM LN MEN 1. Ln men ethanol

Trang 4 5 7 3

8 13 21 32 33 34 47

48 52 55 61 62 66 70 74 75 76 80 82 92 98 99

2. Ln men lactic 3.Ln men 2,3 butadiol 4. Ln men butanol -aceton Cu hi n tp Chng 7: CC CHT TRAO I BC MT 1. Nguyn l ca s tng hp tha 2. Cc phng php to ra th t bin tng hp tha 3. Aminoacid 4. Sn xut cc purin nucleotit 5. Vitamin Cu hi n tp Chng 8: CC CHT TRAO I BC HAI 1. Cc cht khng sinh 2. Cc c t nm Cu hi n tp Chng 9: CC SN PHM CHUYN HA 1. S chuyn ha cc steroit 2. S to thnh phenyl-axetylcacbinol 3. Sn phm t vi khun axetic 4. Sn xut vitamin C 5.Sn xut destran Cu hi n tp Chng 10 : X L NC THI BNG BIN PHP SINH HC 1. Vi sinh vt hc ca cc ngun nc ung 2. X l nc thi 3.Ln men methane Cu hi n tp Chng11 : S TUYN KHONG NH VI SINH VT 1. Cc vi khun ngm chit 2. C ch tc ng ca vi khun 3. Mt s qu trnh thy luyn kim sinh hc 4. S tch ly kim loi nh vi khun v to Cu hi n tp Chng 12: CC BI TP C S V NNG CAO 1. Phn cu hi 2. Tr li mt s cu hi

114 120 125 127 129 134 138 146 150 151 153 172 180 182 187 187 190 198 202 204 208 210 218 220 221 223 233 234 236 245

NHNG CH VIT TT
ADP AMP APG A-1,3-DPG ATP A-6PA CoA CKS DNA R-1,5-DP R-5-P RNA VSV F-6-P FAD G-6-P GAP KDPG N NAD NADH NADP NADPH PP X-5-P Adenosine diphosphate Adenosine monophosphate Acid 3-phosphoglyceric Acid 1,3 diphosphoglyceric Adenosine triphosphate Acid 6-penicillanic Coenzyme A Cht khng sinh Deoxiribonucleic acid Ribulose-1,5-diphosphate Ribulose-5-diphosphate Ribonucleic acid Vi sinh vt Fructose-6-phosphate Flavin adenine dinucleotide Glucose-6-phosphate Glyceraldehyde phosphate 2-Keto-3-deoxi-6-phosphogluconate Nitrogen Nicotinamid adenine dinucleotide dng oxi ha Nicotinamid adenine dinucleotide dng kh
Nicotinamid adenine dinucleotide phosphat dng oxi ha

Nicotinamid adenine dinucleotide phosphat dng kh Pentose phosphate Xylulose-5-phosphate

Ngi bin son 1. PGS.TS. Kiu Hu nh 2. TS. Bin Vn Minh (Ch bin) 3. TS. Phm Ngc Lan 4. ThS. Bch Thy

Bin son cc chng 7, 8, 9, 10 1, 2, 3, 11 v 12 4, 5 6

Chng 1

M u
1. i tng, nhim v, ni dung ca vi sinh vt hc cng nghip
Vi sinh vt hc cng nghip (Industrial Microbiology) l mt ngnh ca Vi sinh hc, trong vi sinh vt (VSV) c xem xt s dng trong cng nghip v nhng lnh vc khc nhau ca k thut. Vi sinh vt hc cng nghip (VSVHCN) gii quyt hai vn chnh tri ngc nhau: Mt mt, n dn ti lm r hon ton nhng tnh cht sinh hc v sinh ho ca nhng c th sng l nguyn nhn c bn v trc tip ca s chuyn ho ho hc, nhng cht c c cht ny hay c cht kia. Trong trng hp ny, VSVHCN s dng nhng VSV thu nhng sn phm quan trng v c gi tr thc t bng con ng ln men. Phng php sinh ho thu nhiu sn phm l phng php duy nht c li v kinh t. Mt khc, chng ta cng bit s ln men do VSV gy ra khng lun lun din ra theo mt hng nh mong mun. S ph hu mt qu trnh ln men thng xy ra do s hot ng ca nhng VSV l. Trong trng hp ny, iu rt quan trng l khng nhng phi bit nhng VSV gy ra qu trnh cn thit m cn phi bit c nhng VSV c hi gy tn tht cho sn xut. Nh VSVHCN c kinh nghim phi khm ph ra chng, lm r tnh cht c hi do chng gy ra v tm ra nhng phng php u tranh vi chng. 1.1. Mc tiu mn hc Sau khi hc xong hc phn ny, sinh vin cn hiu c cc ng dng cng nghip quan trng ca vi sinh vt, s khc bit gia cng ngh sinh hc vi sinh vt hin i v vi sinh vt hc truyn thng, phn bit c cc nhm sn phm v qu trnh cng nghip, vai tr ca vi sinh vt trong tuyn khong v trong x l nc thi bng con ng sinh hc. 1.2. M t mn hc VSVHCN l mt b phn quan trng trong cng ngh sinh hc, l mn khoa hc nghin cu nhng hot ng sng ca vi sinh vt p dng n mt cch tt nht vo cc quy trnh sn xut quy m cng nghip v cc lnh vc khc nhau ca k thut. VSVHCN l mt ngnh khoa hc mi pht trin, nhng do ngha quan trng ca n v mt l thuyt cng nh thc tin nn pht trin ht sc nhanh chng.

2. Lc s pht trin ca vi sinh vt hc cng nghip

S pht trin ca VSVHCN c chia thnh 3 giai on chnh: Giai on u, tnh n na sau th k 19. Vic ng dng tim nng ca VSV c t bui u ca nn vn minh nhn loi nh sn xut ru vang, bia, dm. Vit Nam ngh nu ru, lm dm, lm tng cng c t rt xa xa. Tuy mt s qu trnh c thc hin quy m rng ri, nhng nhng s thnh cng cn ph thuc vo s ngu nhin hay kinh nghim ca nhng ngi th gii truyn cho cc th h sau. Vai tr ca VSV trong s chuyn ho cc cht hu c c con ngi bit n khong hn 100 nm trc y. Nhng cng trnh nghin cu VSVHCN bt u t Pasteur (1878). Nh ta thy Pasteur nghin cu nhiu qu trnh ln men p dng trong sn xut v hc thuyt v mm bnh. Pasteur cng ra phng php thanh trng Pasteur tit trng ru nho, bia m khng lm hng phm cht. Phng php ny hin nay c ng dng rt ln. Bi vy Pasteur c coi l ngi sng lp ra VSVHCN. Vic nghin cu v s dng cc chng nm men thun khit trong sn xut bia (Hansen, 1886) c th xem l bc m u cho cng nghip ln men da trn c s khoa hc. Nm 1898 Buchner cng nghin cu tc dng ln men ca nhiu nm men, vch ra mi lin h gia nm men v ho hc v men, v ng dng hot ng ca nm men vo sn xut tip ging ngoi. ng nghin nm men ly ra dung dch c men zymase v cho ln men ru. Nh vy giai on th nht l giai on s dng cc hot tnh ca VSV- giai on ny c nh du bng vic t c s khoa hc cho qu trnh sn xut ung cha ru.

Hnh 1.1: Cc nh vi sinh vt hc cng nghip tin bi 1. Louis Pasteur (1822-1895); 2. Gerhard H. A. Hansen (1841-1912); 3. Eduard Buchner (1860- 1917)

Giai on th hai ca VSVHCN c tnh n gia th k XX, bao gm s xut hin ca cc cht khng sinh, nhng tin b v di truyn hc trong vic chn lc cc th t bin vi khun, s nghin cu cc iu kin ln men ti u, k thut hc ln men, vic tch v tinh ch sn phm... Giai on th ba c c trng bi s pht trin ca mt nn cng nghip VSV c lp. Ngi ta iu khin c cc qu trnh siu tng hp VSV v to ra c hng lot cc chng t bin VSV. Nh cc thnh tu ny m ngi ta sn xut quy m ln m chnh, lysine v nhiu loi aminoacid khc.

Hnh 1.2: 1. Alexander Fleming(1881-1955); 2. Francis Crick (1916-2004); 3. James Dewey Watson (1928-); 4. Joshua Lederberg(1925-)

Giai on th t (giai on hin nay) c nh du bng s pht hin ra cc enzyme ct gii hn restrictase v cc plasmid vi s gn cc gene l mang cc thng tin tng hp cc protein c bit vo mt c th tr thnh mt phng php thng dng v s kim sot ngy cng tt hn s biu hin ca cc gene ny.

Hnh 1.3: 1.Daniel Nathans (1928-1999); 2.Werner Arber(1929-); 3.Hamilton Othanel Smith (1931-); 4.Herbert Boyer (1936-).

3. V tr v yu cu mn hc
Mn VSVCN nhm cung cp cho ngi hc nhng kin thc v k nng ng dng VSV trong mt s quy trnh cng ngh phc v khoa hc v i sng con ngi, ngoi ra cn gip cho sinh vin phng php nm bt c mt s quy trnh k thut v gii thch c qu trnh sn xut trn c s khoa hc, tin ti c th ch ng hng dn gup mt s c s sn xut trong nhng trng hp cn thit, ng thi cung cp thm nhng kin thc su, rng v VSV hc ng dng, gp phn y mnh sn xut, tng thm ca ci vt cht, ci thin i sng cho nhn loi.

Cu hi n tp
1. i tng nghin cu ca vi sinh vt hc cng nghip l g ? 2. Cc giai on pht trin ca vi sinh vt hc cng nghip ? 3. Giai on pht trin u tin ca vi sinh vt hc cng nghip c nh du bng cng trnh ca Pasteur (1878) chng minh vi sinh vt l tc nhn ca s ln men, v sau l cc cng trnh ca:.............(1886) dng cc chng nm men thun khit trong sn xut bia, v ...............(1898) pht hin ra dch chit nm men c kh nng gy ra qu trnh ln men ru ( chng minh ln men thc cht l mt qu trnh enzyme). 4. Ti sao c ngi ni vi sinh vt va l ngi bn thn thit, va l k th nguy him ca con ngi.

Chng 2

C s khoa hc ca vi sinh vt hc cng nghip

I. c im cu to v hot ng sng ca vi sinh vt
Vi sinh vt (Microogranisms) l tn gi chung ch tt c cc loi sinh vt nh b, ch c th nhn r di knh hin vi quang hc hoc knh hin vi in t. Vi sinh vt bao gm nhiu nhm khc nhau: Cc virus (nhm cha c cu to t bo), cc vi khun v vi khun lam (nhm sinh vt nhn s), cc vi nm (nhm sinh vt nhn chun) v c mt s ng vt nguyn sinh cng nh to n bo cng thuc nhm ny. Gia cc nhm trn khng c mi lin h cht ch v mt hnh thi hay phn loi, nhng ngi ta gp chng li v chng cng c mt s phng php nui dng, nghin cu v hot ng sinh l gn ging nhau v u c cc c im chung. 1. c im chung ca cc vi sinh vt 1.1. Kch thc nh b

Hnh 2.1: Cc phng php quan st th gii sng (t nguyn t n t bo)

Cc Vi sinh vt c kch thc rt b, o bng n v nanomt (1nm = 10-9 m) nh cc virus hoc micromet (1m = 10-6 m) nh cc vi khun, vi nm. Chng hn:

- Cc th thc khun (hay phage) T2, T4, T6 c kch thc bin thin trong khong: (65 - 95) x (25 - 100) nm. - Cc vi khun c kch thc thay i trong khong (0,2 - 2) x (2,0 - 8,0) m; trong vi khun Escherichia coli rt nh: 0,5 x 2,0m. - Cc t bo nm men Saccharomyces cerevisiae c ng knh 5 10m. Kch thc cng b th din tch b mt ca vi sinh vt trong 1 n v th tch cng ln. Chng hn ng knh ca 1 cu khun (Coccus) ch c 1mm, nhng nu xp y chng thnh 1 khi lp nhng c th lch l 1cm3 th chng c din tch b mt rng ti ...6 m2 ! 1.2. Hp thu nhiu, chuyn ha nhanh Tuy vi sinh vt c kch thc rt nh b nhng chng li c nng lc hp thu v chuyn ho vt xa cc sinh vt khc. Chng hn 1 vi khun lctic (Lactobacillus) trong 1 gi c th phn gii c mt lng ng lactose ln hn 100-10 000 ln so vi khi lng ca chng, tc tng hp protein ca nm men cao gp 1000 ln so vi u tng v gp 100 000 ln so vi tru b. Nng lc chuyn ha sinh ha mnh m ca VSV dn n cc tc dng v cng to ln ca chng trong thin nhin cng nh trong hot ng sng ca con ngi. 1.3. Kh nng sinh sn nhanh Chng hn, 1 trc khun i trng (Escherichia coli ) trong cc iu kin thch hp ch sau 12-20 pht li phn ct mt ln. Nu ly thi gian th h l 20 pht th mi gi phn ct 3 ln, sau 24 gi phn ct 72 ln v to ra (4 722 366. 1017) t bo- tng ng vi 1 khi lng ... 4722 tn. Tt nhin trong t nhin khng c c cc iu kin ti u nh vy ( v thiu thc n, thiu oxy, d tha cc sn phm trao i cht c hi...). Trong ni ln men vi cc iu kin nui cy thch hp t 1 t bo c th to ra sau 24 gi khong 100 000 000- 1 000 000 000 t bo. Thi gian th h ca nm men di hn, v d vi men ru (Saccharomyces cerevisiae) l 120 pht. Vi nhiu vi sinh vt khc cn di hn na, v d vi to Tiu cu ( Chlorella ) l 7 gi, vi vi khun lam Nostoc l 23 gi...C th ni khng c sinh vt no c tc sinh si ny n nhanh nh vi sinh vt. y l c im quan trng c con ngi li dng sn xut nhiu sn phm hu ch nh ru, bia, tng chao, m chnh, cc cht khng sinh...

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Vi kun Escherichia coli

Nm men Saccharomyces cerevisiae

Nm si Alternaria

Vi to Chlorella

Hnh 2.3: Mt s vi sinh vt c s dng trong VSVHCN

1.4. Kh nng thch ng rt cao v pht sinh bin d mnh Trong qu trnh tin ho lu di vi sinh vt to cho mnh nhng c ch iu ho trao i cht thch ng c vi nhng iu kin sng rt khc nhau, k c nhng iu kin ht sc bt li m cc sinh vt khc tgng khng th tn ti c. C vi sinh vt sng c mi trng nng n 1300C, lnh n 0-50C, mn n nng 32% mui n, ngt n nng mt ong, pH thp n 0,5 hoc cao n 10,7, p sut cao n trn 1103 at. hay c phng x cao n 750 000 rad. Nhiu vi sinh vt c th pht trin tt trong iu kin tuyt i k kh, c loi nm si c th pht trin dy c trong b ngm t thi vi nng Formol rt cao... Vi sinh vt a s l n bo, n bi, sinh sn nhanh, s lng nhiu, tip xc trc tip vi mi trng sng ... do rt d dng pht sinh bin d. Tn s bin d thng mc 10-5-10-10. Ch sau mt thi gian ngn c th to ra mt s lng rt ln cc c th bin d cc h h sau. Nhng bin d c ch s a li hiu qu rt ln trong sn xut. Nu nh khi mi pht hin ra penicillin hot tnh ch t 20 n v/ml dch ln men (1943) th nay c th t trn 100 000 n v/ml. Khi mi pht hin ra acid glutamic ch t 1-2g/l th nay t n 150g/ml dch ln men (VEDAN-Vit Nam). 1.5. Phn b rng, chng loi nhiu Vi sinh vt c mt khp mi ni trn Tri t, trong khng kh, trong t, trn ni cao, di bin su, trn c th, ngi, ng vt, thc vt, trong thc phm, trn mi vt... Vi sinh vt tham gia tch cc vo vic thc hin cc vng tun hon sinh-a-ho hc (biogeochemical cycles) nh vng tun hon C, vng tun hon N, vng tun hon P, vng tun hon S, vng tun hon Fe...

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Trong nc vi sinh vt c nhiu vng duyn hi (littoral zone), vng nc nng (limnetic zone) v ngay c vng nc su (profundal zone), vng y ao h (benthic zone)... Trong khng kh th cng ln cao s lng vi sinh vt cng t. S lng vi sinh vt trong khng kh cc khu dn c ng c cao hn rt nhiu so vi khng kh trn mt bin v nht l trong khng kh Bc cc, Nam cc... Hu nh khng c hp cht carbon no (tr kim cng, grapht...) m khng l thc n ca nhng nhm vi sinh vt no (k c du m, kh thin nhin, formol. dioxin...). Vi sinh vt c rt phong ph cc kiu dinh dng khc nhau : quang t dng (photoautotrophy), quang d dng (photoheterotrophy), ho t dng (chemoautotrophy), ho d dng (chemoheterotrophy), t dng cht sinh trng (auxoautotroph), d dng cht sinh trng (auxoheterotroph)... 1.6. L sinh vt xut hin u tin trn tri t Tri t hnh thnh cch y 4,6 t nm nhng cho n nay mi ch tm thy du vt ca s sng t cch y khong 3,5 t nm. l cc vi sinh vt ho thch cn li vt tch trong cc tng c. Vi sinh vt ho thch c xa nht c pht hin l nhng dng rt ging vi Vi khun lam ngy nay. Chng c J.William Schopf tm thy ti cc tng c min Ty Australia. Chng c dng a bo n gin, ni thnh si di n vi chc mm vi ng knh khong 1-2 mm v c thnh t bo kh dy. Trc cc nh khoa hc cng tm thy vt tch ca chi Gloeodiniopsis c nin i cch y 1,5 t nm v vt tch ca chi Palaeolyngbya c nin i cch y 950 triu nm.

Vt tch vi khun lam Vt tch Vt tch Gloeodiniopsis Palaeolyngbya Cyanobacteria cch y 3,5 t nm cch y 1,5 t nm cch y 950 triu nm Hnh 1.2: Cc vi sinh vt ho thch c xa

(cn li vt tch trong cc tng c min Ty Australia)

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2. Nhng im khc bit gia cc t bo sinh vt nhn s v nhn chun Cc sinh vt nhn s v nhn chun c mt s c im ging nhau: u c t bo l n v cu to, chc nng l n v di truyn; vt cht di truyn trong cc t bo l DNA xon kp cng cc sn phm ca n l RNA, protein... Tuy nhin gia chng c nhiu nt sai khc rt r. Thm ch ngay c cc t bo nhn chun nh nht cng khc bit mt cch cn bn so vi cc t bo nhn s trong cu to, cch t chc thng tin di truyn cng nh trong cc kiu tng hp RNA v protein ca chng (bng 2.1).
Bng 2.1: Nhng sai khc gia cc t bo sinh vt nhn s v nhn chun c im Sinh vt nhn s Sinh vt nhn chun 1. T chc di truyn - Mng nhn Khng c C - S nhim sc th khc nhau 1 >1 - Cc nhim sc th cha Khng c C histon - Hch nhn Khng c C - Trao i di truyn Mt chiu, qua plasmid Bng s kt hp giao t 2. Cc cu trc ca t bo - Li ni cht Khng c C - B my Golgi Khng c C - Cc lysosome Khng c C - Cc ty th Khng c C - Cc lp th Khng c C thc vt - Kch thc ribosome 70 S 80 S - Si thoi v sc Khng c C -Vch t bo cha C, ngoi tr Khng c peptidoglycan Mycoplasma v vi khun c 3. Mt s c tnh chc nng - Thc bo Khng c i khi c - m bo (ung bo) Khng c i khi c - V tr vn chuyn in t Mng t bo Mng bo quan - Dng t bo cht Khng c C (Ngun: Stanier et al, 1976; dn theo Watson et al, 1987. p. 97)

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[A]

[B] Hnh 2.2: Cu trc t bo vi khun [A] ; t bo ng vt [B]

II. C s ha sinh ca vi sinh vt hc cng nghip

1. ng phn. ng phn l qu trnh phn hu phn t glucose (C6H12O6) to thnh acid pyruvic v NADH+ H+. im c bit ca ng phn l khng phi phn t glucose t do b phn hu m phn t ng glucose c hot ho bi vic gn gc P vo to dng ng phosphate. Qu trnh ng phn gm 2 giai on vi nhiu phn ng phc tp: - Phn ct phn t glucose thnh 2 phn t triose l AlPG v PDA.

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- Bin i 2 phn t triose thnh 2 phn t acid pyruvic. Qu trnh ng phn c th tm tt theo s sau:

Hnh 2.3. S ng phn

Kt qu ca ng phn c th tm tt l: C6H12O6 + 2NAD+ + 2ADP + 2H3PO4 2CH3COCOOH + 2NADH+H+ + 2ATP Trong h hp hiu kh, acid pyruvic tip tc phn hu qua chu trnh Krebs, cn 2NADH+H thc hin chui h hp to H2O. 2NADH+H+ + O2 2NAD+ + 2H2O Phn ng ny km theo vic tng hp c 6ATP qua qu trnh phosphoryl ho.

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Vy kt qu ca ng phn trong h hp hiu kh l: C6H12O6 + O2 2CH3COCOOH + 2H2O ng thi to ra c 8 ATP Trong h hp k kh, 2NADH+H+ s c dng kh CH3COCOOH (trong ln men lactic) hay kh CH3CHO (trong ln men ru) nn khng thc hin chui h hp. Phn ng ln men lactic nh sau: 2CH3COCOOH + 2NADH+H+ 2CH3CHOHCOOH + 2NAD+ Vy kt qu ca ng phn trong h hp k kh l C6H12O6 2CH3CHOHCOOH Qu trnh ny ch to ra c 2ATP 2. Chu trnh Krebs. Sn phm ca ng phn l acid pyruvic s c decarboxyl ha to acetyl-CoA v mt phn t CO2. Acetyl-CoA tip tc phn hu qua chu trnh Krebs trong h hp k kh. Qu trnh phn hu acid pyruvic qua chu trnh Krebs c thc hin ti ty th do nhiu h enzyme xc tc. Chu trnh xy ra qua 2 giai on: - Phn hu acid pyruvic. Trong qu trnh ny s to ra nhiu coenzyme kh (NADH+H+, FADH2). - Thc hin chui h hp qua cc coenzyme kh c to ra do phn hu acid pyruvic. Chu trnh Krebs c tm tt qua s sau:

(a) (b) Hnh 2.4: a) Krebs, Sir Hans Adolf (1900-1981); b) S minh ho chu trnh Krebs

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T phn t acid pyruvic qua chu trnh to ra CH3COCOOH + 3H2O 3CO2 + 5H2 (4NADPH +H+ + 1FADH2) Cc coenzyme (NADH + H+, FADH2) thc hin chui h hp: 5H2 + 5/2O2 5H2O Vy kt qu chu trnh s l: CH3COCOOH + 5/2O2 3CO2 + 2H2O T 1 phn t glucose qua ng phn to ra 2 phn t acid pyruvic vi kt qu phn tch trn. T 2 acid pyruvic qua chu trnh Krebs to ra: 2CH3COCOOH + 5O2 6CO2 + 4H2O Kt hp vi giai on ng phn, ta c kt qu tng qut ca qu trnh phn hu glucose qua h hp hiu kh l: C6H12O6 + 6CO2 6CO2 + 6H2O Trong qu trnh phn hu acid pyruvic qua chu trnh Krebs s to c 4 NADH+H+ v 1 FADH2. Cc coenzyme kh ny qua chui h hp s tng hp ATP vi kt qu: -4NADFH+H+ to ra 12 ATP -1FADH2 to ra 2ATP -Trong chu trnh to ra 1ATP. Nh vy, phn hu 1 phn t acid pyruvic qua chu trnh Krebs to ra 15ATP. Nu phn hu 2 acid pyruvic s to ra 30ATP, kt hp vi giai on ng phn th phn hu 1 phn t glucose to ra c 38ATP. 3. Chui h hp v phosphoryl ho 3.1. Chui h hp Trong t bo s trao i nng lng lun gn vi phn ng oxi hokh. Trong h thng oxi ho kh hai phn ng oxi ho v kh lun i km nhau. AH2 + B A + BH2 Trong t bo phn ng trn xy ra thng cn h thng cc cht truyn in t v H+ trung gian, l h enzyme oxi ho-kh. Cc enzyme ny cng vi c cht hot ng trong mt chui phn ng cht ch chuyn H2 t c cht n O2 to nn chui h hp. Khi u ca chui l c cht dng kh AH2. AH2 lm nhim v l cht cho H2. H2 tch ra t c cht c h thng cc coenzyme ca h enzymee oxi ho-kh vn chuyn n khu cui cng ca chui l O2 kh O2 to phn t H2O.

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Trong chui h hp in t c chuyn t c cht l cht c nng lng cao nht n oxi c nng lng thp nht, th oxi ho cao nht (+0,81V). Gia hai thnh phn trn l cc coenzyme c th oxi ho-kh trung gian, th kh gim dn t c cht n O2. Bi vy chui h hp l qu trnh gii phng nng lng. Nng lng thi ra trong chui h hp c xc nh theo phng trnh: G = -nF. Eo (kCal/mol) Trong : G : mc bin i nng lng ca phn ng oxi ho-kh n: s in t trao i trong phn ng. F: s Faraday (23,06) (hng s Faraday). Eo: chnh lch th oxi ho-kh ca 2 cht tham gia phn ng. Vi phng trnh trn c th xc nh c nng lng thi ra ca tng phn ng trong chui trn c s th oxi ho-kh ca cc h xc nh. 3.2. Phosphoryl ho Qu trnh tng hp ATP trong t bo l qu trnh phosphoryl ho: ADP + H3PO4 ATP + H2O Phn ng ny i hi nng lng tng ng nng lng ca lin kt cao nng th nht (7,3 Kcalo/mol - trong iu kin chun). Tu ngun nng lng cung cp m c cc hnh thc phosphoryl ho quang ha (xy ra trong quang hp) v phosphoryl ha oxi ha (xy ra trong h hp). Trong h hp c hai hnh thc tng hp ATP - Phosphoryl ho mc c cht: l qu trnh tng hp ATP nh nng lng thi ra ca phn ng oxi ho trc tip c cht.

- Phosphoryl ho qua chui h hp: l qu trnh tng hp ATP nh nng lng thi ra ca cc phn ng trong chui h hp. Chui h hp xy ra nhiu phn ng, phn ng no tho mn cc iu kin ca qu trnh phosphoryl ho th qu trnh tng hp ATP xy ra . Trong chui h hp c 3 v tr iu kin tng hp ATP. Nh vy, c vn chuyn c H2 t c cht n O2 s to ra c 3 ATP cho t bo.

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4. S trao i protein, lipid, acid nucleic trong t bo vi sinh vt 4.1. S trao i protein 4.1.1. S tng hp protein Tng hp protein l qu trnh rt phc tp nhng c vai tr rt quan trng trong hot ng sng ca VSV. Con ng tng hp protein ch yu l tng hp theo c ch khun, tc l qu trnh gii m. Phn t protein c m ho trong gene theo m b ba: c 3 nucleotide trn mch khun ca gene m ho mt amino acids trn cu trc bc I ca protein. B ba m gc trn gene c phin m sang b m ho trn mRNA thng qua qu trnh phin m - tng hp mRNA. T mRNA l khun chui polypeptid-protein bc I s c tng hp ti ribosome. Qu trnh tng hp protein xy ra qua nhiu giai on: - Hot ho amino acids nh ATP v gn amino acids vo tRNA; - Tng hp chui polypeptid ti ribosome; - Hon thin phn t protein. 4.1.2. Phn hu protein Protein trong t bo VSV lun lun c i mi. Bi vy qu trnh phn hu protein xy ra thng xuyn cng vi qu trnh tng hp. S phn hu protein xy ra qua 2 giai on - Thy phn protein thnh cc amino acids nh enzyme thy phn protease. - Cc amino acids khng cn thit s b phn hu tip hay chuyn ho thnh cc amino acids cn thit khc. S phn hu amino acids xy ra bng nhiu con ng: kh amin, kh cacboxyl, chuyn v amin. 4.2. Trao i acid nucleic.

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4.2.1. Tng hp acid nucleic Tng hp acid nucleic l qu trnh quan trng trong c ch truyn t thng tin di truyn. Tng hp acid nucleic gm tng hp DNA v qu trnh tng hp RNA. * Sao chp DNA: T 1 phn t DNA gc qua sao chp to ra 2 phn t DNA mi hon ton ging nhau v ging DNA gc. Qu trnh sao chp xy ra bng nhiu c ch trong c ch bn bo th l ph bin v quan trng nht. Trn DNA khun, hai chui c chiu ngc nhau do vy qu trnh sao chp xy ra trn hai chui khc nhau. - Trn chui c chiu 3-5 ca DNA gc: sau khi enzymee helicase tho xon, tch 2 mch ca phn t DNA gc, trn mch 3-5 tin hnh tng hp mt on RNA mi ngn nh primase xc tc. T RNA mi cc nucleotide mi tip tc n ko di chui theo chiu 5-3 nh DNA-polymerase III xc tc. Qu trnh ni cc nucleotide t do vo mch mi theo nguyn tc b sung vi mch gc. Sau khi tng hp b sung xong c mch, on RNA mi b ct b v thay vo bng mch DNA tng ng nh DNA-polymerase I xc tc. - Trn mch c chiu 5-3 ca DNA gc: do trn mch ny chiu tng hp mch mi ngc chiu tho xon nn din ra phc tp hn. Qu trnh tng hp mch b sung xy ra theo tng on ngn. C m xon mt on vi trm nucleotide qu trnh tng hp xy ra ngc chiu tho xon theo tun t tng hp on RNA mi ri tng hp tip on DNA. Tng hp xong on ny, tho xon tip v li tng hp nh on trc . C nh vy trn mch ny DNA c tng hp theo tng on ngn-on Okazaki. Sau cc RNA mi c ct b, thay vo cc on DNA tng ng, nh ligase ni cc on Okazaki li. * Phin m: xy ra qua 2 giai on. Giai on u phin m t gene thnh pro RNA. Sau t pro-RNA s bin i thnh mRNA tng ng. RNA tng hp trn DNA khun hay trn RNA khun (vi virus cha RNA). DNA khun nh RNA polymerase tho xon cc b to nn bng phin m. Bng phin m c chiu di 30 cp nucleotide. Nh yu t sigma () ca RNA-polymerase nhn bit im m u v chui DNA dng lm khun. Nh li enzyme polymerase tin hnh tng hp chui RNA b sung vi chui khun trn DNA. Qu trnh tip din cho n khi yu t rho () nhn bit im kt thc trn DNA v kt thc qu trnh

20

tng hp chui RNA b sung. RNA tch khi DNA khun to ra proRNA. T proRNA qua nhiu bin i phc tp to mRNA trng thnh. - Ni thm m; - Ni thm ui; - Nh enzyme ct ghp tin hnh ct b cc on khng m ho intron (I) v ni cc on m ho exon (E) li vi nhau s to ra mRNA. Kt qu l t 1 on DNA (gene) to nn mt phn t mRNA. Thnh phn trt t cc nucleotide trn cc on exon ca DNA qui nh thnh phn, trt t cc ribonucleotide trn mRNA. 4.2.2. Phn hu acid nucleic Trong t bo acid nucleic lun bin i, nht l cc phn t RNA. i sng cc phn t mRNA, tRNA rt ngn. Chng thng xuyn b phn hu thay th cc loi mi. S phn hu acid nucleic xy ra qua 3 giai on: - Thy phn acid nucleic thnh nucleotide nh cc nuclease tng ng xc tc; - Thu phn nucleotidee thnh cc n phn (base-nitrogene, ng pentose v H3PO4) nh enzyme thu phn nucleotidease xc tc; - Bin i tip base-nitrogene, ng pentose thnh cc sn phm khc nhau. 4.3. Trao i lipid 4.3.1. Tng hp lipid Trong t bo lipid ph bin nht l cht bo. Cht bo c tng hp t glycerin v cc acid bo qua cc bc: - Glycerin + ATP glycero-P + ADP - Glycero-P + acid bo 1 monoglyceric. - Monoglyceric + acid bo 2 diglyceric. - Diglyceric + acid bo 3 Triglyceric + H3PO4. 4.3.2. Phn hu lipid. Qu trnh phn hu cht bo xy ra qua 2 giai on - Thu phn acid bo thnh glycerin v cc acid bo. - Phn hu tip glycerin v cc acid bo. + Glycerin + ATP glycero-P + ADP

21

+ Glycero-P AlGP ng phn. Cc acid bo phn hu bng nhiu con ng trong ph bin nht l phn hu theo con ng -oxi ho. Cc acid bo phn hu theo con ng -oxi ho to nn cc acetyl-CoA. T acetyl-CoA phn hu tip bng chu trnh Krebs. S phn hu acid bo to ra nng lng ATP kh ln cho t bo VSV. V d khi phn hu acid palmitic to ra c 130 ATP. III. C s di truyn vi sinh vt Nh chng ta u bit, cho n u thp nin 1940 cc VSV mi bt u c s dng trong nghin cu di truyn hc. T y hnh thnh nn di truyn hc VSV v di truyn sinh-ha, m ng cho s ra i ca di truyn hc phn t (1953) v cng ngh DNA ti t hp sau ny (1972). 1. Di truyn hc virus Theo R.M. Atlas (1994) th Virus l mt thc th v bo c cha mt lng ti thiu protein v acid nucleic m ch c th sao chp sau khi xm nhp vo nhng t bo sng chuyn bit. Chng khng c qu trnh trao i cht ni ti, s sao chp da vo vic iu khin trao i cht t bo nh h gene ca virus. Trong t bo ch cc thnh phn ca virus c tng hp ring r, ri lp rp thnh dng thnh thc. 1.1. c im v cu to ca virus - Virus l cc th sng cha c cu to t bo, k sinh ni bo bt buc. Mi kiu virus c mt phm vi vt ch nht nh. Chng nhn din t bo ch theo nguyn tc kha v cha kha gia cc protein v ca n vi cc im nhn trn b mt mng t bo. Cc virus ca vi khun gi l bacteriophage (th thc khun), gi tt l phage. - Virus khng c h thng sinh nng lng, khng c ribosome, khng c h thng bin dng ring v do vy cc virus khng tng trng. - Virus khng to mng lipid ring; - Virus khng c khung sn t bo; - Virus khng b tc ng bi cc cht khng sinh; - Mi ht virus thng gm 1 phn t acid nucleic trong, gi l li, v lp v (capsid) bao bn ngoi l cc protein (t hp theo cc n v gi l capsomere), c mang cc thnh phn khng nguyn. Kt cu c bn gi l nucleocapsid.

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Hnh 2.5. Virus trn v virus c v ngoi

H gene ca virus rt c bit (bng 2.2), mi loi virus ch cha mt loi acid nucleic, hoc l DNA hoc l RNA, chui n hay chui kp, dng si thng hay dng vng. Virus b nht khong 4 gene v ln nht khong vi trm gene. Mt s virus ngoi v protein cn c thm vi lp mng bao khc gm (protein + phospholipid) bt ngun t mng sinh cht t bo ch, hoc c (protein + glycoprotein) t virus HIV, virus SARS, H5N1 l mt v d.
(HIVgy hi chng suy gim min dch mc phi (HCSGMDMP); Virus SARS gy hi chng vim ng h hp cp (VHHC); H5N1 gy bnh cm g) Bng 2.2. Phn bit qu trnh truyn bnh AIDS, SARS, CM G Bnh Acid nucleic RNA RNA RNA Tn virus Vt ch Phng thc lan truyn -Qua mu -Quan h tnh dc -M sang con -H hp -H hp, tiu ha

AIDS SARS CM G

HIV1, HIV2 SARS H5N1

Ngi, kh Cy hng, ngi Gia cm

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Hnh 2.6: Virus v ng truyn bnh AIDS, SARS, CM G Bng 2.3: Dng acid nucleic v kch thc h gene mt s virus Virus Phage MS2, f2, R17 Virus TMV Cc Reovirus Phage X174 Virus SV40 Baculovirus Adenovirus Ad5 Phage (Lambda) Phage T2, T4 Vt ch E. coli Thuc l Th E. coli Th Cn trng Ngi E. coli E. coli Dng acid nucleic RNA si nthng RNA si kpthng DNA si nvng DNA si kpvng DNA si kpthng Kch thc h gene ( s cp base) 3.569 ~ 6.000 ~ 50.000 5.375 5.243 (100-150)x103 ~ 36.000 48.502 ~ 200.000

Ghi ch: Cc virus gy bnh ngi v ng vt c h gene ch yu l DNA si kp-thng, nh: cc virus thuc h Adenoviridae gy bnh ng h hp, h Herpesviridae gy mn rp herpes ngi, bnh u g, h Poxviridae gy u ma, u b ... Hoc l RNA si n-thng, nh cc virus thuc h Retroviridae

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gy ung th, AIDS, bch cu ..., h Paramyxoviridae gy si, quai b, bnh g toi, h Rhabdoviridae vi bnh di, h Orthomyxoviridae vi bnh cm, h Picornaviridae vi cc bnh vim ty xm, vim gan A do virus, bnh l mm long mng tru b...

1.2. Phng thc sinh sn v vng i ca virus y chng ta s tm hiu nhng nt chung nht trong chu trnh sng-sinh sn ca virus qua i din l cc phage k sinh E. coli. Thng thng c chia lm 5 giai on: Hp th Xm nhp Sao chp Thnh thc Phng thch, hoc 3 giai on nh sau: (1) Phage bm bng phn ui trn b mt t bo vi khun v tit enzyme tiu hy vch t bo; (2) Phage dng phn ui tim li acid nucleic (DNA) vo trong t bo ch; (3) Trong t bo ch, ty loi DNA cc phage khc nhau m c th din ra mt trong hai trng thi: gy tan hoc tim tan. + i vi cc phage c nh T2 v T4, lc ny DNA ca chng s sao chp nhiu ln v tng hp cc protein cn cho to v v ui, sau chng lp rp vi nhau to ra cc phage th h con. Sau , di tc dng ca lysozyme lm tan t bo ch v phng thch chng 100-200 phage con.

Hnh 2.7: Chu trnh hot ng gy tan (lytic cycle) v tim tan (lysogeneic cycle) ca phage E. coli.

+ i vi phage n ha nh lambda (), c th gy tan hoc khng gy tan. Trng hp tim tan (lysogeney) xy ra l do: sau khi chui vo t bo ch, DNA ca phage t ng vng ( nh c cc u dnh v xc tc

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ca ligase), ri xen vo nhim sc th chnh ca vi khun bng s ti t hp v tr c hiu v n tim n trng thi , gi l tin virus (prophage). Tuy nhin, cc prophage ny c th hot ng tr li gy tan. Chng hn, nu c tc ng ca tia UV gy tn thng nhim sc th vt ch th mt trao i cho ngc tr li s xy ra, nh vy DNA phage c tch ra khi nhim sc th vi khun ch v bt u chu trnh sinh sn gy tan. + i vi virus thuc nhm retrovirus (virus gy ung th, gy bnh AIDS, gy bnh bch cu t bo T...) c chu trnh sng in hnh (hnh 2.6).

Hnh 2.8: Chu trnh sng ca virus nhm retrovirus

y cn hai im quan trng lin quan cng ngh gene: + Ti t hp: Khi c nhiu virus nhim ng thi t bo ch th gia chng xy ra s trao i gene, to ra cc th ti t hp. Nh vy c th lp bn di truyn virus. Dng ti t hp im c hiu gn DNA phage vo nhim sc th vi khun ch v tch ra ( khi b cm ng tia UV) c xc tc bi cp enzyme tng ng l integrase v excisionase. Li dng c im ny, ngi ta s dng phage lm vector trong k thut gene. + Sao chp: Do vt liu di truyn ca cc virus rt a dng, nn s sao chp/sao chp ca chng cng khc nhau, theo 3 con ng:

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DNA DNA; RNA RNA; v RNA cDNA kp RNA. Trong , cc retrovirus c h gene RNA gy ung th, bnh AIDS (HIV), bnh bch cu t bo T (HTLV-I)...u c cha enzyme phin m ngc (reverse transcriptase) tng hp cDNA si n trn khun RNA ca n, ri sau l cDNA si kp c th xen vo nhim sc th vt ch trng thi provirus n nh. Li dng enzyme ny tng hp v to dng cDNA. + Phin m:

Hnh 2.8: S minh ha qu trnh phin m mt s virus

2. Di truyn hc vi khun 2.1. Vt liu di truyn ca vi khun Vi khun l mt nhm ln ca Prokaryote, c cu trc t bo nhng cha c nhn in hnh. B my di truyn ca vi khun phc tp hn virus nhiu, thng gm 1 phn t DNA si kp-vng kch thc ln (v d E. coli l 4,6 x106 cp base). N lin kt vi cc protein tng t histon to thnh cu trc siu xon tp trung mt vng gi l vng nhn (nucleoid). V trong dch bo c rt nhiu phn t DNA trn si kp, dng vng, kch thc b bng khong 0,05-10% kch thc nhim sc th vi khun v c kh nng sao chp c lp, gi l cc plasmid. E. coli c nhiu loi plasmid vi tnh cht v s bn sao c mt khc nhau trong t bo, y cp 2 loi: - Cc plasmid khng thuc, gi l plasmid R (R-resistance), thng c mang nhiu gene m ha cc enzyme c kh nng phn hy cht khng

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sinh tng ng c mt trong mi trng. V d, plasmid pBR322 (4362 cp base) c 2 gene khng ampicilline (AmpR) v tetracyline (TetR). Loi plasmid ny thuc loi sao chp mnh, c th to ra khong 50 bn sao (copies) trong mt t bo. l nhng c im chnh m ngi ta li dng n lm cng c c lc (vector) trong k thut di truyn. - Cc plasmid gii tnh, tc plasmid F (F-fertility) c cha cc gene truyn v bt buc tham gia vo qu trnh tip hp E. coli. Cc t bo vi khun c mang plasmid F, k hiu F+ v t bo khng c F, k hiu F-. Nhn t F c kch thc ln (khong 100.000 cp nucleotide), c sao chp ch 1 ln trong chu k t bo (nh xen vo trong nhim sc th vi khun) v phn ly v c 2 t bo con. Cho nn trong 1 t bo F+ in hnh thng c 1-2 bn sao ca plasmid F. 2.2. Ba phng thc trao i vt liu di truyn vi khun Ln u tin, nm 1946 Lederberg v Tatum pht hin ra s ti t hp vi khun. Cho n nay, ta bit rng vi khun c cc qu trnh sinh sn tng ng sinh sn hu tnh, gi l cn hu tnh (parasexuality), l: tip hp, bin np v ti np. Cc qu trnh ny c cc c im sau: (1) Truyn thng tin 1 chiu t t bo cho (donor) sang t bo nhn (recipient); (2) To ra hp t tng phn (merozygote), v th cho ch truyn sang th nhn mt phn b gene ca n; (3) V b gene ch l mt phn t DNA trn nn ch c mt nhm lin kt v ti t hp v thc cht l lai phn t. Nhng hiu bit su sc v cc qu trnh sinh sn cn hu tnh vi khun gp phn quan trng trong s pht trin ca di truyn hc phn t cng nh ca k thut gene sau ny. a) Tip hp (Conjugation) Tip hp l s tip xc trc tip gia hai t bo vi khun, trong din ra s truyn i mt phn vt liu di truyn t th cho sang th nhn. y, nhn t F c truyn t t bo F+ sang F- trong qu trnh tip hp, nh kiu sao chp vng ln (rolling circle) hay cn gi l sao chp sigma (). Kt qu ca s tip xc l t bo F- cng tr thnh F+, ngha l c s thay i gii tnh (ci c). Tuy nhin, tn s lai F+ x Frt thp, khong 10-6. V sau, ngi ta cn pht hin mt dng vi khun, trong plasmid F c xen ci vo trong nhim sc th vi khun, dng ny c kh nng lai vi t bo F- vi tn s cao hn khong 104 ln, gi l t bo Hfr (High

28

Frequency of recombination). Khc vi cc t bo F+, cc t bo Hfr cn c th truyn i mt phn nhim sc th vi khun qua ng tip hp. Li dng c tnh ny ngi ta lp bn di truyn bng tip hp cho hn 700 gene ca nhim sc th E. coli v cho thy n c dng vng. b) Bin np ( Transformation) Bin np l hin tng xm nhp ca DNA ngoi bo vo t bo th nhn v gy bin i mt s c tnh ca th nhn bi nh hng ca DNA th cho. Hin tng bin np c Frederick Griffith pht hin ln u tin nm 1928 v v sau c Oswald T. Avery, Colin MacLeod v Maclyn McCarty cng pht hin li nm 1944.

Hnh 2.9: 1. Frederick Griffith; 2. Oswald T. Avery (1877-1955); 3. Colin MacLeod (1909-1972); 4. Maclyn McCarty (1911-)

Khc vi tip hp v ti np, bin np l qu trnh chuyn DNA trc tip tch ra t t bo th cho sang t bo th nhn. c trng ca bin np l ch DNA th cho phi dng xon kp. Hiu qu ca n ph thuc vo kh nng dung np ca t bo nhn, kch thc on DNA v nng DNA. T bo c kh nng tip nhn on DNA ngoi lai gi l t bo kh bin, v c th xy ra lng bi ha mt phn b gene (hp t tng phn). S gn kt on DNA ngoi lai vo DNA t bo nhn c thc hin nh c ch ti t hp tng ng. V sau khi chui qua mng t bo nhn, mt si ca on DNA ngoi lai b phn hy, cho nn nu nh on si n cn li khng c gn vo th s b phn hy lun. Ni chung, tn s xut hin cc t bo kh bin l rt thp (ngay c cc loi vi khun c kh nng bin np) v tn s bin np (i vi cc t bo kh bin) cng ch khong 10-3. V vy, bin np ch c dng lm

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k thut lp bn gene cho 1 s loi. Tuy nhin, ngy nay bin np c ng dng rt rng ri nh l mt khu c bn trong k thut di truyn: cy-chuyn gene, tim lp-ghp gene,... E. coli, nm men bia, thc vt, ng vt v c ngi. c) Ti np (Transduction) Ti np l qu trnh truyn DNA t vi khun cho sang vi khun nhn nh phage. C 2 kiu ti np: ti np chung v ti np c hiu. * Ti np chung l trng hp phage truyn bt k gene no ca vi khun cho sang vi khun nhn E. coli, phage P1 l phage ti np chung c nghin cu k. Trong khi nhim vo vi khun, phage P1 sn ra nuclease ct DNA vi khun ch thnh tng on mt cch ngu nhin. V sau, trong qu trnh lp rp v protein vi DNA phage, c khong 10-3 10-2 ht phage con mang on DNA vt ch. Khi cc phage ti np ny xm nhp vi khun khc, s xy ra s ti t hp tng ng vi NST vt ch mi. on gene ti np thng cha khong 50 gene v ti np c th c dng lp bn gene. * Ti np c hiu l trng hp phage ch truyn i nhng gene nht nh t vi khun cho sang vi khun nhn. Kiu ti np ny c mt s c im sau: c thc hin bi prophage lambda (); Nhng gene c chuyn nm st ch prophage xen vo; Do kt qu ca s ct sai ca phage khi tch khi NST vt ch; Cc vi khun ti t hp c th lng bi mt phn. Tuy nhin, do s ct sai ca phage lambda rt him nn ti np c hiu c tn s thp. 3. Di truyn hc vi nm Sau y l mt vi nt s lc v nm men. Hin nay, S. cerevisiae v Schizosaccharomyses pombe ang c nghin cu rng ri mc phn t. Chng c chu trnh sng n gin c lin quan ti tt c pha n bi (n) v pha lng bi (2n). i vi cc t bo (2n) din ra nhng phn chia gim nhim thng thng to ra cc bo t (n). Ton b 4 sn phm ca gim phn c bc bn trong mt ci nang dy c chia . iu cho php kim tra cc on NST trao i cho thun nghch ring bit. Cc t bo nm men S. cerevisiae phn chia bng qu trnh ny chi lm tch cc t bo cha m ra khi cc t bo con (chi) ca chng. Ngc li, S. pombe l nm men phn i (fission), cc t bo cha m sinh trng v sau ngt i to ra 2 t bo ging nhau v hnh thi.

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Trung bnh h gene n bi ca nm men S. cerevisiae c cha khong 14 x 106 cp base, ngha l c hm lng DNA ln hn E. coli khong 3,5 ln, trong khi rui gim Drosophila ln gp 25 ln v cc t bo thc vt bc cao cha nhiu gp t nht l 1.000 ln. Nh vy, dng nm men trong nghin cu di truyn nhn chun qu l n gin v r hn. S. cerevisiae DNA c phn phi trong 16 NST vi khong 5 - 6 ngn gene, trong khi S. pombe ch c 3 NST khc nhau. Tnh n 1996, c 6000 gene ca S.cerevisiae c lp bn . iu ng ni l nhiu t bo nm men cng c cc bn sao phc ca cc DNA-plasmid ni sinh dng vng 2 m (6300 cp base) c kh nng sao chp c lp, thng c 50 - 200 bn sao trn mt t bo. Bnh thng chng khng mang mt gene thit yu no. y l loi vector quan trng trong k thut di truyn. Trong s rt nhiu loi nm men c ng dng rng ri trong cng nghip sn xut bia, ru, nc gii kht, to sinh khi ..., loi S. cerevisiae hin ang c dng nh mt cng c c lc mang cc DNA ti t hp c cc gene quan trng ca ng vt v ngi, nhm sn xut cc protein c hot tnh sinh hc, nh: cc interferon, insulin, hormon sinh trng, hirudin, cc khng nguyn virus v.v.. dng trong chn nui, phng v cha bnh. 4. Cc sinh vt mang gene ti t hp 4.1. Cc vi sinh vt ti t hp Mt trong nhng ng dng u tin ca k thut di truyn l to ra mt chng Pseudomonas syringae. Chng hoang di ca vi khun ny thng thng cha mt gene to bng, gene ny kch thch s to bng trn cc b mt lnh v m. S bin i gene ny to ra mt th ti t hp c kh nng ngn nga s to thnh bng. c to ra di tn gi l Frostban, vi khun ny mang li mt thnh cng khim tn trong vic chng li vic to thnh bng trn cc cy du ty v khoai ty ngoi ng rung. Pseudomonas fluorescens c ti t hp vi cc gene sn sinh cht dit cn trng sinh hc ca Bacillus thuringiensis. Cc vi kun ny c th vo t, chng s bm vo r v gip tiu dit cc cn trng ang tn cng. Nm 1979 xut hin thng bo u tin v s biu hin gene insulin ngi sau khi c gp vo vi khun E. coli, nm 1982 insulin c sn xut bng k thut ny c bn ra trn th trng vi tn thng phm Humulin (Human insulin). Hin nay ngi ta dn thay th vi khun E. coli mang gene ti t hp bng cc VSV khc nh nm men thuc chi Saccharomyces, x khun- Streptomyces....

31

4.2. Cc thc vt ti t hp Tham gia vo hu ht cc thao tc ca k thut di truyn thc vt c mt loi vi khun hp dn, l Agrobacterium tumefacien. Vi khun ny l mt tc nhn gy bnh t nhin thc vt, n xm nhp vo r v to nn mt khi u c gi l bnh mn tn. A. tumefacien cha mt plasmid ln (Ti), n c gn vo gene nom ca cc t bo r b nhim khun v lm bin i cc t bo ny. Ngay c khi cc vi khun b cht trong khi u th khi u vn tip tc pht trin. Plasmid ny l mt vector hon ho a cc gene l vo gene nom thc vt. Trong a s trng hp, Plasmit Ti c ly ra, ghp vo mt gene c la chn ri li a tr li vo Agrobacterium.

Hnh 2.10: Khi u thc vt cha A.tumefacien cha Ti plasmid

Khi thc vt b nhim khun, cc vi khun ti t hp s t ng chuyn gene mi vo cc t bo r ca cy. Vic gn gene theo cch ny lc u c tin hnh cc cy hai l mm nh khoai ty, c chua, thuc l v bng. Cho n nay, cc thc vt ti t hp, hay chuyn gene, ch yu l cc cy bng hoc thuc l khng vi cht dit c, cc thc vt c kh nng dit cn trng, nhiu loi khng vi virus hoc vi nm v mt loi c chua khng chn qu sm v khng b v khi vn chuyn. 4.3. Cc ng vt ti t hp Khng ch virus, vi khun, nm v thc vt m c ng vt cng c th c thit k v mt di truyn. Trong mt s iu kin ng vt cng c th biu hin cc gene c gn nhn to bi chuyn nhim. Cc ng vt u tin c chuyn gene l nhng con rui gim m nhng khuyt

32

ht v mu mt ca chng c sa cha nh vic a cc gene bnh thng vo trng khim khuyt. Chut l nhng ng vt c bit d bo trong vic chp nhn gene theo cch ny. Phi ca chut th tinh c cy cc gene quy nh cc hocmon sinh trng ca ngi sinh ra cc con chut khng l ln gp hai so vi chut bnh thng. Trong mt th nghim khc, cc hp t ca chut do mang mt khim khuyt di truyn thng lm cho chng b bnh run, c sa cha nh cha gene bnh thng mi c a vo. Ngnh chn nui nhanh chng tip thu mt s trong s k thut ny. Nm 1987, phi ln c cy bng gene hormon sinh trng ca b trong mt th nghim nhm tng tc sinh trng ca ln v ct gim lng m to thnh trong khu phn tht. Nhng con ln u tin c t m hn, song tht khng may, chng cng c nhng vn v sc kho nghim trng: vim khp, bnh tim v bnh thn. Khuynh hng gn y nht trong cc th nghim chuyn gene l thit k trng th tinh ca cc ng vt nui nh ln, cu vi cc gene ca ngi. Cc ng vt ti t hp ny s c dng lm cc h thng sng sn sinh ra mt lng ln cc protein c gi tr ca ngi nh hemoglobin, yu t ng mu v hormon.

Cu hi n tp 1. Vi sinh vt c nhng c im chung no ? 2. Hy gii thch ti sao trong thin nhin vi sinh vt tng ln
khng tng ng vi tc sinh sn v cng nhanh chn ca n. 3. Vt liu di truyn ca virus ? Mi quan h di truyn gia virus v t bo vt ch ? 4. Vt liu di truyn sinh vt nhn s khc vi sinh vt nhn chun nhng c im no ? 5. Th no l tip hp, bin np v ti np ? Ti sao ni cc qu trnh ny tng ng vi sinh sn hu tnh sinh vt bc cao ? ngha ca vic pht hin ra hin tng bin np vi khun.

33

Chng 3

S phn loi sn phm

I.Phn loi theo tnh cht thng mi
Theo Thomas D. Brock (1995), cc sn phm vi sinh vt c ngha cng nghip c phn thnh 5 loi chnh: - Bn thn cc t bo vi sinh vt (sinh khi) l cc sn phm mong mun. - Cc enzyme do vi sinh vt to nn: amylase, protease, lipase... - Cc dc phm: cc cht khng sinh v cc alcaloit. - Cc ho cht c bit v cc cht iu v thc phm: bt ngt nhn to aspartam l mt dipeptide gia aspartic v phenylalanin; acid glutamic, lysine v triptophan, mt s vitamin. - Cc ho cht thng dng c sn xut bng con ng vi sinh vt bao gm ethanol, acid acetic, acidlactic v glycerine 1. Sinh khi t bo l cc trng hp ca nm men dng cho mc ch dinh dng v lm n bt m, trng hp cc nm n dng lm thc n. Cc vi khun v vi to cng c nui cho mc ch dinh dng, song cho n nay cha c ngha ln v mt thng mi. y cn phn bit hai trng hp: Thut ng ''protein n bo'' (SCP) thng c dng ch cc t bo cc vi sinh vt nh l cc sn phm cng nghip vi l do l hm lng protein trong chng cao v c quan tm v mt thng mi. Cn ''ging khi ng'' (starter culture) l cc sn phm cng nghip trong bn thn cc t bo vi sinh vt c dng lm nguyn liu cy. Chng hn cc ging vi khun lactic c bn ra di dng cc nguyn liu cy (inoculants) sn xut cc sn phm sa ln men v xc xch. 2. Cc enzyme do vi sinh vt to nn Nhiu enzyme quan trng c ngha thng mi c sn xut quy m cng nghip nh vi sinh vt, bao gm cc enzyme phn gii tinh bt (cc amylase), cc enzyme phn gii protein- protease, cc enzyme phn gii cht bo (cc lipase) Mt enzyme quan trng v mt cng nghip l gluco-isomerase c s dng vi s lng ln sn xut fructose c ngt cao hn glucose. Mt enzyme vi sinh vt quan trng khc l penicillin -acilase c s dng trong cng nghp sn xut cc penicillin tng hp.

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3. Cc dc phm Cc cht khng sinh v cc alcaloid nn trong nhm cc sn phm bc 2. l cc hp cht khng c to thnh trong pha sinh trng u tin m vo lc sinh trng bc vo pha cn bng. Vic hiu bit bn cht ca s trao i cht bc hai c tm quan trng trong trong vic pht trin cc qu trnh sn xut mi. Cng ty Cng nghip ho cht Takeda (Nht Bn) sn xut v cung cp loi thuc khng sinh validacina dng phng tr nhiu loi bnh hi cy trng do nm gy ra. Validacina c bit c hiu lc vi bnh kh vn la, trc tip bo v, lm tng nng sut v cht lng la. Thuc khng gy bt c hin tng ng c no cho cy trng, rt an ton cho ngi, vt nui, c... v nhng ng vt c ch khc; khng tn d trong t v nng sn sau khi thu hoch. Validacina c th hn hp vi hu ht cc loi thuc khc phun. Thuc validacina c cc dng thng phm: Validacin 3L, Validacin 5L, Validacin 5SP. 4. Cc ha cht c bit v cc cht iu v thc phm Cht iu v thc phm: bt ngt nhn to aspartam l mt dipeptide gia acid aspartic v phenylalanin, c hai amino acid ny u c sn xut bng con ng ln men vi sinh vt. cc amino acids khc cng c sn xut bng con ng ny l: acid glutamic, lysine v tryptophan. Mt s vitamin cng c sn xut bng con ng vi sinh vt, l riboflavin, vitamin B12 v acid ascorbic (vitamin C). 5. Cc ha cht thng dng l nhng cht c gi thnh thp v do vy c bn ra vi s lng ln. Cc ha cht thng c dng lm nguyn liu khi u tng hp ha hc cc phn t phc tp hn. Cc ha cht thng dng c sn xut bng con ng vi sinh vt bao gm ethanol, acid acetic, acid lactic v glycerol. Trong s ny ethanol l sn phm quan trng nht. Ethanol c sn xut nh nm men, bn ny c th to ra mt sn lng ethanol v CO2 t ng. Hin nay ethanol c dng lm nguyn liu tng hp ha hc v lm nhin liu chy cc ng c (gazohol).

II. S phn loi sn phm theo sinh l trao i cht

Khc vi s phn chia sn phm mang tnh thng mi (Fritsche, 1978) chia cc sn phm ca vi sinh cng nghip thnh 3 loi. 1. Vt cht t bo (sinh khi) bao gm cc loi protein n bo v cc loi ging khi ng nh men bnh m cc vi khun lactic.

35

Bng 3.1: Mt s enzyme cng nghp v ng dng Enzyme Amylase Catalase Cellulase Glucooxydase Pectinase Penicillinase Protease Streptokinase Aspergillus, Rhizopus Ngun ng dng chnh

Bacillus, B sung vo bt, cng nghp dt, tr gip tiu ha Phng oxy ha thc phm, sn xut phomat Thy phn g, tr gip tiu ha Loi glucose hoc oxy trong thc phm, xc nh glucose lm sng Lm trong vang, dm, bia, dch qu Loi penicillin trong nghin cu Bacillus, Lm trong sake, ch bin tht, loi gelatin Lm lnh cc vt thng vt bng

Micrococcus, Aspergillus Aspergillus,Trichoderma Aspergillus Aspergillus, Sclerotinia Aspergillus Aspergillus, Streptomyces Streptococcus

2. Cc sn phm trao i cht bao gm: - Cc sn phm ln men. V d ethanol, acid lactic, methane, acetolbutanol... - Cc cht trao i bc 1: V d amino acids, nucleotide, vitamins, ng,.... - Cc cht trao i bc 2: V d cht khng sinh, alcaloid, gibberellin, IAA... - Cc loi enzyme: V d cc enzyme ngoi bo nh protease, amylase; cc enzyme ni bo nh asparaginase, penicillinase. 3. Cc sn phm chuyn ho Cc sn phm chuyn ho bao gm steroids v cc sn phm ca s oxi ho khng hon ton nh s to thnh acid acetic v socbose.

II. Sinh trng v s to thnh sn phm

Trong iu kin nui cy tnh, qu trnh sinh trng ca mt qun th VSV tri qua 4 giai on (Tim pht logarit cn bng ng suy vong). Ton b qu trnh nui gn lin vi s thay i ko di ca cc

36

iu kin nui. Cht dinh dng gim i, s lng t bo tng ln ri gim dn, ng thi hot tnh trao i cht cng thay i. S lin quan ca cc sn phm trao i cht vi t bo c th phn bit thnh 2 nhm: cc sn phm bc mt v cc sn phm bc hai. Mt cht trao i bc mt l mt cht c to thnh trong pha sinh trng u tin ca VSV trong khi mt cht trao i bc hai l mt cht c to thnh gn vo lc kt thc ca pha sinh trng, thng l vo gn chnh pha cn bng.

Hnh 3.1: ng hc ca s sinh trng v to thnh sn phm VSV a. Sinh trng v to sn phm din ra ng thi b. S to thnh sn phm bt u sau khi sinh trng kt thc

*c im ca sn phm trao i bc hai: - Cc sn phm bc 2 ch c to thnh bi mt s tng i t c th v dng nh khng cn tht cho sinh trng v sinh sn. - S to thnh cc sn phm bc hai ph thuc vo cc iu kin sinh trng, c bit l thnh phn ca mi trng. - Chng thng c to thnh di dng mt nhm cc cht c cu trc gn gi. Chng hn, mt chng Streptomyces sn sinh ra 32 cht khng sinh antracyclin khc nhau nhng c cu trc gn ging nhau. - Thng c th nhn c s tng hp tha t ngt mnh cc sn phm bc hai, iu ny thng khng gp cc sn phm bc mt l loi sn phm c lin quan cht ch n pha sinh trng. Trong trao i cht bc hai c hai pha trao i cht khc bit nhau c gi l pha sinh trng (trophophase) v pha sn xut (idiophase). Nu chng ta nh sn xut cc sn phm bc hai th chng ta phi m bo rng cc iu kin thch hp cn c to ra trong pha u sinh

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trng din ra ti u v cc iu kin phi c thay i ng lc s to thnh sn phm c th din ra ti u. Trong trao i cht bc hai, sn phm mong mun c th khng bt ngun t c cht sinh trng ban u m t mt sn phm c to thnh t c cht sinh trng ban u. Nh vy, ni chung sn phm bc hai c sinh ra t mt s sn phm trung gian tch lu trong mi trng nui cy hoc trong t bo trong qu trnh trao i cht bc mt. Mt trong cc c im c trng ca cc cht trao i bc hai l cc enzyme tham gia vo s to thnh chng c iu ho mt cch c lp vi cc enzyme ca trao i cht bc mt. 3. Sinh tng hp tha Qu trnh ln men cng nghp i hi s to thnh sn phm mong mun vi lng cao nht. VSV tn ti trong t nhin sinh ra cc sn phm trao i cht v cc thnh phn t bo ch mc cn thit cho s sinh sn ti u v cho s duy tr loi. C ngha l, trong t nhin khng c s sn sinh d tha cc sn phm trao i cht bc 1, bc 2. Bi vy nhng th nghim nhm phn lp cc chng c kh nng sinh tng hp d tha t ni sng t nhin thng khng em li kt qu mong mun. Ch khi no nhng c th thch hp thu c do x l bng cc tc nhn gy t bin nh hng, c chn lc trong iu kin phng th nghim v nhng kh nng nht nh, th ta mi c th thu c cc chng tng hp tha b sai hng trong c ch iu ho. Nhng chng ny c coi l nhng chng c nng sut cao dng trong cng nghip. 3.1. Nhng nguyn tc iu ho trao i cht - iu ho hot tnh enzyme nh c ch bng sn phm cui cng hay cn gi l s km hm do lin h ngc; - S cm ng v c ch qu trnh tng hp enzyme; - iu ho tng hp enzyme nh s kim ch bng sn phm cui cng v s gii kim ch; - iu ho tng hp enzyme nh s kim ch d ho. 3.2. Nhng sai hng di truyn ca iu ho trao i cht v hin tng tng hp tha Cc c ch iu ho trao i cht c th b thay i do nhng t bin dn n s sinh tng hp tha cc cht trao i. Ton b nhng sai hng c th biu hin s cm ng enzyme. Nh nhng sai hng m cc enzyme cm ng tr thnh cc enzyme cu trc, ngha l chng tn ti trong t bo khng ph thuc vo c cht.

38

Nhiu hot tnh ca cc chng sn xut l do sai hng di truyn. Vic la chn cc chng sn xut trc ht l theo kinh nghim. Nhng thnh tu ca sinh hc phn t nh ch ng gy t bin nh hng, chuyn gp gen... to iu kin cho vic la chn nh hng hn v do c hiu qu cao hn. 4. Cng ngh DNA ti t hp Nh bit, hai s kin ni bt nht trong giai on u ca Sinh hc phn t l s khm ph ra cu trc phn t DNA nm 1953 bi James Watson v Francis Crick (nhn gii Nobel-1962) v s gii m thnh cng h thng mt m di truyn nm1966 bi Marsall Nirenberg v Har Gobind Khorana nhn gii Nobel-1968 cng vi nhiu nh khoa hc khc. Giai on tip theo ca nh cao pht trin sinh hc phn t l s ra i ca Cng ngh DNA ti t hp (recombinant DNA technology), vo gia thp nin 1970, vi cc tn gi khc nh: cng ngh gen, k thut di truyn (genetic engineering). Cng ngh DNA ti t hp bt u nh mt khoa hc t 7/1972 khi nhm nghin cu ca Paul Berg (Vin hn lm khoa hc quc gia M) hon thnh vic ch to b gen lai gia virus SV40 ca kh vi phage lambda. Ln u tin kt ni hai virus khc nhau hon ton v mt di truyn thnh mt chnh th v c nhn ln di dng plasmid trong t bo vi khun. Phn t DNA lai cha cc thng tin di truyn ca virus SV40, thng tin iu khin qu trnh t sao ca DNA phage lambda cng nh ton b chc nng ca operon galactosidase ca vi khun E. coli.

Hnh 3.2: 1. Har Gobind Khorana (1922-); 2. Temin, Howard Martin (1934-1994); 3. Daniel Nathans (1928-1999); 4. Paul Berg (1926-)

4.1. Khi nim cng ngh DNA ti t hp

39

l cng ngh bao gm mt tp hp cc k thut nh phn ct v lai ghp cc phn t nucleic acid nh cc enzyme gii hn, ligase...nhn dng DNA trong cc plasmid hoc virus c tng cng vi khun hoc t bo nm men; xc nh trnh t nucleotide DNA on c nhn dng; biu hin ca gen c nhn dng di dng sn phm protein... S ra i ca cng ngh DNA ti t hp v gn y l cng ngh chp DNA, sinh tin hc, nui cy t bo gc, cng ngh nano gn lin vi cc s kin khoa hc sau: - 1965, Xc lp c cc gen khng thuc vi khun nm trong cc plasmid. - 1967, SB Zimmerman v cng s tch chit thnh cng DNAligase. - 1970, Hamilton Smith v cc cng s phn lp c enzyme gii hn u tin t vi khun Haemophylus influenzae c gi l Hind II. -1970, Har Gobind Khorana tng hp c mt gen nhn to u tin l gen m ha vic tng hp tRNAala vn chuyn amino acid alanine nm men. Gene ny di 77 cp nucleotide, khng c cc trnh t iu ha v v th khng c hot tnh, n 1973 mt gen nhn to khc c th hot ng bnh thng trong t bo sng, l gene m ha tRNAtyr vn chuyn cu tyrosin E. coli c chiu di khong 200 cp nucleotide. - 1972, Paul Berg kin to c phn t DNA ti t hp u tin trong ng nghim. - 1973, H. Boyer, S. Cohen, A. Chang v R. Helling - ln u s dng plasmid to dng DNA E. coli. -1975, Temin, Howard Martin, pht hin ra enzyme phin m ngc (reversetranscriptase) m ra s tng hp gene nhn to.

- 1977, xut cc phng php ha hc (Maxam v Gilbert) v phng php enzyme hc (Frederick Sanger v cng s) xc nh trnh t nucleotide ca nucleic acid. - 1977, Thnh lp hng cng ngh gen u tin M (Genetech) sn xut cc ch phm y hc bng phng php DNA ti t hp.

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- 1978, Gii thng Nobel u tin trong lnh vc khm ph v s dng enzyme gii hn c trao cho Daniel Nathans; Werner Arber;
Hamilton Othanel Smith.

- 1983, Gii thng Nobel c trao cho b Barbara McClintock pht hin ra yu t di truyn vn ng hay gen nhy. - 1986, Binnig, Gerd Karl (Nh khoa hc c, gii Nobel 1986) khm ph ra loi knh hin vi hot ng khng theo nguyn tc quang hc, m u cho cng ngh nano. - Thng 4 nm 1996, men n bt m S.cerevisiae gii m xong gm 6.000 gene - Thng 12/ 2002, tp on Genome Sequencing Chut quc t hon thnh mt bn tho genome chut v so snh vi genome ngi c khong 3.000 gene chung. - Nm 2003, gii Nobel c trao cho hai nh bc hc Paul C. Lauterbur v Peter Mansfield c cng pht minh nhng ng dng ca hin tng cng hng t (magnetic resonance). - Nm 2005, gii Nobel Y hc 2005 c trao cho hai nh khoa hc Australia - Barry J. Marshall v J. Robin Warren - do khm ph ra vi khun Helicobacter pylori v vai tr ca chng trong bnh vim lot h tiu ho (chng vim d dy, lot d dy hoc t trng

Hnh 3.3: 1. Frederick Sanger (1918-); 2. Barbara McClintock (1902-1992); 3. Binnig, Gerd Karl (1947- ); 4. Paul C. Lauterbur (1929-)

4.2. Cc enzyme thng dng trong k thut di truyn Nm 1962, Werner Arber ln u tin chng minh rng c nhng enzyme c bit hot ng trong t bo vi khun, chng c kh nng phn bit DNA "ca mnh" v DNA "l" ca phage. Cc enzyme ny c kh nng hn ch s nhn ln ca phage l trong t bo vi khun bng cch phn hy chng mt cch c hiu, do c gi l "restriction

41

enzyme". Ch restrion (hn ch) c ngun gc lch s t v c dng n nay. Thng thng, khi cp n cc enzyme c s dng trong k thut di truyn, ch yu l ni n cc enzym gii hn (restriction endonuclease, hay gi tt l restrictase), tc cc enzyme t vi khun c kh nng nhn bit v ct DNA ti nhng v tr xc nh v ngoi ra l nhm hn hp cc polymerase, ligase v nuclease. Nhn chung, c hai kiu ct: ct thng v ct so le. + Kiu ct thng to ra cc u bng (blunt ends): Mt s enzyme gii hn ct hai mch DNA ti cng mt im, v d: AluI, SmaI (Bng 3.2). + Kiu ct so le to ra cc u dnh (cohesive ends): Mt s enzyme gii hn ct on DNA nhn bit ti v tr so le trn hai mch. Kt qu l to ra cc u si n cha vi base b sung c th dnh chp tr li. Cc enzyme ny c s dng rng ri trong k thut ti t hp DNA. V d: BamHI, EcoRI, XmaI (Bng 3.2).
Bng 3.2 : Mt s enzyme gii hn v on nhn bit ca chng (mi tn ch v tr ct) Vi sinh vt Enzym gii hn on 5/3/ nhn bit 3/5/ AluI Arthrobacter luteus AGCT TCGA BamHI Bacillus GGATCC amylolyquefaciens H CGTAGG EcoRI Escherichia coli RY13 GAATTC CTTAAG SmaI Serratia marcescens Sb CCCGGG GGGCCC XmaI Xanthomonas CCCGGG malvaccarum GGGCCC

42

* Cc enzyme thng dng khc Trong k thut di truyn, bn cnh s dng cc enzyme gii hn nh con dao m tinh vi, cn phi dng ti nhm enzyme sau: - Cc DNA- v RNA-polymerase: xc tc cc phn ng tng hp DNA v RNA. Chng c dng khi cn thit to ra mt s lng ln cc bn sao acid nucleic ( to cc mu d, xc nh trnh t nucleotide...). Trong phi k n DNA polymerase I ca E. coli, enzyme phin m ngc t cc retrovirus, enzyme transferase u cng. - Cc DNA- v RNA-ligase xc tc phn ng ni hai u ca cc on DNA hoc RNA thng dng trong to dng. - Cc nuclease. y l nhm enzyme phn ct DNA (DNase) v RNA(RNase) khng mang tnh chuyn bit cao nh cc enzyme gii hn. 4.3. DNA ti t hp v cc vector 4.3.1. Khi nim DNA ti t hp DNA ti t hp l phn t DNA c ti to trong ng nghim bng cch kt hp DNA t cc ngun khc nhau theo mt qui trnh k thut nht nh. Thng thng, mt phn t DNA ti t hp gm mt DNA ca plasmid hoc phage nguyn vn (gi l vector) c mang mt on DNA cn cho nghin cu (gi l DNA ngoi lai). 4.3.2. Khi nim vector: Vector (th ti, th truyn) l mt phn t DNA ng vai tr l vt trung gian mang mt on DNA cn nhn dng c kh nng xm nhp vo t bo ch v mn b my t bo ch to ra nhiu bn sao ca vector. Cc ng dng ch yu ca vector l: - To dng nhm khuych i s bn sao ca mt on DNA xc nh (nhiu bn sao ging nhau). - Nghin cu s biu hin ca mt on trnh t ca DNA. - a gen vo t bo hay c th (nhm bin i c tnh di truyn mong mun sinh vt c truyn gen). - Sn xut RNA vi s lng ln t DNA c to dng. - Sn xut protein vi s lng ln t DNA c to dng.4.3.3. Cc c tnh ca mt vector - C kh nng xm nhp vo t bo ch v tn ti c qua nhiu th h. - C kh nng t sao chp mnh v c lp vi s ti bn ca b gen t bo ch.

43

- Phi c cc c tnh (c m ha bi cc gen chn lc) cho php d dng pht hin t bo ch c cha chng. Chng hn, nu vector l plasmid th t bo ch c th c pht hin theo kiu hnh ca plasmid c mt trong t bo ch, tc c tnh khng thuc gip vi khun sng c trn mi trng c cht khng sinh tng ng. 4.3.4. Cc loi vector chuyn gen

Hnh 3.4: Bn enzyme hn ch ca plasmid pBR322 ci bin, mang hai gen khng thuc (tetR khng tetracycline v ampR khng ampicillin) v mt lot v tr ct ca enzyme hn ch trong mi enzyme ny ch c mt v tr phn ct, nh vy plasmid vector ny p dng c vi nhiu DNA khc nhau.

C nhiu loi vector c s dng trong k thut di truyn: plasmid, phage M13, cosmid, vector l virus ca eukaryotes, YAC (nhim sc th nhn to ca nm men). * Cosmid l vector c cu to nn t plasmid c gn thm gene cos ca phage . Gene cos gip cho DNA ca phage t dng thng ni u li thnh dng vng. Cosmid c kh nng cha on gene l di n 45kb hin c dng lp th vin gene rui dm, chut, ngi. * Phage M13 l phage th si c DNA mch n dng lm vector nhm: + Xc nh trnh t nucleotide ca gene; + Sn xut cc mu th (hay mu d); + Thc hin t bin im nh hng.

44

* Phagemid l vector c cu to t plamid gn thm mt on DNA ca phage M13. Vic la chn loi vector ty thuc vo kch thc on DNA cn to dng v mc ch to dng, y ch cp hai loi vector thng dng: plasmid v phage. - Cc plasmid ni chung c kch thc 2-5kb, nn ch cha rt t gene chn lc v ch c th nhn 8-9 kb DNA cn to dng. - Cc phage l nhng virus xm nhim v lm tan vi khun ch. Vic s dng phage lm vector c nhiu u im hn vector plasmid ch hiu qu xm nhim mnh v kh nng sinh si ny n nhanh; kch thc on DNA m vector c th tip nhn c l ln hn rt nhiu. Tuy nhin, thao tc trn phage th phc tp hn trn plasmid. Trong s cc phage c s dng lm vector, phn ln bt ngun t phage lambda (h gene cha 48.502 cp base, c gii xong nm 1983; n c hai u dnh t nhin khong 12 cp base, cho php DNA- ng vng sau khi xm nhp vo t bo ch). 4.4. Cc phng php thnh lp phn t DNA ti t hp 4.4.1. Phng php s dng cc u dnh Bt k on DNA no nu c ct bi cng mt loi enzyme gii hn (v d EcoRI) to cc u dnh th c th dnh lp li vi nhau v c ni bi DNA ligase (hnh 3.4). Nm 1973, H. Boyer v tp th ca S. Cohen to ra c phn t DNA ti t hp u tin gm vector l plasmid nh pSC101 ca E. coli v DNA ngoi lai l mt plasmid khc. Chnh s kin ny t ra trin vng to ln cho k thut DNA ti t hp sau ny. 4.4.2. Phng php ni trc tip hoc tng hp cc u b sung i vi cc on DNA c to ra bng vic s l enzyme gii hn ct thng, nh HindII chng hn, th vic ni cc on DNA u bng c thc hin theo mt trong hai cch sau: (1) Ni trc tip nh DNA-ligase ca phage T4 (2) Tng hp b sung cc u dnh vo cc u 3 mt s nucleotid, v d: poly (dA) vo u 3 ca on ny v poly (dT) vo u 3 ca on kia, nh enzyme transferase u mt. Sau cc on ny c ni li nh xc tc ca DNA-ligase vi khun. 4.5. To dng DNA ti t hp Mc ch ca vic to dng l nhm thu c mt s lng ln bn sao ca mt on DNA xc nh.

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V nguyn tc, mt quy trnh k thut DNA ti t hp (to dng) gm cc bc c bn sau : Bc 1: Tch DNA v plasmid ra khi t bo, chn lc v x l DNA thuc cc ngun khc nhau, mt lm vector v mt cn cho to dng. i vi vector, vic chn lc ty thuc nhiu yu t: kch thc on cn to dng, mc ch to dng... i vi DNA cn to dng, cn chn s khi cc on c kch thc gn nhau v tng ng vi loi vector. Sau , x l c hai loi DNA trn bi cng mt loi enzyme gii hn (v d: EcoRI) to ra cc u (dnh) so le. Bc 2: Kin to phn t DNA ti t hp trong ng nghim. Trn chung vector v DNA cn to dng c x l theo mt t l nht nh vi s c mt ca ligase. Nh s xc tc ca DNA-ligase m vector ti t hp s c to thnh; sau c tinh sch qua tch chit v ta.

Hnh 3.4: S minh ha cc bc c bn ca k thut di truyn

Bc 3: a vector ti t hp vo t bo ch. C hai loi t bo ch thng dng nht l: (1) T bo vi khun, thng l E. coli; (2) T bo sinh vt nhn chun, v d nm men S. cerevisiae hoc t bo ng-thc vt nui cy. Ty loi t bo ch m s dng k thut bin np thch hp. Ni chung, mc ch ca bc ny l li dng c tnh sinh si ny n nhanh ca t bo ch v s dng b my t bo ch ti bn vector ti t hp thnh mt s lng ln bn sao hoc c th cho biu hin gen (protein) mong mun.

46

Bc 4: Pht hin dng DNA ti t hp c hiu. pht hin dng cn tm, ngi ta c th s dng cng c l mu d (probe). Mu d c th l mt khng th c trng cho protein c m ha bi gen cn tm hoc mt on DNA hoc RNA b sung cho gen cn tm. 4.6. ng dng ca cng ngh DNA ti t hp Trong sut hai thp k qua, cng ngh DNA ti t hp mang li nhng tin b to ln cho sinh hc. Nhng ng dng ch yu ca n l: - Sn xut cc protein, enzyme, vaccine... hu ch; to ra cc vi khun, nm men c kh nng tng hp cc cht c ngha v mt kinh t. - Gy ra nhng bin i kiu gen cc c th sinh vt lin quan n nhng c tnh mong mun. To ngun DNA v RNA cho cc nghin cu. - To ra kh nng sa cha cc khuyt tt di truyn ng vt v ngi (liu php gen hnh 3.5) - Chn gene kho vo trong ribonucleic acid (RNA)ca Retroviruses;- Cho restrovirus tip cn t bo b bnh; - Thm gene kho vo deoxyribonucleic acid ( DNA) t bo b bnh; - Tim vo bnh nhn. Cng ngh DNA ti t hp m ra mt trin vng to ln trong tng lai gn gip con ngi khm ph b n ca c th vi sinh vt v con ngi, trn c s c th nng cao nng sut vt nui, cy trng v bo v sc khe con ngi.

Hnh 3.5: Phng php cha bnh bng gen

Cu hi n tp

47

1. Cc kiu phn loi sn phm hin nay ? Theo anh (chi) kiu phn loi no hp l nht ? V sao ? 2. c im ca sn phm bc hai ? 3. ngha thc tin ca vic nghin cu sinh tng hp tha ? 4. Th no l enzyme hn ch ? Chng c cc vai tr v tnh cht no c coi l quan trng i vi k thut DNA ti t hp ? 5. Bng hai v d v enzyme ct hn ch, hy chng t rng t nht c hai phng php thnh lp cc phn t DNA ti t hp trong ng nghim (in vitro). 6. Th no l phn t DNA ti t hp, vector tch dng ? Hy nu cc bc ca quy trnh to dng v cho s minh ha. 7. Anh (ch) hy nu mt s ng dng khc nhau ca cng ngh DNA ti t hp.

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Chng 4

Cc phng php v k thut ln men

I. Qu trnh ln men
1. Quy trnh ln men (hnh 4.1)
Ging ng kh Ch to mi trng dinh dng Nguyn liu (C,N,P,K) du ph bt, mui khong, nc Kh trng thit b, h thng ng ng, h lc kh

Ging thch nghing

Kh trng mi trng

Ging nui trn my lc Ln men Nhn ging trong ni nhn ging Cung cp khng kh v trng

Thu hoch tinh ch to thnh phm Hnh 4.1 S qu trnh ln men

2. Chng ging chn c ging vi sinh vt thun chng, bc u tin phi phn lp chng t cc ngun t nhin nh nc, khng kh, t, cc vt liu hu c, v c b phn hy... T nhng k thut vi sinh vt hc c in t thi L. Pasteur v R. Koch ra, nhiu phng php c bit dng phn lp chng ging thun khit dng cho cng nghip c pht trin, nht l trong vic tm chng sn xut nhng cht khng sinh mi. T nhng sinh thi t nhin s phn lp c cc chng hoang di. Cc chng ny c mt s hot tnh sinh enzyme, tch t cc cht trao i bc 1, bc 2 ... nhng thng cho nng sut thp. Theo k thut vi sinh vt c in vic phn lp cc chng thun khit mt nhiu cng sc v chm. Ngy nay, ngi ta dng phng php sng lc va nhanh va c hiu qu. Phng php sng lc ch yu c s dng trong vic tm chng sn cc cht khng sinh. T nguyn l c bn

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phng php c ci tin ngy mt phong ph. chn cc chng sn sinh aminoacidngi ta s dng kiu chn lc theo k thut penicillin. Trong phng php ny cc iu kin c la chn sao cho cc t bo hoang di c th pht trin trong mi trng dinh dng thiu mt aminoacidno v b git cht bng penicillin. Cc t bo cn aminoacid(t dng acid amin) khng sinh trng c nn s sng st. i vi vi khun khng mn cm vi penicillin th dng cht khng sinh khc, nh canamycin hay xycloserin, i vi nm men th dng nystatin l thch hp. phn lp nhng chng c tnh cht c bit, v d nh chuyn ha steroid, ngi ta dng hn hp ca nhiu chng vi sinh vt em nui cy trong cng mt mi trng c cht m ta mun thc hin s bin i. Sau khi nui, chit xut v tch sn phm phn tch theo phng php sc k. 3. Nguyn liu cy Ging sn xut thng c bo qun trnh gim hot tnh. Do , vic cy ging trn mi trng thch nghing trc khi nhn ging l vic lm rt cn thit. C th coi y l vic nh thc chng ging ng thi kim tra hot tnh ca ging sau mt thi gian bo qun nhit thp. T nhng nhng t bo hoc bo t ring r ca chng bo qun, cy ra mt s culture, nhng culture ny c nhn ging trong phng th nghim v c kim tra hot tnh. Nu c s khc nhau th dng culture c hot tnh mnh nht gy nguyn liu cy v to thnh chng mi. 4. Nhn ging Cng ging nh trong phng th nghim v qui m pilot, mun thc hin mt qu trnh ln men qui m cng nghip phi tin hnh nhn ging, m bo s lng t bo vi tui sinh l ang thi k hot ng mnh nht cy vo mi trng ln men. Nhn ging y c th phi qua 2-3 bc, ta thng gi l nhn ging cp 1, cp 2, cp 3 v.v... tu thuc vo quy m sn xut. Vic nhn ging thng din ra bng cch nui chm. Cc iu kin nui c la chn sao cho ch xy ra s sinh trng ch khng xy ra s to thnh sn phm. Khi s dng nm sinh bo t v x khun, trc khi nui chm, ngi ta thc hin nhn bo t trn mi trng c nh mi trng cm, bt ng... Nhn ging cp 1 c tin hnh trn my lc vi nhit v thi gian tu thuc vo ni nhn ging (tng t ni ln men) c sc kh v n nhit. T l nhn ging t ng nghim vo bnh tam gic c th ch mt vng que cy hoc c dch huyn ph ca mt ng ging. T nhn ging cp 1 sang cp 2 (hoc t cp 2 sang cp 3) t l ging thng l 1- 10%

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th tch dch ln men hoc l cao hn, t dch nhn ging cui cng vo ni ln men khong 0,5-10% tu thuc vo c tnh tng chng vi sinh vt.

Hnh 4.2: My lc n nhit (Personal-11 , TAITEC)

Thnh phn mi trng nhn ging v mi trng ln men gn ging nhau. Thng thng th hm lng carbon mi trng nhn ging thp hn mi trng ln men, nhng cc thnh phn khc th giu hn, c bit l cc cht sinh trng phc v cho sinh sn v pht trin ca ging vi sinh vt. Ch nui c bit l nhit gia nhn ging v ln men cng khc nhau (nu cng ch nhit th khng cn phi quan tm lm). Nhng nu nhit ln men thp (nh ln men bia) th cn phi nhn ging vi nhit gim dn khi vo ln men, ging khng b chong sc. Ch sc kh cc cng on ny cng khc nhau, thng th trong thi gian nhn ging nhu cu v xy cao nh trong pha sinh trng ca ln men hoc cao hn. Ni chung mt chng vi sinh vt nhn ging a vo ln men m bo cc yu cu cng ngh nh sau: - Dch ging khng c tp nhim, c bit l thc khun th (Bacteriophage). - Cc t bo m bo tui sinh l thi gian sinh trng tt nht, c hot tnh cao nht, thng l na sau ca pha ch s. - Cc thng s k thut nh OD, pH, mu sc, mi v... ng nh quy nh ca tng dy chuyn cng ngh.

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5. Ni ln men Cc ni ln men c thit k v ch to sao cho c th to c nhng iu kin ti u cho tng qu trnh ln men. Nhng yu cu c th t c hot tnh ti a ca vi sinh vt c thc hin thng qua mt s nguyn tc k thut. Ni ln men cha mi trng nui c kh nng to thnh sn phm vi nng sut cao. Trong qa trnh ln men cn theo di lin tc s to thnh sn phm v trng thi v trng dng qu trnh ng vo thi im thu hoch tt nht.

Hnh 4.3: Ni ln men 75 lt Trung tm CNSH-HQGHN

6. Thu nhn sn phm v x l sau thu hoch Vic thu nhn sn phm c bt u bng cch tch ring t bo ra khi mi trng dinh dng. Nu l nhng c th c dng h si th ngi ta thng lc, cn i vi vi khun v nm men th li tm. Vic x l tip theo l tu theo sn phm c tit ra mi trng dinh dng hay tn ti trong t bo. Bn cht ho hc ca sn phm quy nh cc bin php x l tip theo. Cc bin php c s dng l chit rt, hp ph, sng phn t v kt ta. Cc bc tinh ch tip theo c tin hnh k tip ngay sau bc tch sn phm thng phi qua nhiu cp, trc khi sn phm cui cng c ng gi. Vic thu nhn sn phm vi hiu sut cao c ngha quyt nh i vi tnh kinh t ca mt phng php. Bi vy, vn tch v c lp sn phm phi c ch ngay t khi chn chng v chn dch dinh dng. Vic ti u ho phng php c lin quan n tt c cc bc. Vic loi

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b v s dng cc ph v ph phm cng cn c ch trnh gy nhim mi trng.

Hnh 4.4: My li tm ( centrifuge)

II. Vi sinh vt
1. Sinh vt nhn chun 1.1. Nm men Nm men (Yeast, Levure) thng tn ti dng n bo, a s sinh sn theo li ny chi, cng c khi theo hnh thc phn ct t bo, nhiu loi c kh nng ln men ng v thch nghi vi mi trng cha ng cao, c tnh acid cao. Nm men phn b rng ri trong t nhin, nht l trong mi trng c cha ng, c pH thp, chng hn nh trong hoa qu, rau da, r ng, trong t trng cc loi cy n qu, trong t c nhim du m. Nhiu loi nm men c ng dng cao trong sn xut cng nghip nh ln men bia ru, glycerine, sn xut nm men bnh m, thc n gia sc... 1.2. Nm si Nm si (Microfilamentous fungi) l tt c cc nm khng phi nm men v cng khng sinh m nm. Nm si cn gi l nm mc, c dng si phn nhnh, khng hoc c vch ngn, li sng hiu kh, ch yu l hoi sinh. Nm si phn b rng ri trong t nhin, tham gia tch cc vo cc vng tun hon vt cht, nht l qu trnh phn gii cht hu c v hnh thnh cht mn. Rt nhiu loi nm c s dng rng ri trong cng nghip ch bin thc phm, cng nghip enzyme, cng nghip dc phm, sn xut thuc tr su sinh hc, kch thch t sinh trng thc vt... Nhiu nm si k sinh trn ngi, ng vt v thc vt gy ra cc bnh nm nguy him. Mt s nm si pht trin nhanh trn cc cht hu c gy h hng lng thc, thc phm, nguyn vt liu...

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1.3. To (Algae) Vi to (Microalgae) gm cc i din c kh nng quang hp, c dng n bo sng thnh tp on, phn b ch yu mi trng nc ngt, nc mn v t m. Vi to c th sinh sn theo hnh thc dinh dng, v tnh v hu tnh. Nhiu loi vi to c ng dng trong sn xut v i sng nh thu sinh khi giu protein lm thc n cho ngi v gia sc (Chlorella), nui to silic (Skeletonema costatum) lm thc n cho u trng tm, tch acid bo khng no. S dng vi to cho x l mi trng (Scenedesmus) hoc lm sinh vt ch th trong mi trng ngho calcium (calcium) (to lc Desmid). 1.4. Nm qu th Nhiu loi nm c qu th c s dng lm thc phm, do nm giu protein, cht khong, cc vitamin A, B1, B2, C, D, E. Ngoi ra chng cn c nhiu c tnh ca bit dc, c kh nng phng v cha bnh h huyt p, chng bo ph, ng rut, h tr cha ung th. a s nm n thuc ngnh nm m (Basidiomycota), thng gp nm n thuc b Agaricales nh nm rm Volvariella volvaceae, Agaricus bisporus... Ngoi gi tr ti nguyn, thc phm v dc phm, nhiu loi nm c ngha trong cng ngh sinh hc v i sng do chng c kh nng sn sinh ra nhiu cht c ch nh eter, acid acetic, acid tanic, cc cht khng sinh... Nhiu loi nm c kh nng hp th v o thi cc cht phng x, mt s loi nm c s dng phn gii cc cht thi c hi v cc ngun ph liu gy nhim mi trng. 2. Sinh vt nhn s 2.1.Vi khun Vi khun (Bacteria) c nhiu hnh thi v cch sp xp khc nhau, kch thc kh nh so vi nm si v nm men. Phn ln vi khun thuc nhm d dng, i sng c th hiu kh, k kh hoc l dng sng tu nghi. Nhiu vi khun c ng dng trong sn xut cng nghip. in hnh nh cc loi vi khun ln men cc acid hu c (lactic, propionic...), sn sinh enzyme, acid acetic, acid glutamic, lysine, vitamin, ln men methane, sn xut phn bn, thuc tr su sinh hc... 2.2. X khun X khun (Actinomycetes) thuc nhm vi khun tht (Eubacteria) phn b rng ri trong t nhin. Phn ln x khun l hiu kh, hoi sinh, c cu to dng si phn nhnh (khun ti). X khun l mt trong nhng nhm vi sinh vt ng vai tr quan trng trong t nhin. Chng tham gia vo cc qu trnh chuyn ha nhiu hp cht trong t nhin. Trn 80%

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cht khng sinh c pht hin l do x khun sinh ra. X khun cn c dng sn xut nhiu loi enzyme, vitamin, acid hu c... 2.3. Vi khun lam Trc y vi khun lam (Cyanobacteria) c gi l to lam (Cyanophyta) hay to lam lc. Thc ra y l mt nhm vi sinh vt nhn nguyn thy thuc vi khun tht. Vi khun lam c kh nng t dng quang nng nh c cha sc t quang hp. Vi khun lam phn b rng ri trong t nhin, nhiu loi c ngha trong sn xut sinh khi giu protein, c nh m hay s dng trong cng nghip x l nc thi... 3. Cc tiu chun ca mt vi sinh vt dng trong cng nghip 3.1. Kh nng s dng nguyn liu Tnh kinh t ca mt qu trnh sn xut i hi phi s dng cc ngun nguyn liu r tin, n gin v cc chng vi sinh vt khng i hi qu cao v nhu cu dinh dng. Thng thng, ngun carbon v nit (nitrogen) dng cho sn xut l nhng ngun thng dng nh cc loi r ng, tinh bt, dch kim sulfid, ngun nit k thut nh cao ng, bt u tng. 3.2. Tnh cht sn phm ph Yu cu cc chng vi sinh vt dng trong sn xut l khng to thnh cc sn phm ph khng mong mun. Thc ra th trong qu trnh sng vi sinh vt lun to thnh nhiu sn phm trao i cht tch lu trong mi trng, mt s sn phm trao i cht c th khng c li cho chnh trao i cht ca t bo v c th gy c ch t bo. S tch lu nhiu cc sn phm ph trong mi trng mt mt lm gim hiu sut to thnh sn phm chnh, mt khc gy nhiu kh khn cho qu trnh thu nhn v tinh khit sn phm. 3.3. Mc mn cm vi s ly tp v kh nng tch sn phm khi mi trng Trong qu trnh ln men thng b nhim cc vi sinh vt l. Ngun ly nhim c th t nguyn liu, khng kh hay t thit b ln men. Nu cc vi sinh vt l cng thch nghi vi iu kin sng trong mi trng ln men th chng s cnh tranh vi chng sn xut ng thi to ra cc cht c tc dng c ch hoc gy cht i vi chng sn xut. Do , cc chng sn xut phi c la chn sao cho khng mn cm vi s tp nhim do cc vi sinh vt l v c bit l vi bacteriophage. Trong trng hp b nhim bacteriophage lng ging trong mi trng ln men c th gim rt nhanh. V d trong mi trng b nhim vi khun butiric c th

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nh hng mnh n sc sng ca ging bi v mt lng nh ca acid butiric to thnh gy c i vi t bo. Ngoi cc ch tiu trn, kh nng tch sn phm ra khi mi trng ln men cng l mt tiu chun chn chng vi sinh vt cho sn xut, bi v c nhiu qu trnh sn xut cho sn phm nhiu nhng vic tch sn phm kh thc hin v hiu qu kinh t thp.

III. C cht dinh dng

1. Nguyn t i lng C, O, N, H, P, S, Mg, Ca, Fe Mi trng dinh dng phi cha tt c cc nguyn t cn thit cho sinh trng v to thnh sn phm. Cc mi trng nui cy vi sinh vt cn thit b sung cc nguyn t khong. Nhng nguyn t khong m vi sinh vt i hi phi c cung cp vi liu lng ln c gi l cc nguyn t i lng, bao gm cc nguyn t nh C, O, N, H, P, S, Mg, Ca, Fe. Nhu cu v cht khong ca vi sinh vt khc nhau ph thuc loi, giai on sinh trng pht trin. Nng cn thit v mui khong ca cc nm, vi khun v x khun thng thay i trong cc phm vi sau (bng 4.1). 2. Nguyn t vi lng Mn, Na, B, Mo, Zn, Cu, Ni, Va, Cl, Si
Bng 4.1: Nng cn thit v mui khong ca nm, vi khun v x khun Nng cn thit (g/l) Mui khong i vi vi khun i vi nm v x khun

K2HPO4.3H2O KH2PO4 MgSO4.7H2O MnSO4.4H2O FeSO4.7H2O Na2MoO4 ZnSO4.7H2O CoCl2 CaCl2 CaSO4.5H2O

0,2 - 0,5 0,2 - 0,5 0,1 - 0,2 0,005 - 0,01 0,005 - 0,01 0,001 -0,005 ti 0,03 0,01 - 0,03 0,001 - 0,005

1-2 1-2 0,2 - 0,5 0,02 - 0,1 0,05 - 0,2 0,01 - 0,02 0,02 - 0,1 ti 0,06 0,02 - 0,1 0,01 - 0,05

Nhng nguyn t khong m vi sinh vt ch i hi vi liu lng rt nh c gi l cc nguyn t vi lng, bao gm cc nguyn t nh Mn, Na, B, Mo, Zn, Cu, Ni, Va, Cl, Si... Nng cn thit ca tng nguyn t vi lng trong mi trng thng ch vo khong 10-6 - 10-8 M.

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Thng thng, khi ch to mi trng nui cy vi sinh vt khng cn b sung cc nguyn t vi lng v trong cc thnh phn khong i lng hoc trong nguyn liu hay nc pha ch mi trng c thnh phn vi lng. Ngoi tr mt s trng hp nh b sung Zn vo mi trng nui cy nm mc, Co vo mi trng nui cy vi khun sinh tng hp vitamin B12 nhng vi hm lng thp (khong 3-5 g/l). 3. Dch dinh dng cho cc vi sinh vt d dng Trong qu trnh ln men, mt mi trng nui cy tt nht phi l mi trng m bo cho sn xut vi hiu sut cao trong thi gian ngn nht v gi thnh thp nht i vi chng ging vi sinh vt. Nhng chng vi sinh vt dng cho cng nghip u l cc ging d dng, tr cc chng to thuc ging t dng. Cc chng ca to thng c nui cy trong cc qu trnh kh bn cho nc thi hoc nui to thu sinh khi. Nhng vi sinh vt d dng ch sn sinh nng lng trong ATP dng cho sinh trng nh qu trnh xy ho nhng hp cht hu c. ATP l thnh phn quan trng nht m t bo dng vn chuyn nng lng. Trong nhng phn ng khng thun li v phng din nhit ng, ATP cho php thc hin nhng phn ng vi tc thch hp. Trong cng ngh vi sinh, mi trng nui cy nhng chng d dng thng c cc thnh phn nh sau: 3.1. Ngun carbon v nng lng Ngun carbon v nng lng thng c s dng l tinh bt, mt r, saccharose, glucose, dch ng thu phn t bt hoc g v.v... Mt s loi vi sinh vt c kh nng s dng cellulose, hemicellulose t bit l carbuahydro (alkan, methane). Ch yu ngun carbon s dng l carbohydrate. Lng carbon c b sung vo mi trng tu thuc chng ging vi sinh vt. Mt s chng hiu kh s dng khong 50% c cht, cn cc chng k kh tu tin ch dng ti 10% cho sinh trng. 3.2. Ngun nit Ngun nit ch yu trong cng ngh ln men l nc amoniac v mui ammon. Dng vo mc ch ny cn c cc ngun nit hu c nh cao ng, dch thu phn nm men, thu phn kh lc, u tng, ht bng, cc b thi ca cng ngh bia (dch ngm malt hoc r mm malt), b thi rau qu, khoai ty, sa loi b m, ph phm khi ch bin pho mt, tht c... Cc ngun nit hu c vi vai tr lm ngun nit v c ngun carbon, ng thi cn cung cp cc cht sinh trng. V vy, khi s dng cc ngun nit hu c, vi sinh vt thng pht trin mnh hn. 3.3. Ngun phospho (phosphorus)

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Ngun phospho cung cp cho mi trng ln men dng mui phosphate hoc acid phosphoric. Phospho hu c (phytin) cn c trong cao ng, bt u tng, trong cc loi cm.... 3.4. Cc ngun khong khc - Ngun lu hunh b sung vo dch ln men dng mui sulfate. - Ngun Mg v K thng c a vo di dng cation ca mui phosphate v sulfate. Trong mt s qu trnh ln men, calcium c a vo mi trng dng mui carbonate duy tr pH vng trung tnh hoc gn trung tnh khi acid c to thnh (v d nh ln men to cc acid hu c) - Ngun Fe: thng thng trong cc nguyn liu s dng c st, nn t khi phi b sung. 3.5. Vitamin v cc yu t sinh trng khc Trong cc mi trng dinh dng dng cho cng nghip, vitamin v cc yu t sinh trng thng c b sung dng nguyn liu lm giu vitamin nh cao ng, r ng, cao nm men. Chng cha hn hp cc acid amin, vitamin v c mt s yu t khc cha bit r, v d nh cc dch chit ng vt hay thc vt. Lng c ch ca cc yu t t bit i khi ch cn rt t. V d - alanine c hiu lc nng 1/100.000.000, cn acid pantotenic c hiu lc nng 1/50.000. Nhng yu t cha bit r gi chung l cc yu t sinh trng. Cao ng, cao nm men, dch thy phn t ngun protein thc vt, ng vt va ng vai tr l ngun nit hu c va l ngun cung cp cc cht sinh trng cn thit. Ngoi ra, trong cng ngh vi sinh cn dng nc chit cm, dch p khoai ty, dch p gi u, dch p c chua, bp ci hoc mt s rau qu khc cng cha nhiu vitamin v dng lm ngun kch thch sinh trng trong nui cy vi sinh vt. Dch dinh dng cha cc cht dinh dng mt nng m bo sut qu trnh nui. Nh vy, ngun carbon v nng lng c a vo phm vi 10-100g/l. nhiu c th, nng cn thit duy tr tc sinh trng cc i l rt nh; i vi ng th khong 1-10mg/l. Vi aminoacid v vitamin th t bo ch cn nng 1-100 g/l. Cc tr s c tnh c hiu c th v c hiu qu trnh. Dch dinh dng dng trong ln men cha cc thnh phn cn thit thng khng mt t l cn i m sinh trng i hi. Nh nhng iu kin nh t l C : N cc tr hoc s thiu phosphate m trao i cht c th c iu khin theo hng c ngha cho vic tng hp sn phm mong mun. iu ng vi cc nguyn t i lng cng nh vi lng. Chng hn bng cch a Co vo

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m t c thu hoch cao v vitamin B12, hay s thiu st kch thch qu trnh tng hp acid citric. 4. Ngun carbon k thut v ngun nit k thut 4.1. R ng Trong cng nghip ng thng thu c mt r l dch ng sau khi kt tinh. Cc loi mt r gm c: - Mt r hydrol: dch thu c sau khi kt tinh glucose cc x nghip thy phn bt bng acid sn xut glucose. Trong hydrol c ti 40 - 50% glucose v c mt hm lng ng k NaCl. - R ng: cc nh my ng sn xut saccharose c mt ph phm vi t l kh ln, l r ng. y l mt loi nc ct c tch ra sau khi kt tinh ng. C hai loi r ng l r ng c ci v r ng ma (ty thuc vo ngun nguyn liu ca nh my ng). C hai loi r ny u c mu nu sm do c nu v c nhiu ln nn c nhiu caramen v melanoid to thnh. Trong r ng c ti 70 - 80% cht kh, trong ch yu l ng saccharose 46 - 54%, ng kh 6 - 9%, rafinose 1- 2%, N tng 0,45 - 2,88% v cht khong 3 - 4%. Cc cht hu c c trong r ng l cc acid, ru, acid amin, purine v cc vitamin. Hm lng cc mui phosphate trong r ng thng rt thp. Phn ln cc hp cht phospho nm phn cn lng. Do , khi dng r ng x l v loi b cn th nht thit phi b sung ngun phospho vo mi trng dinh dng. Trong r ng c cha cc hp cht c tc dng kch thch sinh trng vi sinh vt, nhng nu dng r ng vi nng cao th sinh trng ca cc chng sn xut s b km hm, v trong r ng c cha cc cht c tc dng c ch nh SO2, hydroxy-methyl-fucfural hay kalium - imido - disulfonate, cht ny c th sinh ra t sulfate v nitrate do vi khun to thnh. Trong r ng c cha hm lng ln biotine (vitamin H) l cht sinh trng rt cn thit i vi nhiu loi vi sinh vt v l cht iu ha trong qu trnh sinh tng hp acid amin, hm lng khong 20 - 120 mg/kg trong r ng ma v 0,01 - 0,13 mg/kg trong r ng c ci. Cc vitamin trong r ng ngoi biotine cn c vitamin B1, B2, PP, acid pantotenic... 4.2. Dch kim sulfid Trong cng nghip men cn s dng dung dch thy phn t g dch kim sulfid l ph thi ca cng nghip giy. Thnh phn chnh ca dch kim sulfid l linhosunfonate v cc ng pentose. Thnh phn ca dch kim sulfid t g ca cy l bn v cy l kim l khc nhau. Ngoi ra,

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thnh phn ny cng thay i nhiu tu theo mc khai thc. Dch kim sulfid ca g cy l bn chim t l cao cc ng pentose (khong 80% ng) thng s dng nui cy thu sinh khi nm men. Cn dch kim sulfid g cy l kim th cc hexose li chim u th (khong 70% ng) dng ln men thu ru. Vic tin x l cht thi ny trc ln men l ti thiu. Bm hi nc hoc thng kh pH 1,5 - 3,0 l cn thit loi SO2 l cht vn km hm sinh trng ca vi sinh vt. Sau pH s c iu chnh ti ti u (pH khong 5) v mi trng c b sung cc cht dinh dng cha nit v phosphate. 4.3. Cc nguyn liu thy phn tinh bt cung cp ngun carbon ch yu l glucose th bt sn c l l ngun nguyn liu tt nht. Trong bt sn cha ch yu l tinh bt, hm lng N hu c, cht khong, vitamin c vi lng rt nh. Thy phn cc loi tinh bt thng thc hin theo hai cch: - Thy phn bng acid vi p lc d. Dch thy phn thu c qua trung ha bng Na2CO3 hoc NaOH, nu dng H2SO4 lm tc nhn thy phn th c th dng CaCO3 hoc nc vi trung ha, sau em lc qua lc p khung bn vi than hot tnh kh mu. Dch thy phn ny cha ch yu l ng glucose, mt lng nh cc acid amin, c mt cc cht bn, khong c dng chun b mi trng nui cy hoc em c c ti 60 - 70% cht kh s dng dn. - Thy phn bng enzyme: Cc ch phm enzyme ch yu l t nm mc c nui cy b mt hoc b su, dng vi t cch l phc h amylase gm c , - amylase v glucoamylase. Sn phm thu c l hn hp maltose v glucose. Cng c trng hp dng phi hp ch phm enzyme t mc v ch phm enzyme t vi khun nui b su ( - amylase chu nhit) nn hiu qu ca qu trnh s cao hn. Phng php thy phn cc loi bt (bt sn, go, ng, bt m, cao lng, khoai ty) bng cc ch phm enzyme ny c dng trong cng nghip sn xut ru cn. 4.4. Ht v bt ng cc Cc loi bt ng cc thng c dng l bt go, bt ng c tch phi, bt m, bt i mch... Ngoi thnh phn ch yu l tinh bt, cc loi bt ny cn cha khong vi phn trm cc hp cht protein, cc cht x (ch yu l cellulose) v cc cht khong. Bt sn l loi nguyn liu kh r tin so vi cc loi bt khc hin nay ang c s dng nhiu cho cng ngh ln men c bit l ln men cn.

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Cc nguyn liu tinh bt trong ln men c th dng trc tip lm thnh phn ca mi trng dinh dng cho cc chng sinh ra enzyme amylase ngoi bo, c bit l trong phng php nui cy b mt. Ngoi ra, ngun nguyn liu ny cn qua mt giai on thy phn thnh dung dch cc loi ng ri mi dng chun b mi trng dinh dng. Ngoi ra, cc parafin c mch t C8 - C18, kh methane... c th c s dng lm ngun carbon nui cy vi sinh vt thu cn hoc protein n bo cho sn xut thc n gia sc. 4.5. Ngun nit k thut - Bt u tng c dng nh l mt ngun nit k thut tng i ph bin trong nhiu mi trng dinh dng. Trong bt u tng c ti gn 40% l protein v khong 19% cht bo, c cc acid amin. i vi sinh tng hp nhiu cht khng sinh khng nhng ch c cc hp cht protein mi c tc dng m cn phi k n cc cht bo c trong u tng. Cng vi bt u tng hoc kh du u tng ngi ta cn dng bt kh lc (lc sau khi p du), bt hoc kh du cc loi ht bng, ht hng dng... vo trong mc ch ny. - Nc chit ng v cao ng l sn phm ph trong cng nghip ch bin bt ng. Trc khi xay, ng c ngm vi dung dch natrium sulfid. Trong khi ngm, cc acid amin, vitamin c chit ra v ha tan vo dung dch. Nc chit ng c th dng trc tip pha ch mi trng dinh dng, nhng thng thng ngi ta c trong iu kin chn khng ti 50% cht kh dng st gi l cao ng. Trong cao ng c 6,4 - 8% N tng s, khong mt phn na l cc acid amin, phn cn li l peptide v protein, hm lng carbohydrate dao ng trong phm vi kh rng do c lin quan n ln men lactic, hm lng tro 15 - 20%, nhiu vitamin c bit l vitamin nhm B v biotine. - Nc chit nm men v cao nm men: sinh khi men bia hoc men ru c ra sch v cho t phn 48 520C trong 2 - 3 ngy, lc b b, thu c dch thy phn gi l nc chit nm men, c c c cao nm men. Cc sn phm ny giu acid amin, peptid cng nhiu vitamin nhm B v cc cht khong. Trong cao nm men c 40 - 50% cht kh, 0,6 - 1,5 N tng s, 0,3 - 0,5 N - amin (trong c mt 18 loi acid amin). - Dch thy phn cc loi kh du (u tng, lc, bng, hng dng) v tht, c giu nit hu c, nhiu acid amin. Nu thy phn bng acid th nhiu vitamin v mt hay hai aminoacidb ph hy (nh triptophan v mt phn cysteine), cn thy phn bng enzyme th cc thnh phn ny y hn. Dng cc dch thy phn ny lm ngun nit

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hu c trong mi trng nui cy vi sinh vt v mt phn l ngun cht dinh dng.

IV. Nhu cu v xy v s thng kh trong qu trnh ln men

1. ho tan ca xy trong nc T bo s dng xy h hp v lm gim lng xy trong mi trng. V th trong nui cy hiu kh phi cung cp xy mt cch u n. Thiu xy nht thi ti mt thi im no trong mi trng s dn n s ph v qu trnh trao i cht ca t bo. Vi sinh vt s dng xy trong mi trng lng. Lng xy ho tan trong nc thng l rt t. Phi cung cp xy sao cho tc ho tan ca n bng tc tiu th xy ca vi sinh vt. Tc ho tan ca xy vo mi trng lng c tnh theo cng thc: dc R= = K. (C - C1) dt Trong : R- tc ho tan xy, C - nng xy bo ho p sut ring bit, C1- nng xy ho tan thi im la chn, K - hng s t l, t - thi gian. ho tan xy cn ph thuc vo nhit khi nui cy, vo nng cc cht hp phn v nht ca mi trng. Khi nhit tng ln th ho tan ca xy gim. ho tan ca xy trong mi trng gim i 2 ln khi nhit tng t 30 - 370C. iu ny c th khc phc bng cch cho sc kh mnh hn trong qu trnh ln men. Nng xy ho tan cng s gim khi dng cc cht hot ng b mt, cc cht ph bt v hm lng sinh khi vi sinh vt tng. Trong qu trnh nui cy khng kh nn c thi vo thng ln men c h thng cnh khuy. Tc sc kh mnh s tng tc ho tan xy v trn u c cht dinh dng trong mi trng. Nhng khng nn khuy qu mnh v c th dn n s h hng c hc cc t bo v dn n hin tng t phn. 2. Nng xy gii hn xy rt cn i vi i sng ca vi sinh vt hiu kh. Tng thng kh n gii hn nht nh th s pht trin ca vi sinh vt cng tng ln theo. i vi nhiu vi sinh vt, thng kh s lm tng tc sinh trng, rt ngn pha tim pht, nng cao lng sinh khi. Khi tng tc ho tan xy t 0 - 5 milimol O2 /l.pht, lng sinh khi cui cng ca Serratia marsescens s tng mt cch ng k; sinh khi cc i t c khi

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cng thng kh khong 5 milimol O2/l.pht. Nu tip tc tng thng kh hn na th lng sinh khi cui cng s gim. Hin tng ny cn gp rt nhiu ging vi sinh vt. duy tr vic cung cp xy ti thch cho t bo khng cn thit phi lm bo ho mi trng bng xy ho tan. Ch cn mt nng xy nh hn rt nhiu cng cung cp cho cc enzyme phn ng vi c cht . Nng xy gy ra h hp ti a c gi l nng xy ti hn (hoc p sut ring phn ca xy). khng phi l mt i lng c nh m l mt hm s ca tc sinh trng hoc ca tc h hp c lin quan vi n. Tr s ny vo khong 10 mol/l. Khi s vn chuyn xy b cn tr bi nhng tp hp t bo (cc cc si nm) hoc bi lp cht nhy bao quanh cc t bo th nng xy gii hn c tr s cao hn. 3. S cung cp xy cho cc t bo chm S cung cp xy cho cc t bo chm l mt qu trnh chuyn dch cht, trong xy c chuyn t bng khng kh vo mi trng dinh dng v t vo t bo. Qu trnh xy ra nh dng chy v s khuch tn. Lc y l s chnh lch nng xy. S chuyn dch cht t tng kh sang tng lng c quy nh bi b mt gii hn gia hai tng v do vy bi s lng v kch thc cc bng khng kh. Ch mt phn nh ca khng kh c cung cp i vo dung dch. Bi vy trong thc tin, ngi ta thng s dng t s ca th tch khng kh/th tch ni ln men/pht. i vi mi qu trnh ln men cn phi nghin cu nh hng ca cng thng kh i vi hiu sut to thnh sn phm. Trong sn xut cng nghip khng kh c nn qua my nn, qua mt h thng lm ngui, qua h thng lc loi ht tp khun ri thi vo cc thng ln men. Trong cc thng ln men v cc thng nui cy nhn ging u c h thng khuy tu thuc vo yu cu ca tng loi vi sinh vt, vo tng iu kin nui cy nhm thu c hiu sut ti a.

V. Kh trng
1. Ln men khng v trng cho qa trnh ln men din ra c kt qu th cn phi ngn cn s pht trin ca cc c th l. Trong nhiu qa trnh ln men (sn xut acid amin, enzyme, cht khng sinh...) iu c thc hin thng qua vic kh trng mi trng dinh dng, khng kh v cc thit b ln men (ln men v trng). Tri li trong vic sn xut sinh khi nh sinh khi nm men, sinh khi vi khun, to thng tin hnh ln men khng v trng. S pht trin ca cc c th l b ngn cn mnh m bng cch to ra nhng

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iu kin nui sao cho chng sn xut c th sinh trng tri hn, v d nh c cht c hiu hay pH mi trng. 2. Ln men v trng Nhim v thanh trng l dit ht vi sinh vt c mt trong mi trng do c sn t trong thnh phn nh nc, nguyn liu, khng kh v trn b mt cc thit b tip xc vi mi trng. Nhng vi sinh vt ny cn sng st s pht trin cnh tranh vi chng sn xut, lm hng qu trnh ln men. Cc vi sinh vt thng c sc bn vi nhit, mt s c mt trong nguyn liu di dng bo t, mun dit c chng phi gia nhit ti 120 1210C trong vi chc pht. 3. Kh trng bng ni hp

Hnh 4.5:Ni hp p lc cho ln men xp ( Autoclaver)

Hnh 4.6:Thit b kh trng Pasteur

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Hin nay phng php thanh trng trong cng ngh vi sinh ph bin l dng hi nc qu nhit do ni hi cung cp. Trong ln men tng m cc thit b sau khi lm v sinh c kh trng bng hi nng ti 120 1300C, sau mi cho mi trng lng vo cc ni ln men kh trng mi trng cng vi c h thng khuy v cc on ng ng, van tip cn. Vic gia nhit cao c th dn n s ph hu cc thnh phn dinh dng mn cm vi nhit v caramen ho cc ngun ng cng nh xy ra cc phn ng melanoid gia ng vi aminoacidhoc cc vitamin b ph hng. Nhng thnh phn mn cm vi nhit c khi phi kh trng ring, sau mi trn ln hoc kh trng theo phng php khc nh lc qua phin v trng. Ngoi cch kh trng theo phng php hi nc gin on ny, ngi ta cn dng phng php kh trng bng hi lin tc bng cch cho mi trng chy qua thit b kh trng chuyn dng nhit 1400C trong vi pht. * Kh trng mi trng nui cy b mt Mi trng nui cy b mt thng l nhng hp cht rn, gm c cm, bt v cc cht dinh dng. Trong sn xut cng nghip mi trng rn c kh trng bng hi nng trong thit b chuyn dng vi p sut d 0,05 Mpa t nhit 104 - 1100C. iu chnh pH mi trng thch hp bng acid clohydric, sulfuric hoc lactic. Thanh trng bng hi nng c th qua 2 giai on. Giai on u nng nhit ti 1000C v o khi mi trng lin tc trong 15 - 20 pht. Giai on sau nng nhit ti 1100C khong 60 - 90 pht v c sau 15 pht li o mi trng 3 - 5 pht. * Kh trng mi trng lng ln men b su Kh trng mi trng lng c th chn mt trong nhng phng php gin on hoc lin tc. Phng php gin on thng dng trong trng hp khi dch khng ln, th d dch dng trong cc bnh ln men th nghim, cc ni nhn ging v cc ni ln men khng qu ln. Tin hnh ln men tng m ngay trong ni theo nguyn l ni hp p lc qua mt s giai on: - Kh trng ni ln men v h thng ng ng tip xc vi mi trng bng hi nng trc tip hoc gin tip. - Cho dch mi trng pha ch vo ni (lng dch bng 3/4 th tch ca ni v phi tnh thm phn nc ngng khi cho hi trc tip vo mi trng).

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- Gia nhit ti nhit thanh trng. C 2 bc gia nhit: cho hi vo v ni hoc ng xon trao i nhit khi dch ti gn 1000C th cho hi vo ni trc tip vi dch nng nhit n nhit ti hn. - Gi nhit ny trong khong thi gian thanh trng cn thit. - Lm ngui dch ngay trong ni bng cch cho nc vo v hoc ng xon trao i nhit cng vi h thng khuy lm vic. Phng php kh trng ny tng i lu v trnh bin i trong thnh phn dinh dng ca mi trng nn ch tin hnh p sut 0,05 0,1Mpa vi nhit 110 - 1200C trong khong 1 - 1,5 gi t lc t c nhit ti hn. Ngoi ra, c th tin hnh thanh trng lin tc nhit cao hn (140 - 1450C) v gi thi gian ngn hn (5 - 15 pht) nhit ny. 4. Kh trng bng ho cht i vi nhng cht km bn nhit d b phn hu nhit cao th vic kh trng c th bng cch lc qua phin lc hoc bng cc ho cht dit khun. Nhng vic s dng cc ho cht dit khun cn phi cn nhc k cng v tnh gy c cho vi sinh vt nui cy, cho ngi s dng sn phm cng nh v v sinh an ton lao ng. Mt s ho cht c dng kh trng trong mt s trng hp ngoi l, nh etylenoxyl, propiolacton rt thch hp cho vic kh trng cc cht km bn nhit nh enzyme chng hn. Ethylenoxyl hn hp vi khng kh theo t l 3 - 8 % s gy n, v vy khi dng phi trn ln vi CO2 hoc N2. - propiolacton c tc dng mnh hn nhng c c tnh (c th gy ung th i vi ngi). 5. Lc kh trng Khng kh dng cung cp oxy c kh trng bng cch lc kh trng. Nguyn liu lc thng dng l bng , bng thy tinh hoc bng. Hin nay trong cng ngh vi sinh cn ph bin loi lc mng. Lc kh trng c th s dng lm sch khng kh. Cung cp kh sch cho nui cy b mt l mt b phn nhng my iu ho v lm sch kh t bn trn hoc bn cnh phng nui cy. Trong cc x nghip sn xut quy m ln c th thit k s dng kh tun hon. Khng kh sau khi qua phng nui cy c a tr li cc my iu ho ring ch bay ra kh quyn khong 10%. ng thi vi vic cung cp kh sch cn c b phn iu nhit v iu m m bo nhit v m ti u cho qu trnh nui cy trnh lm kh mi trng. Cung cp kh sch cho mi trng nui cy chm kh phc tp. l h thng cc my nn kh v phin lc, ngoi ra cn c cc b phn khc

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nh lc s b, lm ngui kh, thng cha kh. Khng kh sch v trng c a vo ni ln men qua cc b phn phun tia kt hp vi khuy o hoc ng hi lu tng thm ho tan ca xy trong mi trng.

VI. Phng php nui

1. Nui khng lin tc Trong phng php nui khng lin tc (batch - culture) hay cn gi l nui gin on, thng thng vi sinh vt sinh trng n chng no mt thnh phn ch yu ca mi trng dinh dng b gii hn. Khi culture chuyn t pha lu tha sang pha cn bng. Sinh trng gn lin vi s thay i ko di ca iu kin nui, s gim cht dinh dng v s tng khi lng t bo. Trong qu trnh trng thi sinh l ca t bo cng thay i. Thng thng vic to thnh sn phm mong mun lin quan vi mt trng thi sinh l nht nh trong pha sinh trng. Khng th duy tr c trng thi ny trong mt thi gian di. Phng php nui gin on c s dng trc ht cho s ln men v trng,v cch nui ny l d dng v mt k thut. 1.1. Nui chm Phng php ny dng cho c vi sinh vt k kh v hiu kh. i vi nui vi sinh vt k kh trong qu trnh nui khng cn sc kh ch thnh thong khuy trn cn vi vi sinh vt hiu kh th phi sc kh lin tc. y l phng php hin i c dng trong khong na cui th k XX v cho kt qu rt to ln i vi cng ngh vi sinh. Nui cy chm hay nui cy b su dng mi trng dch th. Chng vi sinh vt cy vo mi trng c phn tn khp mi im v chung quanh b mt t bo c tip xc vi dch dinh dng. c im ny i hi trong sut qu trnh nui cy phi khuy v cung cp xy bng cch sc kh lin tc. Ngy nay phng php nui cy chm c dng ph bin trong cng ngh vi sinh sn xut men bnh m, protein n bo, cc ch phm vi sinh lm phn bn, thuc tr su, cc enzyme, cc acid amin, vitamin, cc cht khng sinh, cc cht kch thch sinh hc v.v... Phng php nui cy chm c mt s u im: - Tn t mt bng trong xy dng v lp t dy chuyn. - Chi ph in nng, nhn lc v cc khon ph cho mt n v sn phm thp. - D t chc c x nghip c sn lng ln. - Cc thit b ln men chm d c kh ho, t ng ho . Song phng php chm cng c mt s nhc im sau:

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- i hi trang b k thut cao, d b nhim trng ton b. V vy, nhng thit b ln men chm cn phi ch to c bit cn thn, chu p lc cao, i hi kn v lm vic vi iu kin v trng tuyt i (trong nui cy b mt c th loi b phn nhim trng, cc phn khc vn cn dng c).

Hnh 4.7: S nui chm

1.Thng trn mi trng, 2.Ni thanh trng, 3.Thng cha, 4.Van x, 5.Thit b trao i nhit, 6.Bm li tm, 7.Thng cha dch, 8.Thng ln men, 9.Lc kh s b, 10.Nn kh, 11.Lc kh bc mt, 12.Lc kh bc hai, 13.Thng canh trng thnh phm.

- Trong ln men chm cn phi khuy v sc kh lin tc v vi sinh vt ch s dng c xy ho tan trong mi trng. Kh c nn qua mt h thng lc sch tp trng, h thng ny tng i phc tp v d gy nhim cho mi trng nui cy . 1.2. Nui b mt

Hnh 4.8: S nui b mt

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1.Gu ti cm, 2.Thng cha cm, 3.Thit b kh trng, 4.Ni hp, 5.T cha khay mc ging v trng, 6.Bn trung gian, 7.Phng nui ging, 8.Thng chun b mi trng, 9.Phng hp khay ng ging, 10.Bn trung gian, 11.T cha khay cy ging, 12.Phng nui ging sn xut, 13.T ng khay ging sau sn xut, 14.My nghin, 15.Phng sy,16.Lc kh, 17.Qut nn kh.

Trong phng php nui b mt hay nui ni cc c th tn ti b mt mi trng, do m cc t bo hng v khong khng kh c cung cp y xy. cc vng nm, cht dinh dng ca mi trng ch c hp thu nh cc t bo chm v c chuyn vo si nm kh sinh. S to vng trong phng php nui b mt dn ti mt trng thi sinh l c ngha quan trng i vi vic sn xut cc cht trao i nht nh ca nm, v d nh sn xut acid citric hay cc enzyme. Tuy nhin ngi ta vn c gng t n trng thi sinh l tng ng vi nui cy chm (hnh 4.9). 1.3. Nui cy xp Phng php nui cy ny thng thch hp cho mt s nm mc v x khun. Vic nui thng c tin hnh trn cc khay phng xp chng ln nhau v trong cc bung cha v trng ng kn, ging c cy vo bng cch thi bo t vo bn trong bung cha. Mt hnh thc khc l nui h si nm trn cc c cht rn nh la m, cm hoc la nc trong cc thng quay chm. Phng php ny c dng sn xut mt s enzyme. Theo phng php ny, ging vi sinh vt hiu kh sau khi cy s pht trin trn b mt v dn dn lan xung pha di theo cc k h gia cc cu t thnh phn mi trng. Vi sinh vt s dng xy ca khng kh h hp ng thi thi CO2 ra mi trng xung quanh v to nhit. Phng php ny thng thch hp cho cc qu trnh nui cy nm mc, mt s x khun v vi khun cng c th sn xut theo phng php ny. Nui cy vi sinh vt trn b mt mi trng rn hoc bn rn c c cht dinh dng l cm c trn cc loi bt ng cc, u tng v mt s thnh phn dinh dng khc. Ngun carbon cho mi trng dinh dng l cc loi ht nh ng, go, m, i mch, u tng... c nghin v thnh mnh kch thc khong 1 - 3 mm. m ca mi trng khong 55 - 60%. Khi vi sinh vt pht trin s thi CO2 gy hin tng to nhit lm nng v kh mi trng. Cn phi thng gi, phun m hoc lm m trc tip gi cho m tng i ca khng kh khong 90%. Nhc im ca phng php: - Tn nhiu din tch mt bng, kh c kh ho v t ng ho. - Chi ph nhn cng, in nc... cho mt n v sn phm cao.

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2. Nui cy lin tc Cc phng php nui cy lin tc c th l: - Phng php n cp: nui vi sinh vt trong mt ni ln men, mi trng dinh dng c b sung cng nh mi trng ln men rt ra khi ni ln men mt cch lin tc vi cng mt tc . Phng php ny n gin, d ng dng vo sn xut i vi t bo nm men thu sinh khi hoc sn phm l cc cht chuyn ho gn trc tip vi s pht trin ca t bo. - Phng php nhiu cp: Vi sinh vt c nui h thng ni ln men t lm nhiu cp. Ni th nht c dng cho vi sinh vt pht trin tt nht, cc ni sau cc t bo tit ra cht chuyn ho. Mi trng dinh dng mi c b sung vo ni th nht v t ln lt chy vo ni tip theo. Trong cc h thng h ca phng php nui lin tc th ni ln men thng xuyn c cung cp thm dung dch dinh dng mi, v cng vi mc nh vy, mi trng b s dng mt phn v cc t bo c rt i. Vic khuy v thng kh nhm trn u cht cha trong ni ln men (h thng ng nht). Nh vy cc t bo trong ni ln men lun lun sinh trng theo hm s m v lun lun tn ti trong cng nhng iu kin sinh l. Tuy nhin, cc t bo ang phn chia v cc t bo khng phn chia cng tn ti v khng c s sinh sn ng b. H thng lin tc c iu khin bi cc yu t ho hc chemostas. Khi chuyn t trng thi ny sang trng thi khc th trng thi cn bng mi t c sau mt thi gian. Nh vic tng tc dng vo m sinh trng c th c tng gn tc cc i. Tc pha long (D) v tc sinh trng () l bng nhau trong phm vi ca tc pha long tiu chun. Cc h thng lin tc c ngha cng nghip. c im ca nhng h thng ny l ch, cc t bo li trong h thng hoc a tr li , trong khi mi trng chy i khng ngng. V cc t bo ch hot ng trong mt thi gian nht nh nn sau mt thi gian no cn phi thay th hoc b sung chng. Thc cht th h thng ny l s ko di pha cn bng ca s nui gin on nh vic a c cht vo mt cch lin tc. Cc t bo, hoc c gi li trong h thng nh trng hp ca vi khun acetic sinh trng trn v bo g ca phng php ln men nhanh, hoc c tch ra v a tr li nh trong sn xut bia ru. Trong vic lm sch nc thi, cc m vi khun cng b gi li trong cc b bn sng. Mt kiu khc l cho dng dung dch dinh dng chy vo nhng vng nm. l kiu nui ni ca h thng lin tc kn. quy m phng th

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nghim, cc h thng c kim tra, trong t bo v c cht c tch ring bng cc mng hoc dng c lc (ni ln men - mng). Hin nay vic nui lin tc c ng dng nhiu trong cng nghip sn xut sinh khi v cc sn phm ln men. Vic sn xut cc cht trao i bc mt v bc hai cng nh cc enzyme thng c tin hnh theo cch khng lin tc. u im ca phng php nui cy lin tc: - Gim bt thi gian lm v sinh thit b, kh khun v lm ngui. - Gim bt th tch ca ton b thit b. - Lao ng d dng v c kh nng t ng ho cc thao tc. - Tng hiu sut ca ton b qu trnh cng ngh nh chn lc tt nht cc iu kin thao tc. Nhc im: - i hi cn b v cng nhn thnh tho chuyn mn. Khi hot ng, cng mt lc phi c cc dng nng lng cn thit, gi thnh cao i vi t ng ho v dng c o lng hin i. - Trong qu trnh nui cy t bo vi sinh vt c th c nhng t bin bt ng xy ra lm hng c qu trnh. - Phi v khun tuyt i trong ton b thi gian thao tc. V trong qu trnh nui lin tc to ra cc iu kin ti u cho chng nui cy th cng ti u i vi nhiu loi tp khun. - i vi cc vi sinh vt sinh h si nh nm mc v x khun rt kh tch h si mt cch v khun v c bit l hiu sut chuyn ho thng thp hn so vi nui cy tng m vi nhng chng sn ra cht chuyn ho khng gn vi s pht trin.

VII. Ni ln men
1. S chung Ni ln men cng nghip c hnh tr, c t l chiu cao vi ng knh ca ni thch hp, th c th t vi m3 n khong 100m3 hoc c khi n 500m3 tu thuc vo quy m sn xut. Ni thng c lm bng thp khng r. i vi qu trnh ln men v trng th ni ln men v c cc van ng ng phi c tnh chu p sut c th kh trng p sut cao v trong thi gian ln men p sut d c duy tr sut c qu trnh ngn cn s xm nhp ca vi sinh vt l. Cc ni ln men c thit k v ch to sao cho c th to c nhng iu kin ti u cho tng qu trnh ln men. Nhng yu cu c th t c hot tnh ti a ca vi sinh vt c thc hin thng qua mt s

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nguyn tc k thut. V chi tit c s khc nhau rt ln gia cc kiu ni ln men. Mi mt loi ln men u c mt s yu cu ring bit. i vi ln men chm v trng nhng yu cu l ging nhau. V vy, y ch yu gii thiu ni ln men thuc dng nui cy hiu kh (chm, v trng v sc kh). Ngoi h thng thng kh v h thng lm nng v lm lnh, trong ni ln men cn c cc h thng ng cy, ng np mi trng, ng ly mu, ng np cht chng bt hoc cc cht khc. Ngoi ra cn c mt s my o nh cc in cc o pH, in cc o xy ho tan, in cc chng bt, ng nhit k ni vi nhit k t ghi v mt p k t trn ng thot. Sau cng c cc ca quan st kim sot bn trong ni v mt ca thng k tng v sinh ni .

Hnh 4.9: Ni ln men lin tc thu sinh phm

y di phn cui cng l ng thi c ng knh ln tho b dch, gm b mi trng v xc vi sinh vt. Ni ln men c t trn cc chn ch y, bng st hoc cc b gn vo nn nh v hn chc vo v ni. Chiu cao ca chn ny phi m bo cho ngi ta vo xem xt phn y ni mt cch d dng. phn trn, c sn lng vi lan can v cu thang cho php nhn vin i li kim tra s hot ng ca thit b, gi l gin thao tc. 2. H thng thng kh v h thng khuy S thng kh c thc hin nh vic cung cp v phn b khng kh cng vi s khuy trn. Cc h thng thng kh v khuy trn rt a dng. Nhiu kiu thng kh nh cc trc khuy rng, ng thng kh c

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bit, phn tn kh thnh cc tia nh nh cc ng hnh vnh khuyn hoc lp thnh gin c khoan cc l nh... Cng c ni ln men khng b tr cnh khuy m dng nguyn l bm vng lin tc khi dch kt hp vi sc kh c ng hi lu. Khuy trn v thng kh cng lm cho cc t bo v c cht c phn b ng u v cc bt sinh ra b ph v. Trong ln men k kh vic khuy trn cng cn thit m bo s phn b ng u v ngn cn s lng ng ca t bo. Bn trong ni ln men, ngoi trc khuy c gn my khuy nm ngang cn c thit b thng kh. Nhng bt khng kh thot ra t cc l khoan trn mt ng hnh vnh khuyn c phn tn vo mi trng nh cnh khuy nm ngang t pha trn. Ngoi cng c , ng hnh vnh khuyn dng thng kh cng cn c dng dn hi vo ni khi kh khun. Cc tm chn hn dc theo thnh trong ca ni c dng tng khuy trn v thng kh. 3. H thng lm nng v lm ngui Nhit ln men ti u c iu chnh bng cch cho nc nng hoc nc lnh vo v kp hay a vo trong ni nh cc ng xon rut g. Trong ln men cng nghip thng phi bm nc lnh lm ngui khi dch v trong qu trnh hot ng ca vi sinh vt to nhit rt ln. Nhng ni ln men hiu kh lm vic iu kin v trng c ch to bng thp cun trn, b dy chu ng p lc ca hi nc dng kh trng, hn vi y bnh bn cu nh trong ch to ni hi. V dng hi nc nn cc ni ln men cng c kim tra nh ni hi. Np trn thng hn gi kho ng c my khuy v hp gim tc. Trc khuy ng c lp ghp vi b phn gim tc v i vo ni ln men thng qua mt hp m trc c giong hi nc ngn tp khun gy nhim mi trng. Trn nhng ni ln men th tch nh, c lm thm v dn nc lm ngui, cn nhng ni ln hn 10m3 c cc ng cun trn theo thnh ni dn cht lng iu chnh nhit v dn hi nng dng kh trng. nhng ni c th tch ln, ng rut g cun theo chiu ng ca thnh ni. 4. Cc thit b kim tra - Kim sot s to thnh bt: Nhng mi trng dng trong cng nghip vi sinh, khi thng kh v khuy mnh lin tc to ra rt nhiu bt, c th trn ra khi ni ln men v gy nhim tp mi trng. Vic s dng nhng thit b ph bt l nhng cnh khuy t trn trc khuy phn trn ca ni ln men khng em li kt qu, nn ngi

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ta thin v dng nhng cht ph bt. Nhng cht chng bt thin nhin hay tng hp l nhng du thc vt, m c voi, nhng alcol ng ng cao, nhng silicon hay cc cht hot ng b mt. Nhng cht c dng ring hay hn hp hoc pha thnh dung dch. Trc khi c cc my t ng ph bt, bt buc phi thng xuyn theo di s to bt v cho cht ph bt vo mt cch th cng. Hin nay tt c cc ni ln men u c lp thit b t ng, ngay khi bt bt u hnh thnh l my t ng np vo ni mt lng nht nh cht ph bt. - o cng sut tiu th: Trong cc c s cng nghip, cng sut tiu th thng c o bng mt ot-mt biu th cng sut ca ng c my khuy. Cng sut tiu th ny thay i trong qu trnh ln men cng vi s gia tng ca nht mi trng. Trong nhng ni ln men quy m phng th nghim, vic o lng ny cng c bit quan trng bi v n cho php xc nh nhng tr s cn thit cho vic tnh ton thit k nhng ni ln men cng nghip. Nhng s lng o c bng ot-mt i vi ni ln men nh khng chnh xc v ma st ca trc vi hp m trc, v th ngi ta dng nhng lc k da theo nguyn tc ca cu Uytson cho php o rt chnh xc lc ko, in tr ca nhnh cu t trong trc bin i vi s bin dng rt nh do lc ko trc gy nn. - o xy ho tan: o xy ho tan trong ni ln men c tm quan trng hng u. o xy ho tan theo phng php cc ph rt chnh xc nhng i hi phi lm trn nhng mu v khun ly t ni ln men. Vic s dng cc mng mng bng teflon c th kh khun nhit cao v c thm xy kh , cho php thit k nhng thit b o xy ho tan trong mi trng. Trong qu trnh ln men, nhu cu xy ca mi trng nui cy lun thay i theo thi gian, v vy c th iu chnh thng s ny bng my t ng thay i lu lng khng kh v tc khuy gi cho lng xy ho tan cn thit trong mi trng. - o nht: trong qu trnh pht trin ca vi sinh vt, nht l ca cc nm si, mi trng lng tr thnh nht v c c tnh gi do gy cn tr cho vic khuy trn v thng kh. nht ca mi trng c o bng mt my quay c tc khng i mt nhit xc nh, khi tc gim l do nht tng ln. -o mt quang hc c thc hin bng quang k in. Mt trong hai cc ca my so mu c y kn v c dng mi trng v

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khun i qua ri tr v ni nh mt bm nhu ng. Mt khc, ngi ta th dng my o c c b phn quang in bc kn, t ngay bn trong ni ln men o mt quang. Cng theo nguyn tc , vic o lng lin tc s lng ng do vi sinh vt tiu th trong qu trnh pht trin c thc hin bng khc x k.

Hnh 4.10:My so mu t ngoi (spectrophotometter Shimazdu)

Cu hi n tp chng 4
1.Nhng iu kin cn thit cho mt qu trnh ln men trong sn xut cng nghip? 2. Yu cu chung ca ging vi sinh vt dng cho cng ngh ln men? 3. Cc phng php kh trng thng s dng trong sn xut cng nghip? 4. Nhng u v nhc im ca phng php nui cy gin on v lin tc ?

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Chng 5

S thu nhn sinh khi t bo

I. Tiu chun v chng
1. S dng nguyn liu r tin Cc nguyn liu thch hp l carbohydrate (r ng, dch kim sulfid, cellulose, tinh bt, cn sa), cacbuahydro (parafin, methane, cc ho cht t du ho nh methanol). Ngoi ra, nng lng bc x ca mt tri cng c to s dng vo vic c nh CO2 t dng. Vi sinh vt c th s dng ngun carbon v nng lng vi hiu sut cao. Carbohydrate c chuyn ti 50%, cacbuahydro ti 100% thnh cht kh ca t bo. 2. Tc sinh trng Vi sinh vt khc vi sinh vt khc ch thi gian nhn i rt ngn. vi khun, thi gian ny khong 0,3 - 2 gi, nm men v to l 2 - 6 gi. Do vy, vi vi sinh vt c th sn xut c nhiu sinh khi trn n v thi gian. i vi tnh kinh t ca mt phng php th sinh khi c tng hp trn mt n v thi gian c ngha c bn. 3. Hm lng protein cao Vi sinh vt n bo c hm lng protein khong 50 - 60% cht kh. Hm lng ny c tnh c hiu loi v chu nh hng nhiu bi iu kin nui. Cn to ra cc phng php nhm duy tr nhng thnh phn khc ca t bo, v d cht d tr, mc cng thp cng tt t c mt hm lng protein tng i cao. Cn ch rng, hm lng protein ch bao hm protein tht s ch khng gm cc thnh phn nit phi protein khi xc nh nit theo Kjeldahl, nh nucleic acid v cc peptide ca thnh t bo vi khun. 4. Cht lng protein cao Hm lng cc amino acid khng thay th qui nh cht lng protein. Tiu chun ny cng c tnh c hiu loi v mc nht nh chu nh hng ca cc iu kin nui. Nhiu amino acid c mt trong cc protein vi sinh vt vi hm lng cao ging nh trong cc sn phm tht, sa. Protein vi sinh vt c bit giu lysine. Tri li, hm lng cc amino acid cha lu hunh thp. 5. Kh nng tiu ho cao ca protein Kh nng c tiu ho ca protein vi sinh vt mt mt b hn ch

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bi cc thnh phn nit phi protein (v d nucleic acid, peptide ca thnh t bo vi sinh vt), mt khc bi s bao bc protein bng thnh t bo ca vi sinh vt, thnh ny rt kh cho cc enzyme tiu ho i qua. i vi protein tch khi t bo th khng cn quan tm n vn ny. Tuy nhin, tch ring protein th vn c bn li l kh nng ho tan ca thnh t bo. V th bn cht ca thnh t bo l mt tiu chun cho vic la chn vi sinh vt thch hp. 6. An ton v c t Vi sinh vt gy bnh v cc c th cha nhng thnh phn c hoc nghi ng v sinh l dinh dng dng kh tch ring th khng c dng sn xut protein n bo. Vic chng minh rng nhng thnh phn nh vy khng tn ti dng vt l rt kh khn v i hi s xt nghim ko di nhiu nm v mt c t v sinh l dinh dng. V th cho ti nay protein n bo hu nh ch c dng trong dinh dng ng vt. 7. Thun tin v k thut Vi sinh vt s dng cho sn xut cng nghip phi d tch v d x l. Cc t bo ln nh nm men c tch bng ly tm d hn nhiu so vi t bo vi khun. Ch khi no vi sinh vt sinh trng tt mt cao th mi c th t c nng sut cao. Kh nng sinh trng tt nhit cao hn ca cc c th a nhit v chu nhit lm gim chi ph cho vic lm ngui. Tnh khng mn cm vi s nhim tp l mt tin cho vic sn xut protein khng v trng.

II. Mi quan h vi sinh trng

1. Tc sinh trng Tc sinh trng c tnh c hiu loi v ph thuc vo iu kin nui. Ni chung vi khun c tc sinh trng cao l do kch thc nh nhng t l gia din tch b mt vi th tch t bo ln. S phi hp cc qu trnh trao i cht ring r c iu khin bi nhiu c ch iu ho c tnh c hiu loi. Ngoi ra, tc sinh trng cc i c th c ca mt chng trn cc mi trng khc nhau trong nhng iu kin nui nh nhau thng khng ging nhau. Chng hn, Escherichia coli trn mi trng phc hp nh canh thang c thi gian th h l 20 pht, trn mi trng tng hp cha glucose - 50 pht, cha acetate - 80 pht. S khc bit ny c quyt nh bi h enzyme cn cho sinh trng trn cc c cht khc nhau. Trn mi trng mui khong vi glucose l ngun carbon v nng

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lng, t bo sinh trng vi tc nh hn so vi trn mi trng phc hp hu c. Khi nui cy trong mi trng acetate th vi sinh vt cn phi tng hp cc enzyme ca qu trnh ti to glucose. Trong vic s dng nhng c cht nh vy v nhng c cht khc bn ngoi trao i cht trung gian trung tm ca cc c cht thng thng (v d cacbuahydro) th mt phn ng enzyme trong phm vi trao i cht ngoi vi cng c th c vai tr ch cht hn ch tc sinh trng. Chng hn s hot ho acetate c th l mt phn ng cht. c th nui cy vi sinh vt vi tc sinh trng cc i th tin c bn l nghin cu nhng iu kin nui ti u (thnh phn dch dinh dng, s thng kh, nhit ). 2. Trao i protein v nucleic acid Khong 90% protein ca t bo vi sinh vt l cc protein enzyme, 10% cn li l cc protein cu trc ca mng v thnh t bo. Cc protein c kh nng hot ng c mt cu trc xc nh do trt t cc amino acid quy nh. Do , trong tng hp protein, vn khng ch l s ni cc amino acid vi nhau nh lin kt peptide m s ni cn phi din ra theo mt trt t xc nh. Thng tin v trt t amino acid ca protein c cha trong cht liu di truyn ca t bo, tc l trong DNA, dng trt t cc baz ca nucleotide. truyn thng tin di truyn t nhn sang ribosome l ni tng hp protein, thng tin v mt hoc nhiu protein c ghi ln RNA thng tin (RNAm). S phin m ny c thc hin bng vic tng hp RNAm. RNAm mt si cha thng tin di truyn ca mt operon. l thng tin v mt hay nhiu enzyme hoc v cc di n v ca cc enzyme, nhng di n v ny to thnh mt n v chc nng. Operon lactose ca Escherichia coli cha thng tin v ba enzyme. Salmonella typhimurium th operon tham gia vo tng hp histidine cha 9 enzyme. Trong qu trnh phc tp ca tng hp protein, mt qu trnh gn lin vi s phi hp ca ba thnh phn phn ng c tnh c hiu, tc l ca 20 amino acid khc nhau, ca aminoacyl - RNAt - synthetase v ca phn t RNAt, ngoi ra cn c c s nhn bit v phn ng ca codon v anticodon th rt c th s tng hp protein l phn ng quy nh tc sinh trng cc i. Trong qu trnh sinh trng khng phi mi gen u c biu hin v t bo c cc c ch iu ho hot ng sao cho ch nhng enzyme cn thit cho t bo trong nhng iu kin nui nht nh mi c tng hp. Cng khng phi mi gen u l gen cu trc. Tc l gen cha thng tin cho mt protein hay cho chui polypeptide ca mt

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protein enzyme. Nhiu gen c chc nng iu ho. Khong 1000 gen c biu hin tc dng. T bo cha khong 1000 protein khc nhau. C mt s loi protein m t bo ch cha vi phn t (v d protein cht kim ch) nhng t bo li cha ti hng nghn bn sao cc enzyme ca cc con ng trao i cht trung tm. Nhng enzyme xc tc tng hp cc cht trao i m t bo ch cha mt lng nh (v d vitamin) th tn ti vi khong mt trm bn sao. 3. S ng ho cc ngun carbon khc nhau 3.1. Carbohydrate Nhng ph phm v ph phm sau y ca cng nghip c dng sn xut protein n bo: - Cc sn phm cha saccharose ca cng nghip ng (r ng c ci v r ng ma, b ma, cn r ng, nc ra th) v nhng qu khc cha nhiu ng. - Nc thi ca x nghip sa cha lactose. - Dch kim sulfid cha pentose v hexose l dch thu phn g cng nh cn b ca chng. - Vic s dng cc phn thc vt giu tinh bt (khoai lang, sn, khoai ty) v cellulose (v d rm r) khng qua thu phn trc thnh monosaccharide hoc disaccharide cha c ph bin trong sn xut. c th s dng cc c cht giu tinh bt ngi ta m t mt phng php nui hn hp gia Endomycopsis fibuliger v Candida utilis (phng php Symba). Cc nguyn liu cha cellulose (rm r c x l bng NaOH) c chuyn ho c kt qu thnh protein qui m phng th nghim nh s phi hp Trichoderma viride vi Candida utilis hoc Saccharomyces cerevissiae. 3.2. Hydrocarbua - Methane l thnh phn chnh ca kh m, n cn c to thnh qua con ng vi sinh vt nh s ln men methane v sinh ra trong cc b cha bn mc nt ca cc thit b lm sch. Vic ng ho methane nh vi khun ang c nghin cu mnh m nhm mc tiu sn xut protein qui m cng nghip. S dng methane c mt s u im so vi alkan mch di. Chng hn khng phi ra t bo mt cch tn km bng hexan hoc cc dung mi khc cn thit cho vic loi b cc parafin cn st li. Vic cung cp ti u c cht dng kh cho cc t bo nui chm gp nhiu kh khn. Vic cung cp methane cn kh khn hn do nhu cu

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v xy xy ho CH4 l c cht c tnh kh mnh nht trong tt c cc ngun carbon. V th ngi ta nghin cu s dng hn hp xy - methane (c b sung CO2 v N2) nhng cn phi trnh mt t l hn hp gy n. Do tnh ho tan km ca O2 v CH4 cho nn nu ch a dng kh vo mt ln th c cht khng c s dng hon ton; vic ti tun hon dng kh s nng cao mc s dng c cht. C th trnh c nhng vn nu ln nu thay methane bng methanol thu c t methane nh s xy ho ho hc. Tuy nhin methanol t hn nhiu so vi methane hoc kh m. Thu hoch t bo t methanol cng thp hn. Nhng v methanol d tan trong nc nn c th c dng nng cao hn (2 - 3%). - Alkan: Vic sn xut protein vi sinh vt trn c s alkan c thc hin nh nm men thuc ging Candida (v d C. utilis, C. tropicalis, C. guilliermondii). Vi khun, c bit l i din ca cc ging Pseudomonas, Flavobacterium, Mycobacterium v Nocardia c kh nng s dng cc alkan (C6 - C18) v nhng hydrocarbua khc, nhng kh nng ny hin nay cha c s dng rng ri trong cng nghip. 3.3. S c nh CO2 t dng CO2 l ngun C c ti sinh khng ngng trong vng tun hon ca t nhin, sm c ch c bit i vi vic sn xut protein n bo. Hai qu trnh c ngha i vi vi sinh vt hc cng nghip l: - S c nh CO2 quang hp nh to. - S c nh CO2 ho tng hp vi hydro l ngun nng lng nh vi khun hydro. S xy ho hydro: nhng loi c tp hp di tn chung l vi khun kh n c kh nng xy ho hydro dng kh to ra nng lng thc hin qu trnh ng ho CO2 theo con ng Calvin. Thuc v nhm vi khun ny c nhng i din ca ging Hydrogenomonas, thuc loi t dng khng bt buc. Mt s loi vi khun khc nh Nocardia opaca cng c kh nng ny. Trong vic nui to th cc loi to lc n bo nh Chlorella v Scenedesmus c ch hn c. Sn lng protein to trn mt ha c th t c 10 - 15 tn/nm, cao hn nhiu ln so vi cy nng nghip. Mun t c sn lng ny cn c iu kin kh hu thun li, nh sng mt tri mnh v ko di c y nng lng. Ngoi ra, qu trnh nui to i hi nhng thit b nui cy c bit. Thng thng ngi ta dng nhng b phng hoc nhng mng phng un khc c cc h thng lt o nhm hn ch s lng ca t bo v a t bo lun tr li b mt

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chiu sng. Vic cung cp nng CO2 ti u (4 - 5%) l cn thit. S pht trin sn xut protein n bo t to c y mnh nh vic dng to lam Spirulina maxima. To ny sinh trng thnh si v c th tch ra bng phng php lc. Chng cho sn lng cao, protein c gi tr v khng ng ngi v mt c t. V phng din kinh t th vic nui to c th gp phn lm sch mi trng trong cc thu vc trnh c hin tng ph dng

III. Cht lng sn phm

1. Cht lng protein Cht lng protein c quy nh trc ht bi hm lng v t l cn i ca cc amino acid khng thay th trong protein ca t bo. Thng tin di truyn ca DNA quy nh thnh phn amino acid. Tng ng vi nhng sai khc trong thng tin di truyn ca cc c th ta thy c s khc nhau v thnh phn amino acid gia cc nhm c th ring bit cng nh gia cc loi ca mt ging. V vy, vic tm kim nhng c th c hm lng c bit cao v cc amino acid khng thay th l c trin vng. Cng c s khc bit nh v thnh phn amino acid gia cc culture ca mt chng trn nhng mi trng khc nhau v nhng pha nui cy khc nhau. Trong nhng iu kin khc nhau s din ra s to thnh nhng h enzyme khc nhau. Thnh phn amino acid ca cc protein enzyme ring bit l khc nhau. Chng hn pyrophosphatase ca nm men bnh m cha 1,6% methionin, cn triosophosphatdehydrogenase cha 2,8%. Nhng s khc nhau nh vy ch biu hin yu trn ton b protein v chng c san bng bi v s cc protein enzyme khc. Nh vy, s khc nhau ch biu hin khi cc protein tng ng chim t l phn trm ln trong tng s protein. Nh t bin c th tng lng mt enzyme ln khong 5% hoc hn trong protein tng s ca t bo. Nhng th t bin nh vy ch c duy tr trong qu trnh nui lin tc nu tc sinh trng ca n khng b gim so vi chng ban u. Vic nghin cu di truyn vi khun c kh nng a vo vt liu thng tin di truyn i vi mt protein c hiu giu cc amino acid khng thay th. Tnh n nh v tnh thch hp ca cc chng nh vy i vi s ln men cng nghip cn cn c kim tra. Sinh hc phn t v di truyn hc nu ra nhng im ta nhm ti u ho cht lng protein ca vi sinh vt trong phm vi nht nh. Khi xem xt hm lng amino acid ca t bo cng cn lu , cc amino acid ho tan chim khong 5% cht kh ca t bo. i vi cht

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lng protein n bo th cc vitamin cng c ngha; mt s chng tn ti trong phn protein nh nhng cofactor ca cc enzyme. Vitamin chim t l nht nh so vi protein trong t bo. Hm lng vitamin ecgosterine ca Candida utilis c th c chuyn thnh vitamin D2 (calciferol) khi chiu tia UV ln chng. Thng thng, hm lng ecgosterine ca nm men chim khong 0,5 - 1% cht kh. 2. Hm lng protein Mt t bo vi khun ang sinh trng nhanh di nhng iu kin nui ti u c hm lng protein khong 50%, t bo nm men l khong 40%. Khng th nng cao t l ny, bi v phn cn li ca vt cht t bo l cn thit cho kh nng sng ca t bo. Trong s ny c RNA (10 20%), DNA (2%), cc thnh phn ca thnh t bo (20% l murein vi khun, polysaccharide v kitine nm men v nm si), lipid ca mng t bo (5 - 10%), cng nh cc vin gch cu trc c phn t lng thp v cc cht trao i ca trao i cht trung gian (5 - 10%). Trn nhng mi trng khng cn i hm lng cc cht d tr tng ln mnh, chng c th chim ti hn 50% cht kh. Khi tha carbohydrate v thiu nit th vic tng hp m nh vi sinh vt (s tch lu m ni bo) c th din ra. vi khun, cht d tr thng gp l acid poly - - hydroxybutyric, nm men, nm si v to l m hoc glycogen. S tch lu cht d tr c th trnh c, ph bin l thng qua qu trnh nui cy. Cng c th do t bin m kh nng tng hp cht d tr b ngng. Hm lng cc thnh phn ca t bo cn cho s sng nh nucleic acid, polymer thnh t bo, lipid ca mng khng th gim i mt cch ng k m khng gy tn hi hoc lm mt i nhng hot tnh trao i cht cn thit cho s sinh trng. Trong phm vi nht nh th thnh t bo l mt ngoi l. Cho ti nay vn cha r liu thnh phn v dy ca thnh t bo cc chng tn ti trong t nhin c cn thit i vi cc chng sn xut hay khng. C l do t bin m thnh phn t bo b gim nhng khng nh hng n sinh trng. Lm gim dy ca thnh t bo v thay i thnh phn ca n khng nhng c tc dng dng tnh n phn protein, m cn n s hp th cht v n tnh b phn hu ca t bo trong vic tch protein, hoc n tiu ho ca protein. 3. Hm lng nucleic acid Hm lng cao v nucleic acid ca vt cht t bo vi sinh vt khng nhng lm h thp phn protein m cn bt li cho vic s dng vt cht

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t bo vo dinh dng ca con ngi. Khi tiu ho nucleic acid th cc nucleotide c gii phng, v sau khi c hp th li chng s b phn hu qua adenine hoc guanine ti acid uric. S phn hu tip tc acid ny khng xy ra ngi v khng c uricase. Nu nng nucleic acid trong c th cao s gy ra chng thp khp, ngoi ra cn c nguy c to si thn v si bng quang do ho tan thp ca acid ny. Lng nucleic acid hp th qua dinh dng khng c qu 2g mi ngy. Trong dinh dng ng vt vn ny t quan trng hn, v chng c kh nng phn hu tip tc acid uric. Hm lng nucleic acid ca sinh khi c th b gim nh cc bin php sau y: - Gim mnh tc sinh trng. Mc d iu ny c th thc hin c thng qua giai on th hai ca s nui lin tc nhng khng kinh t. - Chit rt RNA, v d bng dung dch NaCl 10% nng. - Thu phn RNA bng kim v tch protein ho tan trong bng kt ta. - Phn hu RNA bi enzyme nh cc nuclease a vo hay nuclease ca bn thn t bo. Cc nucleotide sinh ra khi phn hu nucleic acid c th c dng nh nhng cht thm v cht gy v (IMP, GMP), hoc nh nhng cht ban u ca cc hot cht (v d kinetine v cc hp cht sinh hc).

IV. Ging khi ng

1. Nm men bnh m 1.1. Vi sinh vt Nm men s dng trong cc x nghip sn xut men bnh m l loi Saccharomyces cerevisiae. Trong cng nghip tuyn chn chng nm men phi m bo cc t tnh sau: - C kh nng n bt tt, lm khi bt nho nh v xp. - D ho vi nc. - C t tnh sinh ho n nh v bn vng tt (kh b t phn). - Bn nhit, c th ko di hot tnh enzyme nhit cao. - C kh nng ln men cc ngun ng glucose, fructose, maltose, saccharose. - Kh nng sinh sn nhanh, cho nng sut cao trong qu trnh ln men.

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- D tch sinh khi v d b gy sau khi p. 1.2. Sinh ha v iu ho - Kim ch d ho bi glucose: trong s sinh trng hiu kh ca nm men bnh m, cc nng 20 - 40 mg/l glucose trong mi trng, hot tnh ca cc enzyme h hp b c ch. Nh xy hiu ng ny s c gim nh hoc b trit tiu. Khi c mt ca xy th ngay c nng glucose cao (30%) s to cc cytochrome vn khng b c ch hon ton. - Cnh tranh v ADP v phosphate v c: qu trnh phosphoryl ho c cht v phosphoryl ho chui h hp u cn n ADP v Pvc. Do hai phn ng ny s c s cnh tranh v ADP v Pvc. Khi gim nng ADP v Pvc trong t bo s dn n s gim tiu th glucose v ngc li. S iu ho phosphofructokinase: enzyme ny S. cerevisiae b km hm hiu qu bi ATP, ITP, GTP, CTP l cc cht cho P song khng phi l cc cht km hm ging nh ATP, chng cng khng c vai tr iu ho. Trong khi tnh c hiu trung tm xc tc nh th trung tm iu ho tnh c hiu li cao i vi enzyme d lp th ny. AMP l mt cht hiu ng dng, n trit tiu s s km hm bi ATP. Escherichia coli, ADP c tc ng nh mt cht hiu ng dng. Phosphofructokinase cn b km hm bi citrate, ATP tng cng s km hm ny. S. cerevisiae cn c mi ph thuc gia hot tnh ca enzyme vi t l ATP/AMP. Khi cc t bo nm men sinh trng k kh, do b loi xy m s phosphoryl ho qua chui h hp khng th xy ra th c th quan st thy mt s bin i t l ATP/AMP v pha tng AMP. Kt qu l phn ng phosphofructokinase s c thc y. - Tc dng ca nng CO2 cao: nng 40% CO2 c mt trong khng kh s bt u km hm s sinh trng v nng 50% th s km hm ny biu hin r rt. - nh hng ca xy: s tng hp cytochrome S. cerevisiae ph thuc vo xy. nng xy ho tan 0,05 M/l hoc thp hn cc cytochrome a, b, c s khng c tng hp. Tuy nhin, nm men bnh m l loi vi sinh vt tu tin c kh nng sinh trng c trong iu kin c v khng c xy. Khi sinh trng trong iu kin c xy, nm men cn b sung biotin, cn nhu cu v inositol v acid pantotenic thay i tu chng. Ngoi cc nhu cu ny, khi sinh trng trong iu kin k kh nghim ngt nm men cn thm cc thnh phn khc nh cc acid bo cha bo ho, ergosterol v mt s chng cn cn c acid nicotinic. Thiu cc thnh

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phn ny trong mi trng sinh trng ca nm men ch din ra sau vi th h sau s dng hn. Quan h ca nm men i vi xy rt phc tp. Ngoi hiu ng Pasteur cn c hiu ng Pasteur ngc (s kim ch d ho) v hiu ng Pasteur m. Nng ng l nhn t c bn qui nh s sinh trng v tc ln men ca nm men. Sinh trng ca S.cerevisiae trong mi trng cha glucose c thng kh c xem nh mt s sinh trng kp trn glucose - ethanol. Glucose trc ht c chuyn ho thnh ethanol trong pha sinh trng logarid th nht trong t bo c tc sinh trng cao. Pha sinh trng th hai bt u khi ng trong mi trng gn nh b tiu th ht. Pha ny c tch bit vi pha u bng mt thi gian pha lag kh r. Tc sinh trng ca t bo trong pha th hai thp hn trong pha ln men v th hin trn mt ng cong sinh trng c dc t hn. Ngc li, tc sinh trng trong pha h hp ph thuc r rt vo mc thng kh. Trong sn xut cng nghip men bnh m ngi ta quan st thy mt di th h l 3 - 4 gi khi ru c to thnh nhiu v 5 6 gi trong mi trng khng c ru. Trong nui cy tnh, cng thng kh ch gy hiu qu nh hoc khng gy hiu qu g ln giai on ln men hoc ln v tr ca pha lag. Trong pha sinh trng logarid u tin, chu trnh TCA hot ng ch to ra b khung carbon cho cc mc ch sinh tng hp. Hot tnh ca cc enzyme thuc chu trnh xy ho l thp, hot tnh ny s tng ln trong pha th hai ca sinh trng kp. Nu nng glucose vt qu mc nht nh th cc enzyme ny s khng c to thnh hoc c to thnh mc rt thp. C th trnh hin tng kim ch d ho bng cch nui cy nm men theo phng thc nng ng c duy tr mc rt thp nh trong mt h thng nui cy lin tc. Nm men cha cc enzyme hot ng cho trao i cht xy ho s chuyn trao i cht ca n t xy ho sang ln men khi nng ng vt qu mt gii hn nht nh. Ngoi kh nng c ch s to thnh cc enzyme xy ho (kim ch d ho), glucose cng c kh nng kim ch hot tnh ca cc enzyme ny. 1.3. K thut sn xut * C cht: Nm men bnh m c th pht trin tt trong mi trng c ngun carbon l glucose, fructose, maltose, saccharose, nhng trong cng nghip thng dng r ng ma hoc r ng c ci sn xut

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sinh khi nm men. Ngun nit v phtpho c b sung vi t l ph hp (t l P2O5/N tng s khong 1/3). Trong sn xut thng dng superphosphate hoc ammonphosphate lm ngun cung cp phtpho. Cc nguyn t khong khc ni chung trong r ng cung cp cho nm men pht trin ngoi tr Mg l phi b sung di dng MgSO4. Khi s dng ng ma cng cn b sung thm K v cc mui ammon nh (NH4)2SO4, urea, NH4OH hoc (NH4)2HPO4. Dung dch mui khong cn b sung tng dn trong sut qu trnh ln men thnh phm nhng cn phi b sung ht trc khi kt thc ln men 3 - 4 gi. Ngoi glucide, hp cht nit v cc mui khong cn thit khc, nm men bnh m cn cn mt s vitamin tng sinh khi nhanh nh biotin, inositol, acid pantotenic... Thng thng r ng ma c biotin hoc thay th 20% r ng c ci bng r ng ma. Ngc li, trong r ng c ci thng c nhiu inositol v acid pantotenic, cc amino acid nn nng sut sinh khi vn cao hn so vi khi dng r ng ma. Ngoi thnh phn mi trng dinh dng, cc yu t nh nhit , pH, nng xy ho tan c nh hng rt ln n s pht trin ca nm men bnh m. Nm men pht trin tt pH 4,5 - 5,0. Trong cng nghip sn xut men bnh m thng ln men pH 4,2 - 4,5 hn ch vi khun tp nhim. Vo gi cui ca giai on ln men thnh phm cn phi tng pH ln 4,8 - 5,2 nm men c mu sng p (v nm men d hp ph mu ca r ng khi pH thp). Trong sn xut men bnh m cn thit phi b sung r ng t t trong qu trnh ln men nhm hn ch s to thnh ru, gim nng glucose trong dch ln men nng cao hiu sut sinh khi. Vi nng glucose khong 5% trong dch ln men, cc enzyme h hp hot ng km lm gim sinh khi t bo. Cn phi thng kh mnh mi lng xy cn thit cho sinh trng. Trc khi ln men r ng cn c x l loi b cc tp cht, dit cc vi sinh vt gy nhim, pha long n 40-500 Brix v dng acid H2SO4 h pH xung 4,5, un nng 900C trong 30 pht v khuy trn. Sau r ng c lm lnh v lng trong, c th dng my li tm tch chuyn dng hoc p lc khung bn lc r ng. * Phng php sn xut: Sn xut men bnh m l qu trnh nui cy qua nhiu giai on: nhn ging trong phng th nghim, nhn ging thun khit v ln men trong iu kin sn xut.

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T ng ging thch nghing ly mt vng que cy chuyn vo mi trng lng nhit 25 - 280C trong 24 - 48h. Ban u c nui cy tnh sau nui cy lc ri chuyn sang giai on nhn ging thun khit trong nhng thng nui cy nh v c sc kh nh bng khng kh v trng nhng khng c b sung mi trng trong qu trnh nui. Sau dch ging c bm sang thng nui cy ln hn c trang b h thng b sung mi trng v sc kh mnh hn. Tu thuc vo quy m v yu cu ca x nghip c th tip tc nhn ging trong nhng thng nui cy ln hoc rt bt s thng nhn ging c lng ging va .
Quy trnh sn xut men bnh m:

ng ging Nhn ging nh 1 lt Ging 50 lt Ging 200 lt Ging 1000 lt Ging 15000 lt Nui men ny chi 90 000 lt Li tm tch sa men, bo qun lnh Thng nui men thng phm 200 000 lt

ng ging Nhn ging nh 1,8 lt Ging 12 lt Ging 36 lt Ging 200 lt Ging 300 lt Ging 30 000 lt Nui men ny chi 116 000 lt Li tm tch sa men, bo qun lnh Thng nui men thng phm 116 000 lt

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Trong qu trnh nhn ging ta thu c men ging th h A. S dng men th h A sn xut men th h B hay cn gi l men ny chi. Sau khi nui cy th h B, dng my li tm tch men ging di dng sa men v bo qun lnh 2 - 40C chun b cho sn xut men thng phm. Men thng phm c pht trin trong nhng thng nui cy ln. R ng, dung dch mui khong, khng kh c cung cp tng dn trong qu trnh ln men v mc ln nht. Khi kt thc ln men pH khong 5-5,5, ru khong 0,03 - 0,05%. Mc sc kh cng gim mnh trong gi nui cy cui cng. Thi gian nui men thng phm ph thuc vo cht lng v s lng nm men, nhit , ch sc kh, mi trng dinh dng v nhiu nhn t khc, trong cng nghip thng l 12 gi. Sau khi nui men thng phm, dch men c lm lnh nhanh, li tm v ra nc vi ln, men c tch ra di dng sa men. Sa men c ra nc vi ln na cho tht sch ri lc p v cui cng l p khun, ng gi v bo qun lnh vn chuyn n cc c s sn xut bnh m hoc dng sn xut men kh (c m khong 8 -10%) d vn chuyn v bo qun di ngy. Gim st k thut: Trong khi theo di qu trnh b sung cn tin hnh cc bc kim tra sau: - Kim tra pH - Xc nh hm lng ng v ru trong dch ln men. - Xc nh s tng trng ca nm men. - Xc nh hm lng cht dinh dng ca dch ln men. C s thng kh cng phi c iu chnh v n c nh hng r rt ln hot tnh sinh ho ca nm men. T lng ru to thnh v lng t bo nm men c mt ngi ta c th xc nh cc qu trnh ln men v cc qu trnh h hp ang din ra theo t l no. bo qun, men p cha 67 - 71% m c nn trong cc thng g hoc nhm trnh khng kh xm nhp v gi trong cc phng lnh. Dch to ra do h hp ca nm men trong qu trnh bo qun phi c loi b ngay, nu khng bn ca men p s b gim r rt. Trong qu trnh bo qun, carbohydrate d tr s b phn gii, kh nng ln men s b gim i. Khi xut xng, hm lng nc ca nm men c nng ln 72 -75% v nm men s c ng gi. Bng cch sy kh nh khng

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kh nng ngi ta thu c men kh c trng lng kh 95 - 96% c hot tnh ln men cao. Cht lng ca men c xc nh bng thi gian lm n bt (thng t 45 - 60 pht), thi gian bo qun nhit 350C l 2 - 3 ngy. Men p tt c mu xm nht, c mi v c trng, khng ng, cu to cht ch, d b gy v khng c chy nho trong khong 40h sau khi ti tay ngi tiu dng. * Vi sinh vt tp nhim: S xut hin cc vi sinh vt sinh acid nh cc vi khun lactic (a s trng hp l cc vi khun lactic d hnh), cc vi khun acetic v t bit l cc vi khun butiric thng c nh hng ti s pht trin ca nm men mt cch r rt. Do s to thnh acid butiric ca Clostridium trong men p m xut hin mi thiu kh chu. Trong vic nui cy nm men cn ngn cn s xut hin ca cc loi nm men khc nh Torula, Candida... Bn ny thng pht trin rt nhanh v ln t Saccharomyces cerevisiae. Cc loi nm men k trn thng kh p v lm gim ng k nng lc ln men ca men p. Hng lot vi sinh vt xm nhp v sau vo men p v gy nn cc hin tng h hng. Nguy him nht l Oidium lactis thng gy cho men p mi ng. Cc nm khc nh Penicillium to nn m mu lc trn men p, Aspergillus to nn cc m mu vng nht ti mu xm, Mucor v Fusarium to cc vt vng v trn men p. Dematium pullulans to nn cc vt mu nu bn, cc vi khun acetic to nn nhng vng trn b mt, Serratia marcescens to nn cc vt trn men p. Bacillus mesenteroides l bn thng gy ra hin tng ko si ca bnh m khi chng c mt vi s lng ln trong nm men. C th ngn nga hin tng ny bng cch b sung acid propionic. 2. Vi sinh vt dng cho k thut v y hc 2.1. Ging khi ng trong cng nghip thc phm T lu trong ngnh sa ngi ta s dng cc loi ging thun khit v ging hn hp sn xut cc sn phm khc nhau nh phomat, cc loi sa chua v.v... Vo khong t nm 1955 ngi ta bt u s dng vi sinh vt cho cc sn phm tht khc nhau, c bit l xc xch th v cc thc phm p mui. Cc vi sinh vt ny c cy vo thc phm tin hnh qu trnh ln men nht nh. Chng hn Pediococcus cerevisiae c dng lm ging khi ng cho loi xc xch ma h ca M, hay cc chng Micrococcus c dng cho cc loi xc xch ca chu u. Thng thng, cc vi khun ny c cy di dng hn hp vi cc loi

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Lactobacillus, c bit l L. plantarum. Cc vi sinh vt ny gi vai tr to mu, lm gim pH v to thnh cc cht thm c th. Ngoi ra, chng cn ngn cn s pht trin ca cc vi sinh vt khng mong mun. Khi s dng cc chng nm si cho cc qu trnh lm chn cc sn phm tht th iu quan trng l phi loi tr s c mt ca cc loi nm to thnh c t trong ging khi ng. Penicillium nalgioversis c coi l loi thch hp c th dng lm ging khi ng cy vo cc loi xc xch. Loi ny c nh hng n v ngoi, mi, v v bn trong bo qun ca cc loi sn phm tht p mui v xc xch th sn xut theo kiu Hungari v kiu . 2.2. Ngun b sung thc n gia sc Brevibacterium divaricatum do giu hm lng acid glutamic v protein nn c ngh b sung vo thc n ca gia cm, ln, cu, b, ch v cc ng vt khc. Bacillus megatherium do giu cc vitamin nhm B cng c s dng lm ngun b sung di dng sinh khi kh. Vi khun, to hin vi v cc ng vt ni cng c s dng lm thc n trong cc trang tri nui ng vt bin nh tm, s, trai v.v... v c cc loi. 2.3. Dng cho iu tr iu tr cc bnh v ng rut bng vi sinh vt sng ngi ta thng dng cc t bo ca Lactobacillus acidophillus hoc ca cc loi Lactobacillus khc, cc chng khng gy bnh ca Escherichia coli hoc hn hp ca cc loi ny. Mc d kt qu ca nhng s iu tr ny vn cn tranh ci, song c nhiu cng trnh c bit ch ti vic nui cy thu nhn sinh khi ca cc loi vi khun ni trn v ca cc vi khun khc nh cc chng khng gy bnh ca Streptococcus, Enterococcus, Enterobacter aerogenes, Bacillus subtilis v Clostridium cng nh ca nm men Saccharomyces cerevisiae, Dispora caucasica v.v... Ngoi ra, ngi ta cng tm cch s dng cc chng Lactobacillus acidophilus hoc E. coli khng khng sinh phc hi li khu h vi sinh vt ng rut b hu hoi do vic iu tr bng khng sinh. Nhiu hn hp khc ca cc loi vi sinh vt khng khng sinh cng c sn xut, chng hn nh hn hp ca cc vi khun lactic vi Streptococcus faecalis, ca cc vi khun lactic, acid lactic v tanin, ca Saccharomyces frogilis v Streptococcus lactis vi Lactobacillus acidophilus, Streptococcus lactis v S. thermophilus.

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Ngoi vic s dng vi khun cho vic iu tr qua ng ming, t lu con ngi s dng cc vi sinh vt cn sng hoc b git cht, c bit l cc vi khun, lm khng nguyn hoc lm c t. Trong vic sn xut cc vaccine, cc vi khun gy bnh thng c nui i tr. Trc y sn xut vaccine thng dng phng php b mt, ngy nay hay dng phng php chm, sau vi sinh vt c git cht bng nhit, acid, formalin, phenol... sao cho tnh khng nguyn ca chng vn c gi li, ri c ch thnh thuc tim. Cc vaccine t vi khun c sn xut vi s lng ln. sn xut cc c t nhm to ra cc khng c t thng l trong cc c th ng vt, ngi ta cng thng nui chm cc vi khun chng hn Clostridium tetani hay C. botulinum, sau tim vo cc ng vt thch hp, chng hn nga. Khng c t ly t huyt thanh ng vt s c thun khit bng bin php ho sinh hc mc cao nht ri em iu tr. 4. Dng cho bo v thc vt Gi vai tr quan trng trong ngnh bo v thc vt l cc sinh vt gy bnh cho cn trng v cc sinh vt gy hi khc cho cy trng. Chng bao gm c vi rt, vi khun, nm v ng vt nguyn sinh. Trong nhiu trng hp, chng gi vai tr iu ho t nhin trong ni b cc qun th cn trng. Vi khun gy bnh i vi cn trng bit r nht l Bacillus thuringiensis. Tc dng gy bnh da trn cc tinh th km (hay ni c t) m s xut hin ca n c lin quan vi s to thnh ni bo t. Cc tinh th protein ny trong rut cn trng, chng s ho vo dung dch v trong vng vi pht s lm ngng qu trnh n cn trng do vic ph hu cc biu m rut. Liu lng c nut vo cng cao th biu m cng nhanh b ph hu v cn trng s cng nhanh b tiu dit. Di liu lng gy cht, ni c t s gy nn s ngng n tm thi. C th cc biu m li c phc hi v u trng ly c kh nng n bnh thng. Vi khun c th sn xut i tr bng phng php nui chm quy m cng nghip trong iu kin hoi sinh. Cc bo t cng vi tinh th c th gi c kh lu. Loi ch phm cha bo t ny c s dng thnh cng chng li nhiu loi cn trng nh Colias eurytheme, Pieris brassicae v cc loi cn trng cnh vy khc hi bp ci, ci du v mt s cy thn g cng nh su c thn ng (Ostrina nubilalis) v loi su hi Datana integerrima.

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Bacillus popilliae var popilliae l mt vi khun gy bnh cho b hung Nht Bn (Popilla japonica) v cng c s dng chng li su c thn ng (Melolontha melolontha). Vi khun ny c nui cy i tr bng phng php chm. Cc loi Bacillus khc nh B. alvei, B. circulans, B sphaericus c tc dng dit u trng cn trng. Serratia picatorum, Streptococcus faecalis v Enterobacter aerogenes lm gim pH trong rut u trng bm, qua c tc dng gy bnh, trong mt s trng hp c th git cht chng. Mt s nc s dng Salmonella typhimurium v S. enteritidis dit chut. Chng c nui cy chm v trn vi thc n cho chut. Trong khi an Mch v mt s nc ng u ch phm ny c dng nhiu th c v M, do mc cm v v sinh ngi ta li cm dng. Trong s nm, Beauveria tenella l loi gy bnh cho su c thn ng. Mt qun th ca u trng trong t cng cao, mc nhim nm cng mnh. Ti 80% u trng c th b nhim. Cng c cc th nghim v nm ny chng cn trng cnh cng trn khoai ty nhng hiu qu cn thp. gy nhim, nm phi ny mm trn cn trng. Sau khi ny mm, nh nhiu loi enzyme trong c kitinase, si nm s m su vo c th cn trng v pht trin mnh m. Cn trng lc ny b p xc hon ton, sau si nm s pht trin ra pha ngoi. Beauverixin l mt ch phm chng cn trng mnh thu c t Beauveria bassiana. Nhiu loi nm khc cng c pht hin l c kh nng dit cn trng, chng hn Asperginllus flavus, A. versicolor, Penicillium rugulosum, Mycrothecium verrucaria c tc dng km hm s pht trin ca rui dm (Drosophila melanogaster). Spicaria rileyi v Cordyceps militaris cng l nm gy bnh cho cn trng. Entomophthora thaxteriana l loi nm gy bnh cho rui nh c th to thnh ti 50.000 bo t/ ml khi c nui chm trong mi trng cha dung dch peptone - cao nm men - glucose sau 15 ngy nhit 280C. 5. Phn bn vi sinh vt nui cy i tr cc t bo Azotobacter c kh nng c nh nit dng lm phn vi sinh vt cn phi s dng cc c cht rt ngho nit. Cc t bo c nui cy nhit 25 - 300C, pH 6,5 - 7,0 v thng kh lin tc trong mt mi trng cha glucose. Sau 36 - 58 gi sinh trng kt thc. T bo c li tm v ng kh trong chn khng ri trn vi t v trng. Vi khun cng c th c lm kh trong khng kh 30 - 350C sau c hp ph trn bt lignin.

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Phn vi sinh vt c bit l vi Rhizobium leguminosagum hoc R. phaseoli l mt loi phn thng dng nhiu nc t cui th k trc. Sn lng ca cc cy h u tng ln rt nhiu nhng vng t mi khai ph, chng hn vng Poldern ca H Lan. sn xut phn vi sinh vt, cc chng Rhizobium mi phn lp, c nh nit tt c nui chm trong cc dch dinh dng hu c ri sau c trn vi cc c cht nh than bn hoc cui. Trong qu trnh ny vi khun phi sng c cng lu cng tt. Phn c s dng di dng bt, di dng dch bn hoc dng thuc phun phun trn cc din tch cn thit.

V. Protein n bo (SCP: single cell protein)

1. Tm quan trng Tuy sn xut protein ng vt ca th gii hon ton c th p ng c nhu cu ca dn s th gii hin ti nhng 2/3 protein sn xut ra ch c tiu th bi 1/3 dn s th gii. cc nc ang pht trin tiu th protein ch nm mc 12g protein ng vt/u ngi/ ngy. Tc tng dn s cc nc ang pht trin ln khong gp i so vi cc nc pht trin. Ch da vo vic ci thin cc phng php nng nghip th khng lng protein cung cp cho nhu cu thc phm. R rng, vic tm ra cc ng cc c nng sut cao hoc cc protein c gi tr sinh hc cao l nhng bin php mang li nhng ng gp c tnh cht quyt nh trong vic gii quyt vn thc phm ca nhn loi. y, sn xut protein nh vi sinh vt trn quy m cng nghip chc chn s gi mt vai tr quan trng. cc nc c mc sng cao, cc thc n hn hp dnh cho ng vt i hi phi cha cc ngun protein c cht lng cao sn xut ra trng, tht tiu chun. Cc thc n ny phi tho mn hon ton cc nhu cu dinh dng ca ng vt thng cha 10 - 30% protein tnh theo trng lng. Trc y ngi ta thng dng bt cc loi ht cha du nh u tng hay bt c p ng cc nhu cu ny. nh dng SCP thay th cc loi bt ni trn s gii quyt c hai vn : - Tng ngun u tng, c (v c ng cc) cho dinh dng ngi. - Cc nc chu u, Nga, Nht v mt s vng khc vn khng trng c u tng do vy sn xut c SCP s lm cho chn nui khng ph thuc vo vic nhp khu protein. So vi sn xut cc ngun protein truyn thng, sn xut SCP c nhng u th sau:

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- Tc sn xut cao. - Hm lng protein cao (30 - 80% tnh theo trng lng kh). - C th dng cc ngun C khc nhau (mt s c th l cc cht thi). - Chng sn xut c nng sut cao v thnh phn tt, d kim, d chn. - Din tch sn xut khng ln, cho sn lng cao (tr to). - Khng ph thuc vo ma v, kh hu. 2. Cc bc chnh ca mt qu trnh sn xut SCP - Chun b ngun C, thng l phi qua x l vt l v ho hc cc nguyn liu th. - Chun b mi trng thch hp cha ngun C, N, P v cc cht dinh dng khc. - Ngn nga s nhim tp mi trng hoc thit b sn xut. - Cy vi sinh vt mong mun. - Tch sinh khi t bo vi sinh vt khi mi trng tiu dng. - Hu x l sinh khi tinh khit hoc khng. Trong , trc ht cn lu nhng vn sau: * Tuyn chn vi sinh vt: mt vi sinh vt s dng cho mc ch sn xut SCP lm thc n protein cho ngi hoc ng vt cn c mt s c im c bn nh khng gy bnh cho ng vt, thc vt v ngi, c gi tr dinh dng cao c chp nhn nh l mt loi thc phm hoc thc n gia sc, khng cha cht c, gi thnh sn xut thp. * Nui cy: d l s ln men din ra di iu kin v trng hoc iu kin sch u cn phi c cc bin php trnh nhim tp nh: - un nng hoc lc cc thnh phn mi trng v kh trng thit b ln men. - Khc vi to, cc qu trnh sn xut SCP khc u cn thng kh mnh. - Nhit to ra c loi b nh h thng lm lnh. * Thu hi sinh khi

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- Nm men v vi khun thng c thu hi bng li tm, cc vi sinh vt dng si c th thu hi bng li tm vt (lc vt). - Phi loi bt cng nhiu nc trc khi lm kh trnh tn km tr nhng ni c th phi nng v s dng lao ng gi r, tuy nhin sn phm s c cht lng thp hn. - Ty thuc vo loi c cht v loi sn phm, sinh khi cn c hu x l loi cc thnh phn c cht hay thng gp hn l lm gim hm lng cc cht khng mong mun (v d nucleic acid) hoc thm ch tch ring phn protein. Mt nhc im ln ca SCP l thng cha hm lng nucleic acid cao. Nu cc loi SCP ny c s dng cho ngi th s l mt vn v con ngi thiu uricase xc tc cho s xy ho acid uric thnh allantoin. Khi n nhiu cc dn xut ca purine s lm tng hm lng acid uric trong mu. Acid ny s kt ta to thnh tinh th trong cc khp v ng gp vo vic gy si ng niu o. c nhiu phng php ra nhm lm gim hm lng acid uric trong SCP nh thu phn bng kim, chit bng ho cht, iu khin v sinh trng v sinh l t bo, hot ho RNA-ase (bng x l nhit ngn). Nhng thng th cc phng php y s dn n gim gi tr sinh hc ca protein n bo. - phng khng loi ra mi trng ngoi lng ln vi sinh vt k c dng cht ln dng sng. Khi dch thi c hm lng BOD cao th cn phi x l trnh nhim mi trng. Sau khi x l, mi trng c th c ti s dng nhm ng thi hai mc ch l gim lng nc mi cn thit v gim gi thnh sn phm. 3. u nhc im ca cc vi sinh vt dng cho SCP 3.1. S dng to sn xut SCP thng dng ba chi to l Chlorella, Spirulina v Scenedesmus. Chng c th c phng thc sinh dng l quang hp, ho tng hp hoc l d dng. Phng thc hay s dng nht l quang hp. Trong trng hp ny nhn t gii hn s l nh sng v vy kinh t nht l dng cc h h di nh sng mt tri. Tuy nhin, khi tin hnh nui to cc h thng c qui m ln th kh c th gi c cc iu kin v trng vi gi thnh thp v trong nhng trng hp ny nguy c nhim tp l nghim trng. 3.2. S dng vi khun Vi khun c th c dng sn xut protein n bo, nhng cn

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lu n nhng u im v nhc im ca chng: - Tc sinh trng nhanh. - S dng c nhiu loi c cht. - pH cn duy tr 5 -7 nu khng s c nguy c nhim cc vi khun gy bnh. - Thu hi bng li tm kh khn. - Hm lng protein th c th rt cao (80%) nhng hm lng ca cc nucleic acid c bit l RNA cng cao (20%) v cn phi c loi b. - Thnh phn amino acid cn i nhng hm lng cc amino acid cha S hi thp. - Khi dng cc vi khun Gram m sn xut SCP cn lu ti kh nng c t ca chng. 3.3. S dng nm men Sn xut nm men qui m cng nghip c pht trin t hn mt th k nay c bit l vic s dng cc loi ca chi Saccharomyces, Torulopsis, Candida. Thng th tc sinh trng nm men l tng i cao tuy nhin khng bng cc chng vi khun sinh trng nhanh nht. Hm lng protein ca nm men chim khong 55 - 60%, tuy nhin hm lng nucleic acid c th ln ti 15%, v vy tin hnh cc bin php lm gim nucleic acid l cn thit. Thnh phn amino acid ca nm men cn i nhng so vi vi khun th hm lng cc amino acid cha lu hunh thp hn, s dng cn b sung thm methionin. Trong nm men giu cc vitamin nhm B. 3.4. S dng nm si Ni chung tc sinh trng ca nm si thng thp hn vi khun v nm men nhng gn y c th phn lp c cc chng c tc gn vi nm men. Gii hn pH thch hp cho sinh trng kh rng (3 - 8), tuy nhin trong sn xut cn gi pH < 5 trnh nhim vi khun. Vic nhim nm men rt hay xy ra tr khi mi trng c kh trng tt. Khi nui cy chm, nm si thng to thnh cc m si nm, iu ny c u im l vic thu hoch c d dng, song li c nhc im l hn ch s phn b ng u ca khng kh trong ton b h si. Hm lng protein th ca nm si nm trong khong 50 - 55%, song khi sinh trng nhanh, hm lng nucleic acid s cao (RNA ti 15%). Thnh phn amino

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acid ca nm si cn i, tuy nhin cc amino acid cha S cng c mt nng thp. Nhiu loi nm si c th sinh c t nm. 3.5. Sn xut nm n Cc ph ph phm nng, lm nghip nh rm r, b ma, li ng, mn ca, tru... vn cha c s dng trit . Bng cc bin php x l thch hp chng c th c chuyn thnh c cht nui nm n. Qu th nm c dng lm thc phm, b thc vt hoi sau khi trng nm c dng lm phn hu c, cc c cht c nm mc c gi tr nh mt loi thc n nng cp cho ng vt. Vic chuyn ho b thi hu c nh nm n c nhiu u im: - Cht thi c loi b mt cch c li v c ho nhp tr li vo h sinh thi nh cc qu trnh chuyn ho t nhin. - Cht thi rn v lng u c th tham gia trc tip vo s chun b c cht. - Lignin khng tiu ho c v cc thnh phn ca thnh t bo b lignin ho cao nh cellulose v hemicellulose u c th c huy ng v c khong ho hon ton. - Cc ngun carbon thng thng hu nh khng c s dng s c chuyn ho thnh sinh khi giu protein. - Vic thu hoch tht qu th t b mt c cht l cch tt nht tch sinh khi vi sinh vt thun khit khi c cht ca chng bng tay hoc bng my. - Nm n l mt loi sinh khi vi sinh vt c xc nh k cng v c ngi tiu dng chp nhn rng ri. trng nm, ch c cc nm m hoi sinh l thch hp v mt sinh thi hc. Trong t nhin, chng gi vai tr quan trng trong s phn gii cellulo-lignin. Mt s nm c v ngon thch hp vi vic dng lm thc phm. Nm n c trng khp th gii di vi cc iu kin kh hu khc nhau v c th s dng cc cht thi nng nghip v lm nghip l nhng c cht thch hp c th nhn c vi nhng s lng ln.

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Mt s loi Nm n v Nm dc liu c th trng Vit Nam

Hnh 5.1: Nm s ( Pleurotus ostreatus )

Hnh 5.2: Mc nh (Auricularia auricula)

Hnh 5.3: Nm rm (Volvariella volvacea)

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Hnh 5.4: Nm m (Agaricus bisporus)

Hnh 5.5: Linh chi ( Ganoderma lucidum)

Cu hi n tp chng 5
1. Tiu chun ca chng vi sinh vt cho sn xut sinh khi ? 2. Yu cu v ging v k thut sn xut men bnh m ? 3. ngha v cc bc chnh ca sn xut SCP ? 4. Cc u v nhc im ca vi sinh vt s dng cho sn xut SCP ?

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Chng 6

Cc sn phm ln men
I. Ln men ethanol
1. Ln men ethanol nh nm men 1.1. C s ho sinh: Ln men ru l qu trnh phn gii ym kh ng di tc dng ca enzyme vi sinh vt c hiu. Trong s ln men ru, u tin pyruvic acid c to thnh qua s Embden-Mayerhoff-Parnas, b decarboxyl ho to thnh acetaldehyt v CO2 nh xc tc ca pyruvat decarboxylase. CH3 - C - COOH O Pyruvic acid Sau , acetaldehyt b kh thnh ru etylic di tc dng xc tc ca ancoldehydrogenase ca nm men: CH3CH2OH + NAD (2) CH3 -CHO+ NADH+ + H+
alcoldehydrogena Pyruvat decarboxylase

CH3CHO + CO2

(1)

y NADH2 to thnh trong phn ng oxy ho glyceraldehyd 3 P ca qu trnh ng phn ng vai tr cht cho hydro, cn acetaldehyd l cht nhn. Tu iu kin ca mi trng, s ln men ru c th tin hnh theo cc kiu sau:

1.1.1. S ln men ru trong iu kin bnh thng:

Xy ra khi pH = 4-5, v c th chia lm hai thi k: a. Thi k cm ng: Trong thi k ny, lng acetaldehyd to thnh theo phn ng (1) cn t, khi hydro 3 P to thnh c chuyn t NADH2 ti aetaldehyd glyccerin 3 P. Cht ny b kh gc P nh enzyme phosphatase to thnh glycerin: NADH2 CH2O P CH2OH CH2O P NAD C6H12O6 CHOH CHO Glyceraldehyd 3 P CHOH CH2OH Glycerin 3 P CHOH

CH2OH Glycerin

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Vy, glycerin l sn phm ph ca qu trnh ln men ru trong mi trng acid. b. Thi k tnh: Khi lng acetaldehyd t ti mc no th cht ny tip nhn hydro t NADH2 chuyn ru thnh etylic theo phn ng (2): C6H12O6 CH3CHO + NADH2 CH3CH2OH + NAD+

Phng trnh tng qut ca s ln men ru bnh thng nh sau: C6H12O6 + 2H3PO4+2ADP = 2CH3CH2OH + 2CO2 + 2ATP + 2H2O 1.1.2. Ln men ru v s to thnh glycerin tng cng s to thnh glycerin ( sn xut glycerin hoc to v cho ung c ru), ngi ta a ra hai dng ln men khc vi ln men ru dng trn: a. S ln men ru trong mi trng c bisulfit: Nguyn tc ca phng php ny l chuyn acetaldehyd thnh acetaldehyd hydrosulfonad kh tan bng cch b sung NaHSO3 vo mi trng. Khi , NADH2 cho dihydroxyaceton phosphat to thnh glycerin 3 P , cht ny b kh phosphat to thnh glycerin di tc dng ca enzyme phosphatase: SO3Na CH3CHO + NaHSO4 CH3 - CH - OH acetaldehyd hydrosulfonad NAD

CH2O P C =O CH2OH

NADH2

CH2O P CHOH CH2OH glycerin 3 P P

CH2OH CHOH CH2OH glycerin

dihydroxyaceton phosphat

b. S ln men ru trong mi trng kim: Trong qu trnh ln men kiu ny, acetaldehyd c loi b nh phn ng ho hai thnh ethanol v acid acetic trong iu kin mi trng kim: 2CH2 - CH = O + H2O CH3CH2OH + CH3COOH

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Khi NADH2 chuyn hydro cho dihydroxyaceton phosphat to thnh glycerin 3 P , cht ny b kh phosphat to thnh glycerin nh enzyme phosphatase: NAD CH2O P NADH2 CH2O P CH2OH C =O CH2OH dihydroxyaceton phosphat CHOH CH2OH glycerin 3 P P CHOH CH2OH glycerin

1.1.3. S c ch ln men ru khi c mt oxy S ln men xy ra mnh m trong iu kin ym kh. Khi c oxy, qu trnh ln men b c ch v chuyn sang c ch h hp. S c ch ln men khi c mt oxy gi l hiu ng Pasteur. Trong qu trnh ny, ATP c tng hp mnh m nh phosphoril ho oxy ho lin hp vi qu trnh vn chuyn proton v electron qua dy h hp ti oxy. Kt qu l trng thi tch lu nng lng ca t bo tng ln, vi sinh vt ch cn mt lng glucose khng nhiu cng duy tr s sng v pht trin ca chng. Hiu ng Pasteur l mt c ch iu ho quan trng i vi qu trnh ln men. Trong thc t khi sn xut sinh khi th ngi ta cho nm men pht trin trong iu kin thong kh, cn khi cn thc y s ln men th cn gi mi trng trong iu kin ym kh. 1.1.4. S to thnh du kht (du fusel) Du kht l sn phm ph ca qu trnh ln men. Thnh phn ch yu l cc ru cao nh ru propylic, amylic, isoamylic, butylic, isobutylic, tyrosol...Chng l nhng cu t to nn mi thm c trng cho cc sn phm ln men. Ngoi ru cao, cc sn phm khc ca qu trnh nh acid amin, acid bo...l cht dinh dng v nguyn liu cho cc qu trnh tng hp ca t bo vi sinh vt. S to thnh ru bc cao trong qu trnh ln men l kt qu ca mt dy phn ng ho sinh ht sc phc tp. C ch to thnh ru bc cao cho n nay c rt nhiu nh nghin cu quan tm nhng cha gii quyt hon ton tho ng. Chung quy li v cc l thuyt gii thch v c ch to thnh ru bc cao theo hai ng hng: - Do phn ng chuyn amin, chuyn carboxyl gia cc acid amin ca mi trng v ca t bo vi acid pyruvic c sinh ra khi ln men ru:

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- Do s trao i cht bnh thng ca vi sinh vt b thay i khi mi trng thay i (tha acid amin), kt qu dn ti s tch t mt s cht trao i trong c cc ru bc cao. V d: khi a vo mi trng alanin, acid aminobutyric, glycin, valin, leucin, isoleucin...th s tng hp cc ru cao tng ng. 1.2. Sn xut bia Bia l loi ung c ru nh v c tnh gii kht cao. Thnh phn ca bia gm c: 80-90% nc, 1.5-7% cn, 3-10% cht ho tan, 0.30.4% CO2. Cht ho tan ch yu l hydratcarbon (dextrin, maltose, glucose v mt t pentose), cc protein v sn phm thu phn ca n (albumose, pepton, cc acid amin), cc cht khong (mui kali, natri, phospho, nhm, canxi, mangan...), mt s acid hu c, cc vitamin (B1, B2, B5, B6, PP, biotin) v cc cht ng, cht thm ca hoa houblon. 1/ S cng ngh sn xut bia: Quy trnh cng ngh sn xut bia hin nay c p dng trong nc cng nh trn th gii u theo s 6.1.
B

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Malt Nghin

Nguyn liu thay th (go)

Nghin

Nc

Nu nguyn liu

Lc dch Houblon ho
Hoa houblon (cao hoa)

Nm

Nui cy Lc dch houblon ho Ln men Nhn ging sn xut

Tng tr
Lc dch ln men Chit chai

Thanh trng Dn nhn Thnh phm Hnh 6.1: S cng ngh sn xut bia

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* Malt i mch: Malt i mch l ht thc i mch c ny mm trong nhng iu kin nhn to v nhit v m theo s cng ngh trn hnh 6.2: Ht i mch

X l (Lm sch, ra v kh trng)

Ngm

m mm

Sy malt

Tch mm v r

Bo qun
Hnh 6.2: S sn xut malt i mch

* Hoa houblon l nguyn liu khng th thiu c trong sn xut bia. Mi thm c c ca bia mt phn l do tinh du c trong hoa houblon.. Cc cht ng c trong hoa houblon truyn cho bia v ng d chu, ng thi pectin trong houblon c vai tr gi bt, tng bn sinh hc cho bia v c ch s pht trin ca vi khun. * Nc l mt nguyn liu quan trng trong cng nghip sn xut bia. Trong nh my bia, nc c dng vi nhiu mc ch khc nhau t

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giai on x l nguyn liu cho n cc giai on thnh phm. Chnh v vy, nc nu bia khng ch i hi y cc tiu chun ca nc ung m cn phi c yu nhng yu cu ring p ng vi cng ngh sn xut bia v cng, pH v hm lng cc ion kim loi. b. Nghin malt: Nghin malt vi mc ch ph v cu trc ca t bo, to iu kin thun li thc y qu trnh sinh ho xy ra trong nguyn liu khi nu nhm thu c dch ng c nng cc cht cao nht t nguyn liu ban u.Yu cu khi nghin malt l gi v malt cng nguyn cng tt v cn c mt t l thch hp gia tm th, tm mn v bt, t l ny ph thuc vo thit b lc dch ng ho. c. Nu nguyn liu (ng ho nguyn liu): Mc ch ca qu trnh nu bia l chuyn cc cht ca malt v nguyn liu thay th t trng thi khng ho tan sang trng thi ho tan nh tc ng ca h enzyme thu phn. tng hiu sut nu ngi ta p dng nhiu phng php ng ho khc nhau. Tuy nhin, tt c cc phng php u theo mt nguyn tc chung v iu khin nhit thch hp cho cc enzyme thu phn, ch yu l h amylase v protease, hot ng. -amylase hot ng thch hp trong khong 70-750C, -amylase: 63-650C, protease: 50-600C. d. un si dch ng vi hoa houblon (qu trnh houblon ho): Mc ch ca qu trnh houblon ho l n nh thnh phn dch ng, truyn cho bia mi v v ca hoa houblon, l keo t cc protein, v hot enzyme v thanh trng dch ng. Trong qu trnh ny phi m bo khng nhit ca dch ng h xung di 700C trnh oxy khng kh tip xc vi dch ng xy ra cc phn ng oxy ho lm gim cht lng ca bia. Hoa houblon dng nu bia c th dng loi hoa nguyn, hoa p hoc cao hoa v phi cho hoa chia lm nhiu t nhm tng mi thm cho bia.Lng hoa s dng nu bia ph thuc vo dng v loi bia, vo lng acid ng c trong hoa m dao ng 1-4g/l bia. e. Ln men bia: Trong qu trnh ln men bia, mt lng ln c cht, ch yu l ng v dextrin bc thp b nm men hp th to thnh ru etylic, kh carbonic v cc sn phm ph. * Cc yu t nh hng n qu trnh ln men: Qu trnh ln men chu nh hng ca rt nhiu yu t nh: cht lng ca nm men, lng nm men gieo cy ban u, nng cht ho

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tan ca dch ng houblon ho, nhit ca dch ln men, p sut b mt, hm lng oxy, cng khuy o dch ln men v nng cc sn phm ln men. - Cht lng nm men: Trong sn xut bia thng dng cc ni men chm thuc ging Saccharomyces carlbergensis c khi cng dng loi men ni thuc ging Saccharomyces cerevisiae. Men chm thng ln men nhit 6-100C, cn men ni 14-250C. Khi chn men bia cn da vo cc ch tiu nh: Tc v mc ln men; Hm lng sn phm bc hai to thnh; Tc v kh nng kt lng; Mc thoi ho; Kh nng chng chu iu kin ngoi cnh; Mt s chng nm men ph hp c cht lng tt c th ti s dng n i th by, th tm. - Lng nm men gieo cy ban u lin quan mt thit n qu trnh ln men. Ni chung, lng nm men gieo cy ban u qu t th thi gian ln men ko di. Mt nm men gieo cy ban u ln th cht lng bia c nng cao do lng cc sn phm bc hai gim. Tuy nhin, mt ti a ca lng nm men gieo cy ban u nu vt qua ngng 70.106 t bo/cm3 th hiu qu mang li khng r nt. - Nng cht ho tan ca dch ng houblon ho 11-12% lm cho qu trnh ln men tt hn so vi cc loi dch ng c nng cao hn hoc thp hn. - Nhit ca dch ln men v mi trng xung quanh c nh hng kh mnh n tin qu trnh ln men. Nu nhit cao th thi gian ln men nhanh, mt ti a t c cao hn so vi nhit thp, ln men trit hn; tuy nhin cc sn phm bc hai (c bit l diacetyl) to ra nhiu hn, lng t bo cht nhiu hn v tc suy thoi nhanh hn nn cht lng bia gim. - p sut b mt ca dch ln men xc nh mc bo ho CO2, l hp cht gy c ch qu trnh ln men, nh hng n lng sinh khi to thnh, trng thi sinh l cu nm men, v vy n nh hng trc tip n qu trnh ln men. Trong thc t sn xut, ngi ta thng khng ch p sut khng tng qu 1 kg/cm2. - Lng oxy trong dch ln men rt cn thit cho qu trnh pht trin sinh khi giai on u ca qu trinh ln men ng thi cng l yu t tc ng mnh n tc suy thoi ca nm men. Chnh v vy, lng oxy ho tan trong dch ng houblon ho ch nn khng ch nng 6,7-7mg/l.

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- Cng khuy o dch ln men l mt trong nhng yu t thc y qu trnh ln men; tuy nhin n lm cho bia cha qu nhiu sn phm bc hai v vy cn phi chn mt phng n v ch khuy dch ln men thch hp. Hin nay, trn cc thng ln men ngi ta trang b nhiu o lnh c th c nhit khc nhau v s i lu ca dch ln men thc hin c nh vo s chnh lch nhit gia cc vng. - Nng cc sn phm ln men: Sn phm chnh ca qu trnh ln men l ru etylic v kh carbonic. Khi nhng hp cht ny tch t n mt nng nht nh trong dch ln men th cc hot ng sng ca nm men b c ch. Nu nng cn ln n 12% th qu trnh ln men b nh ch hon ton. * Nui cy nhn ging men bia: Nm men bia t ng ging gc (mi trng thch nghing) trc khi a vo sn xut c nhn ging theo th tch tng dn nhm lm cho chng gc quen dn vi mi trng sn xut. Trc ht, ging gc trong ng nghim c chuyn vo cc bnh 300ml dch nc malt, gi 18250C. Sau cy chuyn tip qua cc bnh 3-5 lt c 2-3 lt dch ng malt c hoa houblon v nui nhn ging 18-250C. Tip nui ging cc thng 200-400 lt c 100-200 lt mi trng 120C. T l tip ging l 10%. n khi ging sinh trng mnh cc thng ny s tip ging vo cc thng ln men. * S dng li nm men sau khi ln men chnh: Cn men sau khi ln men chnh chia lm ba lp: Lp di cng l lp t bo gi c nng lc ln men yu, lp gia l nhng t bo tr hn c kh nng ln men mnh v lp trn cng l nhng t bo c kch thc nh kh nng kt lng km cng vi nhng vn cn protein v houblon. V vy trc khi ti s dng nm men sn xut cn phi qua x l thu lp gia a vo sn xut. * Ln men: Ln men bia c thc hin theo hai giai on: giai on ln men chnh v giai on ln men ph - tng tr. Hin nay, c hai phng php ln men c s dng ph bin cc nh my l ln theo phng php c in v ln men theo phng php hin i. i vi ln men theo phng php c in qu trnh ln men chnh v qu trnh ln men ph-tng tr l hai giai on tch bit nhau, din ra trong hai loi thit b khc nhau, sp xp cc mt bng khc nhau v c gi l cng ngh ln men hai pha.Theo phng php ny, qu trnh ln men chnh c tin hnh trong cc thng ln men kn hoc h nhit

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80C hoc ln hn, thi gian ko di 6-10 ngy. giai on ny, nm men ln men mnh, dch si bt nhiu. Cui giai on ny, s ln men gim xung, phn ln nm men lng xung y thng. Ta c c sn phm bia non bao gm ru etylic, CO2, cc sn phm ph v cn mt t ng (khong 1.5-2.5%). Bia non tip tc c bm vo cc thng ln men ph v tng tr. Nhit ln men ph gi 1-40C. Giai on ny, nm men ln men phn ng cn li b sung CO2 cho bia, ng thi hon thin cht lng ca bia do qu trnh lng cn nm men cng nh cc kt ta mi to thnh lm cho bia trong hn ng thi xy ra phn ng oxy ho kh gia cc cht c trong bia lm tng hm lng este, ru bc cao v gim hm lng diacetyl. S t phn ca nm men sinh ra peptit, acid amin, vitamin trong giai on ny cng gp phn tng cht lng ca bia. Sn xut bia theo cng ngh c in c u im l sn phm c cht lng cao, nhng nhc im ln nht ca n l chu k ln men qu di. Sn xut bia theo cng ngh hin i c th rt ngn 50-70% chu k ln men m cht lng ca bia c th tim cn c vi phng php c in. cng ngh ln men hin i, hai giai on ln men chnh v ln men ph c thc hin trong cng mt thit b nn cn gi l ln men mt pha. Thit b ln men theo cng ngh ny l tank thn tr-y cn c kch thc ln. Th tch ca chng dao ng t 100-1500m3; c ch to bng thp khng g vi gc cn y l 700. Trn thn thit b c trang b 3-5 o lnh c cht lm lnh vi nhit -60C, thng cht lm lnh l glycol. Cc o lnh ny va c tc dng iu ho nhit trong qu trnh ln men ng thi cn c tc dng gip cho dch ln men trong thit b i lu do chnh lch nhit cc vng khc nhau c to ra khi thay i nhit ca cc o lnh. f. Hon thin sn phm: Bia thnh phm thu c sau qu trnh ln men trong cc tank ln men c lc, chit chai, thanh trng, dn nhn hon thin sn phm. Lc bia thng c thc hin trn cc thit b lc khung bn c diatomit l cht tr lc. Qu trnh chit chai thng c thc hin qua h thng my chit trong thc hin nhiu giai on nh ht chn khng, thi CO2, cn bng p sut, chit bia, phun nc nng ui O2 cn li v ng np. Bia chai t my chit ra c thanh trng trn h thng tunen c vi phun nc pha trn theo cc khong nhit tng dn sau gim dn nhm hn ch s pht trin ca vi khun trong qu trnh bo qun bia.

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1.3. Sn xut ru vang Ru vang l mt loi ung c cn thu c nh qu trnh ln men dch qu khng qua chng ct. Trong ru vang cha y cc cht dinh dng c sn t qu v mt lng cn va phi cng cc cht thm, acid hu c, cht khong v vitamin...Trc y, ru vang thng c ch bin t qu nho, v vy, ru vang cn c gi l ru nho. Hin nay, trn th gii c ti hng trm loi ru vang khc nhau, mi loi c c trng bi mt phng thc sn xut ring tu vo c im ca ru v tnh cht ca cng ngh. Quy trnh ch bin ru vang theo s chung trn hnh 6.3: Nguyn liu qu

p ly nc

Nghin

p ng

Chun b dch ln men

Ln men chnh

Ln men ph

Lng trong v tng tr

Hnh 6.3: S cng ngh sn xut ru vang

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Qu hi v c ra sch p ly nc hoc nghin nh ri cho vo thng chun b dch ln men. Cng c trng hp ngi ta khng p hoc nghin m cho qu vo ngm vi ng theo t l khi lng 1:1 trch ly thnh dng xiro v bo qun ln men dn sau ny. Chun b dch ln men: cc loi qu thng c acid cao v ng thp so vi nc nho. Trc khi cho ln men cn iu chnh pH v khong 3.2 - 3.8 v b sung thm ng. Trng hp l dch xiro (dch qu ngm ng) th pha gp 2 hoc 3 ln dch ln men c khong 1618% ng.. C th trong qu trnh ln men cn b sung thm ng. Ln men: Trng hp ln men t nhin, ngi ta cho khi dch qu t ln men vi cc ni nm men c sn trong v qu t ngoi ng rung mang v hoc b sung cc dch ang ln men cc m trc. Trong ln men cng nghip, trc khi cy chuyn nm men vo dch ln men, ngi ta tin hnh nhn ging cc chng thun khit trong phng th nghim v nhn ging trung gian trong phn xng. Nm men sau khi nhn ging c cy vo dch ln men vi t l 6 -10% th tch thc hin ln men chnh. Nhit ln men vo khong 22-280C (tu thuc vo loi ru vang), thi gian ln men di hay ngn ph thuc vo nhit ln men v thng ko di 7-20 ngy. dch ln men chnh t c 8-10% cn. Ln men ph 15 - 180C trong 15-20 ngy. Sau khi ln men ph, cn 140 th phi thm cn cho ti nng ny hoc cao hn ri chuyn sang tng tr nhit thp hn 100C. Tng tr t nht 10 ngy, sau tch cn v c th hon thnh sn phm hoc tng tr tip tc. Trong thc t c hai cch ch bin ru vang ph bin nht l cch ch bin ru vang trng v cch ch bin ru vang . Ru vang c c khi thc hin ln men nc qu nho ln vi xc. Trong v qu c cha nhiu cht mu, tanin, cht thm. Nhng cht ny phn hu trong qu trnh ln men lm tng lng cht ho tan v to hng thm, mu sc p cho ru. Ln men c xc qu thng tin hnh nhit cao hn nhm trch ly trit cc cht mu, cht thm v tanin. Khi kt thc ln men cn tch xc qu ra khi ru. Trong sn xut ln, trc ht ngi ta cho ru chy qua mt tm li gi xc qu li. Ru thu c lc ny c cht lng cao nht gi l ru vang git, vang chy. Khi ru ngng chy, xc qu trn li c p trn my p thu ru vang p. Ru vang p chim t l khong 15% tng s ru ch c v c th em trn vi ru vang chy. Ru vang c hm lng tanin nhiu hn ru vang trng nn c v cht. Do , sau khi ln men

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ru cn tin hnh ln men malolactic nhm chuyn ho acid malic thnh acid lactic to cho ru c v chua du cn i vi v cht ca tanin. C th s dng vi khun lactic hoc mt lng nh nm men Schizosaccharomyces c kh nng ln men malolactic trit . Ru vang trng c ln men t qu c tch b nn khng c mu. Hng v ca ru vang trng ch yu do nc qu. Ru vang trng thng c ln men nhit thp hn ru vang (khong 15 200C) gi hng v cho ru. Thi gian ln men 6-7 ngy hoc lu hn tu theo nhit v yu cu cng ngh. Kt thc qu trnh ln men khi nhn thy ru khng cn si bt ln na, cn v xc men lng xung y thng. Khi tin hnh gn cn, ru tr c chuyn sang thng mi, tip tc lng trong ri a i tng tr. 1.4. Sn xut ru mnh v cn Quy trnh sn xut ru etylic bng phng php ln men vi sinh vt theo s hnh 6.4: Nguyn liu Nguyn liu tinh bt r ng Nu nguyn Ch phm amylase

X l nguyn liu

ng ho nguyn liu

Ln men

Nm men

Chng ct v tinh ch

Cn
Hnh 6.4: S cng ngh sn xut ru etylic

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Ru etylic (cn) c con ngi xem l sn phm thc phm nhng cng li l sn phm c nguy c c hi i vi c th. Tuy nhin, sn lng ru etylic c sn xut trn th gii cng ngy cng tng. cc nc c cng nghip ru vang pht trin nh Italia, Php, Ty Ban Nha...Etylic c dng tng thm nng ru. Mt lng kh ln etylic c dng pha ch cc loi ru mnh nh Whisky, Martin, Brandy, Napoleon, Rhum....Ngi ta c th sn xut ru etylic bng phng php ln men vi sinh vt hoc tng hp ho hc. i vi nguyn liu tinh bt, trc khi ln men cn qua giai on chuyn ho tinh bt thnh ng c thc hin bng nhiu phng php khc nhau, cn giai on ln men v chng ct tinh ch th c nguyn l ging nhau. V vy, tn gi khc nhau ca phng php ln men bng vi sinh vt chnh l tn ca phng php chuyn ho tinh bt. + Phng php maltase: l phng php s dng enzyme ca malt chuyn ho tinh bt thnh ng. Phng php ny lm cho cht lng ca ru c hng v c trng d chu nhng hiu sut khng cao do qu trnh thu phn tinh bt khng trit . + Phng php acid: l phng php s dng acid (HCl, H2SO4) chuyn ho tinh bt thnh ng. Phng php ny thu phn rt trit nn hiu sut cao. Tuy nhin, n li to ra nhiu sn phm ng khng ln men khc do c qu trnh thu phn cellulose v hemicellulose, ng thi nhiu acid amin b ph hu, thit b s dng t tin do phi chu c acid. + Phng php men thuc bc: l phng php s dng bnh men thuc bc sn xut ru. Phng php ny c c im l qu trnh ng ho v ru ho c tin hnh cng mt lc do s hot ng ng thi ca nm mc v nm men c gieo cy t bnh men thuc bc; tinh bt khng h ho m ch cn lm chn. V vy, n c nhc im l d b nhim tp, tinh bt st nhiu, hiu sut tng thu hi thp. + Phng php amylose: c im ca phng php ny l s dng nm mc v nm men c nui cy thun khit thc hin hai qu trnh ng ho v ru ho ring bit. Phng php ny c u im l d c kh ho v t ng ho, cho hiu sut thu hi cao. Tuy nhin n i hi nguyn liu phi ng u v yu cu v trng tuyt i. Mc ch ca nu nguyn liu l ph v mng t bo tinh bt v bin tinh bt thnh trng thi ho tan trong nc. Hin nay, trn th gii c hai xu hng v nhit nu l 145-1550C trong thi gian di hoc 170-1800C trong thi gian ngn. Trong qu trnh nu tinh bt s c trng n v h ho.

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Nguyn liu tinh bt sau khi c h ho c lm ngui v nhit 60 20C thc hin qu trnh ng ho. Ch phm amylase c s dng trong qu trnh ng ho c sn xut t cc chng nm mc khc nhau nh: Rhizopus, Mucor rouxii, Aspergillus (A. niger, A. flavus, A. awamori, A. oryzae ...) Qu trnh ng ho c thc hin trong trong 5 pht. Sau ,dch ng c lm ngui n nhit ln men (36 10C). Nguyn liu r ng c nng cht kh v hm lng ng rt cao (80-90Bx). Trc khi ln men, ngi ta phi x l nhm to pH thch hp, st trng, ng thi b sung cht dinh dng v pha long n nng yu cu ph hp cho qu trnh ln men. Nm men dng trong sn xut ru l chng Saccharomyces cerevisiae. Cc loi nm men ny cn c cc tnh cht c bn nh : Tc pht trin nhanh; Ln men c nhiu loi ng khc nhau v t c tc ln men nhanh; Chu c nng ln men cao, ng thi t b c ch bi nhng sn phm ca s ln men; Thch nghi vi nhng iu kin khng thun li ca mi trng, c bt i vi cht st trng, acid, nhit cao. Cc chng nm men thng dng ln men dch ng ho tinh bt l: S. cerevisiae Rasse II (chng II), S. cerevisiae Rasse XII (chng XII), chng M, chng MTB Vit nam (c phn lp t men thuc bc). Cc chng nm men dng ln men dch r ng l: chng 396 Trung Quc, chng I-A Lin X c, chng T Vit Nam. Chng nm men gc trc khi a vo sn xut ln men c nui cy nhn ging theo th tch tng dn cho n khi t c 10-15% th tch thng ln men trong sn xut Qu trnh ln men dch ng ho c th c thc hin bng phng php ln men gin on, bn lin tc hoc lin tc. Phng php ln men gin on l c qu trnh ln men t u n cui c thc hin trong cng mt thit b; thi gian ln men khong 68-80 gi nht 36370C. c im ca phng php ln men bn lin tc l giai on ln men chnh thc hin lin tc v xy ra trong nhiu thng ln men (thng l 6 thng) v thi gian ny ko di 60-62gi, giai on cui gin on. Bn cht ca phng php ln men lin tc l ri u cc giai on ln men m mi giai on c thc hin trong mt hoc nhiu thit b ln men c lin h vi nhau. H thng ln men lin tc thng c 11-12 thng c ni vi nhau bng cc ng chy chuyn v van iu chnh. Kt thc qu trnh ln men ta thu c dm chn vi nng ru khong 7-9%.

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thu c cn tinh ch t dm chn, ngi ta thc hin hai qu trnh l chng ct v tinh ch. Hai qu trnh ny c thc hin trn cc thp chng ct v thp tinh ch. Qu trnh chng ct l qu trnh tch cn cng vi cc tp cht d bay hi ra khi dm chn; kt thc qu trnh chng ct ta c cn th. Qu trnh tinh ch l qu trnh tch tp cht ra khi cn th v cui cng ta nhn c cn tinh ch. Qu trnh chng ct v tinh ch cn c gi l qu trnh chng luyn. 1.2. Ln men ethanol nh vi khun: Nhiu loi vi khun c kh nng ln men to ethanol. Tuy nhin, bn cnh ethanol chng cn t ra nhiu sn phm ph trong qu trnh ln men nh cc ru bc cao, cc acid hu c, cc polyol, keton v cc cht kh khc. 1.2.1. C s ho sinh Khc vi c ch ln men ethanol ca nm men, nhiu vi khun chuyn ho glucose thnh pyruvat theo con ng Entner-Doudoroff ( ED). Theo con ng ny, glucoso-6-phosphat b oxy ho thnh 6phosphogluconat ri sau chuyn ho thnh 2-ceto-3-deoxy-6phosphogluconat. Cht ny, sau c phn ct trc tip thnh glyceraldehyd-3-phosphat v pyruvat. 1.2.2. Sn xut ethanol nh Zymomonas mobylis Theo kt qu nghin cu ngi ta thy rng Zymomonas mobylis ch c kh nng chuyn ho cc loi ng theo con ng ED. Tc hp th ng v sinh ethanol nhiu nht khi chng ny c ln men trong mi trng c glucose, ng thi n c kh nng ln men nng ng trong mi trng cao (40%). Vi khun ny cng i hi c y thnh phn dinh dng trong mi trng nh C, N, P. II. Ln men lactic: 1. Sa v cc sn phm t sa: 1.1.Cc thnh phn ca sa Sa l mt trong nhng sn phm thc phm c gi tr dinh dng cao. Trong sa c y tt c cc cht dinh dng cn thit v d hp th nh protein, glucid, lipid, vitamin, mui khong v cc enzyme. Protein ca sa c cha nhiu v hi ho cc acid amin v c hai kiu protein khc nhau l protein ho tan (albumin, imunoglobulin, lisozim, lactoferin, lactoperoxydase...) v protein trng thi keo khng bn bao gm mt phc h mixen ca caseinat v canxiphosphat c b mt rt ho nc. Sa ti lun c pH xp x 6,6-6,7, ng thi do cc mixen mang in tch m nn y nhau nhm m bo bn keo ca

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dung dch. Khi gim pH (do kt qu ca qu trnh ln men to acid lactic), cc ion H+ ca acid s lin kt vi cc mixen lm gim in tch. n mt gii hn nht nh, casein s ng t. Casein ng t tt nht pH =4,5-4,7. Lactose chim v tr hng u trong glucid ca sa. Lactose tn ti hai dng t do v lin kt vi protein v cc glucid khc. Khi sa b nhim vi sinh vt, nhiu loi vi khun c kh nng ln men lactose to thnh acid v lm thay i tnh cht, cu trc ca sa. Ngi ta li dng tnh cht ny ch bin ln men sa thnh nhiu sn phm khc nhau. Trong sa c mt nhiu cation nh K+, Na+, Mg2+, Ca2+ v cc anion ca acid phosphoric, limonic, clohydric nn trong sa c nhiu loi mui khc nhau. Trong , mui canxi c ngha ln i vi ngi. Mui canxi cn c ngha quan trng trong ch bin cc sn phm sa. Khi sa c hm lng canxi thp, sa s khng b ng t hoc ng t rt chm. Cht bo trong sa tn ti di dng huyn ph ca cc ht nh hnh cu hoc hnh ovan vi ng knh 2-10m. Cc ht nh hnh cu ny c vy quanh bi mt mng protein, gm hai phn: phn c th ho tan c v phn khng th ho tan c trong nc. B mt bn trong ca mng c lin quan mt thit vi mt lp ph c bn cht phospholipit vi thnh phn ch yu l lexitin v xephalin. Trong lng cc ht nh hnh cu c cha glycerit vi im nng chy thp, giu acid oleic, lun trng thi lng trong iu kin nhit mi trng. Phn tip xc vi mng l cc glycerit c im nng chy cao hn, c th ng c nhit mi trng. Chnh nh c cu trc nh vy m cht bo n nh trong sa. 1.2. Cc sn phm ln men t sa 1.2.1. Cng ngh sn xut sn phm sa ln men (sa chua): Sa ln men l kt qu ca qu trnh hot ng ca vi sinh vt lm thay i cc thnh phn c trong sa m c trng l qu trnh ln men to thnh acid lactic t ng lactose. Trong mt s sn phm c bit cn c c s to thnh ethanol. Vi sinh vt c s dng trong sn xut sa chua l vi khun lactic vi hai loi c trng: + Lactobacillus bulgaricus (L. bulgaricus): l vi khun ln men in hnh, pht trin tt nhit 45-500C trong mi trng c acid cao. Loi ny c th to ra trong khi sa n 2,7% acid lactic t ng lactose. + Streptococcus thermophilus(S. thermophilus): Pht trin tt nhit 500C v sinh sn tt nhit 37-400C. y cng l vi khun

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lactic chu nhit ln men in hnh, c th chu c nhit un nng n 650C trong 30 pht nhng ch pht trin c trong mi trng acid thp hn L.bulgaricus. Hai loi vi khun nu trn thuc loi vi khun hiu kh v chu c mi trng c acid thp (pH = 4-4,5). Trong sn xut sa chua, vic cy hn hp hai loi vi khun ny cho kt qu sinh ra acid lactic tt hn l ch s dng ring tng loi. L. bulgaricus c kh nng thu phn casein thnh mt s acid amin do to iu kin cho S. thermophilus pht trin. Trong sn xut sa chua bng nui cy hn hp hai loi trn cho thy: giai on u ca qu trnh sn xut, pH ca sa thch hp cho loi Streptococcus hot ng chim u th v m bo cho qu trnh ln men lactic c bt u. Hot ca cc enzyme phn hu casein ca Lactobacillus kch thch s pht trin ca Streptococcus, acid tng ln. pH ca sa thay i lm cho Streptococcus kh pht trin. Lc ny Lactobacillus s thay th ch v s vn cc ca sa bt u. Sa chua c th c sn xut t sa ti t nhin hoc sa bt ho tan vi sa ti. Sau khi thanh trng nhit 950C, sa c lm lnh n nhit 42-460C v cy hai loi vi khun trn thc hin qu trnh ln men nhit 40-500C trong thi gian 2-3h. sn phm c chua nh v thm, ngi ta c th s dng t bo ca vi khun Streptococcus giai on tr v khi mi trng ln men c acid thp. Ngc li, mun sa chua c acid cao th cn s dng t bo Streptococcus gi hn hoc s dng Lactobacillus. Quy trnh cng ngh sn xut sa chua theo s hnh 6.5.

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Nguyn liu Phi trn Nc (t0 = 60-650C)

Nguyn liu Phi trn

Thanh trng (t0 = 95oC, 15pht)

ng ho ln 1 (p =150 bar)

5 h )
Lm mt (t = 42- 46oC)
o

Thanh trng ln 1 (t0 = 750C, 5 pht)

Lm lnh nhanh (t0 = 2-60C)

Cy men ging

hon nguyn (t0 = 2 - 60C, 6 - 8h)

ng ho ln 2 (p = 200 bar) Lm lnh (t =2-5 C) Thanh trng ln 2 (t0 = 95oC, 5pht) Men ging Lm mt (to = 42- 46oC) men (t = 4-8 h, t0 = 42 - 46oC) Lm lnh (to = 15- 20oC) Ch rt Chit h ng gi Hnh 6.5: S cng ngh sn xut sa chua
0 0

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1.2.2. Cng ngh sn xut phomat Phomat l sn phm c ln men hay khng oc ln men (tc l loi sn phm chu tc ng t nht ca qu trnh ln men lactic) ch yu t thnh phn casein ca sa to thnh dng gel mt nc. Phomat gi li hon ton lng cht bo ban u. Ngoi ra trong sn phm cn cha mt t lactose di dng acid lactic v mt t l khc nhau v cht khong. Sn xut phomat gm ba giai on chnh nh sau: Quy trnh sn xut phomat theo s hnh 6.6. Nguyn liu sa Tiu chun ho Cy men ng t

Tch nc

Lm chn

ng gi
Hnh 6.6: S cng ngh sn xut phomat

- To gel casein hay cn gi l qu trnh ng t sa: Qu trnh ny xy ra c th l do tc dng ng thi ca men dch v d dy b v acid lactic sinh ra t trnh ln men lactose bi vi khun. i khi mt trong hai dng ng t chim u th. - Qu trnh tch nc tng phn ca gel c thc hin theo nhiu phng php khc nhau tu thuc vo bn cht ca qu trnh ng t. - Qu trnh lm chn phomat nh h enzyme vi sinh vt.

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Trong thc t vi khun lactic v nm mc cc s dng trong cng nghip sn xut phomat nhiu hn c. Cc loi vi khun lactic thng c s dng trong sn xut phomat nh: Streptococcus lactis, Streptococcus thermophilus v Lactobacillus helveticus. Cc ch phm nm mc thng c s dng trong sn xut phomat l: Penicillium candidum v Penicillium glaucum. 2. Acid lactic: 2.1. Sinh tng hp Acid lactic tn ti rng ri trong t nhin v ng vai tr quan trng trong nhiu ngnh cng nghip khc nhau nh: ch bin thc phm, y dc, thuc da... Tu theo chng vi khun lactic s dng trong sn xut m c hai kiu ln men lactic l ln men ng hnh v ln men d hnh. Trong kiu ln men ng hnh thc t ch sinh ra acid lactic. ln men cng nghip, ngi ta thng dng cc ging Lactobacillus delbrueckii v Lactobacillus bulgaricus. Trong s ln men ng hnh, s phn gii glucose ging nh ln men ru ti giai on to thnh pyruvat (theo con ng Embden Mayerhoff - Parnas). Sau , nh Lactat dehydrogenase, pyruvat c kh bi NADH2 (coenzyme kh ny c to thnh do s tch H2 ca glyceraldehyd 3 P trong qu trnh ng phn) to thnh acid lactic. S ln men lactic ng hnh c biu din theo s sau: CH2O P C6H12O6 CHOH CHO Glyceraldehyd 3 P NAD CH2O P CHOH CH2OP COOH C=O CH3 Acid pyruvic NADH2 COOH CHOH CH3 Acid lactic

Trong s ln men lactic d hnh, nhiu sn phm khc nhau c to thnh. Cc tc nhn gy ln men lactic d hnh l Lactobacillus brevis, Leuconostoc mensenteroides. S ln men lactic d hnh xy ra khi vi khun gy ln men thiu enzyme c bn ca s ng phn l aldolase v trioso isomerase. Khi , glucose c chuyn ho theo con ng pentosophosphat t glucoso 6 phosphat n xiluloso 5 phosphat. Sau , di tc dng ca enzyme

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pentosophosphat cetolase m xiluloso 5 phosphat b phn ly to thnh glyceraldehyd 3 phosphat v acetyl phosphat. Acetyl phosphat c th c chuyn ho tip tc thnh ethanol hoc acid acetic tu theo h enzyme ca tng vi sinh vt. Cn glyceraldehyd 3 phosphat th b kh H2 chuyn thnh acid lactic ging nh trng hp ln men lactic ng hnh. S ln men lactic d hnh c ng dng trong ln men thc n gia sc v mui chua rau qu. Cc vi khun c kh nng gy ln men cc ng mono v disaccharid (sau khi thu phn thnh monosaccharid nh enzyme tng ng ca vi khun. S s ln men lactic d hnh xy ra theo s hnh 6.7 2.2. Cng ngh sn xut Quy trnh cng ngh sn xut acid lactic c tin hnh theo s hnh 6.8. Nguyn liu c s dng sn xut acid lactic c th l ngun tinh bt hoc ng. Nu nguyn liu l tinh bt th trc ht phi c thu phn bng enzyme hoc acid. Dch ln men c cha ng cn c b sung cc cht dinh dng l cc ngun nit nh cao ng, mm i mch, (NH4)2SO4. Sau khi thanh trng, dch ln men c lm ngui xung 500C v b sung CaCO3. Qu trnh ln men c thc hin lin tc nhit 48-500C v ko di 2-4 ngy. Acid lactic c to thnh di dng mui canxi. Do mui lactat canxi c tnh tan thp, nn trc khi tch sinh khi v mt s cht khng tan khc bng phng php lc, ngi ta cn un nng ho tan ton b mui ny. Dch lc c x l vi H2SO4 gii phng acid lactic. Tu thuc vo mc ch s dng m c th tinh ch acid lactic bng nhiu phng php khc nhau nh s dng than hot tnh, trao i ion, thm tch, chit bng dung mi...

III. Ln men 2,3 butadiol

1. Vi sinh vt C rt nhiu chng vi sinh vt c kh nng ln men butadiol vi hiu qu cao nh Klebsiella pneumoniae, Pseudomonas hydrophila, Bacillus polymyxa, Bacillus subtilis, Serratia marcescens. Trong Klebsiella pneumoniae v Bacillus polymyxa c s dng mt cch ph bin trong sn xut thng mi butadiol.

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Glucose ADP Hexokinase ATP Glucoso 6 P Glucoso P dehydrogenase NAD NADH2 Acid 6 P Gluconic NAD Phosphoglucobat dehydrogenase NADH2 Ribuloso 5 P Epimerase Xiluloso5 P CH2 - CH - CHO O P Glyceraldehyd 3 P NAD+ 2ADP + 2P OH

CH3 - C P O
Acetyl phosphat HS CoA

ADP + P ATP
CH3 - COOH Acid acetic

L.brevi
CH3 - CO SCoA Acetyl CoA HS CoA CH3 - CHO Acetaldehyd NADH2 NAD CH3 - CH2OH Etanol

2ATP NADH2 CH3 - C - COOH O Acid pyruvic CH2-CHOH - COOH Acid lactic

Hnh 6.7: S s ln men lactic d hnh

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Nguyn liu

X l nguyn liu

Ln men

Gia nhit

Tch acid lactic

Tinh ch

Acid lactic tinh khit

Hnh 6.8:Quy trnh cng ngh sn xut acid lactic

2. C ch sinh ho ca qu trnh ln men butadiol C ch trao i cht to thnh butadiol ca vi sinh vt t glucose xy ra theo s hnh 6.9. i t glucose, acid pyruvic c to thnh theo con ng phn. Nu c cht ban u l ng pentose th n i theo con ng

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pentosophosphat t c glyceraldehyd 3 phosphat, sau cht ny s chuyn thnh pyruvat theo con ng ng phn. Tip theo , hai phn t pyruvat c ngng t ng thi loi b CO2 to thnh acetolactat. Di tc dng ca enzyme acetolactat decarboxylase, acetolactat to thnh acetoin, cht ny tip tc b oxy ho khng c mt enzyme thnh diacetyl hoc b kh di s xc tc ca butadioldehydrogenase thnh butadiol. Glucose

CO2 CH3COCOOH Acid pyruvic

CH3CO lipoat

OH CH3CCOCH3 COOH Acetolactat CO2 CH3CHOHCOCH3 Acetoin O2 CH3COCOCH3 Diacetyl

CH3CHOHCHOHCH3 2,3 butadiol

Hnh 6.9: C ch trao i cht to thnh butadiol ca vi sinh vt

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Phng trnh tm tt ca qu trnh ln men butadiol nh sau: Glucose Xylose 2CO2 + NADH2 + 2ATP + butadiol 5/3CO2 + 5/6 NADH2 + 5/3ATP + 5/6butadiol

3. C cht v hiu sut ln men C cht v hiu sut ln men ph thuc vo tng chng vi sinh vt gy ln men. V d: Klebsiella pneumoniae c kh nng ln men hng lot ng nh glucose, manose, galactose, xilose, arabinose, xenlobiose v lactose nhng khng c kh nng ln men cellulose v hemicellulose. khc phc vn ny, ngi ta a ra quy trnh ng ho v ln men ng thi bng cch s dng thm chng nm mc Tricodema harzianum thu phn cellulose v hemicellulose. Bng quy trnh ny, hiu sut ln men t 5060%, tng ln 30-40% so vi qu trnh ln men v ng ho ring bit. Bacillus polymyxa, ngoi ln men cc loi ng trn, cn c kh nng ln men cc hp cht cao phn t nh xilan, inulin, tinh bt. 4. Cc yu t nh hng n qu trnh ln men * Nhit : Nhit ti u cho sinh trng ca vi khun c kh nng sinh ra butadiol nm trong khong 30-370C. Nu tng nhit t 33 ln 370C th kh nng ln men butadiol ca Klebsiella pneumoniae gim 66% nhn i vi chng Enterobater aerogenes th hiu qu ny khng th hin r. * pH: Mi chng vi khun thch hp vi cc gi tr pH khc nhau sinh tng hp butadiol l cao nht. Klebsiella oxitoca sinh tng hp butadiol cc i pH=5,2-5,6; Klebsiella aerogenes thch hp pH =6,2 trong khi Enterobater cloacae thch hp pH=5,0-5,5. *Oxy: y l thng s mi trng quan trng nht nh hng n qu trnh ln men. Nu cung cp oxy qu ln th s to thnh sinh khi s chim u th. Ngc li nu oxy c cung cp t th kh nng sinh butadiol cao cng vi s tiu th mt lng ng ln. Tuy nhin qu trnh ln men tt th trong mi trng ln men cn c mt mt t bo vi sinh vt ban u nht nh.

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Nng ng ban u: nh hng ca nng ng ban u n s ln men ph thuc vo thnh phn mi trng. Chng hn nh khi i vi mi trng sn xut, s tng nng ng ko theo s tng hm lng mt s thnh phn khc m trong c th c cc cht km hm, do , nng ng ban u cn c gii hn. Tuy nhin i vi mi trng tng hp th nng ng ban u khong 200g/l qu trnh ln men vn xy ra. 5. Tim nng ng dng Butadiol c ng dng trong nhiu lnh vc khc nhau. N c th dng lm nhin liu hoc c th c dng iu ch cc hp cht hu c khc nhau nh etyl keton v 1,3 butadien. Etyl keton l mt dung mi cng nghip, cn 1,3-butadien dng sn xut cao su.. Butadiol cho phn ng vi aceton to nn hp cht tetrametyl c th phi trn vi xng. Cc este ca butadiol c dng sn xut cc cht do polime chu nhit. IV.Ln men butanol-aceton 1. C s ho sinh ca qu trnh ln men butanol-aceton Tc nhn gy ln men butanol-aceton l chng vi khun Clostridium acetobutilicum. y l vi khun ym kh do sn phm ln men ch thu c trong iu kin ln men khng c oxy. C ch ha sinh qu trnh ln men butanol-aceton theo Cleuberg theo s hnh 6.10 Nh vy, thng qua s trao i cht ca vi khun m trong mi trng tch t c sn phm aceton, butanol, ethanol, CO2 v H2. Ngoi ra cn c cc sn phm trung gian nh acid buttyric, acid acetic, acid formic...

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C6H12O6 CH3COCH(OH)3 Hydrat metylglioxal CH3CHO Aldehyd acetic H2O H2 CH3CH2OH Etylic HCOOH Acid formic

Ngng t

CH3CHOHCH2CHO CH3CH=CHCH(OH)2 Aldehyd crotonic CH3CH2CH2COOH Acid butyric 2H2 CH3CH2CH2CH2OH Butanol

CH3CH(OH)3 H2 CH3COOH Acid acetic CH3C(OH)2CH2CO O HO

2

CH3CHOHCH3 isopropylic

CO2

CH3COCH2COOH

CH3COCH3 Aceton

Hnh 6.10: C ch ha sinh qu trnh ln men butanol-aceton 2. Quy trnh sn xut cng nghip Nhn chung, quy trnh sn xut aceton-butanol c th chia lm ba giai on: Chun b dch h ln men; Ln men; Chng ct tch cc sn phm; Nguyn liu sn xut aceton-butanol bng phng php ln men l tinh bt (cc nguyn liu c tinh bt), khng cn ng ho trc v vi khun c amylase kh hot ng. Yu cu ca nguyn liu l c glucid, nit v phospho. Do , bt cc ng cc l nguyn liu hon ho c cha tt c cc thnh phn cn thit cho s ln men. R ng v cc dch thu phn g, rm, b ng...l nguyn liu khng y bt buc phi trn vi ng cc.

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Nng dch h ln men khong 8-10% tnh theo cht kh hoc 4-6% tnh theo tinh bt. Nu nng cao hn s tha v khng c ln men. Khi nng butanol t xp x 1,5% (tng ng vi nng ban u ca glucid l 6%) th qu trnh ln men b ngng. Nhit ln men ca vi khun aceton-butanoll 36-370C. Qu trnh ln men aceton-butanol c th chia lm hai thi k: thi k u, trong mi trng ln men tch t acid (nn c tn gi l ln men acid), song thi k th th hai th trong mi trng tch t butanol, ethanol v aceton (nn c tn gi l ln men ru). Trong thi k th hai ny, acid chun trong mi trng ln men b gim nhanh: gim i gn mt na so vi gi tr cc i cui thi k u. ng thi cng xy r chuyn ho mt cch nhanh chng cc acid thnh nhng hp cht trung tnh tng ng: acid butyric b kh thnh ru butylic, cn acid acetic b chuyn ho thnh aceton. Tip , acid chun c tng ln mt t, cn vn tc to ra dung mi dn dn b chm li v n cui thi k ln men th ngng hon ton. Trong qu trnh ln men cn km theo s thot kh CO2 v H2. Qua 5-6h u, s thot kh cn chm chp, sau th tng nhanh ri t n cc i, tip n li gim nhanh chng v qu trnh ngng. Cc kh ny c dn t cc thng ln men vo cc thit b gom kh ri sau thi ra khng kh. Sau khi ln men xong th dm chn c bm vo thit b chng ct tch mi th ra khi hn hp.

Cu hi n tp chng 6
1. Hy trnh by s bin i ca phn t glucose n etylic xy ra trong t bo nm men. 2. Trnh by nhng im ging v khc nhau ch yu trong cc qu trnh sn xut bia, ru vang v ru etylic. 3. Phn tch vai tr ca cc nguyn liu nh hng n thnh phm trong sn xut bia. 4. Phn tch cc yu t nh hng n qu trnh ln men bia. 5. So snh hai phng php ln men c in v ln men hin i trong sn xut bia.Phn tch u nhc im ca hai phng php . 6. So snh phng php sn xut vang v sn xut vang trng. 7. Chng nm men dng trong sn xut ru cn c nhng yu cu g? 8. Phn tch thnh phn v tnh cht ca sa c ng dng sn xut cc sn phm t sa. Cho v d minh ho.

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9. C ch v quy trnh sn xut sa chua v phomat ging v khc nhau nhng im c bn no? Hy phn tch. 10.C ch ln men lactic d hnh v ng hnh ging v khc nhau nhng im no? 11. Trnh by quy trnh sn xut acid lactic cng nghip. 12. Trnh by c ch ho sinh v cc yu t nh hng n qu trnh ln men butadiol. 13. C ch qu trnh ln men aceton-butanol din ra nh th no? Trong qu trnh ny ngoi hai sn phm chnh l butanol v aceton cn c nhng sn phm ph no? 14. Phn tch quy trnh sn xut aceton-butanol quy m cng nghip.

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Chng 7

Cc cht trao i bc mt
I. Nguyn l ca s tng hp tha
Vi sinh vt tn ti trong t nhin sinh ra cc sn phm trao i cht v cc thnh phn t bo ch mc cn thit cho s sinh sn ti u v cho s duy tr loi. S trao i cht t bo c tnh kinh t nh vy c m bo nh cc c ch iu ha pht trin cao. Chng hn nhng c ch ny hot ng sao cho cc aminoacid khng c tng hp qu nhu cu ca s tng hp protein. Kt qu l, trong iu kin t nhin khng c s sn sinh d tha cc cht trao i bc mt, bc hai v cc enzyme. Nu trong t nhin c ch iu ha ny b ri lon, v d do kt qu ca mt t bin th cc th t bin sai hng trao i cht thng sinh trng km hn v b cc chng hoang di ln t. Ch trong iu kin phng th nghim v trong ng ging thun khit mi c th duy tr c cc th t bin nh vy. Trong t nhin nhng chng nh vy thng t xut hin trong cc sinh thi. Bi vy nhng th nghim nhm phn lp cc chng c kh nng tng hp tha t ni sng t nhin thng khng em li kt qu mong mun. Ch khi nhng c th thch hp, thu c do x l bng cc tc nhn gy t bin c chn lc trong phng th nghim th ngi ta mi c th nhn c cc chng tng hp tha b sai hng trong c ch iu ha. Nhng chng ny c coi l nhng chng c nng sut cao v c dng trong sn xut cng nghip. 1. Nhng nguyn tc iu ha trao i cht C ba c ch chu trch nhim trc ht v iu ho trao i cht (hnh 7.1): (a) iu ha hot tnh enzyme nh s c ch (inhibition) bng sn phm cui cng (cn c gi l s km hm theo lin h ngc). (b) iu ha s tng hp enzyme nh s kim ch (repression) bng sn phm cui cng v s gii kim ch. (c) iu ha s tng hp enzyme nh s kim ch d ha (catabolic repression). Trong c ch c ch bng sn phm cui cng (a), sn phm cui cng ca mt qu trnh sinh tng hp gy ra s c ch qu trnh tng hp chnh sn phm cui cng . y, sn phm cui cng d c hnh thnh trong t bo hay c thu nhn t mi trng dinh dng, u c cng ngha. Trong c ch ny, sn phm cui cng ni chung nh hng n enzyme u

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tin ca chui sinh tng hp. Enzyme c tnh cht quyt nh ny l mt protein d lp th. N c c im l thay i cu hnh khng gian khi c mt sn phm cui cng nhm gim bt hot tnh xc tc. S c ch ny xy ra nhanh v rt c hiu qu. Trong c ch kim ch (b), sn phm cui cng c ch s tng hp ca enzyme cn cho s to thnh sn phm y, trong vic c thng tin di truyn cn cho qu trnh tng hp enzyme (s phin m) b phong ta. nng cao ca cc sn phm cui cng, s tng hp cc enzyme tham gia vo chui phn ng b ngng hoc b ko di mt cch ng k. Nu nng sn phm cui cng gim xung di mt mc no th s xy ra s gii kim ch, ngha l cc enzyme c to thnh vi tc cao hn. S iu ha theo kiu ny xy ra t t, v n gn lin vi s tng hp enzyme.

Hnh 7.1: Cc c ch i ho trao i cht

a. Kim ch s tng hp enzyme b. c ch hot tnh enzyme u tin trong con ng trao i cht(s c ch ngc)

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Do s khc nhau v tc phn ng v s mn cm, s c ch ngc nhy cm hn s kim ch tuy rng chc nng ca c hai c ch u cn thit cho s iu ha mt cch kinh t qu trnh sinh tng hp cc vin gch cu trc trong t bo. Trong khi s kim ch enzyme iu ha qu trnh tng hp cc enzyme ng ha, ngha l cc enzyme chu trch nhim v tng hp th s tng hp cc enzyme d ha (xc tc s phn hy c cht) li c iu ha bi s cm ng enzyme. l c ch tng t nh kim ch, cng xy ra mc phin m. Trong s cm ng enzyme, mt cht dinh dng ng vai tr cht cm ng kch thch s tng hp enzyme xc tc cho s phn hy chnh n, ngha l cht ny cm ng s tng hp. Do vic tng hp cc enzyme cm ng ch xy ra khi c mt c cht tng ng trong mi trng dinh dng (hnh 7.2).

Hnh 7.2: iu ho s tng hp cc enzyme cm ng

a) Khi khng c mt cht cm ng b) Khi c mt cht cm ng

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Nu trong mi trng dinh dng c mt nhiu c cht th trc ht xy ra s tng hp ca enzyme no xc tc s phn hy c cht d s dng nht. S tng hp ca cc enzyme khc b c ch bi s kim ch d ha (c). Thng thng th glucose l c cht thch hp nht. Bi vy hiu ng trong khi c mt glucose, nhiu enzyme khc ca qu trnh d ha cng nh ca qu trnh trao i cht trung gian v trao i cht bc hai khng c tng hp cng cn c gi l hiu ng glucose. Khng phi bn thn glucose m l mt cht trao i hnh thnh trong qu trnh d ha glucose hay d ha mt c cht khc s dng, c trch nhim i vi s c ch ny. Nhiu kt qu thc nghim cho bit AMP vng c tc ng n giai on phin m. 2. Nhng sai hng di truyn ca iu ha trao i cht Cc c ch iu ha trao i cht c th b thay i do nhng t bin dn n s tng hp tha cc cht trao i. 2.1. t bin dn n nhng thay i trn enzyme d lp th

Hnh 7.3: C ch ca s c ch enzyme nh cht hiu ng d lp th

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Cc enzyme d lp th chu trch nhim v s c ch bng sn phm cui cng, hay c ch ngc, hot tnh ca chng b gim do sn phm cui cng. Ngoi v tr phn ng vi c cht, chng cn c mt v tr khc i vi sn phm cui cng, v tr th hai ny c gi l trung tm d lp th. Hai v tr phn ng tch bit nhau v khng gian v khc nhau v cu trc; cng nh vy, c cht ca enzyme v sn phm cui cng ca chui sinh tng hp c cu trc khc nhau. Ngoi ra, cn lu rng cc enzyme d lp th bao gm nhiu di n v ging nhau. Trng thi hot ng ca enzyme c c trng ch n c th phn ng vi c cht. Nu bn cnh c cht cn xut hin sn phm cui cng nh mt cht hiu ng d lp th th s xy ra s bao vy d lp th. Trong trng hp ny trung tm xc tc b bin i v mt khng gian n mc n khng th phn ng vi c cht c na. Mt t bin c th dn n kt qu l protein enzyme d lp th b thay i bng cch mt i kh nng phn ng vi cht hiu ng nhng vn cn hot tnh xc tc. Mt protein b bin i nh vy vn cn hot ng ngay c khi c mt sn phm cui cng; n dn n s tng hp tha sn phm cui cng tng ng. 2.2. t bin dn n s tng hp tha cc enzyme sinh tng hp Trong s kim ch tng hp enzyme s xy ra nhng phn ng quyt nh trong phm vi thng tin di truyn, s phin m. Nu trong t bo tn ti mt sn phm cui cng gi nh no , mt aminoacid chng hn, ch nng rt thp, th cc enzyme cn thit cho s tng hp aminoacid ny s c to thnh. Mun vy, on DNA cha thng tin di truyn v cu trc ca enzyme c iu ha phi c c. l do cht kim ch (repressor) vi cu hnh khng gian hin c, biu hin nh mt cht aporepressor, khng th phn ng vi gen iu khin (gen operator) ca operon aminoacid. Cht kim ch ny c sinh ra do tc dng ca gen kim ch (gen repressor) nm trong mt on DNA bn cnh. T operator din ra vic c thng tin di truyn; thng tin ny c chuyn n v tr tng hp protein di dng tRNA, v to kh nng cho s tng hp enzyme tng ng. Tri li, nu aminoacid trong t bo c nng cao hn, v d do c a vo t dung dch dinh dng th s xy ra s km hm qu trnh tng hp cc enzyme tng ng. Do phn ng vi cc aminoacid hay vi dng hot ng ca chng (aminoacyl-ARNv) m cht aporepressor b thay i cu trc khng gian ti mc c th chim gen operator nh mt cht holorepressor. Kt qu l s tng hp tRNA tng ng v do vy c s tng hp protein enzyme, cng b phong ta (hnh 7.4).

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S iu ha tng hp enzyme c th b ri lon do nhng t bin khc nhau. Nhng s sai hng dn n hu qu l, ngay c khi c mt aminoacid, cc enzyme cn thit cho s tng hp chng vn tip tc c to thnh. S tng hp enzyme khng th kim ch ny c gi l s tng hp mt cch cu trc. Mt mt, nhng t bin c th ng chm n gen kim ch dn ti mt sai hng ca cht kim ch hoc lm bin mt n, hay mt khc ng chm n gen iu khin v lm cho gen ny mt kh nng tc dng vi cht kim ch. Trong c hai trng hp hiu qu l nh nhau. Thng thng nhng th t bin nh vy, s xy ra s tng hp tha cc enzyme sinh tng hp v do vy c s tng hp tha cc aminoacid.

Hnh 7.4: V d v s kim ch s tng hp enzyme bi sn phm cui cng (chng hn mt acid amin)

a) Khi sn phm cui cng c mt nng thp b) Khi sn phm cui cng c mt nng cao. 2.3. t bin tc ng ln kim ch d ha Ton b nhng sai hng tng ng cng c th biu hin s cm ng enzyme. Nh nhng sai hng y m cc enzyme cm ng tr thnh cc enzyme cu trc, ngha l chng tn ti trong t bo khng ph thuc vo c cht. S kim ch d ha cng c th b mt i do t bin.

II. Cc phng php to ra th t bin tng hp tha

1. Cc c ch iu ho enzyme m u

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Nhiu cht trao i bc mt c tng hp nh cc con ng trao i cht phn nhnh m c nhiu sn phm c to thnh. S c ch bi sn phm cui cng do mt sn phm ca mt con ng phn nhnh d n hu qu l c nhng sn phm khc cng khng c to thnh. Cc con ng trao i cht phn nhnh i hi mt s iu ha phn ha. Ph hp vi iu ny l ba nguyn l c chng minh. Chng lin quan ti c ch iu ha enzyme m u ca mt chui phn ng : - S c mt ca nhiu izoenzyme, mi enzyme b c ch bi mt sn phm cui cng. - S c ch tch ly trong mt enzyme b c ch tng phn bi mi sn phm cui cng. Tc dng ca cc sn phm cui cng cng li s dn n s c ch hon ton. - S c ch a tr (phi hp hay hp tc), trong mt enzyme ch b c ch bi tc dng kt hp ca tt c cc sn phm cui cng. Mt sn phm cui cng ring s khng c tc dng c ch. Cc enzyme d lp th ng u cc chui sinh tng hp phn nhnh r rng c nhiu trung tm iu ha hoc c cc trung tm iu ha rt phc tp. S c ch bi sn phm cui cng khng ch gii hn i vi enzyme ng u cc chui sinh tng hp phn nhnh. Thng thi sau s phn nhnh cn c mt s c ch do lin kt tr li na. Cng tn ti nhng s lin kt tr li k tip nhau, trong mt sn phm trung gian do mt s c ch m tch ly li, by gi li c ch mt phn ng nm trc . Cc nguyn l v s c ch do lin kt tr li ng vi c s iu ha hot tnh enzyme cng nh s kim ch. Trong cc chng tng hp tha, s ph v c ch do lin kt tr li t c nh nhng kiu sai hng v di truyn sau y: - Sai hng trong s to thnh sn phm cui cng nh khuyt mt enzyme (cc th t bin tr dng). Bng cch b sung hn ch sn phm cui cng, sinh trng s xy ra, song s tch ly ni bo ca cht trao i gy ra s c ch tr li b ngn cn. - Sai hng v iu ha. Cc th t bin sai hng v iu ha c th b sai hng trong trung tm iu ha ca enzyme d lp th hoc sai hng c ch kim ch do mt t bin ca gen iu ha hay gen operator. Kt qu l gy ra s tng hp enzyme mt cch cu trc. Thng th cc chng nng sut cao l nhng th t bin nhiu ln c c hai loi sai hng. C nhiu phng php chn lc c hiu qu dng to ra v phn lp cc th t bin thuc c hai kiu.

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2. K thut penicillin lm phong ph cc th t bin tr dng vi khun ngi ta s dng k thut penicillin. Trong phng php ny cc iu kin c la chn sao cho cc t bo kiu hoang di, v d pht trin c khi khng c mt mt aminoacid no , b git cht nh penicillin. Penicillin ch tc dng ln cc t bo ang sinh trng. Cc th t bin tr dng cn aminoacid ny s khng sinh trng c v do sng st. i vi cc vi khun khng mn cm vi penicillin c th dng cc cht khng sinh khc, v d kanamycin hay cycloserine. Vi nm men th dng nystatin l thch hp. Bng vic dng cc tc nhn gy t bin c th nng tn s t bin ti mc th t bin cn tm c th xut hin trong s 105 -108 t bo. gy t bin c th dng nh sng t ngoi, cc tia ion ha hoc cc tc nhn ha hc nh nitrite, hydroxylamine, nitrosomethylguanidine hay ethylmethansulfonate. Dng hai loi cui cng c th t c mt tc t bin c bit cao trong c c cc th t bin kp. V d khi x l bng ethylmethansulfonate thu c 1-10% cc th t bin tr dng trong s cc t bo sng st. Cc t bo ca th t bin tr dng c lm giu nh k thut penicillin sau khi loi penicillin nh penicillinase trc tin c cy ln mi trng thch dinh dng thu nhn nhng khun lc ring bit. Bng cch cy chuyn song song cc khun lc ln mi trng ti thiu v ln thch cha yu t sinh trng c th d dng nhn ra v phn lp cc th t bin tr dng. Vic cy chuyn din ra nh k thut ng du tc l nh mt con du nhung a cc khun lc t mt hp ptri ny sang mt hp ptri khc. phn lp cc th t bin sai hng v iu ha c hai phng php thch hp: phng php dng cht chng cht trao i v phng php phn lp cc th hi bin t cc th t bin tr dng. 3. Phng php dng cht chng cht trao i Cht chng cht trao i l nhng hp cht tng t v cu trc vi mt cht trao i no , v d methionine v ethionine. Do s ging nhau ca chng, cc cht chng trao i cht c tham gia vo qu trnh trao i cht. Song v khng th hon thnh cc chc nng ca cht trao i nn chng gy tc dng c ch trao i cht v thng git cht t bo. S c ch c th do nhiu nguyn nhn gy ra. (a) Cht chng cht trao i, v d mt cht tng t ca aminoacid, do c gn vo mt protein enzyme, s lm cho enzyme ny bt hot.

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(b) Cht chng cht trao i cnh tranh vi cht trao i v trung tm hot ng ca mt enzyme v do vt ngn cn s chuyn ha ca cht trao i. Trng hp malonate c ch s loi hydro ca succinate l mt v d. (c) Cc cht chng cht trao i xut hin v tr ca sn phm cui cng ca mt chui tng hp, do kt hp vi enzyme d lp th m lm gim hot tnh ca n hay do phn ng vi cht kim ch m lm gim s tng hp cc enzyme. V vy s tng hp ca cht trao i tht s b ngn cn v do sinh sn ca t bo b c ch. Cc cht chng trao i m tc dng c ch do trng hp th ba gy ra c ngha i vi vic chn lc cc th t bin sai hng v iu ha. chn cc th t bin kiu ny ngi ta cy ln mt a ptri cha mi trng dinh dng c b sung cht chng cht trao i mt huyn dch t bo m c (khong 108 t bo/a) c x l trc bng mt tc nhn gy t bin. Tuy c s b sung cht chng cht trao i song trn a thch vn xut hin mt s khun lc t bo. y l cc t bo khng cc cht chng cht trao i. Nguyn nhn ca s khng ny c th l, cc t bo, do mt s sai hng v iu ha, tng hp nn mt s lng ln cc cht trao i tht s, ging vi cc cht chng cht trao i. iu ny khin cho cht chng cht trao i b pha long ti mc tr nn v tc dng. Khun lc ca cc th t bin kiu ny thng d nhn ra do c bao quanh bi mt vng cc khun lc v tinh. Cc khun lc v tinh pht trin c l do cc t bo khng tit ra cht trao i bnh thng lm gim nng cht chng cht trao i i vi cc t bo nm xung quanh. Phng php dng cht chng cht trao i gip ta phn lp cc th t bin b h hi trong s c ch bi sn phm cui cng cng nh cc th t bin b ri lon v c ch kim ch. 4. Phng php phn lp cc th hi bin ca cc chng tr dng Trong phng php ny ngi ta i t cc th t bin l tr dng i vi sn phm cui cng, v d mt aminoacid. y ngi ta quan tm trc ht n cc th t bin m tnh tr dng ca chng do mt s h hi trong enzyme d lp th gy ra. T y c th thu c nhng th hi bin vi trung tm xc tc tr li hot ng bnh thng, song trung tm iu ha, ni sn phm cui cng tn cng vo, th khng. phn lp nhng th t bin nh vy ngi ta cy khong 1010 t bo ca th t bin tr dng ln mt mi trng dinh dng ti thiu. Tuyt i a s cc t bo s khng sinh trng. Song mt s khun lc ca cc th t bin, do ly li c kh nng tng hp sn phm cui cng, sinh trng c. iu c ngha l enzyme b h hi thu li hot tnh

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xc tc nh mt t bin th hai. Trong mt vi trng hp, tuy t bin th hai hi phc c hot tnh enzyme song khng hi phc c tnh mn cm vi cht hiu ng d lp th. Nh vy s xut hin mt th t bin c hot tnh enzyme y nhng li khuyt v kh nng iu ha d lp th. Thng thng cc th t bin ny tit sn phm cui cng tng ng ra mi trng v ngi ta c th nhn bit c iu nh mt vng cc khun lc v tinh. T v d v s chn lc nhiu bc cc th t bin cha cc enzyme mt tnh mn cm d lp th c th thy r nhng bin php no l cn thit nhm thu c cc chng c nhng tnh cht mong mun. Tin ca iu l phi hiu bit chnh xc cc con ng trao i cht dn n s to thnh cc cht trao i, k c s iu ha chng. iu ny quan trng trc ht i vi cc sn phm m s to thnh chng do nhiu sai hng v iu ha gy ra. Trong nhng trng hp ny cn phi tm cch gy t bin nhiu ln i vi mt chng no hoc s dng cc phng php truyn thng tin di truyn. III. Aminoacid Cc aminoacid, cng vi cc nucleotide v vitamin, l nhng nhn t sinh trng thng c s dng nh cc loi dc phm hoc cht b sung cho thc phm. Cc aminoacid c s dng rng ri trong cng nghip thc phm, lm cc cht b sung trong y hc, v lm nguyn liu khi u trong cng nghip ha hc (bng 7.1) .
Bng 7.1. Cc aminoacid c s dng trong cng nghip thc phm
Aminoacid Glutamic acid L-aspartic v alanine Glycine L-cysteine L-Tryptophan + L-histidine Aspartame (phenylalanine +L-Aspartate) Sn lng th gii hng nm (tn) 370.000 5.000 6.000 700 400 7.000 b sung vo Nhiu loi thc phm Dch qu, ngt ngt Bnh m Dch qu Nhiu loi thc phm, Sa bt ung nh Mc ch Tng v, lm mm tht Lm du v Tng v Tng cht lng Chng oxy ha Chng oxy ha, chng i, tng dinh dng Gim calo

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L-lysine DL-methionine

70.000 70.000

Bnh m (Nht), thc phm Sn phm u tng, Thc phm

Tng dinh dng Tng dinh dng

Aminoacid thng mi quan trng nht l glutamic acid c s dng lm cht tng v (bt ngt). Hai aminoacid quan trng khc, aspartic acid v phenilalanine to ra ng nhn to aspactame, mt thnh phn quan trng ca cc loi ung nh dng trong n king v cc thc phm khng cha ng khc. Lysine, mt aminoacid khng thay th cho ngi v mt s ng vt nui c sn xut thng mi nh vi khun Brevibacterium flavum dng lm thc n b sung vo bt ng cc hoc vo bnh m. Mc d hu ht cc aminoacid u c th tng hp c bng con ng ha hc, song s tng hp ha hc thng to ra cc hn hp D,L khng hot ng v mt quang hc. thu c dng L l dng quan trng v mt ha sinh, cn phi dng phng php xc tc sinh hc. 1.Sn xut cc aminoacid nh vi sinh vt Hng lot vi sinh vt tch ly cc aminoacid trong dch nui cy. Tuy nhin ch c vi khun mi c nng sut m bo sn xut cc aminoacid quy m thng mi. V rng cc aminoacid l cc thnh phn khng th thiu ca cc t bo vi sinh vt v s sinh tng hp chng c iu khin c mc ch duy tr mt mc ti u, thng thng chng c tng hp mt s lng hn ch v b kim tra bi c ch iu chnh theo kiu lin h ngc m. V vy cn phi vt qua s iu chnh t c s sn xut tha cc aminoacid. Vic sn xut tha aminoacid vi sinh vt c th t c bng cch s dng cc phng php sau y : 1. Kch thch t bo thu nhn nguyn liu ban u; 2. Cn tr cc phn ng ph; 3. Kch thch s to thnh v nng cao hot tnh ca cc enzyme sinh tng hp; 4. Km hm hoc hn ch cc enzyme tham gia vo s phn gii cc aminoacid c to ra; 5. Kch thch s tit sn phm vo vng khng gian ngoi bo. Nhng yu cu trn t c bng cch s dng cc k thut t bin. 2. Sn xut cc aminoacid nh cc th t bin tr dng Cc th t bin tr dng, vn khng c kh nng sinh ra cht hiu ng theo kiu lin h ngc m, s c th sn xut tha tin cht hoc cc

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cht trao i gn gi ca phn ng b phong ta khi cc t bo ca th t bin sinh trng trn mt mi trng cha hn ch mt cht dinh dng cn thit. S sn xut cc aminoacid bng cc th t bin tr dng da trn nguyn tc ny. L- ornithine c sn xut nh mt sn phm trung gian trong qu hnh sinh tng hp L-arginine. Corynebacterium glutamicum cc enzyme u tin v th hai trong con ng sinh tng hp b km hm theo kiu lin h ngc bi L-arginine, nh ch ra trn (hnh 7.5). S to thnh tt c cc enzyme tham gia vo s sinh tng hp u b c ch bi L-arginine. Nh vy, mt c th tr dng cn xitrulin thuc Corynebacterium glutamicum thiu enzyme ocnithine cacbamoyl transferase xc tc cho s chuyn ho ornithine thnh citruline s ngn cn s to thnh arginine v s tch lu L-ornithine vi s lng ln. Cc iu kin ln men ging vi cc iu kin dnh cho s sn xut L-glutamat tr mt iu l mi trng phi cha mt lng thch hp L-arginine v mt lng ln biotin. Mt th t bin tr dng cn citruline thuc Brevibacterium lactofermentum cng c pht hin l c kh nng tch lu mt nng cao L-ornithine t ng (sn lng trn 40% tnh theo ng lng phn t). L-citruline th c sn xut bi cc th t bin tr dng cn arginine ca Corynebacterium glutamicum v Bacillus subtilis theo cch tng t. Cc th t bin tr dng cng c s dng trong qu trnh sn xut cc sn phm cui cng ca cc con ng trao i cht phn nhnh. Ln men lysine l mt v d in hnh. cc c th nhn s L-lysine c tng hp. t L-aspactate vi s to thnh mt sn phm trung gian l ,diaminopimelate. Ngoi L-lysine, L-threonine, L-methionine v L isoleucine cng c sinh ra t L-aspartate (hnh 7.6).

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Hnh 7.5: iu ho sinh tng hp arginine vi khun Corynebacterium sinh glutamate

Mt im kim sot quan trng trong con ng diaminopimelate l aspartokinase, n xc tc cho bc u tin trong s tng hp tt c cc aminoacid thuc h ny. cc chi Corynebacterium v Brevibacterium, enzyme ny b km hm theo kiu lin h ngc bi L-lysine cng vi Lthreonine. Do vy mt th tr dng cn homoserine v mt th tr dng kp cn threonine-metionin c phn lp t Corynebacterium glutamicum v Brevibacterium flavum, theo th t, gim d tr ni bo ca L-threonine, mt cht km hm r rt i vi aspactokinase. C hai th t bin u c kh nng tch ly mt nng L-lysine cao; mt lng biotin y c a vo mi trng ngn cn t bo to thnh L-glutamate.

Hnh 7.6: iu ho sinh tng hp aminoacids thuc h aspartate Brevibacterium flavum v Corynebacterium glutamicum

3. Sn xut cc aminoacid nh cc th t bin v iu ha

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Phng php dng cc th t bin tr dng nu trn khng thch hp i vi vic sn xut cc aminoacid l sn phm cui cng ca cc con ng trao i cht phn nhnh, nh arginine v histidine. Chng c th c sn xut nh cc th t bin v iu ho b mt i mt kh nng iu ho sinh tng hp no . Cc th t bin nh vy c to ra di dng cc th t bin khng vi cc cht tng t aminoacid hoc di dng mt th hi bin t th t bin tr dng song b khuyt mt enzyme iu ho. Cc hp cht ging cc amino acid t nhin v mt cu trc thng gy tc ng ln sinh trng ca mt s vi sinh vt. Trc y ngi ta gi chng l cc "izoster" nhn mnh tnh ging nhau v ho hc khng gian ca chng. Ngy nay, chng c gi l cc cht tng t aminoacid. Tc dng hm hm ca chng s b loi b khi a vo cc aminoacid t nhin tng ng. Mt s cht tng t aminoacid hot ng nh mt corepressor gi hoc mt cht km hm theo mi lin h ngc gi ca mt enzyme tham gia vo qu trnh sinh tng hp aminoacid t nhin tng ng, ng thi cng km hm s gn aminoacid vo protein. Do vy cc chng t bin khng vi cc cht tng t aminoacidt nhin, enzyme iu ha s tr nn mt tnh mn cm. Vo nm 1970 ngi ta phn lp c mt th t bin khng vi cht tng t cha lu hunh ca lysine, S-(2-aminoethyl)-L-cysteine, (SAEC - cng thc NH2CH2CH2SCH2CH(NH2)COOH t Brevibacterium flavum. Khng ging nh s iu ho sinh tng hp lysine Escherichia coli c kim sot bi ba izoenzyme ca aspactokinase v ba sn phm cui cng L-lysine, L-threonine v L-metionin, s iu ha Brevibacterium flavum din ra n gin hn : ch c mt aspartokinase b kim hm theo kiu lin h ngc bi L-lysine v L-threonine. Sinh trng ca Brevibacterium flavum b km hm bi SAEC. S km hm c tng cng r rt bi Lthreonine, v b loi b nh lysine. iu ch ra rng SAEC hot ng nh mt cht km hm ga ca aspartokinase theo kiu lin h ngc. V th mt s th t bin khng vi SAEC s tr thnh cc chng cha mt aspartokinase mt mn cm i vi c ch iu ha theo kiu lin h ngc. Trong thc t, mt s chng c kh nng tng hp tim tng L-lysine c tm thy trong cc th t bin khng vi SAEC, chng c kh nng sinh trng khi c mt c SAEC ln L-threonine. Chng tt nht sinh ra ti 31-33 g/l L-lysine. Aspartokinase trong th t bin ny c tnh mn cm vi s iu ho bi L-lysine cng vi L-threonine theo kiu lin h ngc thp hn khong 150 ln so vi enzyme trong cc chng b m.

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Bin php kt hp tnh tr dng v s sai hng v iu ha hin ang c ng dng rng ri ci thin cc vi sinh vt tng hp cc aminoacid. Brevibacterium lactofermentum, aspartokinase b km hm khi b sung ng thi hoc ring r L-lysine v L-threonine. Ngoi ra, dihydrodipicolinate sinthase, enzyme tham gia vo s sinh tng hp lysine cng b c ch bi L-lysine; nhng mi tng quan v iu ha nh vy c gi l "s kha lin ng trao i cht" (metabolic interlock). Mt th t bin khng vi SAEC phn lp c trn mt mi trng cha SAEC cng vi L-threonine tch lu mt lng ln L-lysine (18g/l). Mt th t bin tr dng cn leucine bt ngun t th t bin khng vi SAEC to ra ti 41g/l L-lysine nh s mt mn cm di truyn ca aspartokinase vi s km hm ca L-threonine v L-lysine theo c ch lin h ngc cng nh nh s gii km hm ca dihydrodipicolinate sinthase trong trng hp thiu L-leucine. Mt pht hin tng t cng gp chng Corynebacterium glutamicum cn L-homoserine v L-leucine v khng vi SAEC. Brevibacterium lactofermentum d tr ca DL-alanine ni bo cao hn d tr ca cc aminoacid khc. Trong t bo ca chng ny, alanine c to thnh bi pyruvate-L-aminoacid transaminase t pyruvate v bi aspartate decarboxylase t aspartate. C hai acid u l nhng tin cht thng thng trong s sinh tng hp L-lysine v L-alanine. Nh vy cc th t bin tr dng cn alanine s c mt nng sut L-lysine cao hn chng b m. Trong thc t, phn lp c mt th t bin tr dng cn L-alanine c ngun gc t th t bin khng vi SAEC sn xut c 39 g/l L-lysine. Mt chng hoang di ca Brevibacterium lactofermentum mn cm cao i vi cc cht tng t lysine, -chlorocaprolactame hoc -methyl-L-lysine ng ring r. Mt th t bin khng vi c hai cht tng t lysine c ngun gc t mt th t bin cn alanin khng SAEC sn xut ti 60 g/l Llysine.

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Hnh 7.7: Sn xut cng nghip lysine nh Brevibacterium flavum

Mt v d khc v vic ng dng s sai hng v iu ho kt hp vi tnh tr dng trong mt vi khun sn sinh aminoacid l s sn xut Lthreonine. Corynebacterium glutamicum v Brevibacterium flavum s tng hp tha L-threonine b ngn cn bng s km hm aspactokinase theo kiu lin h ngc bi L-threonine cng L-lysine, cng nh bng s km hm homoserine dehydrogenase theo kiu lin h ngc bi L-threonine (hnh 7.6), ngay c khi s dng cc th t bin cn isoleucine khng vi SAEC. S sn xut tha L-threonine nh mt th t bin khng vi mt cht tng t threonine, -amino--hydroxyvalerate, CH2CH2CH(OH)CH(NH2)COOH (AHV), c to ra t s mt mn cm di truyn i vi s km hm theo kiu lin h ngc bi L-threonine. C homoserine dehydrogenase ln homoserine kinase tham gia vo s tng hp L-threonine u b kim ch bi L-methionine c hai chng v do vy, c th thu c mt th tr dng cn metionin c nng sut to L-threonine cao hn chng b m. Cc vi khun sn xut c hiu qu L-arginine c chn lc theo cch sau: mt chng hoang di ca Corynebacterium glutamicum th khng cao vi cc cht tng t ca arginine l canavanin v L-arginine hydroxamate. Mt th t bin mn cm vi D-serine c ngun gc t mt th t bin tr dng cn isoleucine th li mn cm vi c hai cht tng t ca arginine. iu ny c th xy l nh tnh thm qua mng t bo ca cc cht tng t ca arginine c tng cng v nh tnh mn cm ca cc enzyme iu ho tham gia vo s sinh tng hp L-arginine i vi cc cht tng t. Th t bin sn xut 1,5 g/l arginine v nng sut ca

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th t bin ny c th c ci thin tng bc nh cc bc t bin v chn lc xa hn. Th t bin cui cng chn c c tnh khng vi Larginine hydroxamate, D-arginine v 2-thiazolalanine, song li mn cm vi D-serine, v n sn xut c 20-25 g/l L-arginine trong mt mi trng cha 15% ng. L-arginine cng c sn xut cng nghip nh cc th t bin v iu ho ca Bacillus subtilis v ca Brevibacterium flavum. Gn y, vic to ra cc th t bin v lu ho nh s dng cc phng php ti np c pht trin trn Serratia marcescens thuc h Enterobacteriaceae. Phng php ti np bao gm hai phn : chn cc t bin c l gy nn s sai hng hon ton cc c ch iu ha khc nhau, v t hp cc th t bin c chn lc bng bin php ng ti np. Chng hn, mt chng Serratia marcescens sn sinh threonine c to ra nh sau: Mt th ti np c cu to t 1) mt chng cn threonine m chng gc cha cc enzyme cu trc aspartokinase mn cm vi threonine v homoserine dehydrogenase v 2) mt phage c nhn ln trn trn mt chng khuyt c ch iu ho theo kiu lin h ngc i vi aspartokinase mn cm vi threonine v homoserine dehydrogenase. Th ti np ny thiu c hai c ch km hm theo kiu lin h ngc v kim ch i vi c hai enzyme, v sn xut c 8,3 g/l L-threonine. Sau , vng threonine ca th ti np ny c chuyn vo trong mt chng cn threonine c ngun gc t mt chng b khuyt v c ch lin h ngc cng nh c ch kim ch i vi aspartokinase mn cm vi lysine. mt trong cc th ti np to nn mt hm lng cao c hai enzyme aspartokinase v homoserine dehydrogenase khng mn cm vi c ch lin h ngc v tit ra khong 25 g/l L-threonine vo mt mi trng cha 16% saccharose. Mt yu t quan trng khc trong sn xut thng mi cc aminoacid l yu t tit. Ni chung, sinh vt khng tit cc sn phm trao i cht m chng phi b cng sc nhiu mi to c. Tuy nhin, s tit cc aminoacid c th c thc hin theo nhiu cch, c bit bng cch lm thay i tnh thm ca t bo. Chng hn, Corynebacterrium glutamicum, mt vi khun sn sinh acid glutamic, tnh thm ca t bo c th c tng cng bng mt s thit ht mt cht dinh dng c bit dn n vic c th to ra mt mng t bo yu, nh cho php n tit ra acid glutamic tha. Chng Corynebacterium glutamicum dng sn xut acid glutamic thiu hn hoc ch c kh nng hn ch i vi vic chuyn -ketoglutaric, mt sn phm trung gian ca chu trnh TCA, thnh succinyl-CoA (hnh 7.8 a).

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(a)

(b)

8. Sn xut acid glutamic

a) S dng con ng ph glyoxylate b p cc cht trung gian thit yu ca chu trnh Krebs. b) Sau khi kt thc sinh trng, hu nh ton b cacbon c cht c chuyn thnh glutamate. Bng vic khng ch nng biotin thp v vic b sung cc dn xut ca cc axit bo c th lm tng tnh thm ca mng v tng s tit nhng nng glutamic acidcao. Cc vi khun b h hi nh vy s s dng chu trnh glyoxylate tho mn nhu cu ca chng v cc cht trao i trung gian thit yu, c bit l trong pha sinh trng. Sau khi sinh trng b gii hn do ngun dinh dng b thay i, mt s chuyn ha isocitrate (gn nh hon ton nu tnh theo mol hoc 81,7% nu tnh theo trng lng) s xy ra (hnh 7.8 b ). IV. Sn xut cc purine nucleotide 1. IMP Sau khi sn xut thnh cng quy m cng nghip cc aminoacid nh ln men, ngi ta tin hnh cc nghin cu rng ri nhm xy dng quy trnh sn xut ln men cc nucleotide tng v, IMG v GMP. Tuy nhin, ln men nucleotit khc ln men cc aminoacid mt s im sau : 1. S sinh tng hp cc purin nucleotide din ra tng i phc tp . Ngoi con ng trao i cht de novo , cc con ng ng cu v

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chuyn ha ln nhau cng tham gia vo s sinh tng hp nucleotide purine (hnh 7.9). 2. Cc enzyme c kh nng phn gii cc nucleotide phn b rng trong cc vi sinh vt. 3. Cc nucleotide khng thm tt qua mng t bo. Do vy vi sinh vt khng d dng tit cc nucleotide vo mi trng nui cy qua hng ro thm thu. Nhng vn phc tp trn c khc phc v t lu IMP c sn xut Nht Bn khng ch bng phng php phn gii ARN nh enzyme m cn bng hai phng php ln men : 1. Sn xut ln men trc tip IMP bng cn ng sinh tng hp de novo. 2. Sn xut inosine bng ln men sau chuyn ho ha hc inosine thnh IMP. y s ni k hn v qu trnh ln men trc tip. khc phc cc kh khn trong ln men sn xut trc tip IMP, hai phng hng nghin cu sau y c tin hnh : 1. Lm mt on di truyn hot tnh phn gii nucleotide ca cc th t bin Bacillus subtilis sn xut inosine. 2. Ci thin v di truyn v v mi trng nhm tng nng sut IMP Brevibacterium ammoniagenes. Sn xut cng nghip IMP hin nay c thc hin nh cc th t bin ca Brevibacterium ammoniagenes theo phng php th hai.

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Hnh 7.9: Con ng sinh tng hp cc purine nucleotide

Chng Brevibacterium ammoniagenes KY3454 (ATCC 6872) c coi nh cy n thn ca Alain v chng ny v cc th t bin sinh ra t n sn xut nhiu loi nucleotid v cc dn xut ca chng theo c con ng de novo ln con ng ng cu. xy dng quy trnh sn xut ln men IMP quy m cng nghip, hng lot chng Brevibacterium ammoniagenes c la chn theo tng bc nhm tng nng sut IMP sau khi x l t bin nhiu ln bng tia t ngoi hoc NTG (N-methyl-N- nitro-N-nitrosoguanidine). Mt th t bin tr dng hng i vi adenine, KY13102, ln u tin c phn lp vo nm 1968 . Khi b sung Mn+2 nng di ti u cho sinh trng, chng ny sn xut c trn 12 g/l IMP. Tuy nhin, hm lng cao ca Mn+2 (trn 20 g/l) s kch thch sinh trng, song li lm gim mnh s tch lu IMP v lm tng s tch lu hypoxanthine. Chng KY13102, do tnh mn cm cao ni Mn+2 nh vy, d khng thch hp cho s sn xut cng nghip IMP. Sau , t chng KY13102, mt chng khc tch lu IMP song khng mn cm vi Mn+2, chng KY13171, c to ra. Sinh trng t bo v s tch lu IMP u tng khi nng Mn+2

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tng. Tuy nhin nng sut IMP ca chng KY13171 khng tho mn cho mt quy trnh sn xut thc t v c hiu qu kinh t. S tng hp cc puri nucleotide Brevibacterium ammoniagenes c iu ha bi aenin v cc dn xut ca guanine. c bit, enzyme cha kha PRPP amidotransferase b km hm mnh bi GMP cng nh bi ATP, ADP v AMP. ci thin nng sut IMP ngtri ta tm cch loi b s kim sot ca GMP theo kiu lin h ngc trong s sinh tng hp de novo purine. Kt qu l vo nm 1982, chng KY13184 thiu ht IMP dehydrogenase v cn guanine cho sinh trng c to ra t chng KY13171. Trc c nhiu cng trnh nghin cu chng minh rng nhu cu v guanin c lin quan ti s tng nng sut IMP v inosine Brevibacterium ammoniagenes. Bng cc chu k t bin v chn lc xa hn, nhiu chng tt hn c to ra t KY13184. Cui cng ngti ta thu c mt chng sn xut IMP c hiu qu, chng KY13369, tch lu ti 20-27 g/l IMP, nhng khng tch lu hypoxanthine. S tng nng sut IMP v s gim nng sut hypoxanthine khng do s bin i v tc sinh tng hp purine nucleotide de novo hay v hot tnh phn gii IMP gy ra m c th l do tnh thm i vi s tit IMP c ci thin. XMP Vo nm 1964, ln u tin ngi ta cho bit chng KY9978, mt th t bin cn guanine ca Corynebacterium glutamicum to ra c 2,57 g/l XMP. Sau , tch ly c mt lng XMP ln hn so vi Corynebacterium glutamicum KY9987. Khi b sung tha cc dn xut ca guanine, s tch lu XMP b c ch. S tch lu XMP ca chng ny hnh nh l do hu qu ca s tit trc tip nucleotide c tng hp de novo ra khi t bo, bi v XMP pyrophotphorylase ch c mt hm lng rt thp hoc hu nh khng c, v xanthine c cung cp t bn ngoi khng c chuyn ho thnh XMP bi cc t bo ang sinh trng. Trong qu trnh ln men ny, nng Mn2+ trong mi trng kch thch r rt s tch lu XMP. Kh c mt 1000 g/l Mn2+, 6,46 g/l XMP c tng hp, trong khi c mt 10 g/l Mn2+, ch c 3,18 g/l c, to thnh. iu ny tri ngc hn vi qu trnh ln men IMP nh cc th t bin tr dng cn aenin cng loi. S tit trc tip IMP ra khi cc t bo Brevibacterium ammoniagenes thng gp kh khn, cho nn t bin v tnh thm l bin php ch yu to ra cc chng sn sinh IMP. So vi cc th t bin sn sinh IMP, cc th t bin sn sinh XMP c th to ra d hn v s tit trc tip XMP xy ra d dng. Theo Fujio v Furuya (1984) mt th t bin cn guanine, hng v adenine ca

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Brevibacterium ammoniagenes c hot tnh nucleotidase yu, chng KY13215, tch ly c vi chc gam XMP trong mt lit mi trng. GMP Vic sn xut GMP bng con ng ln men trc tip cn cha thc hin c Hin nay GMP c th c sn xut cng nghip qua XMP bng cch s dng mt chng tch lu XMP v mt chng chuyn ha XMP. Chng KY13510 c Fujio v ctv to ra nm 1984 t chng Brevibacterium ammoniagenes ATCC 6872 qua mt s bc t bin. Chng ny khng vi decoyinin, thiu thc s hot tnh phn gii 5'nucleotide v giu GMP sinthethase (XMP aminase). Cc t bo KY 13510 chuyn ho c hiu qu XMP thnh GMP. Thnh phn hn hp c bn dng chuyn ha gm KH2PO4, K2HPO4, MgSO4.7H2O, CaCl2.2H2O, FeSO4.7H2O, MnSO4.4-6H2O, Lcysteine-HCl, axit nicotinic, v biotin. Ngoi ra chuyn ho cn cn mt cht hot ng b mt, polyoxyethylene stearilamine (POESA) l cht to iu kin cho XMP v GMP thm qua mng t bo. Nhng lng ln GDP, GTP cng nh GMP c to thnh t XMP khi vic nui cy c tin hnh 32oC. Trong khi , ch c mt mnh GMP c to thnh khi nui 42oC. Tc chuyn ha cao hn ti 88%. Thng kh l nhn t khng th thiu. pH ti u l 7,4. Sau 22 gi 50 g/l XMP.Na2.7H2O vi 120 gam t bo KY13510 /l trong mt ni ln men 5 lt, 43,5 g/l GMP.Na2.7H2O c to thnh. GMP sinthethase (XMP aminase) cn ATP chuyn ho XMP thnh GMP v sinh ra AMP. C l ATP ca t bo c s dng vo giai on u ca s chuyn ha v gn nh ton b ATP c tiu dng trong phn ng chuyn ha u c ti sinh t AMP v glucose. V. Vitamin Mt s vitamin quan trng c sn xut quy m thng mi nh cc qu trnh xc tc sinh hc. Cc vitamin thng c s dng lm cc cht b sung cho thc n ca ngi v ng vt. Tnh v mt doanh thu cc loi dc phm, sn xut cc vitamin ch ng th hai sau cc cht khng sinh - tc l vo khong gn mt t USD mi nm. Hu ht cc loi vitamin c sn xut thng mi nh bin php tng hp ha hc. Tuy nhin, mt vi loi vitamin mun c tng hp ha hc vi gi thnh r phi tri qua nhng qu trnh qu phc tp trong khi li c th c sn xut bng bin php xc tc sinh hc. Trong s ny, vitamin B12 v riboflavin l nhng vitamin quan trng nht. 1. Vitamin B12

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Trong t nhin, vitamin B12 ch c tng hp nh vi sinh vt. L mt coenzyme, trong ha sinh hc ng vt vitamin B12 c vai tr quan trng trong nhiu s ti sp xp ni phn t, mt nguyn t hiro gn vo mt nguyn t cacbon c i ch cho mt gc thay th gn vo nguyn t cacbon bn cnh. ngi, s thiu nhiu vitamin B12 s dn n mt bnh nghim trng c tn l bnh thiu mu c tnh, c c trng bi s sn xut t cc t bo hng cu v bi nhng s ri lon trong h thn kinh. Nhu cu v vitamin B12 ca ng vt c p ng nh thc phm n vo hay nh hp th vitamin trong rut ca ng vt do cc vi sinh vt ng rut sinh ra. Thc vt khng sinh ra v cng khng s dng vitamin B12. sn xut cng nghip vitamin B12 ngi ta s dng cc chng vi sinh vt c chn lc mt cch c bit, c sn lng cao v vitamin B12. cc thnh vin ca vi khun thuc chi Propionibacterium cho sn lng giao ng trong khong t 19-23 mg/l trong mt qu trnh-hai giai on, cn mt vi khun khc, Pseudomonas denitrificans, c th sn xut 60 mg/l trong mt qu trnh-mt giai on s dng r ng c ci lm ngun cacbon. Vitamin B12 trong cu trc c cha cobalt l phn c bn, v vy sn lng ca vitamin ny s tng ln ng k khi cobalt c b sung vo mi trng nui cy (hnh 7.10). 2. Riboflavin Riboflavin l hp cht m ca flavin, FAD v FMN tc l ca cc coenzyme gi vai tr quan trng trong cc enzyme tham gia vo cc phn ng oxi ha-kh trong hu ht mi sinh vt (hnh 7.10). Riboflavin c tng hp bi nhiu vi sinh vt, gm c vi khun, nm men v nm si. Nm Ashbya gossypii sinh ra mt lng ln vitamin ny (ti 7g/l) v do vy c s dng i vi hu ht cc qu trnh sn xut nh vi sinh vt. Tuy c sn lng cao nh vy, song gia qu trnh sn xut nh vi sinh vt v s tng hp ha hc lun lun c s cnh tranh kinh t ln.

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Hnh 7.10: Tri -Cyanocobalamin (vitamin B12), Phi -Riboflavin (Vitamin B2)

Cu hi n tp chng 7
1.Trnh by: a.Cc c ch iu ha hot tnh v s tng hp enzyme cc chng hoang di b.Nguyn tc ca s tng hp tha. c.Tnh cht v vai tr ca cc enzyme d lp th trong iu ha hot tnh enzyme. d.Cc phng php to cc th t bin tng hp tha e. Nguyn tc ca k thut penicillin 2. Lysine c th c to ra nh E. coli bng cch loi CO2 t mt acid amin cha 7 cacbon, acid amin ny c tn l: a.guanine b.proline c.acid diaminopimelic d.acid glutamic e. acid -ketoglutaric 3. Sn xut cng nghip acid glutamic s dng cc loi thuc mt trong cc chi sau y: a. Corynebacterium b. Pseudomonas c. Escherichia d. Acetobacter e. Clostridium 4. Ezyme DAP decarboxylase chuyn ha acid diaminopimelic thnh: a. ethanol b. acid acetic

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c. acid glutamic d. acid lactic e. lysine 5.Trong s sinh tng hp tha acid glutamic, hy nu vai tr ca chu trnh glyoxylate, biotin v cc dn xut ca cc acid bo. chng sn xut, nhng enzyme no hot ng mnh, enzyme no thiu hoc hot ng rt yu ? 6. Trong s iu ha sinh tng hp cc nucleotide purine Corynebacterium glutamicum (tr li bng ng hoc sai): a.Cc th t bin tit IMP mnh l cc th t bin tr dng v adeni v xanthine b.Vic to ra cc th t bin tha GMP d dng hn v ch cn mt bc to s sai hng v iu ha ca IMP- dehydrogenase l .

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Chng 8

Cc cht trao i bc hai

I. Cc cht khng sinh
Hin bit trn 8000 cht khng sinh v mi nm c khong vi trm cht khng sinh mi c pht hin. Trong tng lai chc chn cn c nhiu cht khng sinh khc na cng s c tm ra v a s cc vi sinh vt c kh nng to thnh cht khng sinh c nghin cu cho ti nay u ch thuc v cc chi Streptomyces v Bacillus. Nhiu nh nghin cu v cc cht khng sinh tin rng s c nhiu cht khng sinh mi c pht hin nu tm thm cc nhm vi sinh vt khc. Mt khc cc k thut ca cng ngh di truyn s cho php thit k mt cch nhn to cc cht khng sinh mi khi m cc chi tit v bn gen ca cc vi sinh vt sn sinh cht khng sinh c bit r. Tuy nhin, phng thc ch yu c s dng cho n nay pht hin cc cht khng sinh mi vn l phng php sng lc. Trong phng php sng lc, mt s lng ln chng vi sinh vt c tim nng to thnh cht khng sinh s c thu thp t t nhin di dng ging thun khit, sau cc chng ny s c kim nghim kh nng sinh khng sinh qua vic xem xem chng c to ra bt c cht khuch tn no c nng lc c ch s sinh trng ca cc vi khun kim nh hay khng. Vi khun kim nh c la chn t nhiu loi vi khun nhng phi l cc chng i din hoc gn gi vi cc loi vi khun gy bnh. Quy trnh c in kim nghim cc chng vi sinh vt mi c kh nng sinh khng sinh l phng php cy vch do Fleming s dng ln u trong cc nghin cu tin phong ca ng v penicillin. Nhng chng no c chng minh l c kh nng sinh khng sinh s c nghin cu tip xc nh xem cht khng sinh m chng snh ra c phi l mi hay khng. Khi mt c th sn sinh mt cht khng sinh mi c pht hin, cht khng sinh s c sn xut s lng ln, tinh khit, kim nghim v c tnh v hot tnh iu tr cc ng vt b gy nhim. a s khng sinh mi s khng cho kt qu dng tnh cc php th trn ng vt m ch mt s mi t kt qu ny. Cui cng, ch mt s rt t cc cht khng sinh mi ny c cng dng trong y hc v c sn xut quy m thng mi. 1. Cc cng on sn xut thng mi Mt cht khng sinh mun sn xut c quy m thng mi trc ht phi c sn xut thnh cng cc ni ln men cng nghip

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dung tch ln. y, vic hon thin cc phng php tinh khit c hiu qu gi vai tr quan trng nht. S d cn to ra cc phng php tch chit v tinh khit v lng khng sinh c mt trong dch ln men thng rt nh. Nu cht khng sinh ho tan trong mt loi dung mi hu c khng trn ln trong nc th c th d dng tinh khit n bng cch chit cht khng sinh vi mt th tch nh dung mi v qua c c cht khng sinh. Nu cht khng sinh khng tan trong dung mi th phi loi n khi dch ln men bng cch hp ph, trao i ion hoc kt ta bng con ng ho hc. Trong mi trng hp, mc ch u l thu c mt sn phm kt tinh vi tinh khit cao d rng mt s cht khng sinh khng kt tinh d dng v kh tinh khit. Mt iu c quan h khc l vi sinh vt thng sn sinh ra khng phi ch mt loi sn phm cui cng m trong c ln c cc cht khng sinh khc. t c mc tiu cui cng l ch c mt cht khng sinh duy nht, cc nh ha hc pht trin cc phng php loi b sn phm ph trong khi cc nh vi sinh vt li tm kim cc chng sn xut khng sinh ra cc sn phm ph khng mong mun. Him khi c th phn lp c t t nhin mt chng vi sinh vt to thnh cht khng sinh mong mun vi hm lng cao t yu cu ca sn xut thng mi. Do vy, mt trong nhng nhim v ch yu ca cc nh vi sinh vt hc cng nghip l phn lp cc chng mi c sn lng cao. Ngy nay, sn lng penicillin tng ln hn 50.000 ln so vi chng ban u nh vic chn chng v to ra mi trng thch hp. Vic la chn chng bao gm gy t bin ging ban u, nui cc th t bin v th nghim cc th t bin v kh nng sinh cht khng sinh. Trong a s trng hp, cc th t bin thng to ra t cht khng sinh hn b m, do vy vic nhn c cc chng c sn lng cao l rt him hoi. Trong nhng nm gn y, cc k thut ca cng ngh di truyn ci thin dng k cc quy trnh tm kim cc chng cho sn lng cao. K thut khuch i gen cho php a cc bn sao b sung ca cc gen mong mun vo mt t bo nh mt vect nh plasmit. Vic ci thin cc qu trnh iu khin cng cho php lm tng sn lng. Tuy nhin, kh khn ca vic s dng cc quy trnh di truyn lm tng sn lng l ch cc con ng sinh tng hp a s cc cht khng sinh thng bao gm rt nhiu bc vi rt nhiu gen v khng r gen no cn c ci bin nng cao sn lng. Do vy, nhng nghin cu c bn c ngha rt quan trng. Trong nhiu trng hp, tng s tng hp mt cht khng sinh no , vic ng dng cc kt qu nghin cu trao i cht c bn mang li nhiu li ch ln hn so vi cc chng trnh gy t bin v tuyn chn m mm.

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2. Cc nhm cht khng sinh ch yu dng trong iu tr cho con ngi C hai cch phn loi nhm cc cht khng sinh dng iu tr cho ngi. Cch th nht da vo ngha kinh t ca chng. Theo th vo nm 1997, cephalosporine c doanh thu cao nht, cn trong s cc penicillin th ch c amoxycilin (amoxil) l bn chy nht. T hp cc cht kim ch -lactamase vi cc cht khng sinh -lactam nh augmentin (amoxycilin + clavulanate K), primacin (imipenem + xilastatin) v unasyn (ampicillin + sulbactum) cng vi nhm cc cht khng sinh -lactam to c doanh thu khong 6 t USD vo nm . Hai cht khng sinh macrolide ph rng thuc nhm erythromycin (biacin v zithromax) t doanh s gn 2 t. Nhm th ba l cc quinolone do ciprofloxacin lm i din t 1 t. Ba nhm dc phm ny, theo th t, tn cng vo s tng hp thnh t bo, s sinh tng hp protein v gyrase, enzyme sao chp ADN . Trong khi vo nm 1995, th trng cht khng sinh li c thng tr bi ba nhm thuc khng khun khc hn, l cc tetracyclin, cc aminoglycozide v cc glycopeptide vi doanh thu trn 400 triu USD. Khng sinh chng lao rifampin v dn xut mi ca cacbapenem l imipenem cng nm trong danh sch. Doanh s ton cu ca cc cht khng sinh vo nm 2000 t khong 24 t. Phng php phn loi th hai da vo cc loi bnh nhim khun m cht khng sinh c s dng iu tr. Cc bnh ny c xp thnh hai nhm do vi khun Gram dng hoc Gram m gy ra. Do cc vi khun Gram m c tnh thm mng ngoi nguyn vn, cn vi khun Gram dng th khng, nn ni chung cc cht khng sinh nh vancomycin ch c ch sinh trng ca cc vi khun Gram dng m khng c ch cc vi khun Gram m. Cc lin cu khun Gram dng l nhng tc nhn gy bnh quan trng trong cc bnh vim phi, vim mng no v vim tai gia, trong khi cc t cu v cu khun ng rut (Enterococcus) li l tc nhn ca cc ca nhim trng sau m. Trc khun lao Mycobacterium tuberculosis vn gy nn hng triu ca t vong mi nm. Cc i dch t v dch hch trong lch s tng do hai loi vi khun Gram m, Yersinia pestis v Vibrio cholera gy ra, trong khi cc chng E. coli, Salmonella v Shigella thng xuyn l nguyn nhn ca cc bnh tiu chy. Trc khun Gram m Pseudomonas aeruginosa thng c m t nh mt tc nhn c hi gy bnh nhng ngi b tn thng v min dch hoc b bnh u x nang. Ging nh cc bnh dch hch, t, tiu chy, mt s bnh nhim khun nh vim phi thng d b mc nht trong mi trng cng ng,

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trong khi cc bnh khc li b nhim trong mi trng bnh vin, thng c gi l cc bnh nhim trng bnh vin. Cc bnh sau m do t cu v cu khun ng rut thuc nhm th hai, v v chng tn ti trong mi trng thng xuyn s dng cc cht khng sinh nn nhiu chng t cu v cu khun ng rut c tnh khng vi cc cht khng sinh v tr thnh cc vi khun c bit phin h. Cc t cu khng cc penicillin v c bit l khng methicilin c th c mt vi t l rt cao (40% t l nhim trng gp Staphylococccus aureus khng methicilin v 50% gp S. epidermidis khng cht khng sinh ny) mt s bnh vin. Chng gy t vong cao (25-63%) trong cc trng hp nhim trng mu. Vo cui nhng nm 1990 cc cu khun ng rut chim ti 12% tng s ca nhim trng bnh vin mt s thnh ph ca M trong trn 15% trng hp vi khun khng vi vancomycin. Nhim trng do cu khun ng rut khng vi vancomycin gy nn t l t vong t 42 n 81%.
Bng 8.1: Phng php iu tr hiu qu nht bng cht khng sinh Bnh nhim trng Vim phi cng ng Vim phi bnh vin Tc nhn gy bnh Phng php tr liu hiu qu nht

cephalosporin ph rng th h 4, Streptococcus macrolide hoc fluoroquinolone pneumoniae Vi khun Gram m cephalosporin ph rng th h 4, hoc t cu imipenem v aminoglycozide, vancomycin Vim mng S. pneumoniae hoc cephalosporin ph rng + no vancomycin + rifampin Neisseria meningitidis Hi chng Trc khun Gram cephalosporin + aminoglycoside + nhim trng m, song c cu vancomycin khun Gram dng Nhim trng Vi khun Gram m sulfametoxazol + trimetroprim, ng niu nh E. coli fluoroquinolone, fosfomycin Lao isoniazid + rifampin + pirizinaid + Mycobacterium etambutol tuberculosis

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Nhiu cht khng sinh khc cng c s dng trong nhng trng hp c bit v chng li nhng vi khun c bit, chng hn bacitracin c s dng ti ch chng li cc bnh nhim khun da cn cc tetracyclin c dng iu tr cc bnh do Helicobacter, Vibrio cholerae v cc bnh nhim khun do Brucella. 3. ch tc dng ca cc cht khng sinh vi khun Cc c ch tc dng ca hu ht cc cht khng sinh khng khun u c lm sng t sau khi ngi ta pht hin ra rng cc phn t ny gy hiu qu ln sinh trng ca vi khun, hoc bng cch lm chm t ngt sinh trng (c khun) hoc git cht vi khun (dit khun). Hnh 8.1 trnh by bn ch ch yu cc vi khun gy bnh c tm ra sau nhiu thp k nghin cu v c ch tc dng ca cc cht khng sinh : s sinh tng hp thnh t bo, s sinh tng hp protein, s sao chp v sa cha ADN v s sinh tng hp coenzyme folate.

Hnh 8.1.- Bn ch tc dng ch yu ca cc cht khng sinh ln vi khun

4. Cc cht khng sinh -lactam : penicillin v cephalosporin Mt trong nhng nhm khng sinh quan trng nht c v mt lch s ln mt y hc l nhm -lactam. Nhm ny gm cc penicillin, cephalosporin v cc cephamycin, c gi l -lactam v chng cha h thng vng -lactam (hnh 8.2) l mt h thng d vng phc tp.

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Cc cht khng sinh -lactam phn ng vi hai loi enzyme ch yu vi khun. Khi biu hin hot ng khng khun ca mnh, cc cht khng sinh -lactam phn ng vi cc enzyme tng hp thnh t bo lm gin on s tng hp thnh t bo ca vi khun. Trc y ngi ta cho rng mt cht khng sinh -lactam km hm hot tnh ca transpeptidase v n ging v mt cu trc vi phn D-alanyl-D-alanin ca peptidoglycan mi sinh ra. Song trong nhng nm gn y, ngi ta m t v nhng v tr a th th trn mng t bo cht vi khun c th lin kt cng ha tr vi penicillin. Mt s protein lin kt vi penicillin (penicillin binding proteins = PBP) hot ng nh cc enzyme v biu hin hot tnh ca carboxypeptidase, transpeptidase, endopeptidase v transglycosilase. Vai tr chnh xc ca cc PBP, bt k thuc hot tnh enzyme no, trong s sinh trng v phn chia t bo, vn cha c bit r, v v vy c ch phn t ca hot ng gy cht ca cc cht khng sinh -lactam vn cha th gii thch c. Nhm enzyme th hai phn ng vi cc cht khng sinh -lactam c vai tr sinh l i lp vi cc enzim tng hp thnh t bo, tc l, chng thu phn vng -lactam v hot ng bo v t bo khi s tn cng ca cc -lactam. Vic lm bt hot cc cht khng sinh -lactam bi cc lactamase c bit ti t cch y hn 50 nm, v iu ny l mt yu t quan trng ng gp vo vic pht trin khng ngng nhm cht khng sinh ny. -Lactamase l cc enzyme c hiu qu cao xc tc cho s thu phn hng lot cc -lactam. C cht cho cc enzyme ny bao gm cc penicillin v cc cephalosporin nh vng cng nh cc -lactam mt vng v cc -lacton. 4.1. Cc loi penicillin Phn nhm Cht khng sinh -lactam c pht hin u tin, penicillin G, ch yu hot ng chng li cc vi khun Gram dng. S d nh vy l v cc vi khun Gram m khng cho cht khng sinh ny i qua. V sau ngi ta pht hin ra nhiu penicillin mi trong mt s chng li rt c hiu qu cc vi khun Gram m. l nhng pht hin c ngha nht trn lnh vc cht khng sinh trong vi thp k qua. Penicillin l cc dn xut acyl ca acid 6-aminopenicillanic (nhn 6-APA - hnh 8.3). Chng c sn xut nh hai kiu ln men : kiu 1 to ra cc penicillin vi chui bn khng phn cc tc l ch c cc -lactam, v kiu 2 sinh ra penicillin N cng vi cc -lactam khc (hnh 8.4). Kiu 1 ch gp nm v bn cht ca chui bn khng phn cc c th xc nh c bng cch b sung

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cc tin cht chui bn thch hp vo mi trng ln men. Chng hn, nu acid phenylacetic c b sung vo mi trng ln men kiu 1 th benzylpenicillin s c to thnh. C nhiu v d v cc penicillin sinh tng hp c to thnh theo cch nh vy v mt s c trnh by trong hnh 8.2. Penicillin N l mt penicillin cha chui bn D--aminoadipyl lin kt kiu , c to ra bi 8 loi nm khc nhau trong Cephalosporium acremonium (tn khc Acremonium chrysogenum) l loi bit r nht. Cc vi sinh vt ny cng sn sinh cephalosporin. Nhiu loi Streptomyces cng sn sinh penicillin N cng vi cc cephalosporin, cephamycin, clavam, hay cacbapenem. Cc vi sinh vt ca c hai nhm ln men u sn sinh izopenicillin N trong acid -aminoadipic c cu hnh L song cht ny thng khng c tit ra mi trng ln men m ngc li nm di dng mt sn phm trung gian ni bo ca sinh tng hp. Ngoi cc penicillin sinh tng hp, cc penicillin bn tng hp c sn xut bng cch acyl ha nhn 6-APA. Nh cch ny sn xut c cc penicillin nh methicillin, nafxilin, v cc penicillin isoxazolyl, cc penicillin ph rng ampicillin, amoxycilin, epicilin v cyclacilin; cc penicillin khng Pseudomonas nh cacbenicilin v ticarcilin, cc penicillin ureido, pipeacilin, azocilin, v mezlocilin.

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Hnh 8.2: Cu trc c s ca mt s loi cht khng sinh -lactam gp trong t nhin

Penicillium chrysogenum l mt v d bit r nht v ln men kiu 1, song mt s loi Penicilium khc cng nh hng chc chi nm khc cng sn sinh cc penicillin mang chui bn khng phn cc.

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Hnh 8.3:

Nhn 6-APA

Hnh 8. 4:

Penicillin N

Hot tnh sinh hc Nng c ch ti thiu (MIC) ca mt penicillin no c th c coi nh mt i lng o kh nng thm nhp vo mng ngoi t bo, kh nng khng vi s tn cng ca cc lactamase nu chng c mt, v th ba, kh nng c ch ca n i vi cc enzim tham gia vo s tng hp thnh t bo. Penicillin G ch ra kh nng chng li mnh cc vi khun Gram dng tr cc chng sn sinh penicillinase. Khng sinh ny c hot tnh yu hoc khng c hot tnh chng li cc vi khun thuc h Enterobacteriaceae v cc chng Pseudomonas. Hnh 8.5, Cc penicillin bn tng hp methycilin, khng sinh bn vi penicillinase c hot tnh chng li vi khun sinh penicillinase song c hot tnh yu chng li Streptococcus faecalis v khng c hot tnh i vi cc vi khun Gram m. Tt c cc penicillin c ph rng, ampicillin, ticarcilin, v piperacilin u mn cm vi penicillinase song li c hot tnh chng li cc vi khun Gram m mnh hn. Temocilin ch hot ng chng li cc vi khun Gram m. S lin kt ca ca penicillin vi cc protein PBP l tiu chun tt nht hiu v hot tnh khng khun mc phn t. Cc PBP 1a, 1b, 2 v 3 l nhng PBP ch cht Escherichia coli v vic lin kt vi mt hoc nhiu trong cc protein ny s dn n s bin i hnh dng ca t bo v cui cng dn n t vong.

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Hnh 8.5: Cc penicillin bn tng hp

Penicillin G v ampixilin c i lc cao i vi PBP 2 v 3 v hu qu l lm xut hin mt s lng thp cc t bo b phng ln v ko di.

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Amoxycilin c i lc vi cc PBP 1a, 1b v 2 v cho t bo t c hnh si hn so vi ampicillin. R rng rng tnh lin kt ca cc penicillin khc nhau vi cc PBP thay i rt nhiu v bn thn cc PBP cc vi sinh vt khc nhau cng c s khc bit v kiu v s lng. Ln men v tch sn phm Penicillium chrysogenum l c th vn c s dng sn xut cng nghip penicillin. Cc chng cho sn lng cao thu c nh gy t bin v chn lc bng nhiu cch qua hn bn chc nm nay. Nng sut 60 mg/l chng u tin nay ln ti 85000 mg/l nhng chng hin ang c dng trong sn xut. Qu trnh sn xut penicillin c trnh by theo s hnh 8.6. Ging thng c gi di dng bo t ng kh hoc gi trong nit lng. Ging trong cc bnh nui lc (4 ngy) c cy vo cc b nhn ging th tch c th ln ti trn 300.000 lit. Phn chnh ca qu trnh sn xut l cc ni ln men. Mi trng thng dng nht bao gm glucose, phenylacetate natri, cao ng, sunfat amon v phosphate kali acid. pH ca mi trng ln men c gi 6,87,4 bng cch b sung NaOH hay acid sulfuric v nhit thng thng l 25oC v thng kh l nhn t gi vai tr rt quan trng trong sn xut penicillin. Thi gian cn thit sn sinh mt lng penicillin ti a thng thng l 8 - 10 ngy. Qu trnh ln men t khi din ra trong iu kin tnh thun ty. Vo ngy th 5 n ngy th 6, mi trng ln men c loi khi ni ln men v mi trng mi c b sung. Trong mt phng php khc, phng php tnh c b sung, tin cht chui bn, glucose, v.v.. c a vo mt cch lin tc hay nh k. Theo tnh ton, khong 70% lng glucose tiu th c dng trc tip cho sinh trng, 26% dnh cho s duy tr c th v 6% cho s tng hp penicillin. Trong sn xut penicillin, cn xem xt cc thng s sau y : tc sinh trng ca vi sinh vt, nhu cu duy tr c th (g glucose/ g t bo/ h), tc sn sinh penicillin ring phn (q pen) i vi mt c th (mg penicillin /g t bo/ h) v nng sut penicillin trn g glucose. Ngoi ra, thi gian lu trong ni ln men, dung tch b, thi gian ch bin, mi trng v chi ph nhn cng l nhng nhn t b sung c tm quan trng cng nghip. Khi ln men kt thc, ton b mi trng ln men c lc v si nm cng nh mi cht rn khc u c loi b. Dch lc c acid ha n khong pH 4 ri penicillin c chit trong mt loi dung mi nh butyl acetate. Sau penicillin c kt ta khi dung mi nh b sung

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acetate kali v c th hi nh bin php lc. bc tip theo, n c th c kt tinh li t isopropanol.

Hnh 8.6: Quy trnh sn xut penicillin

Mui kali ca penicillin G c th s dng c sau khi tin hnh cc quy trnh kim tra cht lng, hoc thng thng hn, c chuyn ha thnh nhn 6-APA cho cc qu trnh ch bin tip theo. Nhn 6-APA c sn xut t penicillin G bng cch cho penicillin G i qua mt ct cha penicillin acylase bt ng. Acylase s ct penicillin G thnh acid phenylacetic v 6-APA : pH ca ct c gi trung tnh bng cch b sung NaOH. Nhn 6-APA ri khi ct c thu hi bng cch kt ta pH 4. Cc penicillin acylase c nhiu loi vi khun, nm si, nm men v x khun sinh ra. Hai loi penicillinase c ch trong qu trnh sn xut penicillin l cc penicillin G acylase, ch yu do vi khun sinh ra, c pH ti u khong 7,5 v cc penicillin V acylase thng do nm sinh ra. Sinh tng hp Penicillin l cc dn xut ca 3 acid amin, acid L-a-aminoadipic, Lcysteine v L-valine. Trc ht, mt peptit (L)--aminoadipoyl-Lcisteinyl c to thnh, cht ny, sau khi c b sung L-valine v c epime ha s tr thnh tripeptide L--aminoipyl-L-cysteine-D-valine, tc l c cht ca penicillin synthase. Trong ln men kiu 1, isopenicillin N c to thnh bng cch nh vy s b loi axyl to thnh nhn 6APA nu khng c mt mt tin cht chui bn. Tuy nhin, nu c mt

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phenylacetyl-CoA th nhn 6-APA s c acyl ha to thnh penicillin G. Trong kiu ln men 2, chui bn L-a-aminoadipyl ca isopenicillin N c epime ha to thnh chui bn D--aminoaipyl ca penicillin N. 4.2. Cephalosporin v cc cht khng sinh -lactam khc Cephalosporin l cc cht khng sinh -lactam cha mt vng ihyrothiazin thay vo h thng vng thiazolidin. Cephalosporin ln u tin c tm thy Cephalosporium acremonium song hng lot cc nm khc cng sn sinh cht khng sinh ny. Hn na nhiu cephalosporin bn tng hp c sn xut. Gi tr ca cc cephalosporin khng nhng nm ch chng c tnh c thp m cn v chng l nhng cht khng sinh c ph rng. Qu trnh sng lc quy m ln nhm tm ra cc cht khng sinh lactam mi dn n vic pht trin cc hp cht mi m cu trc ca n khc vi c penicillin ln cephalosporin. l cc cht khng sinh nocardicin, acid clavulanic, v thianemycin. Acid clavulanic l mt cht khng sinh c ch c bit bi v, mc d n khng c hiu qu nh mt cht khng sinh song n li km hm hot tnh ca cc -lactamase. Cc enzyme ny do mt s vi khun to thnh, chng ph hu cc cht khng sinh -lactam lm cho chng tr nn v hiu khi c dng cha mt bnh no . Do vy, khi c s dng t hp cng vi cc penicillin v cephalosporin mn cm vi -lactamase, acid clavulanic s lm tng r rt hot tnh ca cc cht khng sinh ny. 5. Cc cht khng sinh do cc sinh vt nhn s sinh ra Nhiu cht khng sinh hot ng mnh chng li cc sinh vt nhn s cng li c sinh ra bi chnh cc sinh vt nhn s. Thuc v nhm ny c cc aminoglycoside, cc macrolide, cc tetracyclin v nhiu loi khc. Trong s chng, nhiu cht khng sinh c cng dng y hc ch cht v v vy vic sn xut chng gi vai tr quan trng trong cng nghip dc. 5.1. Cc cht khng sinh aminoglycoside Aminoglycoside l nhng cht khng sinh cha cc ng amin ni vi cc ng amin khc bi cc lin kt glycoside. Nhiu cht khng sinh c cng dng iu tr cao thuc v cc aminoglicozit, l streptomycin v cc cht h hng, kanamycin, gentamycin v neomycin. Chng c s dng trong thc tin y hc trc ht chng li cc vi

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khun Gram m. Streptomycin cng c s dng rng ri iu tr bnh lao. ng v mt lch s, vic pht hin ra gi tr ca streptomycin i vi bnh lao l mt bc tin quan trng trong y hc bi v l cht khng sinh u tin tm thy c kh nng khng ch bnh nhim trng ng s ny. Tuy nhin, khng c cht khng sinh aminoglycoside no c s dng rng ri nh trc y chng tng c s dng. Streptomycin b thay th bi mt s ho cht tng hp mt mt v streptomycin gy nn mt s hiu ng ph nghim trng, mt khc ngy cng c nhiu vi khun khng vi cht khng sinh ny. Vic s dng cc cht khng sinh aminoglicozit chng li cc bnh nhim khun Gram m tr nn t c ngha hn k t khi xut hin cc penicillin bn tng hp v cc tetracyclin. Ngy nay cc cht khng sinh aminoglicozit c coi l cc cht khng sinh d tr, ch c s dng khi cc cht khng sinh khc khng mang li hiu qu. Mt trong nhng c im ng ch ca cc cht khng sinh aminoglycoside l s iu khin sinh tng hp chng. Nh c th thy trn hnh 8.9, ba thnh phn ca phn t streptomycin c tng hp t 3 con ng tch bit nhau ri cui cng cc di n v c hp nht vi nhau. Sn phm trung gian cui cng, streptomycin-P, khng c hot tnh sinh hc nhng c hot ho khi loi b mt phn t phosphate. Streptomycin c tng hp nh mt sn phm bc hai in hnh. Mt s enzyme tham gia vo qu trnh ny ch c tng hp vo cui pha sinh trng. Mt phng thc trong s iu khin l s sn sinh mt cht cm ng c tn l nhn t A. Nhn t A khng ging v mt ho hc vi streptomycin, song bng cch no n tham gia vo s trao i hydrat cacbon. Cc enzyme ch cht ca s sinh tng hp ch c tng hp khi mt nng nhn t A nht nh c tch lu, iu ny gii thch vai tr ca nhn t ny trong trao i cht bc hai. Trong pha sinh trng, nhn t A c tng hp s c tit ra v tch lu dn dn trong mi trng. Ch khi nng t ti mt mc ti hn nhn t A mi bt u hoi ng v cm ng s tng hp cc enzim ch cht cn cho s sinh tng hp streptomycin. ngha ca nhn t A i vi s tng hp streptomycin th hin ch l cc th t bin nhn t A(-) mt i kh nng sinh tng hp streptomycin, song nn nhn t A c a vo cc th t bin trn th s sinh tng hp streptomycin li c phc hi. Tuy vy, bn thn nhn t A khng phi l mt tin cht ca streptomycin, iu ny c ch ra qua hin tng l ch cn b sung mt lng rt nh

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nhn t A, 1g, c th dn n vic to thnh 1 gam streptomycin. Song cng cn lu rng, s sinh tng hp streptomycin khng ch c iu khin bi mt mnh nhn t A v cc qu trnh iu khin khc cng tc ng ln s sinh tng hp streptomycin.

Hnh 8.7.- Sinh tng hp cc penicillin

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Hnh 8.8: Cc cht khng sinh -lactam mi

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Hnh 8.9: Sinh tng hp streptomycin Streptomyces griseus

5.2. Cc cht khng sinh macrolide Cc cht khng sinh macrolide cha cc vng i lacton ni vi cc thnh phn ng Nhng s thay i trong cu trc ca c hai thnh phn vng lacton v ng lm xut hin nhiu loi khng sinh macrolide. Cht khng sinh macrolide bit r nht l erythromycin, cn cc macrolide khc l oleandomixin, spiramixin v tyloxin. Erythromycin thng c s dng trong iu tr thay th penicillin i vi cc bnh nhn b d ng vi penicillin hoc vi cc cht khng sinh -lactam khc. Erythromycin c gi tr c bit trong iu tr cc bnh do Legionella gy ra v tnh mn cm cao ca vi khun gy bnh, Legionella pneumophila, i vi cht khng sinh ny.

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Tnh phc tp ca cc cht khng sinh macrolide th hin trong cu trc ca chng. Ngi ta bit ti trn 25 bc phn ng do enzyme xc tc tham gia vo s sinh tng hp erythromycin. Vic iu khin sinh tng hp xy ra theo nhiu cch. Glucose v phosphate km hm sinh tng hp v erythronolit B, mt trong nhng sn phm trung gian trong sinh tng hp erythromycin cng c ch s tng hp cht khng sinh ny. Bn thn erythromycin cng gy nn s c ch bi sn phm cui cng i vi mt s enzyme ch cht. 5.3. Cc tetracyclin Cc tetracyclin l mt nhm cht khng sinh quan trng c ng dng rng ri trong y hc thc hnh. l nhng cht khng sinh ph rng u tin c ch hu ht cc vi khun Gram dng v Gram m. Cu trc c s ca cc tetracyclin bao gm h thng vng naphtacen. B sung vo vng ny l cc thnh phn khc nhau tu tng loi. Chng hn, clotetracyclin cha mt nguyn t clo trong khi oxytetracyclin cha mt nhm hydroxyl v khng c clo). C ba cht khng sinh ny u c sn xut t con ng vi sinh vt hc, song trn th trng cng c bn cc tetracyclin bn tng hp trong cc thnh phn khc c gn vo h thng vng naphtacen nh phng php ho hc. Cc tetracyclin v cc cht khng sinh -lactam l hai nhm cht khng sinh quan trng nht trong lnh vc y hc. Tetracyclin cng c s dng trong ngnh th y v mt s nc c dng lm thc n b sung cho gia cm v ln. c mt thi, chlotetracyclin c s dng bo qun c bng cch a n vo dng p c bin va nh bt c, song vic s dng cc cht khng sinh c cng dng y hc hin nay khng c khuyn khch v n cha ng nguy c to nn hin tng khng khng sinh. S sinh tng hp mt tetracyclin bao gm nhiu bc phn ng do enzyme xc tc. Trong trng hp ca chlotetracyclin c ti 72 sn phm trung gian tham gia vo s tng hp m a s trong chng ch c bit mt cch rt i cng. Cc nghin cu v di truyn hc vi khun sn sinh chlotetracyclin Streptomyces aureofaciens cho bit rng c trn 300 gen tham gia vo ! Vi mt s lng gen ln n nh vy, s iu khin sinh tng hp cht khng sinh r rng l rt phc tp. Tuy nhin mt s tn hiu iu ha c lm r v cc quy trnh sn xut ang c ci thin.

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Chng hn, s tng hp cc tetracyclin b kim ch bi c glucose v phosphate. S kim ch bi phosphate c tm ngha c bit ln bi vy mi trng nui cy trong sn xut thng mi phi c s dng vi nhng nng phosphate hn ch. Cng ging nh trong sn xut penicillin, cao ng c s dng trong sn xut chlotetracyclin quy m ln, song ngi ta trnh dng glucose v glucose gy nn hin tng kim ch d ha. Ngun cacbon tt nht nn dng y l saccharose.
Quy trnh sn xut chlotetracyclin nh sau:

Nguyn liu cy (bo t trn thch nghing hoc trong t v trng)

cao tht 2%; asparagin 0,05%; glucose 1%, KH2PO40,5%; thch 1,3%

a thch
Dng bo t cy

Bnh lc 24 gi Tin ln men

5% nguyn liu cy

cao ng 2%; saccharose 3%; CaCO3 0,5%

ging nh bnh lc
ng knh 1%; cao ng 1%; (NH4)2HPO4 0,2%; CaCO3 0,1%; MgSO4,ZnSO4; CuSO4; MnCl2

Ln men chnh
2-10% nguyn liu cy

Tinh khit phn dch trong sau khi loi t bo

(R1 = H; R2 = CH3; R3 = OH; R4 = Cl) Hnh 8.10: Quy trnh sn xut chlotetracyclin

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II. Cc c t nm (mycotoxin)
Mycotoxin l mt thut ng chung dng miu t cc c t c to thnh trong qu trnh sinh trng ca nm mc. c t nm l mt vn khng cn mi m v vic ng c bi thc phm v thc n gia sc b nhim nm mc c bit n t nhiu th k nay, song vic pht hin ra aflatoxin B1 (AFB1) nh l mt trong nhng nhn t gy ung th c hiu lc cao nht gp trong t nhin vo u nhng nm 60 thc y mnh m cc nghin cu trn lnh vc ny. Mi nguy him do cc c t nm v cc bnh do chng gy ra trong mt thi gian di khng c nh gi ng mc, l nguyn nhn ti sao chng c mnh danh l "cc bnh b lng qun" . Tnh trng ny thay i t ngt k t khi ngi ta pht hin ra hin tng nhim c hng lot i vi g ty cc trang tri min Nam v .mn ng nuc Anh do lc dng lm thc n b nhim mc gy ra. Thc n cha lc b nhim Aspergillus flavus, nm ny sinh ra cc aflatoxin rt c (k c vi ngi) v gy nn bnh ung th gan. T cc nghin cu mnh m thuc nhiu lnh vc c lin quan pht hin ra cc c t nm khc na cng gy nn nhng bnh rt nguy him.

AflatoxinB1(Aspergillus flavus)
LD50(ng ming, chut cng) 7,2mg/kg

Roquefortin (P. roqueforti)

LD50(trong mng bng, chut nht) 10-20 mg/kg

LD50(ng ming, chut cng) 4-13mg/kg Hnh 8.11: Cu trc ca hai loi c t nm in hnh Aflatoxin B1 v roquefortin
B

LD50(ng ming, chut cng) 15mg/kg ;

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1. Tnh hnh nghin cu v c t nm

Cho n gia nhng nm 1980 ngi ta bit ti trn 300 c t nm, chng c th xp thnh 25 kiu cu trc v do kbong 350 loi nm khc nhau to thnh. Hnh 8.10 trnh by hai loi c t nm in hnh, aflatoxin B1 v roquefortin. c ca hai loi cht c ni ting l asen v paration cng c nu ra tin so snh. Penicillium roqueforti, nm si sn sinh roquefortin, thng c s dng chun b nhiu loi phomat mu lam. Cng may l hm lng roquefortin c trong phomat mu lam thng thng rt thp (< 7ppm) cho nn liu lng gy cht i vi ngi trng thnh ch t c sau khi n 200 kg loi phomat ny. C th tm thy t cc loi nm si gp trong thc phm ca ngi cc c t nm mang trn 20 loi hot tnh khc nhau, chng thng xuyn e da tnh mng con ngi v nhiu trong s chng gy nn bnh ung th. Theo tnh ton ca T chc Quc t v Ung th th 80% cc bnh ung th l do cc yu t mi trng gy ra, trong s c t nm c mt vai tr quan trng. Con ngi b e da bi cc c t nm theo hai cch. Th nht, con ngi tiu th trc tip cc nguyn liu b nhim nm sinh c t. Th hai, thng qua tht, sa v trng ca cc ng vt n phi thc n b nhim cc khun lc nm, con ngi hp thu c t nm c trong cc thc phm ny, c bit l trong gan v thn ng vt l nhng ni c t nm c tch ly. Gy xut huyt, nhim trng, gim hng cu, teo ty sng,
km hm tng hp protein

[A]

[B]
Hnh 8.12: Bnh nm ca: c t v phng thc tc dng

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[A]Ecgotamin LD50(IV, chut nht) 62mg/kg, co mch ngoi vi, hoi th v hoi t chn tay [B] Acid secalonic A LD50(IV, chut nht) < 50mg/kg, hoi t gan.

Hnh 8.13: c t T-2 (Fuarium culmorum)

Aflatoxin B1( A. flavus)

Aflatoxin M1 (Sa ca cc ng vt m thc n ca chng c cha Aflatoxin B1)

Hnh 8.14: Bnh do aflatoxin ngi: c t, ngun gc v phng thc tc dng

Hnh 8.15: Citreoviridin (P. citreoviride, A. terreus) gy kh th, bun nn, nn ma ngng th, km hm tng hp ATP ti th

2. Cu trc, hot tnh sinh hc v cc bnh do c t nm gy ra

L loi sn phm bc hai in hnh gp trong t nhin, cc c t nm tn ti di rt nhiu dng cu trc v c c tnh ha hc a dng. Chng hn

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monoliformin do Fusarium moniliforme sinh ra c cu trc n gin, rt d tan trong nc v c c tnh cao, cn penitrem A do Penicillium crustosum sinh ra gy nn bnh run th li c cu trc rt phc tp v k nc. Bnh nm ca Vic lm sng t cu trc ca cc c t nm c bt u vi tc nhn gy bnh nm ca, mt bnh do c t nm gy ra c bit t thi k rt xa xa. Bnh ny do mt loi nm si, Claviceps purpurea gp trn cc bng la gy ra, chng to thnh cc phn x cng thng c gi l ca g. Bng cch , chng thm nhp vo cc ng cc dng lm bnh m v gy nn cc dch ng c gp Chu u t thi Trung c. Cc nghin cu ha hc trong vng trn 100 nm gn y lm sng t c hai nhm c t nm - l cc alcaloid ca nm ca g (ergotamin) v cc ergocrom (acid secalonic) Bnh mt bch cu do nhim c thc n (ATA) y l mt bnh nghim trng khc do c t nm gy ra, nguyn nhn l do mt loi nm si mc trn ng cc. Bnh ny c c trng bi s ph hu dn dn h thng to mu chu trch nhim i vi s to thnh cc tiu th hng cu v bch cu. ATA bt u bng s ph hu da, xut huyt, vim, v nhim trng. Trong cc giai on cui, tu sng b teo i v lng hng cu v bch cu b gim mnh. T l t vong do bnh ny gy ra c th ln ti 60%. Bnh ny c xc nh l mt bnh do c t nm gy ra nh cc nghin cu v v dch n ra nm 1944 Uran thuc min Nam nc Nga tri rng trong mt vng di trn 500 km. Thc phm b nhim cc loi Fusarium sinh c t, bn ny tng hp loi c t nm "kiu T2" (hnh 8.12). c t T2 km hm s tng hp protein theo mt c ch ging nh c ch ca kh m tt cha nit. V rng i din u tin ca nhm c t nm ny c phn lp t Trichothecium roseum nn chng cng c gi l "tricothecen". Cho n nay trn 50 tricothecen bit lm thnh mt trong nhng nhm c t nm quan trng nht. c bit, cc vng kh hu nhit i tricothecen l mt mi nguy him i vi dn c trong vng, r rt nht l trng hp xy ra Campuchia vo nhng nm 1980. Bnh do cc aflatoxin y l bnh dch th ba c nguyn nhn t c t nm c nhn ra sau bnh nm ca v ATA v l bnh nguy him nht. Ngay sau khi cc aflatoxin ca loi nm si Aspergillus flavus c xc nh l nguyn nhn ca "bnh X g ty", hng lot nghin cu c tin hnh nhm xc nh xem cc loi c t nm hoat lc cao ny c gy nn cc bnh nhim c ngi hay khng. iu ny i hi mt chng trnh sng lc

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ton din v phc tp. Chng hn, s hp thu aflatoxin c xc nh bng cch phn tch mt s lng ln cc mu thc phm thu thp t th trng v cc gia nh. T thit lp c mt mi tng quan mang tnh thng k quan trng v t l mc bnh ung th gan v vic hp thu aflatoxin. T l mc bnh ung th gan bnh thng l hai trng hp trn 100.000 dn trong mt nm. T l ny quan st c nhng vng cao nguyn kh hu kh, ni c th tng i d dng gi thc phm khng b nhim mc v do vy hm lng aflatoxin ca chng nm mc thp. S hp thu aflatoxin hng ngy cc vng ny nm di 5 mg/kg th trng. Nu s hp thu hng ngy i vi aflatoxin tng gp i ti 10 mg/kg, nh trong trng hp cc vng thp c m cao hn ca Kenya th t l mc bnh ung th gan trung bnh s tng 100%. Mc d cc mc hp thu aflatoxin cao hn, t l mc bnh ung th khng tng theo t l thun, song hiu qu ca cc c t ny vn biu hin r rt. Ging nh trng hp ung th gan ngi ta cng quan st c mt mi tng quan tng t gia vic hp thu aflatoxin v t l mc bnh "x gan thiu nin" gp n . Mc d ngi ta tm thy nhiu loi mycotoxin trn cc loi nng sn khc nhau, song ch c mt s hn ch, chng hn cc aflatoxin v ochratoxin, l c kh nng gy ung th. Mt s khc, chng hn cc mycotoxin do cc loi Fusarium v Alternaria sinh ra th c th gy t bin trn mt s c th, song hiu qu gy ung th ca chng th cha c xc nh. Do hiu qu gy ung th mnh v hay gp trn thc phm v thc n gia sc, AFB1 l mt trong nhng mycotoxin c nghin cu nhiu nht. Aflatoxin gm mt lot cht trao i c tc dng c v gy ung th do Aspergillus flavus v A . parasiticus sinh ra trn ng rung v trong qu trnh bo qun mt s nng sn quan trng nh lc, ht bng ng, la, ht bu b, ht hng dng v ht ca cc loi qu hch. Trong s gn 20 aflatoxin bit, ngi ta quan tm nhiu nht n aflatoxin B1v aflatoxin M1, cht ny cng l mt tc nhn gy ung th. Aflatoxin Ml l mt sn phm hiroxyl ha c to thnh trong trao i cht ca cc ng vt c v m trong thc n ca chng c cha aflatoxin B1. Aflatoxin trong sa b c th l nguyn nhn ca bnh x gan trm trng gp cc tr em n v ch Phi . C ch tc dng ca cc aflatoxin vi s tham gia ca mt 15, 16-epoxit v cytochrom P450 hin ang c nghin cu mnh m. Mc du AFB1 gy nn c hiu qu cp tnh (ng c gan) ln mn tnh (ung th gan) song hiu qu gy ung th mi l iu ng lo ngi
B

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nht. Khi th nghim trn chut c Fischer ngi ta pht hin ra rng 10% s chut th nghim b ung th gan khi chng c cho n thc n ch cha 1g/kg AFB1 trong mt khong thi gian l 2 nm. Nguy c tim tng ca cc aflatoxin i vi sc khe con ngi dn n vic hnh thnh nhiu chng trnh c quy m quc t nhm gim st s c mt ca c t ny trong cc loi lng thc hu ht cc nc trn th gii. Liu lng cho php i vi aflatoxin tng s nm vo khong t 0 n 50 phn t (ppb). a s cc nc iu chnh mc 20 ppb. Ring i vi AFM1 , liu lng cho php nm gia 0 v 0,5 ppb v do vy, thc n cho b sa cng phi c iu chnh sao cho hm lng aflatoxin tng s thp hn cc loi thc n khc. Bnh " beriberi tim" Loi bnh c t nm th t l do c t ca loi nm Penicillium citreoviride mc trn la thuc vng ng , polyen lacton citreoviriin gy ra. Bnh ny c tn l "beriberi tim" v triu chng ca n ging nh bnh beriberi xut hin khi thiu vitamin B1. Bnh ny m u bng nhng s ri lon chc nng ca tim ri tin trin dn thnh kh th, bun nn v nn. Beriberi tim tng gy nhng v dch trong dn c vng ng Nam cho n lc m nguyn nhn ca n c pht hin v c loi tr v c bn nh vic ngn nga la khi b nhim nm. Trc , chng hn vo nm 1908, ch ring Nht c trn 10000 ngi cht v bnh "beriberi tim ". Di nhng iu kin nht nh c th khc phc dc hiu qu ca citreoviriin, m tc dng ca n r rng l c lin quan ti s km hm qu trnh tng hp ATP ti th, bng cch tim vitamin B1. Cho n gn y ngi ta cho rng citreoviridin ch gp nm la ng Nam , tuy nhin, vo nm 1980, c t nguy him ny v hai c t nm khc c cu trc tng t cng c pht hin loi nm si Aspergillus terreus sng trong t, trn ng cc, v trn cc nguyn liu cha ng. Citreoviriin ca A. terreus c th c sinh ra vi mt nng rt cao, ti 2% trng lng kh ca si nm. Vic pht hin ra Aspergillus terreus sn sinh cc c t nm cng mang mt ngha quan trng v nm ny thng c dng trong sn xut cc sn phm cng nghip, chng hn acid itaconic. Mt trng hp nm c ch khc cng ng thi sn sinh c t nm trng hp ca Aspergillus niger vn c s dng sn xut acid citric. Nm ny tng hp mt nhm cc c t c cu trc cyclopeptide, chng hn malformin A c cu trc lp th. Trong nhng trng hp ny, sn phm ln men c ch phi c thun khit c bit khi cc c t nm.

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3. Sinh tng hp
Cc vin gch cu trc c bn Hin nay ngi ta bit rng s sinh tng hp trn 300 c t nm bit u da vo mt s rt t cc vin gch cu trc c bn c trnh by trn hnh 8.16. Cc ecaketid cha t hai n mi n v acid acetic l nhng hp cht c tm quan trng c bit v cc c t nm c hot tnh sinh hc rng u c th c xy dng t cc polyketid ny theo cc phng thc cc k linh ng. Ngoi ra, nhiu c t nm cng c to thnh t mt mnh isopentenyl pyrophosphate hoc t nhng t hp ca hp cht ny vi tryptophan v cc acid amin khc. Mt trong nhng c im c trng ca s sinh tng hp cc c t nm l, khng ging cc sn phm c ngun gc vi sinh vt khc nh cc cht khng sinh, ng khng c s dung nh cc tin cht hoc vin gch cu trc cho sinh tng hp.

Hnh 8.16:Cc vin gch cu trc quan trng nht trong sinh tng hp c t nm.

Hnh 8.17: Cu trc ca moniliformin (Fusarium moniliforme) [A] v penitrem (Penicillium crustosum) [B]

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Sinh tng hp moniliformin Cho n gn y ngi ta vn cho rng tetraketit l thnh vin u tin ca lot sn phm t nhin c to thnh t s vng ha cc poliketid. Tuy nhin, nhng thc nghim nh du mi y cho thy mt diketit cng c th tc dng nh mt tin cht trong sinh tng hp mt c t nm rt n gin v mt cu trc l moniliformin (hnh 8.18).

Hnh 8.18: Cc con ng sinh tng hp moniliformin

Moniliformin ln u tin c Cole v ctv tch ra (1973) v Springer v ctv lm sng t v mt cu trc (1974), c to thnh nng cao (ti 33g/kg) bi loi nm Fusarium moniliforme rt thng gp trn cc thc n cha ng. Lng c t ny git cht 200000 con g trng non. Fusarium moniliforme v Gibberella fujikuroi ch sn sinh moniliformin trn mi trng rn cha ng. Trn mi trng ny nm sinh trtrng rt mnh v chuyn ha ton b v nhanh chng c cht thnh h si nm mu trng. S sinh tng hp moniliformin t acetate c th din ra qua malonyl CoA v 1,3-butandion (hnh 8.18). S oxi ha nhm methylene phn ng ca 1,3-butanion c th dn n mt hp cht trung gian c vit di cc dng h bin, cht ny sau s b mt nc to thnh moniliformin.

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Cu hi n tp chng 8
1. Hnh sau trnh by cu trc ca mt cht khng sinh thuc h lactam

a. Tn ca cht khng sinh ny l g, do vi sinh vt no sinh ra ? V mt cu trc, n ging v khc cc penicillin ch no ? b. ch tn cng ca cc cht khng sinh thuc h -lactam l g ? N khc vi ch tn cng ca cc aminoglycoside (nh streptomycin), cc macrolide (nh rifamycin, erythromycin) hay cc tetracyclin ch no ? c. Trnh by c ch tc dng ca cc cht khng sinh thuc h lactam; c ch ny ging v khc vi c ch tc dng ca vancomycin ch no ? d. Phn bit cht khng sinh vi sulfa. Sulfa tn cng ln u ? Cc quinolone l g v tn cng ln u ? e. Vic thay i cc chui bn (gc R) ca cephalosporin c th to cho cht khng sinh ny nhng tnh cht mi no ? Cephalosporin th h 3 khc vi Cephalosporin th h 1 c im quan trng no ? 2. Phn bit penicillin bn tng hp vi penicillin t nhin v penicillin sinh tng hp. Cc penicillin bn tng hp c nhng u th g nu em so snh vi cc penicillin t nhin ? 3. Bn va tm ra c mt cht khng sinh mi, nhng cha bit chc chn l mt cht c khun, mt cht dit khun v cht tiu khun. a. Phn bit cht c khun, cht dit khun v cht tiu khun. b. Lm th no xc nh cht va tm ra l cht c khun, dit khun hay tiu khun ? 4. Mycotoxin l ngoi c t hay ni c t ? a. Cho v d v ngoi c t v ni c t.

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b. Nu nhng im khc bit chnh gia ngoi c t v ni c t vi khun (quan h t bo-c t, ngun gc, bn ct ha hc, tnh bn nhit, tnh c, tnh khng nguyn, kh nng chuyn thnh gii c t).

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Chng 9

Cc sn phm chuyn ha
Mt trong nhng pht hin c ngha quan trng ca vi sinh vt hc cng nghip l vic nhn thc c rng vi sinh vt c th c s dng thc hin cc phn ng ho hc c bit vt ra ngoi kh nng ca ho hc h c. Qu trnh s dng vi sinh vt cho mc ch ny c tn l s chuyn ho sinh hc, n bao gm s sinh trng ca vi sinh vt trong nhng ni ln men ln theo sau l s b sung ho cht cn c chuyn ho ti mt thi im thch hp. Tip tc ln men thm mt thi gian na v sinh vt tc ng ln ho cht ri tch chit dch ln men, v cui cng sn phm mong mun c tinh khit. Mc d v ngnyn l chuyn ho sinh hc c th c s dng cho nhiu qu trnh khc nhau song trong thc t n ch c ng dng sn xut mt s hormone nht nh.

I. S chuyn ha cc steroid
Vic s dng vi sinh vt thc hin nhng s chuyn ho steroid c ngha rt ln trong cng nghip dc phm. Steroid hormones iu chnh nhng trng thi trao i cht khc nhau ng vt k c ngi. Mt trong nhng hormone , cortisone, c tc dng lm gim cn au c lin quan n bnh vim khp. Cc dn xut cortisone khc lm du cc triu chng lin quan n cc bnh d ng hoc vim. Nhiu loi steroid hormones iu chnh hot ng gii tnh ngi trong mt s c sn xut thnh dng thuc ung trnh th thai. Cc c tnh sinh l ca mt steroid ph thuc vo bn cht v v tr chnh xc ca cc thnh phn ho hc nm trn cu trc vng ca steroid gc. Vo u nhng nm 1930, Kendall Trng i hc Tng hp Basel tch c cortisone, mt steroid do tuyn thng thn tit ra. Khong mt thp k sau Hench ch ra rng vic ung cortisone c th lm du cn au cc bnh nhn b bnh vim khp. Nhu cu thc t ca hormone tr nn cp bch v cc phng php ho tng hp steroid c pht trin v th trng tim tng l rt ln. Tuy nhin, ho tng hp kh phc tp yu cu ti 37 bc trong nhiu bc xy ra di cc iu kin cc tr. Cortisone tng hp c theo con ng ny tr gi 200 la mt gam.

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Mt trong nhng iu phc tp ch yu gp trong ho tng hp cortisone l vic phi a mt nguyn t oxygen vo mt v tr trong cu trc steroid 4 vng c gi l v tr 11; y l bc quyt nh trong vic to nn hot tnh sinh l ca phn t. Vo nm 1952, Peterson v Murray thuc hng Upjohn pht hin ra rng nm mc mc trn bnh m Rhizopus arrhizus c kh nng hydroxyl ho progesterone, mt steroid khc, bng cch a mt nguyn t oxygen vo v tr 11. Progesterone l mt tin cht trong ho tng hp cortisone, v bng phng php hydroxyl ho nh vi sinh vt (trong cng nghip thng dng cc chng h hng vi R. arrhizus) vic tng hp c rt ngn t 37 xung 11 bc. Nh vy gi thnh gim xung cn 6 la mt gam. S hydroxyl ho progesterone mang li hiu qu kinh t do rt ngn c s tng hp ho hc. S ln men c th thc hin 30oC vi nc l dung mi v p sut ca kh quyn. Cc phn ng di cc iu kin ny r hn nhiu so vi cc phn ng din ra di cc iu kin cc tr v nhit v p sut v dung mi khng phi l nc nh trong ho tng hp cortisone. n nay c mt s qu trnh khc ng dng vi sinh vt tng hp cng nghip cc steroid. Nm Cunninghamella blakesleana cng c kh nng hydroxyl ho steroid cortesolon to thnh hydrocortisone nh vic gn oxygen vo v tr s 11. Nhng s chuyn ho nhn steroid khc do vi sinh vt thc hin bao gm s hydro ho, s loi hydro, s epoxygent ho, v s loi hoc thm cc chui bn (hnh 9.1). Cc steroid t c ngha thng mi l cc cocticosteroid nh cortisone, hydrocortisone (hnh 9.2, hnh 9.3), prednison v dexametazon, kch t tnh c testosteron v hormone ng dc estraiol (hai loi sau dng cho cc loi thuc trnh th thai) v spironolacton (thuc li tiu). Nguyn liu dng cho tt c cc qu trnh trn l cc loi ru phc tp c tn l cc sterol. C hai ngun sterol thng thng : Sn xut du u tng li mt cht thi giu stigmasterol v sitosterol; r ca cy barbasco Mhic cha diosgenin. Ngoi ra, c th s dng ergosterin ly t nm men hoc cc steroid sn xut thun tu bng con ng tng hp. Nhiu vi sinh vt c kh nng thc hin cc phn ng chuyn ho steroid. Tuy nhin iu quan trng l chng c th tin hnh phn ng vi tc chuyn ho cao, hiu sut ln v khng to thnh sn phm ph hay khng. Do yu cu ny m s chng c th s dng cho cng nghip b

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thu hp li rt nhiu. hydroxyl ha ngi ta s dng x khun v nm (c bit l Fusarium v cc loi Curvularia). hydroxyl ho v tr 11-a ca progesterone, thay cho h si nm ngi ta dng bo t trn ca Aspergillus olivaceus. Vic hydro ho c tin hnh vi cc loi Saccharomyces, Streptomyces v Rhizopus. loi hydro ngi ta dng cc vi khun nh Corynebacterium v nm (Fusarium, Calonectria, Cylindrocarpon) cn ct vng c th dng Penicillium chrysogenum hay Pseudomonas testosteroni cha mt steroidizomerase c hiu. Trong mt qu trnh chuyn ho steroid in hnh, vi sinh vt trc ht c a vo mt mi trng thch hp khng cha steroid. Ngi ta thng s dng cc ni ln men nh (10-50m3). Thng vo cui pha sinh trng logarit th steroid mi c a vo vi nng 0,05-0,1%. V cc steroid tan yu trong nc nn ngi ta dng cc dung mi hu c ho tan c trong nc (nh methanol, propylenglycol, dimetylsulfoxygent) lm cht ho tan trung gian. Thi gian chuyn ho ko di t 6 n 48 gi, thng thng th qu trnh c kt thc sau 20 gi bng cch tch t bo v chit sn phm. Hiu sut t c l 60-95%. Cc sn phm ca phn ng nm bn ngoi t bo. Vic theo di phn tch s chuyn ho xc nh thi gian ln men cc thch gi vai tr rt quan trng, v nu vt ra ngoi thi gian s xy ra cc phn ng k tip khng mong mun. Nu khng ch tt th c th tin hnh hai phn ng chuyn ho mong mun k tip nhau trong cng mt ni ln men. Chng hn 6-fluo-21-acetoxygen-16-methyl-4pregnen-3,20-ion trc ht c hydroxyl ho v tr 11 nh Aspergillus ochraceus v sau cng trong mi trng y nh b sung Bacillus lentus m li c loi hydro v tr 1,2. Vo nm 1980 gi cortisone M ch cn 46 cent mt gam, r hn 400 ln so vi gi ban u. Vic tm c cc ng dng khc ca cc steroid (dng cho trnh th thai, bnh thiu hormone, cc bnh v da, vim v d ng) cng vi tnh hiu qu cao ca phng php sn xut to ra mt nhu cu cp thit i vi cc loi dc phm ny. Doanh thu 4 loi steroid chnh (cortisone, aldosteron, prednison v prednisolon) trn th trng th gii vo nm 1987 l 300 triu la.

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Hnh 9.1: Sn xut steroid bng cc bin php ho hc kt hp vi s chuyn ho vi sinh vt

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Acid Deoxycolic

Cortisone

Hnh 9.2: Coctisone c tng hp t deoxycolic acide

Hnh 9.3: S sn xut 11 -hydroxyprogesterone v hydrocortisone

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II. S to thnh phenyl-axetylcacbinol, tin cht ca epherin

Epherin l mt alcaloit c trong cy Ephedra vulgaris. Ging nh arenalin, n c tc dng lm tng huyt p v c dng lm thuc iu tr cc bnh suy nhc tun hon, hen, vim ph qun v.v. .. Cu to ho hc ca n nh sau :

Tch cht ny t thc vt l mt cng vic tn km. Ho tng hp n cng kh thc hin bi v do hai nguyn t C khng i xng ca phn t m xut hin bn ng phn lp th trong ch c dng L l c dc tnh. Nh Saccharomyces cerevisiae m benzaldehyde c th c chuyn ho thnh phenyl-axetylcacbinol, tin cht ca epherin, vi hiu sut 50-60% khi c b sung vo mi trng cha mt nng t bo l 0,8- 1% . Trong sn phm chuyn ho ny, nhm hydroxyl nm cng v tr nh trong L-ephedrin. Trong phn ng ny, cc t bo nm men gn "acetalehyde hot ng" c to thnh trong s ng phn vo benzalehyde va b sung vo mi trng (phn ng cacboligase):

Benzaldehyde

''Acetaldehyde hot ng'' acetylcacbinol

L-phenyl-

Sau phenyl-acetylcacbinol c chuyn thnh L-epherin bng con ng ho hc nh mt s kt hp hydro amin ho. III. Sn phm t vi khun acetic Trong s oxygen ho ethanol thnh acid acetic, chuyn ho thc cht l mt s s dng c cht. y, NADPH2 xut hin c chuyn qua chui h hp thu nhn nng lng, song c cht khng b phn gii

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hon ton do vy qu trnh cng cn c gi l s oxygen ho khng hon ton. Loi chuyn ho ny c th tri qua nhiu bc. Cc i din ca vi khun acetic (Acetobacter, Gluconobacter) oxygen ho khng ch ethanol m c mt ph rng cc ru bc mt v ru bc hai cng nh cc polyol. Cc loi vi khun m sn phm ca chng c gi li c gi l cc loai oxygen ho thp (suboxygendant). Thuc v nhm ny c vi khun acetic dng trong cng nghip l Acetobacter suboxygendans. Cc loi ch tch lu acid acetic tm thi sau li oxygen ho tip c xp vo nhm oxygen ho cao (peroxygendant), chng hn Acetobacter peroxygendans, Acetobacter pasteurianum). Gia hai nhm ny c cc dng chuyn tip. 1. Sn xut dm n Cc phn ng c dng sn xut dm n nh Acetobacter suboxygendans l : CH3 CH2 OH
ase alcohol dehydrogen

CH3 CHO + 2H

Sau s hydrat ho axetaldehyde s din ra mt s loi hydro ln th hai :

Hydro c NADP nhn v qua cc xitocrom c chuyn n O2 l cht nhn in t cui cng. S to thnh acid acetic i hi cung cp oxygen mnh. Khi thng kh khng y c th xy ra s ho hai ca axetaldehyde thnh acid acetic v ethanol theo phng trnh sau y (phn ng Canizzaro) : Ethanol li i vo phn ng th nht v cui cng cng c oxygen ho thnh acid acetic. Ethanol hoc acid acetic khng th c dng lm ngun cacbon duy nht v ngi ta khng tm thy cc enzyme cn thit ca chu trnh glyoxylate Acetobacter xylinum. V vy sinh trng vi khun cn c b sung cc ngun C khc nh glucose.

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S cung cp oxygen c tnh cht quyt nh n k thut sn xut dm. Trc kia qu trnh c tin hnh bng phng php nui b mt nh mt lp vng ca Acetobacter xylinum (phng php Orlans), trong vi khun c c nh trn cc v bo g gi. V bo ng trong mt thng g hnh tr (generator) c thng kh t pha di v ti cc dung dch cha ru (v d cc loi ru vang km phm cht) t pha trn (hnh 9.4). Ngy nay cc phng php chm ngy cng c ngha hn. Hin ti ngi ta dng cc ni ln men c bit (acetator) c thng kh rt mnh trong dch dinh dng c thay th tng phn bng dch dinh dng b sung cng c thng kh y . Sau khi ethanol c chuyn ho thnh acid acetic, phn ln dch dinh dng trong ni c ly i v thay th vo l dch dinh dng mi. Nh c th t c mt phng thc ging nh nui cy lin tc. Qu trnh ln men c tin hnh nh cc chng chn lc ca Acetobacter suboxygendans trong mt dch dinh dng cha glucose vi 10-12% ethanol. Ethanol gn nh c chuyn ton b thnh acid acetic. Nng acid acetic cao nht t c l 13%. Qu trnh din ra 2830oC v ko di khong 48 gi. Trong phng php c in Orlans, s ln men ko di ti 5 tun l. Trong cc phng php hin i vi nng ru hoc acid acetic cao l 12%, th mt s ngng thng kh t 10 n 20 giy s lm cht ti mt phn ba s vi khun. nhng nng c cht thp hn, s ph thuc vo oxygen khng n ni kht khe ti nh vy. Vi sinh vt oxygen ha ethanol thnh acid acetic thng c gi l cc vi khun acetic, cc vi khun ny thc hin trao i cht pH mi trng thp, iu ny phn bit chng vi cc vi khun khc. Cc vi khun acid acetic l bn a hnh, t bo t hnh elip ti hnh que thng hoc hi cong 0,5-0,8 0,9-4,2 m, ng mt mnh, thnh cp hoc thnh chui c dng khng chuyn ng v dng chuyn ng vi tin mao cc hoc vng quanh c th. Chng l bn hiu kh bt buc, mt s to thnh sc t, mt s to thnh cellulose. Ngi u tin tm cch phn loi cc vi khun acid acetic l Hansen (1894). Ngy nay cc vi khun acid acetic mi phn lp c xp vo hai chi chnh, Acetobacter, Gluconobacter. Cc loi ca Acetobacter (trn 60) cha 5 c im: c mt catalase, oxygen ha ethanol qua acid acetic ti CO2 v H2O, oxygen ha lactate thnh cacbonate, oxygen ho glycerol

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thnh DHP v s sn sinh acid gluconic t glucose. Cc vi khun trong chi Acetobacter thng c chia thnh bn nhm : oxygen ho mnh, oxygen ho, oxygen ho trung bnh v oxygen ho yu.

Hnh 9.4: Thit b sn xut dm theo phng php c in

IV. Sn xut vitamin C ( acid L-ascocbic )

a s ng vt tng hp c ton b lng vitamin C cn thit cho nhu cu ca mnh v do vy vitamin ny c tm thy trong cc m ca chng (ch yu trong gan v thn vi nng 10-40 mg/100g). Tuy nhin, ngi v mt s ng vt c xng sng cng nh cn trng li ph thuc hon ton v ngun vitamin C t bn ngoi. Ch yu l rau (bp ci, spinat, c chua, 30-150 mg/100g) v qu (cam, chanh, 40-50mg/100g). cung cp cho con ngi lng vitamin C cn thit (45-70 mg/ngy).

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Mt s vi sinh vt (nm, nm men, to) sn sinh mt lng rt nh acid L-ascocbic cn cho cc qu trnh trao i cht ca chng. Cho n nay cha tm thy acid ascorbic vi khun, hnh nh chng khng cn acid ny. ng vt c v (tr bn linh trng v mt s khc) acid Lascorbic c tng hp t D-glucose trong C1 ca glucose tr thnh C6 ca acid ascorbic v ngc li. S tng hp din ra t D-glucose ti acid D-glucuronic v sau thnh lacton ca acid L-gulonic. S oxy ho sau ca gulonolacton v tr C2, c xc tc bng L-gulonolacton dehydrogenase, mt enzyme khng tm thy ngi, theo sau l s enol ha s cho acid L-ascorbic (hnh 9.5). thc vt, acid Lascorbic c to thnh t D-glucose hay D-galactose qua mt s con ng chuyn ha. Mt trong nhng con ng ny gi khng lm cho trt t ca chui cacbon b thay i, song cc sn phm trung gian ca con ng sinh tng hp th cn cha bit r. C th con ng ny cng ging con ng tng hp ng vt.

Hnh 9.5: Con ng sinh tng hp acid L-ascocbic

T trn 50 nm nay, cng nghip c th p ng nhu cu ngy cng tng v vitamin C ca con ngi nh phng php bn tng hp t glucose. Nhiu qu trnh ho hc v sinh ha k tip nhau tham gia vo qu trnh ny v tt c u qua mt sn phm trung gian l acid 2-keto-L-

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gulonic, t s thu c acid L-ascorbic nh con trng ho hc bng cch lacton ho v ng phn ho (hnh 9.6).

Acid-keto-Lgulonic

Methyl-2keto-Lgulonate Acid L-ascobic

Hnh 9.6: S chuyn ho ho hc acid 2-keto-L-gulonic thnh acid Lascocbic

Cc quy trnh chun b acid 2-keto-L-gulonic c th c xp thnh hai nhm: (1) nhm bt u bng s kh D-glucose v lm o ngc trt t cacbon, v (2) nhm bt u bng s oxygen ha D-glucose trong khng c s o ngc chui cacbon. 1.Cc quy trnh bt du bng s kh D-glucose
Quy trnh cng nghip ny c Reinstein v Gruessner ra t nm 1934 v n nay vn cn s dng. N bao gm 5 bc phn ng k tip nhau, tt c u t sn lng ti 90-95% (hnh 9.7)

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Hnh 9.7: S tng hp acid 2-keto-Lgulonic t D-glucose qua Dsocbitol v L-socboza

1. Kh D-glucose thnh D-sorbitol vi s c mt ca niken Raney, sau loi ti mc ti a niken ha tan bng cch x l dung dch sorbitol vi nha cation. 2. Oxygen ha D-sorbitol thnh L-sorbose nh vi sinh vt : c nhiu chng Acetobacter chu c nng niken ti 10-20 mg/l, thc hin nhanh chng phn ng ny trong cc mi trng cha nng sorbitol cao. Qu trnh ln men din ra 30oC trong mt mi trng cha 200 g/l sorbitol, 10 g/l cao ng, v 0,5 g/l CaCO3 vi mt chng Acetobacter suboxygendans hon tt trong 24 gi v cho khong 180g sorbose trong mt lit. Sau khi lc, loi ion v c c dch nui, sorbose c kt tinh (ti tinh khit 99%) 3. To thnh diacetone-sorbose bo v cc nhm khng tham gia vo giai on sau. 4. Oxygen ha ha hc diacetone-sorbose thnh acid diacetone-2keto-L-gulonic.

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5. Gii phng acid diacetone-2-keto-L-gulonic nh thu phn. Gia nhng nm 1960 v 1975 nhiu c s nghin cu ca M v Nht tm cch gim s bc trong quy trnh Reinstein bng cch oxygen ha trc tip D-sorbitol hay L-sorbose nh mt k thut vi sinh vt thnh acid 2-keto-L-gulonic khi s dng cc chng Acetobacter v Pseudomonas. Tt c nhng nghin cu ny u khng cho kt qu kh quan, nng sut t khi vt qu 5 g/l trong cc iu kin tt nht vi mt sn lng vo khng 10% so vi sn phm ban u. Vo nm 1981, cc nh nghin cu Trung Quc cho bit sn xut c 37 g/l acid 2-ketoL-gulonic t 100 g sorbose khi s dng mt chng Gluconobacter oxygendans. y l mt s ci thin ng k so vi cc kt qu trc . 2.Cc quy trnh bt u bng s oxygen ha D-glucose Hai con ng c nghin cu : (1) ci gi l con ng acid Lionic bt u bng s oxygen ha glucose v tr cacbon s 5 ; (2) con ng acid 2,5-diketo-D-gluconic. a) Con ng acid L-idonic Con ng ny gm ba bc k tip nhau : (1) oxygen ho sinh ha hc D-glucose thnh acid 5-keto-D-gluconic qua acid D-gluconic ; (2) kh ha hc (hoc sinh ha hc) acid 5-keto-D-gluconic thnh acid Lidonic ; (3) oxygen ha sinh ha hc acid L-ionic thnh acid 2-keto-Lgulonic (hnh 9.8). Phn ng u tin d dng c thc hin bi Acetobacter suboxygendans. Chng ATCC 621 chuyn ha 100 g/l glucose trong 33 gi vi mt sn lng l 90%. Mt s phng php c ra nhm kh acid 5-ketoD-gluconic. Trong thc t, ch c phng php ho hc do Grey xut (1947) l c gi tri. Trong phng php ny, s kh t c nh s hiro ho c xc tc di p lc v khng may l dn n vic to thnh hai ng phn, acid L-idonic v acid D-gluconic theo nhng t l tng i m trong cc iu kin tt nht th l 70 v 30%. Hai phng php loi acid D-gluconic va to thnh c ch : (1) nh ln men vi Acetobacter suboxygendans, n c th b oxygen ha tr li thnh acid 5keto-o-gluconic, cht ny sau s c quay vng, hay (2) hn hp c th c ln men vi mt vi sinh vt (Pseudomonas fluorescens, P. aeruginosa, Acetobacter melanogenus), bn ny s dng acid D-gluconic sinh trng v phn gii n hon ton cng mt lc vi vic chuyn ho acid L-idonic thnh acid 2-keto-L-gulonic. Bng phng ny ngi ta

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thu c nhng kt qu tt : 67 g/l acid 2-keto-L-gulonic c to ra t 100 g/l hn hp cha 70% acid L-idonic, song cn c nhng vn k thut cha c chp nhn trn quy m cng nghip.

Hnh 9.8. S chuyn ho D-glucose thnh acid 2-keto-L-gulonic qua acid L-inonic

b) Con ng acid 2,5-diketo-D-gluconic Con ng ny , ch yu c nghin cu t nm 1970, gm hai bc sinh ho hc : (1) oxygen ha D-glucose thnh acid 2,5-diketo-Dgluconic qua acid D-gluconic v acid 2-keto-D-gluconic ; (2) kh acid 2,5-diketo-D-gluconic thnh acid 2-keto-L-gulonic (hnh 9.9). Bc mt khng gp tr ngi g nghim trng. Ngay t nm 1974 cc nh nghin cu Nht Bn cho bit v mt nng sut 220 g/l 2,5diketo-D-gluconat canxi t c t 200 g/l glucose sau 3 ngy ln men nh Acetobacter fragi. Vo nm 1982 cc nh nghin c Shionogi s dng : (1) cho bc mt, mt th t bin ca Erwinia c kh nng sn xut 328 g/l 2,5-diketo-D-gluconat canxi trong 26 gi; (2) trong bc hai, h s dng mt chng t bin ca Corynebacterium to ra 106 g/l 2keto-L-gluconat canxi trong 66 gi.

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D-Glucose

Acid D-gluconic Acid 2-keto- Acid 2,5-diketo- Acid 2-ketoD-gluconic D-gluconic L-gluconic Hnh 9.9: S chuyn ho D-glucose thnh acid 2keto-L-gulonic qua acid 2,5diketo-D-gluconic

Sau bc ln men u tin, mi trng nui Erwinia c kh trng bng cch x l vi 0,25 g/l oecylsulfate natri 28oC trong 6 gi. Sau khi lm lnh, ngi ta b sung glucose (0,2 kg/kg acid 2,5-diketo-Dgluconic) dng lm cht cho hiro trong phn ng kh, t sinh ra acid 2-keto-L-gulonic. Cht ny c a vo mi trng nui Corynebacterium vo cui pha sinh trng. Sn lng chuyn ha tnh theo phn t gam s l 94% v bc hai l 92% so vi 2,5-diketo-Dgluconat canxi s dng. Xt ton b, sn lng phn t gam ca acid 2keto-L-gulonic thu c vo cui bc ln men th hai so vi lng glucose s dng trong bc ln men th nht l 86%. y l quy trnh hiu qu nht i vi s sn xut acid 2-keto-Lgulonic k t khi quy trnh Reinstein ra i vo nm 1934 v n loi b tt c cc bc ha hc. S lng cc bc c gim t nm xung ch cn hai. Vic tch acid 2-keto-L-gulonic vo cui bc ln men th hai t c vi mt sn lng l 90% bng cch x l dch nui vi carbonate natri v bng cch c c ti lc 2-keto-L-gluconat natri kt tinh. c nhng cng b v kt qn nghin cu nhm thu nhn acid 2keto-L-gulonic t glucose ch bng mt ln ln men. Cc chuyn gia ca hng Genetech vo nm 1985 t c iu ny nh vic nhn ln chng Erwinia herbicola ATCC 2 1988, chng ny thc hin tt bc mt trong quy trnh Shionogi, gen m ha cho vic tng hp acid 2,5 diketogluconic reductase tch ra t Corynebacterium sp. ATCC 31090. Gen ny biu hin tt trong t bo ch song quy trnh cn lu mi c th s dng cho sn xut cng nghip. Nng glucose cc i c th ti t hp ny chp nhn cn mc tng i thp v nng sut cn rt ngho nn (0,6 g/l acid 2-keto-L-

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gluconic c to ra sau 57 gi trong mt mi trng cha 20 g/l glucose). V rng s sinh tng hp acid L-ascorbic ng vt c bit r, ngi ta hy vng mt ngy no s c th a cc gen m cho cc enzyme c lin quan vo mt vi sinh vt thch hp cui cng c th thu nhn vitamin C trc tip t glucose.

V. Sn xut destran
a s cc polysaccharide ngoi bo t vi sinh vt u l sn phm ca s chuyn ha ni bo c cht thnh cc sn phm trung gian, v cui cng thnh polime. Destran khc cc polysaccharide ny, c cht khng thm nhp vo t bo vi sinh vt m n c chuyn ha bn ngoi t bo thnh -D-glucan phn nhnh, tc l destran. Ch c saccharose c dng lm c cht cho phn ng ny. Nh vy, polysaccharide c th c sn xut bi cc t bo nguyn vn trong mi trng nui hoc c th c to ra t cc ch phm phi t bo cha phc h enzyme destransaccharase (-1,6-glucan : D-fructose 2glucosyltransferase). Enzyme ny gii phng fructose v chuyn gc glucose ln mt phn t cht nhn cng c lin kt vi enzyme : (1,6--D-glucosyl)n + saccharose (1,6--D-glucosyl)n+1 + fructose Nng lng t do ca ln kt glucoside trong phn t disaccaride nm vo khong 23 kJ trong khi nng lng t do ca lin kt glucoside bn trong destran thp hn mt cht (12-17 kJ). Do vy phn ng din ra theo chiu t tri sang phi i km vi mt s gim nng lng t do. Trong qu trnh polime ha chui destran ang di ra vn lin kt cht ch vi enzyme, mc polime ho tng cho n khi phn t cht nhn (trong trng hp n gin nht l mt phn t c cht) gii phng chui polime khi enzyme. Cc thnh vin thp nht ca cc oligosaccharide khng phi l cc phn t cht nhn c hiu qu, i lc i vi enzyme tng dn theo di chui. Tuy nhin, s hn ch v khuch tn cng tng theo trng lng phn t. Bi vy, di cc iu kin thng thng, cc destran c trng lng phn t rt cao s trc to thnh, c bit cc nng c cht thp. sn xut cc destran c trng lng phn t trung bnh, cn phi tin hnh thu phn cc sn phm c trng lng phn t cao (bng acid

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hoc bng enzyme) v phi phn on sn phm di nhng iu kin kim sot rt cn thn. Trong khi , cc polime trng lng phn t thp v ng nht c th c ly ra dng lm nguyn liu ban u cho mt phn ng khc sn xut cc destran c mc trng hp mong mun. Cc nng saccharose cao v c th c maltose khi c dng lm nguyn liu ban u s to nn cc iu kin thun li tng hp cc destran mi trng lng phn t thp v ng nht kiu . Destran c sn xut bi hng lot loi vi khun, nh Streptobacterium destranicum, Streptococcus mutans v cc loi khc. Streptococcus mutans gi mt vai tr quan trng trong bnh su rng. Sn phm ph thuc c vo chng ln vo cc iu kin s dnng cho sinh trng v tng hp polime. mt s chng, destransaccharase nm dng ha tan trong khi cc chng khc, n lin kt mt phn hay hu nh hon ton vi t bo. Sn xut destran cng nghip dng Leuconostoc mesenteroides to ra mt polime cha khong 95% lin kt -1,6 (phn cn li l lin kt -1,3) v mt trng lng phn t l 4-5 107 dalton. Cc destran c s dng trong ngnh dc lm cht dn mu (cht thay th huyt tng). Tuy nhin nhng destran ny phi c trng lng phn t thp nm trong mt phm vi rt nh (75000 25000). Cc sn phm vi trng lng phn t thp hn c loi khi h tun hon qu nhanh nn khng c gi tr iu tr y trong khi cc sn phm c trng lng phn t qu cao li tham gia vo s ngng kt mu. Cc dung dch cha 6% loi destran ny c gi tr v nht v tp tnh keo-thm thu rt ging vi huyt tng ca ngi. V cc destran khng b phn gii bi cc amylase trong c th ngi nn kh nng ph hoi gan thp hn so vi cc cht thay th huyt tng khc. Cc destran c m c th hp th rt tt, do vy c th c s dng cho nhiu loi dc phm, chng hn st trong khi iu tr bnh thiu mu. Gn y, ngi ta cho rng destran c th c s dng to nn mt lp a nc trn b mt cc vt bng rng v hp th cc cht tit t cc vt thng. Trong vic sn xut cc destran, vic duy tr cung cp oxygen gi mt vai tr quan trng c bit v trong qu trnh ln men dung dch tr nn rt nht. Hin nay ngi ta cha c kh nng tnh ton mi tng quan gia nng lng khuy trn a vo v tc chuyn ng ca oxygen trong

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dung dch vi tp tnh phi Newton ca dch lng nn trong a s trng hp ngi k s sinh hc ch cn bit da vo kinh nghim ca bn thn mnh. Mi trng ln men cha cc mui v c, 2% cao ng v saccharose. St v mangan l nhng nguyn t vt rt quan trng. Dung dch c kh trng bng sc nng, c chuyn sang b ln men v cy ging sau khi lm lnh. Ln men c tin hnh 25oC, trong qu trnh ny, ch yu do s to thnh acid lactic, gi tr pH gim t 6,5-7,0 xung cn 4,0. Dung dch ln men c khuy lin tc v thng kh. Khi destran c to thnh, dung dch tr nn m c cho n khi qu trnh kt thc sau 3 hay 4 ngy, mt khi dng keo s c to thnh. Dch ln men cha ch yu l destran, fructose t do, acid lactic, etanol v cc vi sinh vt. Destran sau s c kt ta bng methanol. Dch trong c cht li v methanol c thu hi bng phng php ct. Nu ln men dn n vic to thnh cc destran c trng lng phn t cao th chng s c thu phn bng acid chlohydric 100-105oC. S hon tt thu phn c xc nh bi nht. Sau mt giai on phn ng nht nh, phn ng lc u c lm chm li bng cch lm lnh sau dng li hon ton bng cch b sung hydroxygenate natri. Dung dch c trn vi cc yu t hp ph v lc qua kizengua. Cui cng destran c kt ta phn on vi methanol, cc phn on mong mun s c ti ha tan, lc, c c v sy phun. V rng vic thu phn bng acid kh kim sot v vic phn on thng khng thu c cc phn c thnh phn mong mun, nn ngi ta tm cch tng hp trc tip cc destran c trng lng phn t thp. lm iu ny cn c kin thc chc chn v ng hc ca phn ng. Trng lng phn t b nh hng bi : a) nng ca saccharose, b) cht nhn glucose, c) nhit phn ng. Chng hn khi vng mt mt cht nhn, mt dung dch saccharose 10%, s thu c mt sn phm vi mt trng lng phn t ln hn 108 dalton. nng 70% saccharose, cc phn t destran ch yu c trng lng phn t thp s c to thnh. Nu cc destran trng lng phn t thp c a vo lm mi th enzyme s c nhiu im khi u v cc polime tng i ng nht s c to thnh. iu quan trng l cc t bo vi khun khng cha cc vt ca cc destran c trng lng phn t cao. V vy nguyn liu cy phi c ra vi ln bng dung dch mui loi cc destran bm vo.

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Nh vy mt hn hp 2% (w/v) mt cht nhn c trng lng phn t thp (1000-25000) vi mt nng ng l 10% 15oC v pH 5,0 s cho mt sn phm m 50% c trng lng phn t trong phm vi mong mun gia 50000 v 100000 dalton. Cc destran ny c s dng trong bnh vin v khng b phn gii bng thy phn. Theo tnh ton sn lng destran vo nm 1980 Ty u l 1000 tn, v trn th gii l 200.000 tn. Trong khi hu ht cc polysaccharide ngoi bo c s dng di dng khng b bin i v mt ha hc th destran dng cho ngnh dc v nhiu mc ch khc li c chuyn ha thnh cc phn on c trng lng phn t tng i thp gia 40000 v 70000 dalton nh cc quy trnh thu phn thch hp (thu phn bng acid long). Th trng ch yu ca cc dn xut destran di dng cc ch phm lin kt cho cha cu trc khng gian ba chiu m cc nhm chc nng c gn vo nh cc lin kt ete vi cc gc glucose chnh l cc phng th nghim sinh ha hc. sn xut cc loi gel cho mc ch sinh ha, destran c trng lng phn t tng i cao c dng tng hp cc sn phm c tnh thu hi nc cao. Phn ng vi destran trong dung dch kim dng epichlorohydrin thu nhn gel, gel ny sau c nghin v trung ha. V phn ng gia epichlorohydrin v polysaccharide l phn ng ta nhit, cn ch nhit ta ra c loi b khi h thng. iu ny l c bit quan trng v gel c to thnh trong phn ng c tnh dn nhit thp. V cc gc ng kh tn cng ca cc phn t destran c tnh phn ng capo v d b phn gii trong phn ng polime nn chng b kh thnh sorbitol v borohydride trong dung dch kim trc khi b sung epichlorohydrin. Mc du cc gel lin kt cho thuc loi sephaex bn thn chng cc k c ch trong vic phn on cc nguyn liu hot ng v mt sinh hc, song s chuyn ha chng thnh cc dn xut ete s lm tng gi tr tim tng ca chng. Cc ete carboxymethyl hay dimethylaminoethyl l nhng sn phm kiu . Nhng hp cht ny khc bit nhiu vi cc destran lin kt cho v tp tnh trng ca chng trong dch lng. Cc sn phm c lin kt cho khc nhau s cho ra cc cht hp ph c l khc nhau v gii hn phn on khc nhau. Vic a cc nhm ha hc khc vo cc phn t s cho ra cc cht hp ph mang c im a lipit. Chng c th c dng phn on

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cc lipit, v.v. v c th dng trong dung mi lng cng nh trong cc dung mi hu c phn cc. Mt s destran khc c dng lm cc cht mang trong cc quy trnh nui cy t bo. Di dng ny (c tn thng mi do hng dc phm AB ca Thy in t ra l Sephaex), destran c ng dng rt rng ri tch v thun khit cc phn t sinh hc khc nhau v in tch, v kch thc phn t v.v.. Protein khng b bin tnh bi mng li polime a nc v s hp ph khng c hiu l rt thp.

Cu hi n tp chng 9
1. Da vo s sau y, hy nu cc nguyn tc ca s chuyn ha steroid:

2. Nu cc bc chnh trong qu trnh sn xut mt steroid. 3. Phn bit cc vi khun acetic peroxydant v suboxydant

hormone

4. Nu s khc bit trong cc phng php sn xut dm (Orleans, Spring, phng php chm). 5. Nu ng dng v nguyn tc ha hc ca s to thnh cc destran.

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6. Mt trong nhng iu phc tp ch yu gp trong ha tng hp cortisone l................ y l bc quyt nh to nn.............Trong vi sinh vt hc cng nghip, c th dng cc vi sinh vt sau y thc hin bc phn ng ny: ........... 7.Destran c sn xut cng nghip nh.......................Trong mi trng cha saccharose, vi khun ny tit ra enzyme.......................c chc nng .........................

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Chng 10

X l nc thi bng bin php sinh hc

I. Vi sinh vt hc ca cc ngun nc ung
Nhng v dch t mi y Nam M lm cht hng ngn ngi, v mt v dch mi bng n Wisconsin do Cryptosporidium gy ra nh hng ti 370.000 ngi u c nguyn nhn t cc ngun nc ung b nhim bn. Trong nhiu khu dn c trn th gii, s thiu nc sch dn n hng t ca tiu chy v cp i khong hai triu tr em mi nm. Ch tnh ring M hng nm cng c gn mt triu ngi mc cc bnh c nguyn do t nc bn. Nc sch (nc ung c) c nh ngha l nc khng cha cc tc nhn gy bnh, cc c t ho tan, c c, mi, mu v v kh chu. R rng l, c c nc sch khng phi l iu d dng v y, vi sinh vt va l nguyn nhn va l gii php ca vn . Do thng xuyn tip xc vi khng kh, t cng nh cc dng chy ca cc h thng thot, nc b mt lun lun thu nhn cc vi sinh vt hoi sinh c hi. Thm vo , n cng tip nhn c cc vi sinh vt gy bnh. Cc vi sinh vt gy bnh ni bt nht ca nc thng gp trong thi gian gn y l : cc ng vt nguyn sinh Giardia v Cryptosporidium, cc vi khun Campylobacter, Salmonella, Shigella, Vibrio, v Mycobacterium, cc virut vim gan A v Norwalk. Mt s trong cc tc nhn ny (c bit l cc ng vt nguyn sinh sinh bo nang) c th tn ti trong cc thy vc t nhin trong mt thi gian di ngoi c th con ngi, trong khi mt s khc ch tn ti mt cch tm thi v nhanh chng bin mt. S c mt ca vi sinh vt trong nc ung phi lin tc c gim st m bo rng nc khng cha cc tc nhn ly nhim. V rng vic xc nh mt s tc nhn gy bnh trong nc thng kh kh khn v tn thi gian, cho nn cch hay dng nht xc nh sch ca nc l pht hin s nhim bn bi phn. Mc nhim phn cao c ngha l nc cha nhiu tc nhn gy bnh v do vy khng an ton. Do vy, cc ging, b cha nc, v cc ngun nc khc phi c theo di v s c mt ca cc vi khun ch th. Cc vi sinh vt vn khng gy bnh ny l nhng k ng c trong ng rut ca chim v ng vt c v, chng d dng pht hin c nh cc phng php thng dng vn dng trong phng th nghim thng thng.

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Cc vi khun ng rut c ch nht trong cc bin php gim st s nhim ca nc bi vi sinh vt l cc coliform v cc lin cu khun ng rut, chng tn ti trong nc nhng khng sinh sn c . Pht hin c chng vi s lng cao cng c ngha l nc mi b nhim phn hoc b nhim phn mc cao. Cc tiu chun ca C quan Bo v mi trng (EPA) v v sinh ca nc da ch yu vo hm lng cc coliform, y l cc vi khun gram m, ln men ng lactose, sinh kh nh Escherichia coli, Enterobacter v Citrobacter. S nhim phn ca nc bin thng d dn n cc bnh ng rut d dy do cc cu khun gram dng gy ra, m trc ht l cc Enterococcus. i khi, cc th thc khun ca coliform v cc reovirut (virut Norwalk) cng c dng lm cc vi sinh vt ch th v nhim bn bi phn, song s pht hin chng kh hn v i hi nhiu vn k thut hn. 1. nh gi chung v cht lng nc Trong k thut xc nh hm lng vi khun tng s, mt mu nc nh c cy ln b mt mt mi trng rn. Con s khun lc ch ni ln qun th vi khun sng st tng s c mt m khng cho php phn bit c cc coliform vi cc loi khc. Thng tin ny c bit c ch trong vic nh gi hiu qu ca cc bc lm sch nc khc nhau. Mt yu t ch th khc i vi cht lng nc l nng oxy ha tan. N ch ra rng nc cha nng cht hu c v vi khun cao s c hm lng oxy thp v oxy b tiu th cho h hp hiu kh. m s lng cc coliform C hai phng php tiu chun dng pht hin v xc nh s lng cc coliform : xc nh hm lng thng gp nht (MPN = most probable number) v phng php xc nh qua mng lc. Trong k thut MPN, coliform c xc nh bng mt lot cc php th : php th s b, php th khng nh v php th hon thin (hnh 10.1). Php th s b s dng 3 dy ng nghim, mi dy cha nhng nng canh thang lactose v lauryl khc nhau. Mt dy gm 5 ng nghim trong cha cc ng Durham t ngc thu kh sinh ra trong qu trnh ln men. Ba dy ni trn ln lt c cy 10, 1 v 0,1 ml mu nc. Sau khi gi 24 gi, cc ng c nh gi theo s sinh kh. Kt qu dng tnh v sinh kh s l bng chng s b v s c mt ca cc coliform. S ng dng tnh s c m v c dng tnh s lng thng gp ca coliform da theo

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mt bng thng k. Php th khng nh c tin hnh bng cch cy mu dng tnh vo mt ng nghim khc. Php th s c hon thin nh phn lp cc loi coliform trn cc mi trng chn lc v phn ha, nhum Gram v khng nh li vic sinh kh.

Hnh 10.1: Quy trnh xc nh hm lng coliform tng s (MPN) trong mt mu nc.

K thut MPN c mt s nhc im. Trc ht n cn vi ngy thc hin. Th hai, n khng phn bit c cc coliform thng gp trong t v nc (Enterobacter) vi cc coliform thuc phn tht s ch yu sng trong ng rut ca cc ng vt (Escherichia). xc nh coliform thuc phn i hi mt php th c hiu hn chng hn php lc qua mng. Phng php lc qua mng i hi t bc v t mi trng hn, r hn, gn nh hn, v c th c tin hnh vi mt lng nc ln hn.

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Phng php ny thch hp xc nh cc dch lng long, nh nc ung, cha t cht ht, v khng thch hp lm i vi nc cha vi sinh vt sinh trng mnh hoc cha cc cht c ch kim loi c th lin kt vi mng lc. Sau khi lc, mng lc c t vo mt a Petri nh cha mi trng chn lc. Sau khi , cc coliform thuc phn c th c m v thng thng c xc nh cc c um khc bit trn cc mi trng ny. Thng th i vi cc k thut xc nh nhanh c tnh c hiu cao ngi ta dng cc c cht, m khi c mt cc enzyme phn gii lactose cha trong cc coliform, s gii phng cc cht mu vo mi trng. Khi mt php th cho kt qu m tnh v coliform th nc c coi l thch hp i vi ngi tiu dng. Tuy nhin, ngay c nhng nng coliform thp cng c th c chp nhn trong mt s hon cnh. Chng hn, nc thnh ph c th cha ti a 4 coliform trong 100 ml, cc ging t nhn c php cha nhiu hn. i vi nc ung th khng cho php cha bt k mt nng coliform, cu khun ng rut, virut hay ng vt nguyn sinh no. Cc thu vc dng nui c hay lm b bi c cho php cha t 70 n 200 coliform trong 100 ml. Nu s coliform cc b nc dng cho gii tr t ti 1000 con trn 1 ml th cc c quan y t thng cm s dng. X l ngun nc Hu ht cc ngun nc ung u c ly v t sng, h cha nc, ging. Ch nhng vng ni cao hay vng xa, km pht trin th cc ngun nc ny mi c s dng trc tip. Ti a s cc thnh ph, nc phi c x l trc khi cung cp cho ngi tiu dng. Cc ngun nc nh ging su, thng tng i sch v khng cha cc cht nhim bn, i hi x l t hn cc ngun nc b mt b nhim nhiu cht thi. Qu trnh x l nc gm nhiu bc. Trc ht nc c gi trong cc b ln va l ni d tr va l b sa lng. Vic a nc vo cc b ny phi c kim tra nghim ngt trnh b nhim bn bi xc ng vt, cht thi v nc thi. Ngoi ra, s sinh trng qu mc ca vi khun lam v to s lm cht lng nc xu i, cn c phng nga bng sulfate ng (0,3 ppm). Qu trnh sa lng loi bt cc ht ln cng c thc y trong giai on d tr. Tip theo, nc c bm vo cc h hoc b, s tip tc xy ra s sa lng , s thng kh v s lc. Nc trc ht c lc qua m ct hoc t bt cha nhiu to ct loi vi khun, virus v cc ng vt nguyn sinh, ri qua than hot tnh loi cc cht bn hu c khng mong mun. Nc thu thp t cc h thng lc ny c a v cc b d tr qua cc ng

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dn. Bc cui cng trong x l nc l ty trng ha hc bng cch bm bng kh clo qua cc b cho n khi t c nng 1-2 ppm, song mt s c s x l nc cc thnh ph cng s dng cloramin cho mc ch ny. M, ngi ta cng s dng c ozon hoc peoxyde cho giai on ty trng, song phng php ny i hi gi thnh cao v khng gi c hiu qu khng khun cho mt thi gian di. Cht lng cui cng ca nc c th khc nhau, song hu ht nc my u hi c mi v v do nh hng ca giai on ty trng.

II. X l nc thi
1. c tnh ca nc thi Nc thi, hay nc cng, l ngun nc s dng ca mt cng ng v bao gm : 1. Cc cht thi sinh hot ha trong nc, bao gm phn ngi v cc loi nc ra i t cc cng rnh ca cc cn h v ca c thnh ph vo h thng cng. 2. Cc cht thi cng nghip ha trong nc nh acid, du, m, cht hu c c ngun gc ng thc vt do cc nh my thi ra. 3. Nc ngm, nc b mt v nc kh quyn thm nhp vo h thng nc thi. c tnh l, ha hc ca nc thi Nc thi cha khong 99,9% nc. Lng cht rn l lng trong nc thi nh n mc ch c tnh bng phn triu (ppm); hm lng cht rn ca nc thi dao ng t vi ppm n 100 ppm. Cc thnh phn ha hc, mc d c mt nng thp song c ngha cc k quan trng v thay i v kiu loi v s lng tu theo cng ng dn c cng nh theo thi gian trong ngy. Cc cht v c c trong nc my thng xut hin trong nc thi. Cc hp cht hu c thng c b sung vo nc thi qua phn ngi v cc cht thi sinh hot khc. Cc cht thi cng nghip b sung cho nc thi c cc hp cht v c ln hu c. Chng hn, cc l git m, cc nh my ng, xng bt giy, xng sn xut cc sn phm sa b sung cc cht hu c, cng nghip ha hc v ch bin kim loi b sung cc acid, mui kim loi v cc cht thi kim loi v c khc. K ngh hin i ang lm thay i c tnh ca nc thi. Bao gi dnh cho rc thi sinh hot lm tng hm lng hu c tng s trong nc thi. Vic cc cht ty ra thay th phn ln cc loi x phng gy nhng

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nh hng c hi cho qun th vi sinh vt cn thit cho vic x l hiu qu nc thi . c tnh vi sinh vt hc Nm, ng vt nguyn sinh, to, vi khun v virut u c mt trong nc thi. Nc thi khng x l c th cha ti hng triu vi khun trong mt mililit, bao gm cc coliform, cc Streptococcus, cc trc khun k kh sinh bo t, nhm Proteus v cc loi khc bt ngun t ng rut ca ngi . Cc ngun b sung vi sinh vt khc l nc ngm, nc b mt v nc kh quyn cng nh cc cht thi cng nghip. Ngoi ra, tnh hiu qu ca mt qu trnh x l nc thi cn ph thuc vo nhng s bin i sinh ha hc do vi sinh vt tin hnh. Typ sinh l ca cc vi sinh vt chim u th tham gia vo cc qu trnh chuyn ha ny c th thay i tu theo tng giai on ca qu trnh x l nc thi. Cc iu kin c th dao ng t hiu kh cao n k kh nghim ngt. Nhu cu oxy sinh ha Nhu cu oxy sinh ha (biochemical oxygen demand - BOD) l lng oxy ha tan m vi sinh vt i hi cho qu trnh phn gii hiu kh cc cht hu c c mt trong nc thi. Mt trong nhng nguyn nhn u tin v sao phi x l nc thi trc khi a n tr li cc thy vc t nhin l lm gim yu cu v cung cp oxy ha tan cho phn nc nhn c. Mc BOD l mt ch s ni ln lng cht hu c c trong nc thi; cng nhiu cbt hu c d oxy ha c mt, BOD cng cao. "Nng " ca nc thi c biu hin bng mc BOD. Gi tr BOD cao c ngha l cht hu c c mt hm lng cao trong khi BOD thp c ngha l cht hu c d b oxy ha c mt vi hm lng thp. S sng ca bt k mt thy vc no cng ph thuc rt nhiu vo kh nng ca n i vi vic duy tr mt lng oxy ha tan nht nh cn thit duy tr s sng ca cc thy sinh vt trong thy vc . Chng hn, thiu oxy c s b ngt th v cc sinh vt nc thng thng s b hy hoi. Cc qu trnh x l nc thi Vic a nc thi khng c x l y vo sng h c th dn n mt hoc nhiu hu qu khng mong mun sau y: 1. Tng kh nng lan truyn ca cc vi sinh vt gy bnh. 2. Tng mi him ha khi s dng cc thy vc t nhin lm ngun cung cp nc ung.

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3. Lm cho cc loi trai s b nhim bn gy hu qu khng an ton khi dng lm thc n cho ngi. 4. Gy tn tht ln trong qun th chim nc do ngun thc n ca chng b nhim. 5. Gy nguy him cho ngi bi v lm gim gi tr ca nhng thy vc dnh cho cc mc ch gii tr. 6. Lm cn kit ngun oxy ca nc do s c mt ca cc cht hu c khng bn trong nc thi, do vy hy hoi s sng trong nc. 7. To ra cc tnh trng khng mong mun nh c mi kh chu hoc tch lu cc cht cn b do vy lm gim gi tr ti sn v tnh nng gii tr. C nhiu phng php x l nc thi, chng c chia thnh hai loi, mt loi p dng cho tng h gia nh hoc cc n v ring l v mt loi cho c cng ng hoc thnh ph. Thit b x l cho cc n v nm c lp Vic x l. nc thi i ra t cc h gia nh hoc cc n v nm c lp nh khch sn hoc trung tm thng mi c th c thc hin nh cc loi b phn gii k kh hay hiu kh. B t hoi l loi b phn gii k kh thng gp nht dng x l mt lng nc thi hn ch. B t hoi gii quyt hai mc tiu : sa lng cc nguyn liu rn v phn gii sinh hc cc cht rn ny. Nguyn liu s c tch lu phn y ca b c gi l bn. Khi nc thi i vo b, s sa lng xy ra phn bn di, cho php loi i mt dch lng cha t cht rn l lng (suspended solid - S.S.) hn. Phn cht rn sa lng s c tip tc phn gii bi cc vi khun k kh, cc sn phm cui cng s l cc hp cht hu c, c BOD cao v c mi. Dch i ra t b t hoi s c dn vo lng t pha di lp b mt, s din ra s phn gii tip tc nh vi sinh vt m ch yu l s oxy ha hiu kh cc cht hu c c trong dch lng. Kiu x l ny khng m bo c th loi tr ton b cc tc nhn gy bnh. Bi th, dch thi i ra t cc b t hoi khng c php dn thng vo cc ngun nc dnh cho vic cung cp nc ung. Cng c cc h thng x l hiu kh nc thi dnh cho cc n v dn c nh. Cc h thng ny bao gm cc b v thit b nhm lm gim kch thc cc cht rn np vo thnh cc ht c nh, mt b thng kh v mt b sa lng. Oxy c bm vo b thng kh cho php xy ra s oxy ha lin tuc v s phn gii hiu kh cc cht rn c trong nc thi. Cc h thng ny

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c bit cn thit cho nhng a phng nm trn cc vng t c tnh thm khng cao (nh t t) hoc cc loi t cha nhiu . Thit b x l nc thi th Cc thit b x l nc thi th thc hin hng lot qu trnh x l. Cc yu cu cho tng cng on x l c trnh by trn bng . 1. X l s cp Vic loi bng bin php vt l cc cht rn dng th c thc hin qua ba bc : a) loi cc vt rn ln nh hp, lp xe, chai l, can; b) sng lc cc vt rn nh nh , si; c) sa lng (s cp) loi cc nguyn liu dng ht nh hn nh phn v giy. Cc nguyn liu dng ht ny (bn hay cht rn sinh hc) thng c x l bng con ng sinh hc nh s phn gii k kh trong mt b phn gii bn (sludge digester)
Bng 10.1:Cc mc v yu cu ca qu trnh x l nc thi Mc x l S cp Yu cu x l Loi khong 30% BOD v 60% cht rn l lng tng s (TSS). Loi c BOD ln cht rn l lng tng s ti ch cn 25-30 mg/l, song phn c loi khng nm di 85%, pH nm gia 6,0 v 9,0. Loi c BOD ln cht rn l lng tng s ti ch cn t hn 9 mg/l, hoc loi trn 95% BOD v TSS; trong nhng trng hp l c bit cn loi b c cc cht dinh dng (nh photphat v nitrat).

Th cp

Tam cp

X l th cp (bng con ng sinh hc) l s phn gii cc cht hu c v qu trnh lm gim BOD. Mt hoc nhiu trong s cc phng php sau y s c s dng: a) Lc trch (trickling filters) : Nc thi c phun (v qua c thng kh) ln mt lp m . Mi vin s c ph bi mt lp sinh khi nhy cha vi khun c g l zoogloea. Chnh zoogloea s phn gii cc thnh phn ca nc thi khi nc thi chy thnh cc dng nh trn cc vin (hnh 10.2) b) Bn hot tnh (activated-sldge) : Nc thi c thng kh mnh dn n vic to thnh cc ht cha y vi snh vt phn gii hiu kh. Qu

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trnh ny din ra trong cc b thng kh, sau nc thi c a sang mt b sa lng loi b cc cht rn sinh hc (hnh 10.3).

Hnh 10.2: X l nc thi theo phng php lc trch

Hnh 10.3: X l nc thi theo phng php bn hot tnh

c) H oxy ho (oxydation ponds hay lagoons) : Trong cc h nng (ch su t 1 n 2 m), to, nh cc loi thuc chi Chlorella s tiu th cc cht dinh dng ca nc thi v sn sinh oxy cn thit cho s phn gii hiu kh (hnh 10.4).

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Hnh 10.4: H oxy ho

d) B phn gii bn (sludge digester) : Ti y xy ra s phn gii cc cht rn c tch lu trong x l s cp v i khi sau s x l th cp. Cc vi sinh vt k kh s phn gii bn trong cc b kn, to ra methane (c th c dng lm nhin liu t nng), CO2 v mt lng nh cc kh nit v hydro. S phn gii k kh nc thi mt qu trnh din ra chm (hnh 10.5).

Hnh 10.5: X l bn trong b phn gii k kh

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X l tam cp Qu trnh ny loi b cc cht nhim cn li sau x l th cp. Nh qu trnh ny, mt loi nc thi c cht lng cao thch hp, vi nhiu mc ch ti s dng s c to ra. X l tam cp c th bao gm mt hoc nhiu cng on sau y : a. Keo t ho hc (chemical flocculation) loi cc cht dng ht cn lu li . b. Bc lc cui cng (final filtration) loi cc cht rn, cc cht ny sau s c lm kh hoc t, hoc c dng lm phn. c. Loi b hoc lm gim hm lng photphat v nitrate. d. Chlo ha dch thi git cht cc nhm vi sinh vt m mt s c th l cc vi khun gy bnh. Dch thi cui cng s phi c loi clo trc khi a vo cc thu vc bi v clo c hi cho cc sinh vt thu sinh.

III. Ln men methane

1. Qu vi sinh vt hc tng th S to thnh methan 1 mt qu trnh vi sinh vt hc tng th trong cht hu c c chuyn ha thnh methane. Sohngen khi khng nh cc nghin cu trc y ca Omelianski, chng minh rng s ln men cc cht hu c mt mt s to thnh cc sn phm cui cng nh H2, CO2 v acid acetic, v mt khc s sinh ra methane nh s kh CO2 bi H2. Vic hai qu trnh ny c quan h vi nhau trong qu trnh to thnh methane tng th b b qua trong nhiu nm v nhng kh khn v mt phng php trong cc thc nghim vi sinh vt hc. Tng t, ngh cho rng H2 di dng phn t hot ng nh mt sn phm trung gian gia hai giai on khng c tha nhn cho n nm 1979. Thm vo , Bryant cn gi thit v s tn ti ca mt giai on th ba trong qu trnh vi sinh vt hc tng th ca s to thnh methane, bng cch cung cp nhng mi lin quan cn thiu gia mt pha l cc acid bo bay hi cha 3 nguyn t cacbon hoc hn v mt pha khc l acetate, H2 v CO2. Dn dn, ngh v mt mi lin quan gia cc vi khun ln men chu trch nhim i vi pha th nht ca qu trnh to thnh methane tng th v cc vi khun sinh methane chu trch nhim i vi pha th hai c chng minh r rng ti mc din bin ca qu trnh ny c th c biu din di dng mt m hnh sinh hc. Bc mt

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Cc loi vi sinh vt ch yu ca tiu qun x ln men c kh nng tn cng cc polime ngay c khi cc cht ny nm th rn. Cc loi vi sinh vt ny cha cc enzyme ngoi bo, tc l cc enzyme c t bo xut ra pha bn ngoi khoang chu cht v thm ch c th c tit vo trong mi trng. Cc enzyme ny s thu phn cc nguyn liu polime thnh cc nguyn liu c trng lng phn t thp, thm ch cc monome: protein thnh cc acid amine, polisaccharide thnh cc oligo- v cc monosaccharide. Cc phn t nh ha tan sau s c cc vi khun cng loi hp thu v s dng cho trao i cht ca mnh. Cc loi vi khun sng bng protein c th khc vi cc loi vi khun sng bng polisaccharide. Khc vi cc vi khun ln men ni trn, mt nhm vi khun ln men khc khng c kh nng thu phn nguyn liu polime, song cng c kh nng hp thu cc phn t ha tan nh hn v dng cc phn t ny cho trao i cht ca mnh. Thng gp bn ny trong trng hp ca ng hn l trong trng hp ca cc acid amine. Do kt qu hot ng trao i cht ca nhm vi khun ln men th nht trong hn dch s xut hin cc loi sn phm cui cng dng kh, l cc acid bo bay hi cha t 2 n 5 nguyn t cacbon hoc hn, ethanol (v cc ru hoc keton khc), v/hoc cc acid hu c nh acid lactic. Do nhiu acid hu c c sinh ra trong qu trnh ln men ny, bc mt ca qu trnh vi sinh vt hc tng th trong s to thnh methane thng c gi l bc sinh acid. Cc acid hu c s xut hin trong hn dch di dng cc anion v thng bt ngun t cc phn t c cht trung tnh. Do vy mt cation cp i s phi c mt trong hn dch m bo tnh trung ha v in. C mt s cation cp i ng c vin. Khi cc hp cht cha nit, c th l protein, c phn gii, cation cp i NH4+ s c to thnh. Khi vng mt cc hp cht cha nit, cation cp i duy nht c th c to ra t cc cht hu c l ion hydro, c k hiu l H3O+. Nh vy trong bc ln men, cation cp i H3O+.thng c sn sinh nhiu. Bi vy, bc sinh acid thng l mt bc acid ha, song cn trnh hiu sai v mt thut ng hc. S sinh acid thc cht l s sinh cc anion acid hu c. Cn s acid ho l s sn sinh cc ion hydro v do vy lin quan n pH ca hn dch. Bc sinh methane Cc vi sinh vt sinh methane khng phi l cc vi khun tht (eubacteria) m l cc vi khun c (archeobacteria). Cc vi khun c khc v

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c bn vi cc vi khun tht v thc cht nm trong s cc sinh vt c nht trn tri t. Ch c H2 cng vi CO2 hoc acetate l c th c dng lm c cht cho cc vi khun c to thnh methane. Song cng c mt vi ngoi l. l trng hp ca cc hp cht gn gi nh methanol, focmiat, CO2 v cc methylamine. Cc loi vi sinh vt u tin c gi l bn dinh dng hydro, l cc vi khun c ha dng v c v chng dng H2 kh CO2 bng nhm thu nng lng v l cc vi khun c t dng v chng s dng CO2 lm ngun cacbon. Cc loi vi sinh vt th hai, c gi l bn sinh acetate, l cc vi khun c ho dng hu c v chng ct acetate thnh methane v CO2, thu nhn nng lng ca mnh t acetate mc d khng nht thit phi ct acetate, v l bn d dng v chng s dng acetate lm ngun cacbon. Acetate l mt anion. Khi b ct thnh methane v CO2, cn c mt cation cp i tham gia vo qu trnh. Khi ion cp i l ion amon, NH4+, c sinh ra trong bc ln men u, th amonium bicacbonate, NH4HCO3, s c to thnh trong hn dch. Khi cation cp i l H3O+.c sinh ra trong bc ln men u, th acid cacbonic, H2CO3, s c to thnh v CO2 s c gii phng trong th tch kh sinh hc thot ra. Kt qu ca c hai trng hp l mi trng tr nn kim hoc kh nng trung ha ca n s c tng cng. Do vy s to thnh methane xut hin nh mt bc kim ha. Bc trung gian Mt vi nhm vi khun c dng lm cu ni gia bc ln men v bc to thnh methane. Khng nhng th, mt s trong chng cn cnh tranh vi cc vi khun ln men v cc c cht monome. Mt s khc cnh tranh vi cc vi khun c sinh methane v acetate hoc v H2 cng vi CO2. Cc acid bo bay hi c chuyn ha thnh acetate, H2 v CO2 nh cc loi vi khun c bit thuc v nhm sn sinh acetate v to thnh hydro bt buc, c thi c gi l cc vi khnn kh proton bt buc. Cc vi khun ny, do cc nguyn nhn v mt nhit ng hc, ch c th sng cng dng m khng phi l cng sinh vi cc vi khun dinh dng hydro. Kt qu l p lc ring phn ca hydro, sn phm trung gian ca chng, pH2 , trong sut qu trnh u gi mc rt thp (khong 10-6 bar). V vy, H2 thuc loi phn t ch pht hin c trong cc hn dch sinh methane

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mt cch c h thng v nh lng c trong thi gian rt gn y. Etanol v lactate u c chuyn ho thnh acetate, H2 v CO2 nh mt nhm vi khun sinh acetate v to thnh hydro bt buc khc. Cc loi vi khun khc c th cnh tranh vi chng. Thc cht, lc nhit ng hc dng duy tr p lc ring phn ca hydro cc gi tr t thp n rt thp trong sut thi gian to iu kin thun li cho cc loi vi khun ny v chng c c ch dinh dng hydro ca bn thn chng. Khi cc anion sulfat, SO4-, c mt trong mi trng, cc vi khun kh sulfate c th tr nn quan trng. Mt s trong chng c th cnh tranh vi cc vi khun sinh acetate to thnh hydro bt buc v cc acid bo bay hi dng lm c cht. Nhm vi khun c kh nng s dng ethanol v lactate c th bao gm ch yu l cc vi khun kh sulfate. Bn ny thm ch c th l bn kh sulfate ty tin. Khi cc anion sulfate c mt, c sinh ra t c cht hu c s c s dng kh SO4- thnh sunfua, HS-. Khi cc vi khun dinh dng hydro c mt, H2 sinh ra t c cht hu c s c chng s dng. Khi cc anion sulfate v cc vi khun dinh dng hydro ng thi c mt, s cnh tranh v hydro c th xy ra, v kt qu s ph thuc vo cc iu kin mi trng. Vic cc vi khun kh sulfate c th b li ko vo qu trnh to thnh methane dn n hai kt lun quan trng. Th nht, chng c th h tr hoc cnh tranh vi cc vi khun sinh methane. Th hai, chng chu trch nhim i vi s c mt ca H2S trong th tch kh sinh hc thot ra. y l hai s kin c tm quan trng k thut c bn . Ngoi cc vi khun kh sulfate, mt nhm vi khun dinh dng hydro khc cng gi vai tr quan trng trong qun th vi sinh vt sinh methane tng th. Cc vi khnn ny c gi l cc vi khun sinh acetate ng hnh, chng thuc bn ha dng v c v thu nng lng ca mnh t phn ng kh CO2 bng H2 to ra mt mnh acetate, tn ca chng bt ngun t . Mt s trong cc vi khun ny l bn t dng v c kh nng ng ha CO2. Cc vi khun sinh acetate ng hnh khc cng cnh tranh vi cc vi khun ln men v chng c kh nng to ra mt mnh acetate t glucose. Trong trng hp ny, chng l bn ha dng hu-v c hn hp, v l bn hn dng, tc l va t dng va d dng. Cn lu rng, cc vi khun sinh acetate ng hnh khng cnh tranh vi cc vi khun sinh methane v c cht (v chng sinh ra acetate) m v nng lng v chng s dng mt phn nng lng tim tng ca hn hp, H2 cng vi CO2.

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Khi qu trnh vi sinh vt hc tng th ca s to thnh methane b ct thnh hai bc tch bit, bc ln men sinh acid acid ho u tin v bc sinh methane kim ha th hai, th cc vi khun sinh acetate ng hnh c th gi mt vai tr h tr v quan trng v chng c th bt gi H2 sinh ra trong bc th nht, trc khi n thot ra, to thnh acetate. V th, acetate s c d tr trong hn dch nh l mt c cht sinh methane tim tng cho bc hai. Hin ti cha c bng chng thc nghim cho gi thuyt ny. Cc acid bo khng th c chuyn ha mt mnh thnh cc sn phm cui cng thng thng c tnh kh do nhng l do v nhit ng hc. Chng cng c chuyn ha thnh acetate, H2 v CO2 bi mt nhm vi khun sinh acetate sn sinh hydro bt buc khc, bn ny phi sng cng dng bt buc vi cc vi khun dinh dng hydro duy tr p lc ring phn ca hydro. pH-thp hoc rt thp trong sut thi gian din ra s phn gii .

Cu hi n tp chng 10
1. Vic a nc thi cha mt hp cht khng c nhng d phn gii, chng hn tinh bt, vo sng hoc ao h s gy nn mt hiu qu xu n cht lng ca nc. Nhng hu qu c biu hin nh th no ? Ti sao ? 2. Trong thc t, 200C, sau bao nhiu ngy th s oxy ha sinh hc cc cht hu c c trong nc thi c th c coi nh kt thc ? Ti sao ngi ta thng ch xc nh BOD5 ? Lm th no xc nh BOD5 ? i vi nc thi sinh hot v nhiu loi nc thi cng nghip, BOD5 tng ng vi khong bao nhiu % BOD tng s ? 3. Ti sao ngi ta ni rng, s c mt ca khu h vi khun nitrate ha trong nc thi c th lm cho cc kt qu o BOD tr nn thiu chnh xc ? Ti sao vic o BOD5 cha b nh hng ca khu h vi khun ny ? 4. Sau 5 ngy mt loi nc thi cng nghip, lng oxy ha tan cn li trong mu i chng l 7,8 mg/l, trong mt dch nc thi pha long 0,1% l 2,8 mg/l a. b. Tnh BOD5 ca loi nc thi cho Trong 1000m3 nc thi ni trn c cha bao nhiu kg BOD5 ?

5. Sau khi mt mu nc thi c k (tc oxy ha sinh hc) bng 0,15/ ngy 7 ngy 200C v ngi ta o c BOD bng 208 mg/l. Tnh BOD5 ,BOD10 v BOD tng s ca loi nc thi ny. 6. Cch xc nh COD khc cch xc nh BOD ch no ? Ti sao gi tr COD thng ln hn gi tr BOD ?

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7. Hn ch ca phng php xc nh COD so vi phng php BOD nm u ? Ti sao trong mt s tnh hung, ngi ta vn phi dng phng php COD thay th cho phng php BOD ? 8. Qu trnh phn gii cc cht hu c thnh methane tri qua nhiu bc, trong c hai bc ln men. a. l nhng bc no ? b. Tuy vy, ngi ta khng th gi s to thnh methane l mt qu trnh ln men, v sao ? Vy l qu trnh g? c. Nu ngha ca hin tng cng dng trong s to thnh methane sinh hc. .

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Chng 11

S tuyn khong nh vi sinh vt

Vic tch kim loi t qung nh qu trnh ngm chit (leaching) khng phi l mt cng ngh mi. Trong thc t, ngm chit qung ng c thc hin t nm 1947 ti min Bc Hungari, t th k 16 ti vng ni Harz ca c, t khong nm 1670 ti Rio Tinto Ty Ban Nha v t th k 18 min Wicklow nc Anh. Vic ngm chit qung c a vo M t u nhng nm 1920. Nga, vic chit ng t cc dng chy qua m pirit ng c thc hin vng Uran t gia nhng nm 1940. Tuy nhin, trong tt c nhng quy trnh tin hnh trc y, vai tr ca vi khun cha c nh gi ng mc v vi sinh vt ch yu chu trch nhim i vi s oxy ha cc sunfua kim loi, Thiobacillus ferrooxidans mi c xc nh u tin vo nm 1947. S tham gia ca vi sinh vt vo vic ngm chit qung c nhiu tc gi chng minh. y l nhng vi sinh vt ha dng v c bt buc hay ty tin, hoc vi sinh vt d dng, chng c th thuc v nhm trung hay a nhit c kh nng oxy ha st hai thnh st ba cng nh cc loi lu hunh dng kh thnh acid sunfuric hoc sunfat kim loi. Ngoi ra, hng lot cc vi sinh vt khc nh nm, to, v ng vt nguyn sinh cng c mt trong s sinh trng cng sinh trong cc dung dch ngm chit tn ti trong t nhin.

I.Cc vi khun ngm chit

Thiobacillus ferrooxidans l vi khun c nghin cu nhiu nht trong mi quan h vi cc bin php x l thy luyn kim sinh hc (biohydromatallurgical treatment) cc loi qung v tinh qung cha sunfua. l mt vi khun hnh que, Gram m, di ng bng tin mao, khng hnh thnh bo t, ng mt mnh hay i khi thnh tng cp. Vi khun ha dng v c ny thu nhn nng lng cn thit cho sinh trng v ng ha CO2 t s oxy ha st hai v cc hp cht lu hunh v c c tnh kh. V mt sinh l, T. ferrooxidans ging vi Thiobacillus thiooxidans, loi vi khun thng thng c mt trong cc loi nc khai m c tnh acid. S khc bit c bn c nhiu ngi tha nhn gia hai loi ny l vic T.thiooxidans khng c kh nng oxy ha st hai v cc sunfua kim loi khng tan. H thng oxy ha st hai ca T. ferrooxidans c lin kt vi lp mng ngoi cha lipopolysaccarit ca thnh t bo. Cc enzyme oxy ha st l Fe2+-citocrom c oxydoreductase, citocrom a, v coenzyme Q...

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S chung ca s oxy ha sinh hc bao gm cc loi sau: S2-, S0.,SO42-. S4O62-, SO32-, v SO42-. Trong dy oxy ha ca s trao i cht S2-, lu hunh nguyn t nm trng thi phn t c polime ha (cu trc vng S8) v sunfit l mt sn phm trung gian cha kha c chuyn ha thnh sunfat. Cc phn ng enzyme tham gia vo dy oxi ha sunfua ny c th c trnh by nh sau:
S2- + O2 + 2H+ S0 + O2 +H2O 2SO3 + O2 S2O322S2O32SO32- + 2AMP 2APS + 2Pvc S0 + H2O SO32- + 2H+ 2SO42SO32-+pesunfua dihydrolipoat O3SSSSO3 + 2e4APS + 4e2ADP + 2SO42(11.1) (11.2) (11.3) (11.4) (11.5) (11.6) (11.7)

Cc vi khun ngm chit khc bao gm Leptospirillum ferrooxidans ln u tin c phn lp t qung sunfua vng. N c th ngm chit pirit, c kh nng sinh trng t dng trn ion st hai, c nhiu s tng ng v mt sinh l vi Thiobacillus thiooxidans v mn cm vi s km hm bi ion st ba. Thiobacillus acidophylus c kh nng i vi c sinh trng ha t dng ln sinh trng d dng. N c phn lp t mt mi trng nui T. ferrooxidans.Vi khun ny oxy ha lu hunh nguyn t, ng, acid amin v cc acid cacboxilic pH 3,0-3,5. Thiobacillus kabobis v Thiobacillus organoporus oxy ha lu hunh nguyn t v sinh trng ph 1,5-5,0. Thiobacillus thioporus oxy ha lu hunh nguyn t, thiosunfat v nhiu loi sunfua k c sunfua km. Sunfobacillus acidocaldarius c phn lp t cc sui nc nng c tnh acid. N sinh trng ha t dng vi cc c cht l lu hunh, cc hp cht kh ca lu hunh v st hai. II.C ch tc ng ca vi khun S chit kim loi ra khi cc qung cha sunfua c th t c nh cc phng thc trao i cht trc tip hay gin tip ca vi sinh vt. C ch trc tip c th biu din bng phng trnh:

MS + 2O2 MSO4 (11.8) Trong M l kim loi nng ha tr hai. C ch gin tip, v d i vi qu trnh ngm chit qung uranium, c th c biu din nh sau:
Vi khun

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UO2 + Fe2(SO4)3 UO2SO4 + 2FeSO4 (11.9) St hai c sinh ra trong phng trnh trn s c ti oxy ha bi cc vi khun thnh st ba theo phng trnh sau:

2FeSO4 + H2SO4 + 0,5O2 Fe2(SO4)3 + H2O

Vikhun

(11.10)

V vy, trong phng thc tc dng gin tip ca vi khun, vi khun ng vai tr cung cp mt cch lin tc cht oxy ha, Fe2(SO4)3. St hai s thu c t s oxy ha sinh hc pirit l cht lun lun kt hp vi cc sun fua v qung uranium: 2FeS2 + H2O + 7,5O2
Vi khun

Fe2(SO4)3 + H2SO4

(11.11)

Bc mt trong s oxy ha trc tip cc sunfua kim loi. MS, l s ha tan c cht trc khi xy ra phn ng trao i cht. iu ny c th t c nh s phn ly ca MS: MS M2+ + S2(11.12) Anion sunfua c gii phng ra sau s lp tc c lin kt vi h thng enzyme ca vi khun v b oxy ha thnh sunfat: S2+ 2O2
Vi khun

SO 2 4

(11.13)

V vy, anion sunfua b loi khi phng trnh (11.12) v cn bng s chuyn v pha bn phi gy ra mt s ha tan mnh hn. V mt l thuyt qu trnh ny c th tip tc n khi ton b c cht (MS) c chuyn ha thnh sn phm (MSO4). Tuy nhin, trong cc h thng khng lin tc s tch ly cc sn phm c th t ti mt nng tr nn c i vi vi sinh vt hoc Jarosit s kt ta trn b mt ca c cht lm cn tr hot ng ca vi khun. S ny gii thch ti sao vi sinh vt li a ngay st b mt khong vt. Hn na ngi ta pht hin thy mt mi quan h trc tip gia tc chit kim loi (dM2+ /dt) ha tan sn phm ca cc khong vt sunfua (Ksp): dM2+ /dtKsp = Ksp = [M2+][S2-] (11.14) Trong l h s t l thun. Theo phng trnh (11.14), tc chit kim loi l cao nht khi ha tan sn phm ca khong vt sunfua ca n l cao nht.

223

III. Mt s qu trnh thy luyn kim sinh hc

1. Ngm chit sinh hc qung ng Hin ti khong 25% ton b sn lng ng min Ty nc M c sn xut nh ngm chit sinh hc cc loi qung ngho. Phn ng tng th ca s ngm chit chancopirit nh vi khun din ra nh sau:

Cu2S + 2,5 O2 + H2SO4 2 CuSO4 + H2O (11.15) Phn ng ny din ra qua 2 bc:
Vikhun

Bc 1: Cu2S + 0,5O2+H2SO4

Vi khun CuS + CuSO4 +

H2O(11.16)

Bc 2: CuS + 2O2 CuSO4 (11.17) CuS c thnh phn ging nh thnh phn ca covenlit, mt loi khong vt t nhin. Cc loi khong vt sunfua ng khc l bocnit (Cu5FeS4), cubamit (CuFe2S3) v enacgit (Cu3AsS4). Tt c cc khong vt ny u c oxy ha nh vi khun. Vic ngm chit cht ng thng c tin hnh gn cc im khai thc gim ti a gi thnh vn chuyn. Qung ngho c vn chuyn bng xe ti hoc bng chuyn ti mt v tr khng thm ri thnh cc ng c dng hnh nn ct. a s cc ng qung c to dng da vo cc a hnh t nhin. Cc ng qung ln c th cao ti 200m, rng 80m pha nh v 250m v pha y v cha ti 50.000-300.000 tn qung. Dung dch ngm chit c phun ln phn nh ca ca cc ng. N thm qua phn thn ng v tch li chn ng. ng c thu hi t dch nh xementit ha vi st (trong qu trnh ny, ng c kt ta di dng kim loi trong khi st c ha tan: Cu2+ + Fe0 Cu + Fe2+ ) hoc bng cch chit chn lc thnh mt dung mi hu c v sau khi tch pha lng khi pha hu c, ng c ra sch khi dung mi hu c bng acid sunfuric long to ra mt dung dch sunfat ng m c v tinh khit, t n c kt ta nh in phn. S ca qu trnh ngm chit kiu cht ng c trnh by trn hnh 11.1. Cc ng chm chm c p dng trong trng hp ng c thu hi t dung dch sau ngm chit (pregnant solution) nh xementit ha vi st ph liu. Trong cc x nghip hin i, ng c thu hi bng phng php chit nh dung mi v tch nh in phn. Ni chung ng xementit cha
Vi khun

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khong 85% ng. N c trn vi tinh qung tuyn ni v c a vo ni nu kim loi trong bc thu hi cui cng. ng thu c nh phng php tch chit bng dung mi -in phn tinh khit ti 99% v y tiu chun a ra th trng. Vic chit ng trong cc qu trnh cng nghip ny t c nh c hai c ch ngm chit vi sinh vt trc tip v gin tip. Cc yu t nh hng n s thu hi ng v n hot tnh ca vi khun l: a) c tnh vt l ca th qung (kch thc ht qung, thm i vi dung dch ngm chit, tnh dn nhit v kch thc ng. b) Thnh phn ha hc ca qung v nguyn liu ngho l ch cha khong vt ng (s c mt v phn b ca pirit v ht qung ring bit bn trong th qung); c) Thnh phn ca dung dch ngm chit i vo (O2 v CO2 ha tan v cc cht dinh dng khc, acid, t l Fe2+ /Fe 3+ , qun th vi sinh vt).

Hnh 11.1. S ngm chit qung ng

Hng sn xut ng ln nht ca M, hng Phelps Dodge Company, vo nm 1986 tuyn b rng h c th sn xut mt pao ng t qung thi ca h vi gi 30 xu bng phng php cht ngm chit ng tch chit bng dung mi - in phn. Gi bn nm vo khong 75-80 xu. Hng ny c k hoch u t trn 50 triu USD m rng x nghip ngm chit ca mnh.

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Vic ngm chit ti ch (in-situ) qung ng cng ging nh ngm chit cht ng nhiu im. Khi qung qu thp khng th dng phng php khai thc thng thng, ngi ta s pha v khi qung trong lng t bng thuc n v ngm chit ti ch. 2.Ngm chit sinh hc qung uranium Uranium c chit t qung nh qu trnh ngm chit vi khun hc quy m cng nghip. Ha hc ca s chit ny c th c biu din bng mt s thay i t uranium khng tan ha tr bn sang dng ha tr su ha tan trong mi trng ngm chit c tnh acid:

UO2 +Fe2(SO4)3+2H2SO4 H4[UO2(SO4)3]+ 2FeSO4 (11.18) Phng trnh ny l phng trnh o ngc ca phng trnh (11.9). Sunfat st hai c ti oxy ha thnh sunfua st ba theo phng trnh (11.10). S bin i nng lng t do chun (86,1 kJ/mol) v th oxy ha kh i vi h thng U4+/U6+ (446mV) cng ging nh s bin i nng lng t do v th oxy ha kh ca h thng Fe2+ /Fe3+ (77,4 kJ/mol v 747mV). Cc qu trnh sinh ha hc v thy luyn kim sinh hc i vi vic chit uranium c bit rt r. 3. Ngm chit sinh hc qung bc S phn b ca bc v cc nguyn t khc trong t b nh hng mnh bi cc chu trnh sinh a ha. Cc kim loi nng v kim loi qu thng to thnh cc phc cht v c duy tr nh cc lp mn ca t. Cc vi sinh vt tham gia vo vic s dng cc nguyn t ny bao gm c vi khun, nm, to v a y. V kh nng ca Thiobacillus ferrooxidans s dng sunfua bc lm ngun nng lng hin ang cn tranh ci. Mt s tc gi cho rng (Ag2S) tng hp v t nhin u khng c vi khun ngm chit phn gii v bc c vi T. ferrooxidans. Tnh c ny c lin quan n s tch ly c hiu i vi bc ca t bo. Song vic nui T. ferrooxidans trong mi trng c nng AgNO3 tng dn li to ra cc chng khng vi bc. Vo nm 1986 mt nghin cu khc pht hin ra rng T. ferrooxidans thc y s chit chn lc bc t mt loi qung sunfua hn hp. Trong 49 ngy ngm chit, khong 75% bc c ha tan t qung hn hp khi c mt vi khun v ch c 50% khi vng mt vi khun. 4.Thy luyn kim sinh hc vng Cng ngh vng thng dng t cc ti nguyn khc nhau rt c pht trin. Vng c thu hi bng phng php clo ha, xianit ha, ngm
Vikhun

226

chit tiosunfat v tioure, to hn hp (amalgamation), tuyn ni, tuyn bng trng lc v nu chy hoc bng t hp cc quy trnh trn. Nu vng phn tn tinh vi bn trong cc loi qung m sunfua nh trong pirit, acxenopirit, pirotit, galen (qung ch sunfua), v sphalerit th nhiu trong s cc phng php tuyn vng k trn s khng mang li hiu qu kinh t mong mun. Cht ngm chit khng xm nhp c vo bn trong khi qung rn tip xc vi cc ht vng c kch thc nh hn mt m. Mun gii phng c vng th cc loi qung cha sunfua ny thng phi c tin oxy ha. Tuy nhin nung l mt quy trnh tn nng lng v lin quan n cc vn v mi trng nh pht sinh acxenic bay hi, ch, lu hunh, v cc cht c khc. Mt bin php c th thay th tin oxy ha l p dng cc phn ng sinh hc gii phng cc ht vng t qung. Ngay t nhng nm 1960 ngi ta pht hin c cc vi khun d dng c kh nng ha tan vng t cc khong vt laterit. Trong cc nghin cu ny, hm lng vng cc i khng vt qu 1,5mg/dm3. Tuy nhin sau 283 ngy ngm chit tch c ti 82% vng cha trong qung. Mt s loi nm c chng minh khng nhng c th chit vng t qung m cn c th loi vng ha tan khi dung dch ngm chit nh hp ph sinh hc ln b mt c th. Nhng t bo nm ny sau s c lc, lm kh v nung. Ngi ta phn lp c hng lot chng nm hot ng c th loi ti 98% vng t dung dch ngm chit sau 1520 gi tip xc. 4.1.Ha Tan vng t qung pirit nh hot ng ca vi sinh vt Cc th nghim ngm chit c tin hnh vi pirit cha vng (150g vng/tn qung) trong cc bnh nn cha 5g pirit v 200cm3 mi trng dinh dng cha 200mg (NH4)2SO4, 50mg KCl, 50mg K2HPO4, 500mg MgSO4 v 10mg Ca(NO3)2 trong mt lt nc ct, v c vi 10 cm3 mt chng Thiobacillus ferrooxidans thun khit. Kt qu ch ra trong bng (11.1) Th nghim s 1 c tin hnh m khng cy vi khun vo dung dch ngm chit (mu i chng v trng). Cc s liu trong bng 11.1 nhn c sau 3 tun ngm chit nhit thng. Hiu qu ca vi khun trong vic ha tan vng t pirit l r rt v trong th nghim nui tnh, vng khng c tm thy trong mu i chng v trng. Trong cc th nghim vi bnh lc, lng vng c ha tan vo cc mu c cy ging tng ln 6 ln. Cc tc gi ch ra rng trong qu trnh oxy ha cc sunfua kim loi nh vi khun (pirit, galen v cc sphalerit), cc loi

227

hp cht oxy ha ca lu hunh c to thnh phc cht vi cc ion ny v c ha tan, chng hn theo phn ng sau y: Au + O2 + H2O + 3S2O32- Au(S2O35- + 2OH(11.19)
Bng 11.1. S ha tan vng t c cht pirit nh Thiobacillus ferrooxidans Th nghim nui tnh s 1 2 3 4 S tch chit vng g/dm3 Khng 150 270 390 Th nghim nui lc s 1 2 3 4 S tch chit vng g/dm3 60 480 490 520

S oxy ha pirit dn n s to thnh acid sunfuric nh ch ra trong phng trnh 11.11. Cc ion hydroxil trong phng trnh 11.19 c trung ha bng acid sunfuric thu c t phng trnh 11.11: (11.20) 2OH- + H2SO4 2H2O + SO42Bng cch s dng pirit, chancopirit, asenopicrit, galen v macasit cha vng c nghin nh (c cy bng T.ferrooxidans) v trong cc mu i chng v trng vi tng loi hoc hn hp sunfua, mt s tc gi khc pht hin ra rng vic thu hi vng t cc mu c cy ging khng cho hiu qu cao hn so vi cc mu i chng v trng. Pht hin ny tri ngc vi cc kt qu va nu trn v cc tc gi ca cng trnh ny i n kt lun rng T.ferrooxidans trong dung dch long khng c tc dng ng k ln s tch ly v s thot vng vo t. Trong khi cc th nghim ngm chit vi ph liu Colorado Mineral Belt ch ra rng khi c mt vi khun v cht dinh dng, s thu hi cc kim loi thng c nng cao ng k so vi cc mu i chng v trng. Cc tc gi ny xut mt quy trnh ngm chit hai bc t mt hiu qu kinh t cao hn. Trong bc th nht, mt qu trnh ngm chit bng vi khun (hoc acid) s c thc hin loi cc kim loi thng ra khi ph liu v gii phng vng v bc. Sau phn cn li s c trung ha bng vi v trong bc th hai c th s dng k thut xianua ha thng thng thu hi vng v bc. Bng cch s dng nguyn l ngm chit hai bc k trn, Klusman v Nelson (1976) cng nh Lawrence v Bruynesteyn

228

(1983) chng minh kh nng p dng ca phng php ny i vi s thu hi vng v bc t cc tinh qung perit chu la. Vic thu hi vng tng theo t l thun vi hiu sut tin oxy ha pirit nh vi khun. Chng hn, nu khng p dng qu trnh tin oxy ha nh vi khun cho cc mu pirit th qua k thut xianua ha vng thu hi ch t khong 25%. Cn nu pirit c tin oxy ha ti 80% th vng thu hi nh bin php xianua ha s vt qu 90%. pH tng i thp (di 1) to iu kin thun li cho vic gi cc ion st ba trong dung dch. Vi khun T.ferrooxidans hot ng tt nht pH 2,3. Cc gi tr pH di 1,5 thng km hm hot tnh ca vi khun. Tuy nhin Lawrence v Bruynesteyn pht hin thy rng vi khun ny c kh nng thch ng tt vi cc gi tr pH thp ti khong 0,6. Trong mt th nghim khc tin hnh vo nm 1985, vic thu hi vng c tin hnh t mt loi ph thi tuyn ni km v ch (cha 1,75g vng/ tn) nh phng php ngm chit kp. Trong bc mt, cc mu tuyn ni tri qua cng on tin khai thc v nghin mn c ngm chit trong mt b c thng kh v khuy trn nh s dng T.ferrooxidans , km, st v ng c ha tan. Sau khi tch bit hai pha rn -lng, phn cn li (c tnh acid) c ngm chit theo k thut tioure thu hi vng. Ngi ta cho rng trong qu trnh ngm chit cht thi tuyn ni cha sunfua nh s dng vi khun, vng s c gi li di dng t nhin trong phn cht thi rn, song mt phn s c gii phng ra di dng t nhin trong phn cht thi rn, song mt phn s c gii phng ra di dng cc ht c kch thc di 1mm t qung pirit ngho. S thu hi vng theo phng php tioure din ra theo phng trnh sau: Au + Fe3+ + 2CS(NH2)2 Au[CS(NH2)2] + Fe2+ (11.21) Ion st ba c cung cp t phn ng oxy ha pirit. u im ca vic s dng tioure thu hi cc kim loi qu t cht thi c tnh acid sinh ra t qu trnh ngm chit nh vi khun l ch n khng i hi phi trung ha cht thi vn l mt cng on tin quyt ca quy trnh xianua ha. Ngoi ra, tioure v cc sn phm phn gii ca n khng gy nn nhng ri ro cho mi trng so vi cht ngm chit xianua. Nhng s lo lng mi y v hiu qu i vi mi trng v gi thnh ca cc phng php vn s dng cng vi nhng thnh cng t c nhhot ng thc y ca vi khun cho php gi thuyt rng cng ngh sinh hc phi c ng dng cho vic ch bin qung vng. Gi thnh chi ph cho s tuyn khong sinh hc cc loi qung vng sunfua chu la s gim do quy m ca nh my s c nng cao.

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4.2.Thu hi vng t cc ti nguyn cha asenopirt S tuyn khong asenopirrit c tin hnh t nhng nm 1960.Vi khun Thiobacillus ferrooxidans c kh nng gii phng vng t qung cha cacbon trong asen chim khong 6%. Cc iu kin ti u cho s ngm chit xc nh c l: t trng bn khong l 20%, pH1,5-2,0 v kch thc ht l 0,05mm. Di cc iu kin ny, 90% acid asen v 60% st c tch ra t qung sau 10 ngy ngm chit. V mt sinh ha hc, s oxi ha asenopirrit cth c biu din bng cc phn ng in ha sau y: -xy ha anot:

FeAsS Fe2+ +As3+ + S +7e-Kh catot:

Vikhun Vikhun

(11.22)

3O2 + 14H+ +7e- 7H2O (11.23) 3+ Ion As c gii phng trong phng trnh (11.22) s b thy phn ngay thnh acid asenic H3AsO3 + 3H+ (11.24) As3+ + 3H2O 2+ Acid asen (phng trnh 11.24) cng nh Fe v S (phng trnh 11.22) s c oxy ha tip tc
H3AsO3 Fe2+ + O2

Vikhun
Fe3+ + eVikhun

H3AsO4

(11.25) (11.26)

Vikhun

S0 + 1O2 + H2O H2SO4 (11.27) Ion st ba c i lc cao vi acid asenic v mt kt ta asenat st ba s c to thnh: (11.28) Fe3+ + H3AsO4 FeAsO4 + 3H+ Tng cc phng trnh t 11.22 n 11.28 s l: FeAsSO + 3O2 + H2O FeAsO4 H2SO4 (11.29) Da trn cc phn ng oxy ha asenopirit k trn, mt nh my sn xut th c xy dng t nm 1975 thu hi vng t asenopirit. Theo quy trnh ny, mt loi tinh qung asenopirit cha 10% asen c ngm chit theo phng php kp nh s dng vi khun.

230

Khong 90% hm lng asen c chit ra trong khong 100 gi. Cc iu kin ngm chit l: t trng bn khong l 20%, 75% c kch thc ht nh hn 0,074mm, nhit 25-280C v pH 1,75-2,1. Sau qu trnh ngm chit nh vi khun v cng on tch bit pha lng rn, phn cn li c trung ha bng vi ti pH 3,5. Phn asenat cha kt ta c loi b v dung dch sau khi c iu chnh pH cng nh nng cht dinh dng s c a tr li bc ngm chit. Mt nghn cu khc (Polkin v ctv, 1985) cng ch ra rng cc phng php ngm chit nh vi khun l cc phng php c hiu qu nht trong vic gii phng vng nm di dng phn tn mn t cc tinh qung asenopirrit, chng hn loi tinh qung cha 8,4% asen, 24,1% st, 26% lu hunh v 290g vng/ tn. Ht ca cc tinh qung ny c kch thc di 0,074mm. Vic ngm chit c tin hnh theo mt h thng ni tip gm ba b phn ng, mi b c dung tch hot ng 1,5 dm3 cha 25% cht rn. Bn khong c cy bng ging Thiobacillus ferrooxidans, c khuy bng bin php c hc v thng kh (vi tc 3 dm3 khng kh/dm3 bn khong/pht). Vic ngm chit c hon thnh sau 30 gi vi mt s thu hi asen ti 90%. Phn cn li sau khi ngm chit c trung ha bng vi v c x l bng phng php xianua ha trong cc dch huyn c t trng bn khong l 20% cha 1% KCN. Hiu sut thu hi vng nm trong khong 88-92%. Nu tinh qung asenopirit cha vng c ngm chit trc tip bng dung dch KCN th hiu sut thu hi cui cng ch nm vo khong 7-10%. Nu asenopirit cha vng trc ht c nung ri sau mi ngm chit bng cc dung dch KCN th vng thu hi cng khng vt qu 77%. Nhng s so snh ny ch ra u th v hiu qu ca phng php thy luyn kim sinh hc. Sau cng on ngm chit nh vi khun, cht rn cha vng v bc c chuyn sang tuyn thu hi kim loi qu. vng v bc c ha tan nh bin php (trung ha vi vi) v xianua ha hc nh x l trc tip bng tiore m khng cn trung ha. T tuyn ngm chit nh vi khun, bt c lng Cuv Zn no gii phng ra t qung u c th c thu hi nhm tng hiu qu kinh t ca qu trnh. Cc c im c bn ca qu trnh ngm chit kim loi qu vi s tham gia ca vi khun c minh ha trong hnh 11.2 di y.

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Hnh 11.2.Qu trnh ngm chit kim loi qu nh vi khun (R= rn; L=lng) 5.Ngm chit sinh hc sunfua km S tch chit km t cc qung sunfua nh Thiobacillus ferrooxidans c thc hin theo phn ng sau y:

ZnS + 2O2 ZnSO4 Cc b ngm chit qung sunfua km bn cng nghip nh Thiobacillus ferrooxidans to c nhng nng km trong dch mang (pregnant solution) ti 120g/dm3 v tc gii phng km vo dung dch ti 1300mg/dm3/gi. Dng php tch chit kim loi bng in phn t dung dch ngm chit nh vi khun (do Cominco, Trail, B.C.,Canada thit k) t c mt hiu qu ti khong 80% vi cht lng km thnh phm loi cao. Cc nghin cu ng hc ch ra rng tc tch chit, V(mg/dm3/gi) l mt hm s ca kch thc ht qung sunfua, d v c biu din bng phng trnh sau: V=Vmexp(-kd)= 569,2exp(-5,24.10-2d) Trong Vm l tc l thuyt cc i c th t c v k l mt hng s t l thun.
Vikhun

232

Trong mt nghin cu khc c tin hnh vo nm 1986, B.Zurita tm thy rng tc i vi cc ht qung nh hn 12m c tnh theo phng trnh: V=exp(-0,136k + 5,452) v i vi cc ht c kch thc ln hn 12m: V=exp(-0,03d + 5,88) Khi s dng phng trnh th hai tnh tc , s tch chit km c c trng bi cc bin ngm chit. Bng cch cc i ha phng trnh hi quy, cc gi tr bin ti u c xc nh l: nhit = 350C; pH= 2,3; t trng bn khong =22%; (NH4)2SO4 = 0,75g/dm3, K2HPO4=0,66 g/dm3, CO2 trong khng kh = 7,9% v din tch ring phn ca tinh qung sunfua km-ch khng th x l c hiu qu kinh t bng quy trnh lm nng chy thng thng. Song cc k thut thy luyn kim sinh hc c s dng thnh cng thu hi km v cc kim loi khc t cc sunfua km phm cht. Trong cc k thut ny, km, ng, v cadmium s c ha tan, trong khi ch vn cn nm li trong qung. V sunfua km khng ha tan trong mi trng ngm chit sinh hc cha acid sunfuric cho nn c th thc hin c d dng vic tch chit chn lc. 6.Tch chit coban v niken t qung sunfua nh bin php vi sinh vt. S trao i niken v c tnh ca niken i vi vi sinh vt c Kaltwasser v Frings nghin cu vo nm 1980. Nng vt ca niken c th kch thch sinh trng ca vi sinh vt trong khi cc nng ln hn li tc ng nh cht km hm. Ngi ta tm thy cc vi khun d dng tit ra cc acid hu c c th dng ngm chit cc qung laterit cha niken (Bosecker, 1986). Chng hn, Penicillium simplicissimum tch chit c ti khong 70% niken t qung laterit nh cc vi sinh vt d dng c mt tng lai y ha hn.S khai thc cc m niken ngho trong tng lai s ph thuc nhiu vo cc k thut r tin nh k thut ngm chit acid vi khun. Cc mui sunfua tng hp hoc tn ti trong t nhin (NiS) c th c oxy ha nh Thiobacillus ferrooxidans theo phng trnh sau:

NiS + 2O2 NiSO4 Pentlandit l loi qung sunfua niken ch yu gp trong hu ht cc m c t tnh. Trong qung ny, niken c thay th bng coban vi t l Ni/C= 50/2. Vic ngm chit sinh hc pentlandit c th biu din bng phng trnh sau:
Vikhun

233

+3 H2SO4 4NiSO4+2 (Ni,Fe)9S8+17 O2 Fe2(SO4)2 + 3H2O Trong cc thc nghim vi cc bnh ngm chit, trn 80% niken c tch khi pentlandit trong 12 tun, cn trong cc thc nghim b ngm chit trn 97% niken c tch chit trong 3 ngy. Cc thc nghim khc cho thy rng s tch chit niken c ci thin v c bn khi c mt vi khun c nh nit Beijerinckia lacticogenes. Ngi ta cho rng sinh trng ca Thiobacillus ferrooxidans c kch thch bi s gii phng ionamon nh Beijerinckia l vi khun nhn c ngun cacbon dnh cho sinh trng t T. ferrooxidans. Theo cc kt qu cng b, qu trnh ngm chit sunfua coban nh T. ferrooxidans trong cc th nghim c cy ging din ra vi tc 75 ln nhanh hn so vi cc mu i chng v trng (Karavaiko, 1985) v nng t c ln ti 30g/dm3 (Torma, 1978).
Vikhun

IV. S tch ly kim loi nh vi khun v to

Vi sinh vt bao gm x khun, vi khun lam, cc vi khun khc cng nh nm si v nm men c kh nng tch ly cc kim loi nng v cc nuclit phng x t mi trng ngoi. S lng tch ly c c th rt ln, v cc c th tham gia vo qu trnh tch ly ny c th dao ng t nhng mi tng quan l-ha nh hp ph v kt ta ti cc qu trnh ph thuc vo trao i cht t bo nh qu trnh vn chuyn. C t bo sng ln t bo cht u c kh nng i vi vic hp thu v tch ly v do vy cc sn phm c to ra s l bn thn cc t bo vi sinh vt hoc cc cht bt ngun t cc t bo nh cc sn phm tit ca trao i cht, cc polisaccarit, cc cu t ca thnh t bo. 1.C ch ca s tch ly kim loi nh vi sinh vt 1.1.S hp ph sinh hc khng ph thuc vo trao i cht Hp ph l s tch ly hay s tp trung cc cht, cht b hp ph, ln mt b mt hoc mt phn pha, cht hp ph. Ngc li, hp th xy ra khi cc nguyn t hay cc phn t ca mt pha xm nhp gn nh ng u vo trong cc nguyn t hoc phn t ca mt pha khc. C ba kiu hp ph chnh. Kiu th nht do lc hp dn in gy ra v thng c gi l s hp ph trao i- l s hp dn ca cc ion tch in dng i vi cc phi t tch in m trong nguyn liu t bo Loi hp ph th 2 , trong cc phn t c hp ph c th c s chuyn dch bn trong bao gm cc lc van der Waal.

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Loi th 3 l lc hp dn ha hc gia cht b hp ph v cht hp ph. Ni chung kh c th tch bit gia s hp ph vt l v s hp ph ha hc v a s cc loi hp ph u bao gm c ba dng m t trn. Thut ng hp thu(sorption)c th bao gm c hp ph v hp th v dng ch mt qu trnh trong mt thnh phn chuyn dch t mt pha sang mt pha khc, thng l mt pha rn. Do vy thut ng hp thu sinh hc c dng m t nhng mi tng quan l- ha khng nh hng c th tn ti gia cc loi kim loi/ nuclit phng x v cc thnh phn ca t bo. *Vi khun v vi khun lam Thnh t bo ca cc vi khun Gram dng hp thu kim loi c hiu qu tuy rng ph hp thu ca chng c th kh rng. i vi B.megaterium, Micrococcus lysodeikticus, v Streptococcus mutans s lin kt kim loi c xp theo th t sau y: La3+ >Cd2+ >Sr2+ >Ca2+ > K+ > Na+ . Thnh t bo ca Bacillus subtilis c i lc chn lc i vi mt s cation v dng nh rng nhm COO ca acid glutamic trong peptidoglycan l v tr ch yu i vi s kt ta kim loi. 1.2. S tch ly ni bo ph thuc vo trao i cht *Vi khun v vi khun lam Ngi ta pht hin c cc h thng vn chuyn c hiu i vi Mn2+ mt s vi khun nh E. coli, B. subtilis v Lactobacillus plantarum. Ngoi ra, Mn2+cng nh (Ni2+ v Co2+) c th c hp thu nh l mt c cht c i lc thp ca h thng vn chuyn Mg2+. L.plantarum, s vn chuyn Mn2+ ph thuc vo gradien H+ xuyn mng v ngi ta pht hin c cc nng ni bo ca kim loi ny cao ti 30mmol/l. vi khun lam Anabaena cylindrica ngi ta ghi nhn c h thng vn chuyn Ni2+ c tnh c hiu cao. Niken c tp trung nhiu hn khong 2.700 ln bn trong t bo v s hp thu ph thuc vo hiu th ca mng v b km hm trong bng ti hoc bi cc cht km hm trao i cht. S hp thu tch cc Cd2+ cng c pht hin Anacystis nidulans v qu trnh ny b km hm cnh tranh bi Ca++ v Zn2+.

Cu hi n tp
1. Cc Thiobacillus l nhng vi khun chu acid: a. Xc nh tip dinh dng ca cc vi khun nhm ny. b. Xt nhu cu oxy, phi xp chng vo nhm no ?

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c. Cho bit mt mi trng nui cy thch hp cho nhm vi khun ny ? 2. Bn cht ca hin tng n mn cc ng dn bng st l: a. S oxi ha H2S thnh acid sufuric. b. S kh acid sufuric thnh H2S. c. S oxi ha H2S thnh lu hunh d. S khong ha cht hu c thnh H2S 3. Trong s n mn ng dn nc, s oxy ha di iu kin k kh din ra vi s tham gia ca cc qu trnh.......................................................... khi c mt ging vi khun c tn l..............................................v.................................trong mi trng. 4. S oxy ha lu hunh nguyn t c thc hin bi cc vi khun nh Thiobacillus thiooxidans din ra theo phng trnh sau y: 2S + 2H2O + 3O2 2...........................................

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Chng 12

Bi tp c s v nng cao
I. Phn cu hi
1. in vo ch trng Cu 1: a. Th gii vi sinh vt v cng a dng. nghin cu chng cc nh khoa hc phi da vo cc tiu ch v ...................,........................,......................... sp xp chng vo bc thang phn loi v t tn. b. Cc vi sinh vt c sp xp vo bc thang phn loi t thp n cao:....................................................................................Loi l bc thang phn loi thp nht.........l bc thang phn loi cao nht. Loi c t tn theo h thng..........................theo ting.....................vit nghing. V d vi khun gy bnh than c tn l Bacillus anthracis. Cu 2: Virus l nhng thc th gy nhim.................... m gen nom ca chng ch cha hoc l................ hoc l ............... . ....Chng ch nhn ln trong ........................... bng cch s dng b my...........................v .............................ca t bo tng hp nn cc bn sao ca bn thn mnh nhm truyn .................. ca bn thn chng sang cc t bo khc. Cu 3: Cu trc in hnh ca mt virus n gin nht bao gm mt............... (..............hoc..... ....) v mt .................c gi l ............... Cc virus phc tp hn, c bit l cc virus ng vt cn cha mt lp v bc c to nn bi mt t hp....................,.................v............................ Cu 4: a. DNA si n ca virus va c transcriptase ngc tng hp nn trc ht c chuyn thnh...... ..............v sau c.................................ca t bo ch. b. mt s virus, lp v bc c ph bi cc................ c bn cht l.................hoc..............m vai tr ca chng l nhn bit (v mt ho hc) cc ........ v gn vo t bo m chng gy nhim. Cu 5: a. La tui b nhim HIV cao nht l la tui ......................... b. Bnh vim gan B c truyn ch yu qua ng............

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c. Bacteriophage nhn ln nh.................v ph hu n. l qu trnh............, cn cc vi khun cha mt prophage v lu gi n vnh vin trong...........ca mnh c gi l cc vi khun.............. Cu 6: a. Prophage, nm trn .....................ca vi khun, chnh l nguyn liu di truyn ca............................ b. Cc ht tng t virus song ch cha ARN thun khit c gi l ..........l bn gy bnh thc vt c ngha kinh t nh............................ c. Cc ht protein thun khit gy bnh thn kinh ng vt c tn l........ Cu 7: a. Hin tng mt loi virus ch nhim bnh trn mt vt ch hay nhm vt ch nht nh c gi l .............ca virus. Tnh ny do cc.............................to nn. b. Bnh di l mt bnh ...................cp tnh thng dn n Cu 8: Trong cc cu trc sau y, hai cu trc no khng gp trong mt ht HIV: a) ARN si n; b) cc glicoprotein b mt; c) DNA si kp; transcriptose ngc; e) protein v; f) DNA si n. Cu 9: a. Nhim sc th vi khun khi nm di dng...................... s khc vi dng ................bi mt cu trc m c hn v t cng knh hn. b. Khi c mt ca lisozim v saccarose, mt trc khun Gram m s b bin i thnh mt t bo......................c tn l........................v mt i dng hnh que ca n. Cu 10: Nm l sinh vt thuc dng t bo ............................C th c th n hay a bo dng si, c thnh.......................(mt s t c thnh..................), khng c...............sng d dng theo kiu................. Sinh sn bng .............khng c...............v ............................................... Cu 11: S khc bit c bn ca cc t bo Prokaryote so vi t bo Eukaryote l ch: a. Nguyn liu di truyn ca chng ..... khng c bao bc bi........ b. Thiu cc bo quan nh:....................c bao bc bi mng.. c. Thnh t bo lun lun cha phc h..............hay cn gi l.......... d. Ribosome ca cc t bo prokaryote thuc loi ............................

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Cu 12: a. Ni bo t l th ............... ca cc t bo vi khun, khng phi l phng tin sinh sn, khi gp iu kin thun li, bo t s ny mm v mi bo t chi cho ........t bo dinh dng. b.Ni bo t c tnh ........cao, do vy thng gy kh khn trong cng tc bo qun thc phm v trong vic kh trng cc bnh vin. Cu 13: a. Nhn t di truyn ngoi nhn ca vi khun c tn l cc ............., chng khng quyt nh s sng cn ca t bo vi khun song mang mt s gen quan trng nh cc gen.........................., ................................ b. Cc vi khun lu hunh mu ta v mu lc s dng.............lm cht cho in t trong quang hp, kt qu l to thnh.......................tch lu bn trong t bo hoc c tit ra bn ngoi t bo. Cu 14: a. C nh nitrogen l qu trnh chuyn ho nitrogen ca kh quyn (N2) thnh........... mt cht cn thit cho s sinh tng hp cc amino acid. b. Trong th gii vi sinh vt, ch c mt s loi.................... l c kh nng c nh nitrogen phn t. c. Enzyme chu trch nhim i vi s c nh nitrogen phn t c tn l .... d. C......nhm vi khun c nh nitrogen ch yu, l.............. Cu 15: a. Nitrogenase d b lm bt hot bi........, v vy cc t bo vi khun c kh nng c nh nitrogen hoc l phi h hp mnh vng b mt t bo gi cho phn bn trong t bo vng mt oxy (chng hn ...........................). b. Protein c chuyn ho thnh cc peptid bi cc enzyme c tn l........, cn cc peptid c phn gii thnh cc aminoacid c tn l................ Cu 16: a. Qu trnh phn nitrat ho l mt qu trnh c hi i vi nng nghip, bi v......... b. S kh nitrogen dng kh thnh amoniac c tn l.................... c. Phc h enzyme chu trch nhim i vi s c nh nitrogen cng sinh c tn l.... Cu 17: a. S phn nitrat ho xy ra mnh nhng ni oxy...........................

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b. Chi vi khun oxy ho amoniac thnh nitrit c tip u ng......, trong khi Chi vi khun oxy ho nitrit thnh nitrat c tip u ng............. c. Mt vi khun t sinh bo t sng k kh c nghin cu nhiu do kh nng c nh nitrogen mnh ca n c tn l.............................. Cu 18: a. Vi khun c nh nitrogen sng cng sinh trong bo hoa du thuc bn ....(1).... sinh oxy v c tip dinh dng l.................. b. Vi khun khng lu hunh mu ta hay gp trong cc ngun nc t nhin thuc bn............. sinh oxy v c tip dinh dng l................ c.Chi no trong cc chi sau y cha cc loi c nh nitrogen sng cng sinh: Rhizobium/ Anabaena/ Clostridium/ Frankia/ Azotobacter. Cu 19: Bt c lc no cng c th tm thy trn c th ca mt ngi trng thnh t nht............. t bo vi sinh vt, a s trong chng l..............v chng c gi l...................ca c th con ngi. Cu 20: Mt s vi sinh vt sng trn da, song a s sng trn cc b mt........ca c th, l.............................bao ph bn trong mi, ming, ng h hp trn, ng rut, v ng niu sinh dc. Cu 21: ................khng c coi l mt b phn ca khu h bnh thng. Cu 22: Nhiu c quan v v tr bn trong ca c th khe mnh nh...................................................khng cha vi sinh vt. Cu 23: Nu hin tng ..............cn s c mt ca mm bnh sau s gii phng c t ca mnh vo cc t chc th hin tng.............ch l hu qu ca s tip xc gia c t vi t chc m khng nht thit cn s c mt ca mm bnh. Cu 24: Trng hp ng c thc n c tn l botulism (lit do ngoi c t tc ng ln h thn kinh) khng phi l mt s nhim trng m l mt s................... Cu 25:

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Mt bnh nhim khun c th c truyn bng ............, tc l s tip xc c hiu qu gia mt c th b nhim v mt c th khe mnh. Khng c nhm ln phng thc truyn bnh ny vi .......hoc ...... l cc phng thc truyn qua mt ng vt trung gian c tn l .............., gia mt c th b nhim khun vi mt c th khe mnh. Cu 26: Nu s...............gy nn mt s tham gia c hiu qu ca c th trong vic hnh thnh khng th, c gi l........................th s..............l mt phng php a vo c th cc khng th c tng hp t trc; l mt s min dch....................Trng hp u l trng hp tim chng .................., cn trng hp sau l trng hp tim chng ....................... Cu 27: Cht khng sinh l cc cht ha hc do VSV tit ra c kh nng......................... hoc ..............................ca cc VSV khc. Cu 28: Penicillin G ch yu hot ng chng li vi khun Gram..............., vi rng .............................Nhc im ch yu ca penicillin G l:................ Cu 29: a. Dng vaccine l a vo c th mt loi.......................ly t vi sinh vt gy bnh. b. Vaccin l vi sinh vt sng c ly t vi sinh vt sng mt...... c. Gii c t c sn xut t................... ca vi sinh vt. d. Mun phng dch c kt qu, vaccin phi c dng cho ....% i tng cm th. h.Vaccine c dng cho nhng ngi............. Cu 30: Tr nh v ngi cao tui d mn cm vi cc bnh nhim trng v h hoc l h thng min dch........................hoc l........................ Cu 31: Giai on pht trin u tin ca vi sinh vt cng nghip c nh du bng cng trnh ca Louis Pasteur (1878) chng minh vi sinh vt l tc nhn ca s ln men, v sau cc cng trnh ca: a. -...............(1886) dng cc chng nm men thun khit trong sn xut bia.

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b. - ...............(1898) pht hin ra dch chit nm men c kh nng gy ra qu trnh ln men ru (chng minh ln men thc cht l mt qu trnh enzyme). c. Giai on th 2 ca CNVS c nh du bng............................................ d. Giai on hin ti ca CNVS c nh du bng s pht trin ca cng ngh di truyn vi: - S pht hin cc enzym........................(cc con giao ct) - S pht hin ra cc ...............................(cc vector) - S gn cc gen l mang cc thng tin tng hp cc........................ vo mt c th. - S kim tra ngy cng tt hn s...............................ca cc gen ny. Cu 32: Theo Thomas D.Brock (1995), Cc sn phm c ngha cng nghip mang tnh thng mi do vi sinh vt to thnh c th c phn thnh 5 loi l: a. ............................................................................. b............................................................................... c.............................................................................. d............................................................................... e.............................................................................. Cu 33: Trong qu trnh nui cy vi sinh vt thu nhn sinh khi t bo, cn lu : a. Cc bin php trnh nhim tp thng dng l...........hoc.................. b.Tr to, s thu nhn sinh khi cc vi sinh vt khc cn mt s........................ c. Nhit to ra phi c loi i bng mt h thng................ Cu 34: a. Mc ch ca kh trng Tyndal l tiu dit cc vi khun mang................... nhit ...........Nguyn l ca phng php l cho cc ...........ny................thnh...............ri sau un si git cht cc th ..................

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b.Trong k thut xc nh s lng t bo bng phng php nui trn mi trng rn, ngi ta gi khun lc l........(l ch vit tt ca.....) v rng khng phi bao gi n cng c mc ln t mt t bo....... Cu 35: sn xut bia ngi ta s dng i mch ny mm (nha), hublon, nm men v.....(a)........ Cc ngun tinh bt khc hoc cc ....(b)........cha ng c th c b sung vo nha i mch l : nha la m (Triticum), cc loi ng cc cha ny mm (i mch, la m, ng, la), cc sn phm phn hu tinh bt v cc ng c th ln men. Vic s dng cc nguyn liu b sung ny i hi phi c s b sung (mt phn) cc ch phm.......(c).......c ngun gc t vi sinh vt. Cu 36: Cm gic hng hi (invigorating) v say (intoxicating) ca bia l do.....(a)......gy ra, mi thm, hng v v v ng ca bia l do......(b).......,Gi tr dinh dng l do hm lng cc cht keo ho tan khng ln men (bao gm......(c)... v cui cng, tc dng gii kht l do.......(d).....gy ra. Cu 37: Tc dng ca hublon l mt cht lm trong v n kt ta.......(a)......trong dch hm, l cht lm thay i c tnh ca dch hm do to nn......(b)........., v cng vi ethanol v CO2 to nn cc c tnh......(c)....... mnh to tnh n nh cho bia. Hm lng........(d)...........cha trong hublon lm tng kh nng to .......(e).......ca bia Cu 38: Dch ng ho sau khi nu, c gi l.....(a)......., dch ny sau khi lc loi tru v b, c b sung hublon v un nng nhiu gi nhm chit cc thnh phn mong mun t hublon v nhm kt ta v loi cc ...(b).......c mt trong hublon tng tnh n nh ca bia Cu 39 : Brandy l ru mnh ct t .................., whysky l ru mnh ct t.............., rum ct t................ln men, cn vodka l ru mnh ct t..........hoc................ln men. 2. Hy tr li bng ng () hoc sai (S) Cu 40: Trong nhng s khng nh sau y, u l s khng nh ng:

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a.Nng lng gii phng trong d ho c s dng trc tip cho s tng hp cc thnh phn t bo. b. ng ho cung cp cc vin gch xy dng tng hp cc thnh phn t bo. c. S tng hp cc thnh phn t bo l mt qu trnh gii phng nng lng. Cu 41: Mt cht khng sinh c hot ph hp l cht khng sinh: a. Ch c tc dng ln cc vi khun Gram m b. Ch c tc dng ln cc vi khun Gram dng c. Ch c tc dng ln cc vi khun Gram dng ln vi khun Gram m d. Ch tc ng ln mt nhm ring bit, thm ch ln mt hoc vi loi vi khun. e. c s dng rng ri hn trong thc tin y hc so vi cc cht khng sinh c hot ph rng. Cu 42: a. Vi khun l nhng t bo b. Tt c cu khun u di ng c. Mi vi khun gy bnh u c th i hnh d. Sau khi b thoi hnh, vi khun c th tr v hnh th bnh thng nu iu kin thch hp. Cu 43: Nhng nhn nh no di y l ng vi t bo vi khun a. Nhn c phn cch vi phn cn li bi mng nhn b. Vt cht di truyn l DNA kt hp vi protein histon c. Khng c mng nhn d. Vt cht di truyn l DNA khng kt hp vi protein histon Cu 44: a. Acid teichoic l thnh phn c trng ca t bo vi khun Gram dng. b. S d c tn gi "sao chp bn bo th" v phng thc ny cho php bo ton nguyn vn 50% lng DNA c mt trong t bo m. c. Tc nhn gy bnh giang mai Treponema pallidum chuyn ng rt nhanh nh h thng tin mao bao quanh c th gi l chu mao.

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d. c im c trng ca mt ni c t l n lin kt vi t bo vi khun v s khng khi no c t bo tit ra. Cu 45: Sn xut cng nghip acid glutamic s dng cc loi thuc mt trong cc Chi sau y: a) Corynebacterium; b) Pseudomonas; c) Escherichia ; d) Acetobacter; e) Clostridium. Cu 46: Hy gi tn cc vi sinh vt c kh nng to thnh cc hn hp sau y nh ln men ng: a. CO2 + ethanol b. CO2 + ethanol + acid lactic c. CO2 + ethanol + acid lactic + hydro + acid axetic d. CO2 + ethanol + acid lactic + hydro + acid axetic + butadion e. CO2 + hydro + acid butiric + acid axetic. f. CO2 + hydro + aceton + butanol + ethanol + 2-propanol. Cu 47: a. H hp k kh l qu trnh oxy ha thu nhn nng lng trong cht nhn in t cui cng l oxy lin kt. b. Cht nhn in t cui cng trong ln men ethanol l acetaldehyte. c. cc vi khun sinh methan thnh t bo cha peptodoglycan. Cu 48: Tm s khng nh sai Phng trnh: Glucose + ADP + Pvc 2 Ethanol + 2CO2 + ATP l phng trnh biu din tng qut qu trnh ln men ru : a. Saccharomyces cerevisiae b. Sarcina ventriculi c. Zimomonas mobylis d. Escherichia coli

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3 Sp xp s m t bn phi sao cho ph hp vi cc thut ng bn tri Cu 49:

Bin np Ti np Tip hp Hfr Alen a. Kiu chuyn gen trong mt t bo th nhn nhn cc gen t cc phn t DNA t do trong mi trng xung quanh. b. Kiu chuyn gen ph thuc vo s tip xc t bo - t bo. c. Cc gen tn ti cng mt v tr tng ng trn nhim sc th tng ng nhng khc v mt trng thi t bin. d. Kiu chuyn gen trong mt phage c dng lm phng tin mang DNA t vi khun ny sang vi khun khc. e. Mt t bo F( trong plasmid F c gn vo nhim sc th ca t bo vt ch. a.Kh nng ca mt VSV gy nn mt bnh bnh b.VSV, c th bt u mt bnh mt c th khe mnh

Cu 50:
c tnh (Virulence) Bnh (Disease)

Kh nng gy bnh c.Mc v kh nng gy ra mt bnh mt tc (Patogenicity) nhn gy bnh Tc nhn gy bnh d. S ph hng cc chc nng ca c th do nhim (Causative agent) trng

II.Tr li mt s cu hi
1. a. cu to, dinh dng, sinh sn. b. Loi(species) Chi (genus)- H (family) -B (order) -Lp (class)Ngnh (phylum)- Gii (kingdom)-Siu gii (superkingdom); tn kp, ting Latinh. 2. khng c cu trc t bo; DNA; RNA; cc t bo vt ch; sinh nng lng; sinh tng hp; genom. 3. li acid nucleic; DNA; RNA; v protein; capsid; lipid, hydratcarbon; protein. 4. a. DNA si kp; gn vo genom ca t bo ch b. gai; hydratcarbon; protein; th th (receptor). 5. a. t 13 n 19; b.mu c. vi khun; gy tan; con ci; tim tan. 6. Nhim sc th; Bacteriophage

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7. chuyn ha; gai nhn bit cc th th ca t bo m chng gy nhim. 8. c; f. 9. a. siu xon; gin xon; b. spheroplas. 10. nhn chun; kitin; cellulose; lc lp; hoi sinh, k sinh, cng sinh; bng bo t; lng; roi. 11. DNA; mng nhn; bo quan; peptidoglycan; histon, 70S. 12. a. ngh; mt; b. khng nhit. 13. a. plasmid, b. khng vi cc cht khng sinh; sn xut cc cht c kh nng gy bnh; sn xut cc cht bacteriocin; to nn mt c tnh trao i cht vi khun. 14. a. amoniac; b. vi sinh vt; c. nitrogenase; d. hai; nhm cng sinh v nhm khng cng sinh. 15. a. oxy; Azotobacter; b. protease; peptidase. 16. lm mt nitrogen ca t v do vy, lm gim cht dinh dng cho sinh trng ca thc vt. 17. a. vng mt; b. nitroso; nitro; c. Clostridium pasteurianum. 18. a. (c); quang dng v c; khng; quang dng hu c; c. Rhizobium, Anabaena, Frankia. 19. 100 nghn t; vi khun; khu h bnh thng. 20. trong; mng nhy. 21. cc virus k sinh ni bo. 22. dch no ty, mu, bng quang, t cung, vi Falop, tai gia, thn... 23. Nhim c; ng c. 24. nhim c. 25. tip xc trc tip; cn hoc t; vector truyn bnh. 26. tim chng; min dch ch ng nhn to; tr liu huyt thanh hc; th ng nhn to; phng bnh; cha bnh. 27. c ch, tiu dit chn lc; km hm s nhn ln. 28. dng; cc vi khun Gr(+) c thnh t bo c cu to ch yu t peptidoglycan. 29. a.khng nguyn; b. c tnh; c. ngoi c t; d. 80; h. kho mnh. 30. cha hon chnh; khng cn hiu qu na. 31. a.Gerhard Henrik Armauer Hansen ; b. Eduard Buchner; c. s xut hin cc cht khng sinh; chn lc di truyn cc th t bin; s pht

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trin ca phng php nui cy lin tc; d. restrictase; plasmid; protein c bit; biu hin. 32. a. Bn thn t bo VSV l cc sn phm mong mun b. Cc enzim do VSV to nn: amylase, protease, lipase. c. Cc dc phm: cc cht khng sinh, c t, alkaloit. d. Cc ha cht c bit v cc cht iu v thc phm. e. Cc ha cht thng dng c sn xut bng con ng VSV bao gm ethanol, axid acetic, axid lactic. 33. a. v trng hoc sch; b. thng kh mnh; c. lm lnh. 34. a. ni bo t; 1000C; bo t; ny mm; t bo mi; dinh dng. b. n v hnh thnh khun lc; CFU (colony forming unit); n c. 35. a. nc; b. nguyn liu cha ng; enzyme. 36. a. ethanol; b. hublon; c. hydratcarbon, protein; d. CO2. 37. a. protein; b. hng thm v v ng; c. khng sinh; d. pectin; bt. 38. a. dch hm; b. protein. 39. a. vang; nha ln men; r ng; ng cc, khoai ty. 40. b. 41. d. 42. d. 43. c,d. 44. a; d. 45. a. 46. a. Saccharomyces cerevisiae, Sarcina ventriculi b. Leuconostoc meseteroides c. Escherichia coli d. Enterobacter aerogenes e. Clostridium butyricum f. Clostridium acetobutylicum 47. a. (); b. (); c (S). 48. c. 49. a. bin np; b. tip hp; c. alen; d. ti np; e. Hfr. 50. a. kh nng gy bnh; b. tc nhn gy bnh; d. c tnh; e. bnh.

TI LIU THAM KHO

I. TI LIU TING VIT 1. Kiu Hu nh. 1999. Gio trnh vi sinh vt hc cng nghip. NXB KHKT. H Ni.

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