Hnh 1: Cu to ca acid citric C6H8O7

1. GII THIU V LCH S PHT TRIN: Forter m u quyn sch Hot ng ha hc ca nm bng mt tuyn b: C l cc nghin cu tp trung vo qu trnh ln men acid citric cn nhiu hn c nhng nghin cu v cc chc nng khc trong qu trnh bin dng nm mc, tuy vy, nhng kin thc c bn c quan tm n c l t hn c nhng qui trnh c miu t trong quyn sch ny tuy nhin nu xt trong lch s nghin cu, ln men acid citric l qui trnh v nm u tin c tim nng trong cng nghip c khm ph. Thc vy, t khi iu ny c cng b th s lng cc bi bo v ch ny tng ln ng k v hi vng rng n s gp phn xng ng vo khi kin thc v cng ngh v sinh ha vi sinh vt. Nm 1891- 1893, ln u tin s to thnh acid citric c quan st thy bi Wehmer, n c xem nh l mt sn phm ph trong qu trnh sn xut acid oxalic bng cch nui cy Penicillium glaucum trn mi trng c ng. Nm 1893, ng phn lp c hai chng mi c kh nng tch ly acid citric v cho rng chng l mt chi mi. Cytromyces c xem nh l chng Penicillia n vng (Wehmer, 1903). Nhng th nghim ca Fabrique de Produit Chimiques de Thann et the Mulhouse nhm sn xut to acid citric trn quy m cng nghip b tht bi. Nguyn nhn dn n tht bi l do qu trnh ln men phi c tin hnh trong vi tun th mi thu c mt sn lng ng k v vy rt d b tp nhim. c bit l v Wehmer tin vo nhng tuyn b mang tnh thuyt phc ti thi im rng to ra v duy tr sn phm acid th acid to thnh phi c trung ha.

Nm 1913, Zanhorsky c cp patent (bng sng ch c bng quyn) cho quy trnh sn xut acid citric khi s dng Sterigmatocytis nigra mt tn gi khc ca cho Aspergillus niger. Tuy nhin chnh kt qu nghin cu ca Currie mi m ra con ng cho nhng thnh cng sau ny ca vic sn xut acid citric quy m cng nghip. Nm 1916, J.N. Currie vi vai tr l cng s ca tin s Ch. Thom - ngi ng u phng th nghim vi sinh Cc Ha Hc ti thnh ph Washington.DC, ln u tin quan st thy mt s lng ln cc chng Aspergillus niger c trong phng th nghim c kh nng sn xut mt lng ng k acid citric trong nhng iu kin thun li. Pht hin c cho l quan trng bc nht chnh l Aspergillus niger pht trin rt tt khi gi tr pH nm trong khong t 2,5 3,5 m acid citric li c to ra nhiu khi gi tr pH ca mi trng mc thp. Hn th na, im ni bt ca pht hin ny chnh l vi gi tr pH thp nh vy th sau t 1-2 tun c th thu c gn 60% sn lng trn mi trng ti thiu vi kh nng b tp nhim l rt thp. Cho n lc ny, tng lng dung dch acid citric c to ra t nc p tri cy (ch yu l t chanh) khong 10.000 t/a, trong hm lng acid citric nguyn cht chim 7-9% v tn ti di dng mui calcium. sn xut ra 1 tn acid citric cn khong 20 - 30 tn chanh (tng ng vi din tch khong 2,7 ha). Lc ny, Italy l nc cung cp hn 80% nhu cu v acid lactic ca th gii. Nhiu cng ty cc nc khc nhau tin hnh nhp t mui calcium citrate n acid citric ch yu l cung cp cho ngnh cng nghip sn xut thc ung. Sau khi kt thc chin tranh th gii th nht c nc xut khu l Italy ln cc nc nhp khu u ri vo hon cnh ti chnh kinh t b hn ch, v vy hnh thc sn xut c khng cn ph hp, ngi ta tin hnh tm kim nhng phng thc sn xut acid citric mi. Chnh lc ny, nhng pht hin trc y ca Currie mi tr thnh nhng cng c quan trng nht cho s pht trin ca ngnh cng nghip sn xut acid citric trn quy m ln bng hnh thc ln men. Vic Currie pht hin ra rng Aspergillus niger c th pht trin v tch ly acid citric ti pH < 2 cho ngha l khi qu trnh ln men c bt u nng pH ny th kh nng ca s xm nhim c th c loi b hon ton. Hn na, ng tm ra rng nng ng cao l iu kin thun li nht cho t l sn phm cao v ng quan st thy rng sn lng ti u ch c th t c di iu kin gii hn pht trin. Nm 1919, nhng n lc khc nhm a hnh thc ln men acid citric vo trong sn xut thng mi b tht bi (Societ des Produits Oganiques de Tirlemont, B), nhng n nm 1923, Chas. Pfizer & cng ty ti New York, thnh cng khi p dng phng php ca Currie v h tr thnh nhng ngi sn xut acid citric ln nht th gii. Sau , ti Anh, J v E. Sturge tin hnh sn xut calci citrate theo quy trnh theo quy trnh tng t da trn bn quyn sng ch ca Fernbach v cs. (1927- 1932). Nm 1928, mt nh my sn xut acid citric da trn ngun carbon r tin l mt ng c ci c xy dng ti Kaznjov gn Plzen, Cng ha Czech (Montan - und Industrialwerke, vorm. J.D. Stark, 1924; Szucs, 1925). Kh khn khi s dng loi vt liu ny chnh l s hin din ca cc ion kim loi, trong ng vai tr quyt nh l cc ion kim loi chuyn i. Theo Leopold v Valtr (1964) th cc nh my ny cn s dng hexacyanoferrate (HCF) trong bc u tin ta hoc to phc hp vi cc kim loi khng mong mun trong mi trng. iu ny c ct gi b mt v c cng b kh tr vo nm 1938 vi bng sng ch c cp cho Mezzadroli (1938).

Nhiu nh my c xy dng ngay sau nhiu nc nh l Lin bang Soviet, c (Joh. A. Benekiser GmbH), B (La Citrique Belge) v mt vi nc khc. K t nm 1933 theo Perquin (1938) tng lng acid citric trn th gii l 10.400 tn vi ch 1.800 tn c sn xut t Italy, phn cn li c sn xut vi quy m ln bng cng ngh ln men ( Chu u: 5.100 tn, M: 3.500 tn). Rt nhiu tc gi tham gia nghin cu gip lm sng t c ch sinh ha ca qu trnh sn xut acid citric nh l Bernhauer (1940), Foster (1949), Prescott v Dunn (1959) hay Johnson (1954). Mt cu hi kh th v c t ra: c bao nhiu gi thuyt c a ra cho ti khi con ng sinh tng hp acid citric t con ng ph v carbohydrate thng thng c cng b v phn ng nn tng ca chu trnh tricarboxylic acid (TCA) s tch t acetyl CoA v oxaloacetate, c hiu mt cch r rng. Tuy nhin, c ch iu ha dn n s tng hp acid citric vi hm lng cao v s bi tit nhanh chng ch mi c bt u nghin cu t thp nin 1970 v n tip tc c nhiu nhm nghin cu quan tm. Ban u vic ln men acid citric c tin hnh theo hnh thc nui cy trn b mt. Sau ny hnh thc ln men chm c ngh tuy nhin hnh thc ny cn ny sinh nhiu vn ch yu l do iu kin ca h thng ln men v c bit chng sn xut nhy cm vi nhiu yu t m cho n nay vn cha c nhn thc r. Tc ng ca cc ion kim loi chuyn i c cng b nhiu hn v iu l cn thit la chn cc loi vt liu th ph hp. Trong sut thp nin 1950 t l ca cc phc cht c bn trong qu trnh ln men c tuyn b. Ch vi hnh thc ln men ny m c rt nhiu chin lc c bit c pht trin tng ng vi cc loi vt liu th. Nm 1944, Szucs a quy trnh hot ng chia thnh 2 giai on chnh: giai on u kch thch s pht trin ca h nm si, giai on sau gii hn s pht trin to acid citric. t c iu ny cn cung cp mt lng phospho (~ 70mg/l) vo trong mi trng giai on pht trin v chun b mt lng phospho t do trong mi trng giai on sn xut sn phm. Nm 1948 - 1949, Shu v Johnson ci tin mi trng ln men c th tin hnh c hai giai on trong cng mt h thng. Cc tc gi ny ch ra rng iu kin ti u ca qu trnh ln men i hi phi c s cn bng gia mangan, km, phospho. Trong ln men thng mi vic b sung cc nhn t cn thit l rt kh khn v quy trnh ny s dng cc vt liu th c thnh phn khng c xc nh r rng (v d mt r ng). Rt nhiu cng trnh nghin cu c thc hin tm kim iu kin ti u khi s dng cc loi c cht r tin nh l mt r ng (Shu, 1947; Karow v Waskman, 1947; Woodward v cs., 1949; Moyer, 1953), tinh bt (Moyer, 1953a, b), tinh bt c thy phn (Schweiger v Snell, 1949). C rt nhiu quy trnh x l v tinh sch mt ng c bit l vic loi b cc kim loi vt. Hu ht chng u c x l bng HCF u tin, hin nay ngi ta ngh vic s dng cc ht trao i ion (Perlman v cs.,1945 Woodward v cs.,1949 .). Tuy nhin hu ht cc th nghim ch ra rng HCF va ng vai tr l cht loi b ion, va ng vai tr l cht ng c ch s pht trin c th tch t c nhiu acid . Mt bc t ph chnh t c ti M bi nhng ngi ngi nghin cu ca tp on Miles Laboratories. Trong qu trnh pht trin k thut cc tc gi gii thiu phng php s dng tinh bt c thy phn nh l mt loi c cht r tin. Khi nghin cu vi nhng vt liu th tinh khit hn, h pht hin ra rng hot ng bt li ca cc kim loi vt, c th b bt hot nu thm vo mt lng va ion ng

(Schweiger 1959,1961). Hn th na, tng t HCF, nu ion ng c thm vo vt qu mt lng nh th s rt thun li cho vic sn xut acid vi sn lng cao. Hon cnh hin ti: nhiu cng ty s dung nhng thnh tu k thut ca ln men chm v sn xut khong 80% lng acid citric trn th gii. Ch mt vi cng ty vn cn tip tc s dng phng php nui cy b mt truyn thng .

Hnh 2: Con ng chuyn ho n gin trong sn xut acid citric t carbohydrate v n-alkan

Vo thp nin 1960 mt quy trnh mi c ra trong khi nghin cu m rng trn vic s dng n-alkan nh l mt ngun carbon cho qu trnh sn xut sinh khi vi sinh vt (protein n bo, SCP) v nhng sn phm khc nh acid amino, vitamin, acid hu c .v.v. Cc nh nghin cu ti Nht Bn l nhng ngi u tin s dng cc chng vi khun Corynephom t bin thay cho Corinebaterium, Arthobater v Brevibacterium trong cng ngh ln men ngay sau tr thnh mt cng ngh c thit lp vi nhng c tnh tiu biu ca nhng thnh tu tr tu. Nm 1966 - 1967 mt vi chng t bin c to ra sn xut acid citric t n-parafins (C9-C30 )v cc c cht lin quan. Mc d n c ng k bn quyn (Tanaka v cs., 1968), tuy nhin n khng c khai thc trn quy m cng nghip. Sau ngi ta pht hin ra rng nm men, chng Candida v cc dng lin quan s dng n-alkan lm ngun carbon cho ngun sn xut sinh khi ng thi n cng c th sn xut ra mt lng ln acid citric t nparafins sn phm c trn ln (isocitric acid) (Fukui v Tanaka,1980). Nhng n lc c lp acid citric nh l mt sn phm ph trong qu trnh sn xut SCP sm c thay th bng vic u t nghin cu sn xut acid citric nh l mt sn phm chnh. iu ny cng do vic sn xut SCP t ngun hydrocarbon khng c chp nhn v nhng nh hng ca n n sc khe vt nui. Mc d sn lng acid citric to ra t cc alkan t C9 C20 trong phng th nghim t hiu sut 100% nhng vic a vo sn xut cng nghip vn b hn ch bi mt vi l do: vic sn xut acid citric t c cht ny lun km theo mt lng ln isocitric acid m s c ca cht ny khng c chp nhn trong nhiu ng dng, v d trong sn xut thc n cng nghip; vic chn dng v xc nh cc thng s ti u ca qu trnh cn c xem xt. Trong thi gian ch i th r rng n khng phi l mt c cht r to ra sn phm cui cng l acid citric. Cho d nh th no th vic nghin cu li ng dng ca nm men hoc vi khun cng em li nhiu hiu qu th v. c bit trong thi k ca vic sn xut acid citric nh l mt sn phm ph ca sn xut SCP. Vic ny cn thit thc y cc quy trnh c lp dung dch acid citric. Phng php li trch bng dung mi c pht trin v ngy nay c ng dng ph bin trong hot ng ln men truyn thng. Mt khc, ngi ta cng quan st thy rng c nhng chng nm men c th sn xut c acid citric khi pht trin trn mi trng c cha glucose hay cc ngun carbon tng t (Tabuchi v cs., 1969; Abe v cs., 1970). Nhng kt qu tm ra c nghin cu m rng v pht trin xa hn trong nc CHDC c. Nhng lin kt vi ngi c c l hn ch cc n lc tin xa hn a cc k thut mi vo sn xut trn quy m ln. 2.C S SINH HA S IU HO TCH LU ACID CITRIC 2.1 Loi v nng ng khi ng con ng tch ly acid citric khng thng qua s iu ha Glycolytic C nhiu thng s nh hng n hiu qu tch ly acid citric khi s dng Asp. niger. Trong s loi v nng ng l yu t nh hng nhiu nht. Trong khi nng ti u ca cc kim loi vt phosphate v nitrogen c mi tng quan vi nhau th nng v loi ng c xem nh l mt thng s c lp. S thch hp ca cc ngun carbon khc nhau trong sn xut acid citric bng Asp. niger c a ra tho lun bi Xu v cs (1989b): ch nhng ngun carbon nh glucose v sucrose mi c

nm chuyn ha nhanh chng cho kt qu sn lng v t l acid c tch ly cao. Nn tng sinh ha ca tc ng ny khi khng thng qua s iu ha glycolytic: c t nht 3 enzyme trong con ng chuyn ha hexose biphosphate ca Aspergilli kim sot phn ln qu trnh l hexokinase (EC 2.7.7.1), 6-phosphofructokinase (EC 2.7.7.11; PFK 1), v pyruvate kinase (EC 2.7.1.40) (Smitt v NG, 1972; Kubicek, 1988b). Cn ch rng c ch iu ha bin dng tt l nguyn nhn ca s iu ha glycolytic tc l khi gia tng hot tnh ca mt vi enzyme glycolytic th khng dn n s gia tng lng acid citric to ra. S khuch i gen v d pki A (gen m ha pyruvate kinase) khng dn n s ci tin ca chng sn xut (l. De Graaff, J.Visser, S. Choojun, M. Roehr v C. P. Kubicek, kt qu ny cha c cng b). Tng t, s iu ha glycolytic nm men khng b tc ng bi s khuch i cc gen chn lc (Heinisch, 1986).

Hnh 3: Quy trnh chuyn ho ca acid citric c im ca PFK1 trong A. niger c hiu r hon ton v rt nhy cm trong kim sot nhiu con ng bin ng khc nhau nh l c hot ha bi Fru2,6-P2 AMP, ion NH4+ v b c ch bi phosphoenol Pyruvate, citrat v ATP (Habison v cs., 1983; Arts v cs., 1987). c nhng bng chng gin tip v s iu ha protein 8

kinase da vo c-AMP (Legisa v Bencina, 1994). S hot ha bi Fru-2,6-P 2 kt ni cc PFK1 vi nhau v iu ha 6-phosphofructose-2-kinase (EC 2.7.1.105; PFK2). N kch thch s t m rng enzyme sau iu ha km trong A.niger. V kt qu ch ra rng hot ng ca enzyme ny c bin i chnh do s gn Fru-6-P ln b mt (Harmsen v cs., 1992). V vy s chuyn i ca Fru-6-P thnh Fru-1,6-P c xem nh l mt im quan trng trong s iu ha dng chy glycolytic. Theo mt bo co cho rng s tch ly acid citric ch c th xut hin di iu kin PFK1 khng th kim sot dng chy glycolytic mt cch hiu qu. S trung ha vai tr ca NH+4 - tch ly di iu kin Mn (Kubicek v cs., 1979a) - s c ch citrate ca PFK1 l mt thng s lin quan trong qu trnh ny (Habison v cs., 1979,1983). Tuy nhin s tch ly acid bi nng ng cao li ln quan ti mt con ng bin dng khc: nng ng cao lm gia tng nng Fru-2,6-P 2 ni bo (Kubicek Pranz v cs., 1990). y ch l mt quan st vi ngun carbon cho sn lng acid citric cao. V nng Fru-6-P ni bo ch cao di mt vi iu kin, nghin cu tm thi cho thy rng n ph thuc loi ngun carbon v nng acid citric tch ly v s khc nhau ca chng nh hng n nng Fru-6-P trng thi bn m iu nh hng n s thnh lp cc nhn t Fru-2,6-P2 hot ha glycolytic. Tuy nhin, ngi ta ra mt gi thuyt m nu ng n s gi ra cc bc bin dng trc khi to thnh Fru-6-P ngha l hexokinase (phosphoglucose isomerase xc tc phn ng cn bng nhanh chng v khng nm trong nhng cht iu ha quan trng) c l tham gia vo trong vic iu ha phn ng thy phn glucose v tch ly acid citric di nhng iu kin ph hp. Bng vic s dng phng php phn tch nhy ca trng thi bn, Torres (1994a,b) cho rng hoc c s chuyn i hoc phosphoryl ha glucose y mnh qu trnh tch ly acid citric. c im sinh ha ca qu trnh phosphoryl ha hexose trong Asp. niger c nghin cu k (Steinbock v cs., 1993): khng ging nh S. cerevisiae (Lobo v Mattra 1977) nhng n li tng thch vi chng Khuyveromyces lactis (Prior v cs., 1993), Asp. niger ch cha mt loi hexokinase. Enzyme ny c to ra khi nng sucrose hay glucose mc cao. c im ng hc ca n gn ging nh enzyme ca ng vt, ngha l n khng b c ch bi glucose-6-phosphate. Vic s dng chng A.niger t bin khng 2-deoxyglucose trong sn xut acid citric c loi b (Fledurek v cs., 1988; Kirimura v cs., 1992) v n kh hot tnh ca hexokinase. th hot ng ca hexokinase chng li s tch ly citrate trong chng t bin c ngh rng hexokinase khng b gii hn trong dng bin dng. Pht hin ny cho rng vic kh hot tnh ca hexokinase c th l h qu ca dng chy carbon t l thp trong chng t bin khng desoxyglucose. Nh vy hu nh cc nhn t kim sot hiu qu cc bc chuyn ha sm ca glucose c khm ph 2.2 Pyruvate carboxylase v vai tr ca s phn ngn chuyn ho c lp vi qu trnh iu ha sn phm cui cng ca qu trnh glyco gii hiu kh l pyruvate. Trong t bo bnh thng cc sn phm ca qu trnh d ha pyruvate i vo trong ti th v c chuyn ha thnh acetyl-coA. Asp. niger v mt vi loi nm khc c cha pyruvate v carboxylase c kh nng bin i pyruvate v CO2 to thnh oxaloacetat (Osmani v Schutton, 1983; Bercovitz v cs., 1990; Jaklitsch v cs., 1991). Enzyme ny c cm ng khi nng carbon hydrat hin din cao (Feir v Suzuki 1969; Hossain v cs., 1984). S lin quan ca pyruvate carboxylase trong qu trnh hnh

thnh acid citric c chng minh bng cc th nghim vi cht c nh du (Cleland v Johnson, 1954) v vic o lng lng kh thot ra t qu trnh ln men (Kubicek v cs., 1979b). im khc bit ln nht gia pyruvate carboxylase ca nm vi hu ht cc sinh vt c nhn chnh l v tr ca n trong t bo: hu ht cc enzyme ca sinh vt c nhn c cha trong ty th (Osmani v Scrutton, 1983; Turohtt v Ratledge, 1988) cn trong Arpergillus spp. n xut hin ti cc v tr chuyn bit trong cc phn cytosolic (Jaklitsch v cs., 1991) y chnh l im thun li c bn nht trong qu trnh tch ly acid: oxaloacetat khng cn chuyn ra t ti th v vy n s khng b kh thnh malate. V cht mang tricarboxylate s dng dicarboxylic acid nh mt ng c cht. Kt qu c trong s to thnh citrate v malate. Hot ng ca citosolic pyruvate carboxylase kt ni qu trnh glyco gii n s to thnh citrat t ti th. 2.3.Hot ng ca chu trnh Tricarboxilic Acid (TCA) v s tch ly acid citric Hu ht nhng nghin cu trc y v s tch ly acid citric u tp trung vo s c ch ca mt bc trong chu trnh TCA v cha nhng im mu thun vi nhau. Hu ht cc nh nghin cu u cho rng nht thit phi c mt bc c ch trong chu trnh tch ly acid hu c, trong khi mt s khc - s dng cc ti th c lp - li cung cp bng chng cho thy s hin din ca mt chu trnh bnh thng ( xem xt li xem Roehr v cs., 1992; Kubicek v Roehr, 1986; Kubicek v cs., 1994). Khng ai dm chc rng c hay khng s gia tng nng citrate l cn thit cho s tch ly acid citric nhng n c th xut hin nh l mt sn phm ca c tnh iu ha cc enzyme trong chu trnh TCA. Trong khi c nhng bng chng cho thy rng aconitase khng b c ch khi lng citrate to ra qu nhiu (Rubicek v Roehr, 1985), iu ny khng th c loi tr i vi mt vi enzyme khc trong chu trnh TCA. V citrate c ch NADP - c hiu cho isocitrate dehydrogenase ( Mattey, 1977; Legisa v Mattey, 1986), t l kh in t trong bin dng [NADH/(NAD + NADH] c ch NAD - c hiu cho isocitrate dehydrogenase (Kubicek, 1988a), oxaloacetate, cisaconitate, NADH c ch 2oxoglutarate dehydrogenase (Meixner Monori v cs., 1985). Tuy nhin iu quan trng i vi s c ch chnh nu chng xut hin trong in vivo th cha c hiu r: v dng ra ca citrate ph thuc vo dng vo malate, nguyn nhn c ch ny c vch ra bi s tch ly citrate l c lp bi s gia tng vn chuyn trong t bo cht. Tht khng may, c tnh ca cht mang tricarboxylase ca Asp. niger, ti th vn cha c nghin cu. Ngi ta vn tip tc quan tm cao n iu ny bi v n gip gii thch ti sao isocitrate v cis aconitate mc d c s cn bng ca aconitase vn khng c tch ly bi Asp. niger. Cn phi ch rng phng php phn tch nhy cm ca trng thi bn cho thy s quan trng ca s tun tro citrate trong ti th i vi t l tch ly citrate (Torres, 1994b). 2.4 S iu ha qu trnh hnh thnh sn phm trong cc iu kin mi trng khc nhau: Bn cnh cc yu t nh nng v loi ng nh hng n s tch ly citrate ( c ni phn trn) th cc thng s mi trng khc cng nh hng n s hnh thnh acid citric i km vi s hnh thnh mt s sn phm ph khc nh l gluconic acid v oxalic acid.

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iu kin mi trng ti u cho s tch ly acid citric l: - Nng ng cao: 120 250 g/l - S thiu ht cc kim loi vt c bit l Mn2+ v Fe3+. - p sut oxy ha tan cao (> 140 mbar) - pH: 1.6-2.2 - Nng phosphate c iu chnh mc ti thiu cho yu cu pht trin. - Lm tng nng NH4+ C s sinh ho v s tc ng ca chng s c cp phn di. 2.4.1 Nhng ion kim loi vt: Rt nhiu kim loi vt c gi tr c bit l Mn2+, tuy nhin trong s tch ly acid bi A. niger th ch Fe3+ v Zn2+ ng vai tr quan trng, s thiu ht Mn2+ l bt buc thu c sn lng cao. C nhiu cch gii thch c ngh quan tm n c im sinh ha trong hot ng ca kim loi vt (Roehr v cs., 1992). Vai tr ca s thiu ht mangan c l tp trung lm gim s tng hp cc i phn t (Kubicek v cs., 1979a; Hockertz v cs., 1989) do cm ng gia tng protein bin tnh (Kubicek v cs., 1979a; Ma v cs., 1985). S gia tng nng NH4+ tch ly trong si nm hot ha PFK1, m s c ch ca n c trung ha bi citrate nh thc y s glyco gii (Kubicek v cs., 1979a). Gi thuyt c a ra cho chng A. niger t bin khi PFK1 nhy cm vi nng citrate ng thi lm gim s nhy cm ca s tch ly acid citric bi Mn2+ (Schreferl v cs., 1986). S gim st trong qu trnh tng hp cc i phn t c cho l s c ch ca ribonucleotide reductase, dn n s gim st trong vic tng hp DNA do thiu tin cht monomeric ca s nhn i DNA (Hockert v cs., 1988, 1989). Mt vi tc ng khc ca s thiu ht DNA nh l nh hng ln sinh tng hp vch t bo v hnh thi hc hyphal, mi lin h ca s ri lon trong nhn i DNA cha c lm r (Kubicek v Roehr, 1986). Tc ng sau cng l yu t ni bt nht v c th c s dng nh l tiu chun d on s thnh cng ca qu trnh ln men acid citric: s thiu ht mangan trn si nm pht trin c khng bo mnh, phn nhnh cao, vch t bo c lm c, xut hin nhng ch phnh. Hin tng ny c th lin quan n s nh hng pht trin ngn bi s mt nh hng vn chuyn cc ti. Kh nng ni bt ca Cu2+ l trung ha nhng tc ng c hi ca s xut hin Mn2+ bi v n c kh nng c ch Mn2+ bng cch hp thu, s xut hin ca n l c hiu trong h thng vn chuyn c i lc cao (Seehaus v cs., 1990). nh hng ca cc kim loi khc trong s tch ly acid citric hu c bi Aspergilli hin nay vn cha c r rng: mt vi nh nghin cu cho rng Fe3+ c nh hng to ln nhng quan im ny b bc b bi nhiu ngi khc (Kubicek v Roehr, 1986). Trong quan im nng cao ca Fe3+ cn thit tch ly citrate, cc tc gi ny cng nhn rng tp cht mangan c th tht s nh hng n kt qu quan st c (Kubicek v Roehr, 1986). 2.4.2 p lc oxy ha tan v s thng kh: S hnh thnh tt c cc acid hu c u tng di iu kin thng kh mnh. S hp thu oxy vi hm lng cao l cn thit cn bng chuyn ha gia s thnh lp citrate vi vic s dng hm lng ng cao. iu ny xut hin khi kh nng chui h hp ca A. niger khng to thnh NADH vi t l cao. Hn th na, s gn kt ca phc hp 1 t chui h hp ( NADH: ubiquinone reductase) tr nn ri lon trong iu kin

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tch ly acid citric (Wallrath v cs., 1991; Schmidt v cs., 1992). Trng thi ny c b p bi s hot ha mt con ng h hp khc m trong s ti oxy ha NADH th khng c kt hp to ATP (Zehentgruber v cs., 1987). Cc thnh phn khng xc nh ca con ng phi ph thuc vo s duy tr p lc cao ca oxy v mt s gin on ngn c th lm gim hot ng ca n ( Kubicek v cs., 1980). Nhng n lc nghin cu c ch quan trng ny gip trnh khi s d tha ATP m khng th s dng trong phn ng sinh tng hp. 2.4.3 pH: S tch ly acid citric bi Asp. niger chu nh hng ca pH tc l acid citric ch xut hin khi gi tr pH<2,5. gi tr pH cao hn th oxalic acid v gluconic acid c to ra nh mt sn phm ph. Gi tr pH bn ngoi ch nh hng rt nh n pH ca cytosolic (Legisa v Kidric, 1989). Khng ai chc rng s thay i pH nh chnh l nguyn nhn lm bt hot cc enzyme ni bo nhng n c th nh hng n qu trnh iu ha: mt vi enzyme c chng minh rng c im iu ha v ng hc thay i khi pH thay i ch mt n v (Banuelos v cs., 1977). Trong khi s to thnh gluconic acid khi gi tr pH ngoi bo cao c th d dng c gii thch bi tc ng ca pH ti bn ca glucose oxidase ngoi bo th s tch ly acid oxalic tip tc c gii thch. Nghin cu sinh ha s dng 14C nh du cho thy oxalate c ngun gc t oxaloacetate v oxaloacetate hydrolase ti cc v tr chuyn bit trong t bo cht (Kubicek v cs., 1988). Enzyme ny cnh tranh vi malate dehydrogenase cho sn phm ca phn ng l pyruvate carboxylase v mi quan h ca 2 enzyme in vivo ny quyt nh loi acid c to thnh. Oxaloacetate hydrolase c cm ng c hiu bi s gia tng nng pH mi trng ln t nht l 5 khi c s hin din ca nitrogen v phosphate (Kubicek v cs., 1988). Tuy nhin, hot ng ca cytosolic malate dehydrogenase isoenzyme ph thuc vo t l cytosolic NADH/NAD, s hnh thnh oxalic acid b nh hng bi hiu qu ca s ti oxy ha NADH. Mt tc ng khc ca pH cng c lm r (Roehr v cs., 1992): s d tha mt ATP v 3NADH xut hin khi qu trnh tch ly acid citric c din ra di iu kin c s chuyn i cn bng gia phn t glucose thnh citrate trong bc cui ca qu trnh ln men acid citric (Roehr v cs., 1981), iu ny l cn thit duy tr gradient pH gia cytosol vi mi trng ngoi bo. Mattey v cs. (1988) thit lp c s lin quan gia s gia tng ca ATPase trn mng t bo vi s duy tr gradient pH trong sn xut acid citric Asp. niger. Cc tc gi ny c mt tng th v l gi tr pH thp l yu cu ca qu trnh sinh tng hp acid citric, n ng vai tr nh l van lm d ATP, m ni theo cch khc n s dn ng cho s chuyn ha khng cn bng v s dng s nhim acid. S cm ng ca qu trnh sinh tng hp oxalic acid ti gi tr pH ngoi bo cao ph hp vi gi thuyt ny v s to thnh oxalic acid ch xy ra khi tha ATP hay NADH. Tuy nhin, gi thuyt ny cha c bng chng thc nghim. 2.4.4 nh hng ca phospho v nitrogen: Nng ca c phospho v nitrogen u c nh hng trong mi trng ti u dng sn xut acid hu c bi Aspergilli. Nhng ch khi tch ly acid trong nui cy m th s cn bng ca N, P v kim loi vt mi ng vai tr quan trng (Shu v Johnson, 1948b). Nn tng sinh ha c bn ca nhng quan st c bit n rt t: Dawson v cs. (1989) s dng hnh thc ln men fed-batch nh l mt th nghim

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khng nh rng vic ngn chn s chuyn ha nitrogen l yu t iu ha s sn xut acid citric. Tuy nhin, kin thc di truyn phn t c th c dng trn cc loi domain m rng trong A. nidulans (Caddick, 1992), khng nh ny cn c xc nhn di truyn. 3. QUY TRNH SN XUT: T khi kin thc v acid citric c cng b ln u tin trn sch Biotechnology (Roehr v cs., 1983) th k thut sn xut acid citric c nghin cu li bi mt vi tc gi nh Bigelis (1985), Milsom v Meers (1985), Milsom (1987), Verhoef (1986), Kubicek v Roehr (1992), Bigelis v Arora (1992), Smith (1994), Noyes (1969) cung cp thm nhng kin thc hu ch v ton din v nhng bn bo co c cp bng sng ch trong sut giai on t 1944 - 1968 (xem Lawrence, 1974). 3.1 Ging sn xut : V c bn s la chn ging khi u bng s phn lp ging t mi trng t nhin theo phng php vi sinh thng thng. Nhiu chng hp thnh b su tp cc chng cng nghip v cc chng c s qun l ca chnh ph. Hu ht cc nh vi sinh u bit rng Asp. niger c th c lm giu t mu t m cha khong 20% tannin c xem l ngun C chnh v tin hnh t 2-3 tun (Rippel v Baldes, 1940). V Asp. niger s hu nhng enzyme cn thit cho vic s dng tanninmt c tnh m ch c s dng trong nn cng nghip c - lm giu mi trng nui cy vi c Hnh 4: To dng bo t A.niger cht ny thng thu c kt qu l chng Asp. niger hoang di, thun chng. Sau khi p dng k thut nui cy thun chng, vic sn xut chng chn c th c tin hnh theo quy tc sau: - Trong mt qun th hp t nm, ch c mt vi bo t c c tnh sn xut mnh m c tch ra. - C 2 phng php chn lc: phng php chn lc bo t n v phng php cy chuyn. *K thut bo t n: Nui cy mt s lng ln nhng bo t n ch nh c pha long vi nng thch hp. Bo t t nhng mi trng v d t l hay ng thu tinh cha mi trng chun v ngun C, c th dng mt r ng lm ngun carbon, c dng cy chuyn. Th nghim ny c th c tin hnh khi nui cy b mt hay l, ng thu tinh c khuy trn, chun lng acid to thnh theo thi gian. Nhm t kt qu mt bo t nh ca Asp. niger s to ra khong 10.000 bo t, c th cy chuyn thu c kt qu cao hn. Trong sn xut, cc ging c hiu sut tng hp cao thng b thoi ho sau mt vi chu k ti s dng. Do , trong k thut nui cy t bo n, cn c bin php chng li hin tng thoi ho, ngha l thay i kh nng sinh acid citric theo thi gian lu li, nhn ging hay l mi trng nui cy.

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Nhiu th nghim tm ra nhng k thut chn ging d dng hn c vch ra. Phng php ca James v cs. (1956) bng cch ci thin th nghim trc y ca Davis (1948), Quilico v cs. (1949), bao gm vic to ra mt s lng ln cc bo t c nui cy n trong a petri s dng a giy lc c lm m vi mi trng. ng thi c cht ch th pH pht hin vng acid c to thnh trong qu trnh pht trin ca si nm. Tuy nhin, phng php ny cn ph thuc vo cc cht dinh dng v c thm vo; phng php ny c th biu hin s acid ha khi s dng cc mui sinh l (v d amoni sulphate) dn n kt qu sai lm, iu ny c pht hin bi Bonatelli v Azevedo (1982). S sai lm ny c th trnh nu la chn cc mui b sung v cht ch th thch hp (Amirimani v Roehr, kt qu ny cha c cng b) hay s dng cc loi thuc th c hiu (p- dimethylaminobenzaldehyde) pht hin s to thnh acid citric (Roehr v cs., 1979). Mt vi v d v cc chng c la chn da trn k thut bo t n: chng Wisconsin 72 (ATCC 1015), chng b m Wisconsin 72- 4 (ATCC 11414) c pht hin bi nhm Wisconsin (Perlman, 1949; Shu v Johnson, 1948a,b) v nhng nhm nghin cu ngi Canada (Martin v Waters, 1952; Clark v cs., 1966). Ti Chu u, chng u tin s dng trong cng nghip c sn xut ti Viena (Szucs, 1925; Bernhaur, 1928) v Prague (Bernhaur, 1928). Cc chng tm thy c p dng ti cc nh my ca Kaznejov gn Plzen, Cng ha Sc; ti Bernekiser GmbH, Ladenburg, c v Citrique Belge, tng t ti Nga v M (ATCC 10577). * Phng php cy chuyn: Dung dch cha bo t c tri thnh lp trn mi trng c cha c cht vi nhng nng khc nhau ty thuc vo qun th nui cy ang giai on ny mm hay pht trin. Kt qu ca phng php chn lc ny ch ng mt phn trn cc qun th c nhng c tnh khc bit, hoc da trn c im khoa hc c bn hoc da trn kinh nghim. V d nh: s chng li gi tr pH thp (Currier, 1917; Bernhaur, 1929), s gia tng nng acid citric (Montan und Industialwerke, vorm. J.D. Stack, 1933; Bernhauer, 1934; Leopold, 1958), nhit (Leopold v Valtr, 1970), desoxyglucoses (Kirimura v cs., 1992), s thch nghi vi vic Hnh 5: Nm giai on pht gia tng nng ng (Doelger v Presscott, trin sinh bo t 1934; Schrefere- Kunar v cs., 1989; Xu v cs., 1989a), s chn la v thch nghi vi ngun C ch nh nh l vt liu th (Pelechova v cs., 1990). Quy trnh ny thng c dng b sung kt hp vi k thut bo t n ci tin ging v cung cp mt vi ging cho nhng ng dng chuyn bit. Nh lu trn, rt kh duy tr nhng c tnh mong mun ca chng sn xut trong mt thi gian di m khng lm mt i kh nng pht trin v sn xut acid. Nhiu phng php khc nhau duy tr ging c vch ra v khng c mt qui trnh chung no c p dng. Mt quy trnh thng xuyn c p dng trong thc tin bao

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gm vic nui cy trn mi trng hnh thnh bo t n nh, thu nhn cc bo t v trng trong giai on sy (v d nh trn ln vi cc vt liu tr dng bt), sy bo t vi khng kh v trng hoc a chng vo trong cc t sy n nhit. Cui cng, bo t c bo qun trong cc ng thy tinh c bt kn hoc trn vi t kh cn, ct kh, carbon c hot ha bng st t do ti nhit thp hay nhit phng. S bo qun bng nhit cc thp c ngh (Symonyan v cs., 1988) nhng ch tr mt vi bn bo co c cp bng sng ch vi rt t thng tin c gi tr. S n nh ca chng ging khi s dng quy trnh ny thng l vi nm nhng phi thng xuyn phn tch mu bi c th xy ra s nhn ln ca bo t v cn tin hnh th nghim ln men quy m nh. Mt vi tc gi ngh ci tin chng bng cch gy t bin gen. Tuy nhin c rt t chng ny c s dng v rt nhiu cng trnh c cng b nhng chng c rt t gi tr thc t. Chiu tia UV l cch thc n gin nht v v vy n l phng php c a chung. Bo t c chiu c nng rt long (10 7- 108 mL-1) hoc nui trn mi trng agar c bng cch s dng n st trng bnh thng. Theo Gardner v cs. (1956), t bin UV mc cao chng b m (Wisconsin 72-4) c pht trin trong cc bc t bin chng. Mt khc, Ilezuk (1969, 1970) a ra bng chng rng s pht trin gii hn dn n t bin khuyt dng l nguyn nhn lm gia tng kh nng sn xut acid. Mt vi tc gi khc vch ra mi tng quan gia s gia tng sn xut acid vi t bin cm ng s thay i hnh thi A. niger. Mt vi phng php ci tin ging khc c th l chu trnh cn gii tnh, c a ra u tin cho A. niger bi Pontecorvo v cs., (1953). Tuy nhin, cc d hp t nh th lng bi gp phn ci tin chng cng nghip (Chang v Terry, 1953; Ciegle v Raper, 1957; Ilezuk, 1971a). Theo bn bo co ca Ilezuk(1971b) v Das, Roy (1978), th lng bi c hiu sut sn xut acid citric cao khi cha m ca chng th n bi, nhng khuynh hng ny thng khng bn (Bonateli v cs., 1983). Dung hp protoplast Aspergillus bao gm c A. niger c vch ra bi nhm ca Ferenczy v Kevel ti Szeged, Hungary (Ferenczy v cs., 1975a,b; Ferenczy v Farkas, 1980, Ferenczy, 1984) v mt nhm khc ca Feberdy ti Nortingham, Anh (Anne v Feberdy, 1975, 1976; Feberdy v Ferenczy, 1985; Feberdy, 1987; Powell v cs., 1993) l mt cng c y trin vng (xem Gadau v Lingg, 1992) iu khin di truyn ca A. niger nhm tng hiu sut sn xut acid citric. Mt vi nghin cu gn y: - Kirimura v cs. (1986, 1988a) nghin cu s dung hp protoplast ca chng sn xut v to ra c chng c kh nng sn xut acid vt tri so vi chng b m khi ln men trn pha rn nhng khng c tc dng trong nui cy chm. S dng phng php dung hp bng polyethylen glycol t l phn chia l vo khong 5% (Martin v cs., 1990). S n bi ho to ra nhng chng tri hn c th s dng trong ln men chm (Kirimura v cs., 1988b; Ogawa v cs., 1989) Tt c cc nghin cu ni ting lin quan n di truyn phn t ca Aspergilli u c bt u t thp nin 1980. Mt vi thnh tu chnh c th c k n: pht trin ca mt vi chng mi c ch nh khng cc thnh phn bt li trong ln men vt liu th, c kh nng s dng vt liu th c bit (tinh bt, cellulose, pectin, mt vi loi vt liu ph thi (v d nh whey- cht lng cn li sau khi sa chua ng), ci tin qu trnh ln men. Nhng mc tiu t ra l cn hiu bit tng tn v c ch iu ha qu

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trnh tch ly v bi tit acid citric, iu ny c th gip ci tin chng vi sinh nh l s kt hp gia nm men mt sinh vt n bo vi A. niger. Nhiu bn bo co c tuyn b hon ton trong thi gian gn y nh l Bennet v Lasure (1985, 1991), Nealeinen v Penttila (1989), Bennet v Klich (1992), Powell v cs. (1994), Martinelli v Kinghorn (1994). 3.2 Nhn ging bo t: Nhn ging quy m ln l mt trong nhng khu quan trng v quyt nh s thnh cng khi vn hnh cc nh my sn xut acid citric. V d mt nh my mun sn xut 20.000ta-1 th i hi cn phi cung cp 30 300kg bo t. Mt s bo t c sn xut qua qu trnh nui cy chng mong mun trn nhng khay cn cha agar c trong phng kn di iu kin kim sot s thng kh v m. Cn duy tr iu kin v trng mc cao bng cch s dng h thng cung cp hi nc v cc loi thuc ty. Mi trng hnh thnh bo t cha mt t l thch hp ca ngun carbon v nitrogen nh mt ng, mui, peptones Cn ch loi tr cc ion kim loi gy bt li cho mi trng. Bo t t dng c bo qun c thi vo mi trng hnh thnh bo t kh (10mg bo t/m2). S hnh thnh bo t bt u ti 300C trn nhng lung khng kh v trng (thp hn 0.5 m/s) m c cung cp khong 70-90%, sau khong 6 ngy thy kt qu bng mt mu thm, l nhng m bo t nm si dy c. Bo t c sy v trng nng cao mc n nh di iu kin bo qun. Sn lng bo t sy kh l vo khong 60 - 120g/m2. Cc bo t sy kh c thu hoch cn thn bng dng c ht kh v c ct tr mt ni lnh kh. Mc d c rt t thng tin v cc th nghim lin quan nhng do duy tr c kh nng sn xut acid vi hm lng cao m phng php bo qun ny vn c duy tr trong nhiu nm. Tuy nhin cn phi tin hnh kim tra li cc bo t bo qun trc khi a vo sn xut. Mt vi phng php nhn ging bo t khc c ngh nh s dng cm (Foster, 1949) hay cc loi ht nh bp (Schweiger, 1965). S hnh thnh bo t trong mi trng lng cng c ngh. 3.3 Vt liu th: C rt nhiu loi carbohydrate c th c s dng trong sn xut acid citric. Chng c chia thnh hai loi: 1.Vt liu th vi hm lng tro thp, trong cc cation c th c loi b bi cc qu trnh chun. Loi c cht ny c th c k n nh ng ma hay ng c ci v tinh bt c lm tng hoc gim tinh khit nh dung dch dextrose v dextrose kt tinh. 2. Vt liu th vi hm lng tro v tng s cc c cht khng ng cao nh mt ng ma v c ci, cc loi ng tri cy. Bn cnh cc loi vt liu th r tin, th cc vt liu ph thi r hn nhiu nh dung dch cn sau khi sa chua ng (whey), khoai lang, bt m, bt la m, hoc cn ng ma c ngh nh l ngun nguyn liu. Vic s dng whey hay whey chy trn c nghin cu ti New Zealand (Hossain v cs., 1983, Maddox v Archer, 1984, Somkuti v Bencivengo, 1981). Trong hu ht cc trng hp cc loi vt liu th khc ch c tc dng cc b v him khi c cht lng ph hp to ln. Thnh phn ca mt ng ph thuc vo nhiu nhn t khc nhau. V d loi c ci hay ma phng php trng (cht dinh dng, thuc tr su), iu kin ct gi, qu

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trnh sn xut, phng php tin hnh (v d s chuyn ch, s khc nhau v nhit ), vic s dng cc thnh tu c trc Nhiu tc gi th phn ct mt ng m t cc tiu phn c hi cho ln men acid citric (nh l Zakowska,, 1991). Mc d xut hin nhiu phng php phn tch khc nhau, nhng cng vic vn rt cn thit nh gi s ph hp ca cc loi mt ng i vi s lng, kh nng sn xut v hot ng iu chnh cho ph hp. Mt ng ma vi hm lng tro cao (7 10 %) v cc cht hu c khng phi l ng (20 25%) c xem l km ph hp cho sn xut acid citric quy m ln. Mt ng c ci, l mt loi vt liu c a thch Chu u c th cha n 80% cht rn vi 50% sucrose v khong 20 30% thnh phn khng ng. Tuy nhin trong thi gian gn y nhng n lc ln sn xut ng c ci trong cng nghip gia tng s lng ng t nhng lng tri cy ph hp bng nhng quy trnh mi, mt ng c a vo th trng (v d mt ng Quentin) vi hm lng ng <50% v hm lng cc thnh phn khng ng tng ln khin n khng cn ph hp cho sn xut acid citric vi quy m ln. Nhiu phng php sn xut v tinh luyn ng ra i nhng u b loi b mc d ng trong cc vt liu th thng khng nguyn cht. V c bn, cc thnh phn trong mi trng do Currie ngh nm 1917 c s bin i. Mi trng ca Currie bao gm 2-2,5g/L NH4NO3, 0,75- 1,0 g/L K2PO4, 0,200,25g/L MgSO4.7H2O. Trong nn cng nghip hin i ngun P, N nh kh NH3, amoni hydroxide v acid phosphoric r hn, v vy, chng c a chung hn. Kim loi vt, c bit l Zn, Fe, Mn c xem nh l cht nhim ho hc. 3.4 Quy trnh Koji y l quy trnh n gin nht cho sn xut acid citric c pht trin Nht Bn (Yamada, 1965). N gn ging nh hnh thc ln men trong mi trng rn, da trn b quyt ca qu trnh Koji truyn thng. Vt liu th l tinh bt tha ti cc nh my sn xut tinh bt, nh ph thi ca khoai lang, go v cc cht tng t. Quy trnh ny c s dng nhiu Nht Bn, khong 60.000 tn phn x cn li ca khoai lang cho kt qu qu trnh tng ng 30.000 - 40.000 tn tinh bt, gi c c tnh tng t nh mt ng. Tinh bt c trn vi nc thu c hm lng nc khong 65-70% v c x l vi hi nc nng lm nho tinh bt. H tinh bt v trng to ra c a ln khay hoc trn b mt ca bung ln men v tin hnh cy bo t A.niger ln trn vt liu. Trong trng hp vt liu c x km, b ma hay b la m c th c thm vo. Khay thng ch cha c 10L( di cc chiu 0,6; 0,4; 0,05 m). V vy, n ch c kh nng s dng trong sn xut nh. 30 oC v gi tr pH khong 5,5 th amylaze ca nm mc tin hnh qu trnh ng ho, sau chuyn i thnh acid citric vi du hiu l s gim pH xung cn khong 2. Sau t 1-2 tun, khi ln men cha acid c p v ly trch vi nc nng trong mt my chit c dng nc ngc n gin. Kt qu, l thu c dung dch cha khong 4-5% acid citric. S thnh cng trong qu trnh ng ho v acid ho to acid citric i hi phi c chng A.niger c bit, c 2 kh nng ny. Trong trng hp ny, cn ch cp n cc chng c chn lc v kim tra trong len men chm (Jin v cs., 1993; Seel v cs., 1993). Thm vo , chng cn chu c nng cao ca kim loi nng trong nhiu vt liu ph thi. Mt vi chng c bit c ngh p ng nhng yu

17

cu ny. Mt khc, nh hng ca ion kim loi thng ch gii hn trong mt vi qu trnh ln men (Shankranand v Lonsane, 1994). Mt vi qu trnh tng t c vch ra khi s dng ng v b ng ma (Lakshminarayana v cs., 1975) v mt ng ma (Chau, 1978) ti n . Mt vi cch thc s dng vt liu thi khc mi c pht trin gn y nh l: da hng (Yo, 1975; Usami v Fukutomi, 1977), nho vn (Hang, 1988), bt to nghin (Xu v Hang, 1988). 3.5 Qu trnh nui cy b mt y l hnh thc sn xut acid citric c in, mi trng nui cy c cha trong nhng b hp v nm pht trin thnh h si trn b mt mi trng. Nn to ra s tip xc ln gia pha lng, h si nm v khng kh cung cp lng O2 yu cu. Khi tin hnh nui cy ln, vic o trn pha lng nh gradient nhit v nng theo chiu su cht lng. Nh vy, trong h thng phng ln men c cha mt s lng ln cc khay ln men cn c trang b mt h thng gi bn vng. Cc khay thng c lm t nhm c tinh khit cao hoc t thp khng r. Khay c th c nhng kch thc khc nhau t 2m x 2,5m x 0,25m n 2,5m x 4m x 0,25m vi mc cht lng thng thng t 0,1 - 0,18m. Th tch cht lng trong mi khay t 0,5 - 2 ln khi lng khay nn cn c cc h thng gi . Cc bung ln men c gn h thng thng kh hiu qu vi nhim v chnh l iu ho nhit , ngoi ra cn cung cp O2 v iu ha m. Khng kh c thi vo phng ln men theo tng lp xuyn qua nhng ng dn ti tt c cc chiu cao ca phng ln men. Tng v sn dng ph vt liu nn d c ra v chu c cht ty cng tt nh chu acid, ca s nn c ng cht. Cc phng ln men nn c v trng bi cc cht ty ra th lng (phun) v th kh (nh kh sulhirdioxid hay folmaldehyde) v thng xuyn ch ra tng v sn vi cc cht ty dng lng (xt hay dung dch folmaldehyde). Tuy nhin theo th nghim ca tc gi ny th vic kh trng trong cc phng ny khng hiu qu v con ngi vn phi i vo phng ln men. iu ny dn n s nhim Pennillia, cc chng Aspergillus khc, nm men v vi khun sinh acid lactic. Mt ng c s dng trong ln men b mt c x l qua cc bc sau: + Vt liu kh c pha long mt ng t khong 120 180 kg/m 3, cc cht dung dch c b sung vo, dung dch c acid ho bng cch s dng acid phosphuric thu c pH l 6.0 - 6.5, un nng khong trong 15 45 pht. Sau , postassium hexacyanoferrate (HCF) c b sung vo dung dch ang nng. HCF c hai tc ng quan trng: gim hoc to thnh phc hp vi cc kim loi c hi (Fe, Mn) v hot ng nh l mt cht c ch chuyn ho hn ch s tng v cm ng s sn xut acid (Martin, 1955; Clark, 1964). V nhu cu HCF ph thuc vo cht lng v loi mt ng s dng, v vy tt hn ht nn tin hnh s dng quy m nh vi nhiu nng HCF khc nhau quyt nh lng HCF cn b sung, lng HCF t do trong qu trnh x l vi lng HCF c th c quyt nh bi phng php ca Marier v Clark (1960). Phng php HPLC cng c xem xt n (Selahzadch v Roehr, kt qu chua c cng b). Lng HCF tha c th nm trong khong t 5 100 mg/l cho mt c ci ng v 5 200 mg/l cho mt ng ma (Clark, 1964). Lng chnh xc c quyt nh bi th nghim ln men quy m nh vi chng c chn.

18

Dung dch mt ng tch tr dng nng hoc c s dng ngay lp tc a vo cc khay khi nhit vo khong 40 oC. Vic phun khng kh m vo h thng c bt u t giai on ny kh hi nc ng. Tc lm lnh quyt nh s lng ln men (Schimitz, 1976), tc ny nu nh hn 1oC/h s lm hn ch ti a thit hi. Vic cy chng cng c tin hnh theo nhiu cch khc nhau: Mt s lng ln ht bo t (khong 100 - 150mg/m 2) c a vo trong h thng bng cch pha long hoc a cc bo t sy kh trn cc khay. S ny mm i hi thi gian t 1-2 ngy v trong sut thi gian qu trnh lm ngui c th b cn tr do s gia tng nhit dng kh a vo. Trong sut giai on ny v bc lm lnh trc vic trnh tp nhim ng vai tr quyt nh. V vy, cn s dng cc phng php v trng c bit v khng kh a vo trong giai on ny (chim 21% tng lng khng kh) phi qua cc thit b lc, vic lc khng kh din ra trong sut qu trnh ln men. Sau khi bo t ny mm, cc h si nm pht trin lan rng thch thc y bin dng cn thit ca qu trnh sn xut acid citric. Mt si nm dy nhng khng bao ph hon ton v iu ny c th lm gim kh nng sn xut acid. Khi mi bt u hnh thnh si nm, s to thnh acid citric ng thi vi s gim gi tr pH xung nh hn 2 v qu trnh ny c th hon thnh trong thi gian t 6-8 ngy. Vic trn ln dung dch ln men l cn thit duy tr nhit cc lp ln men khng cch nhau qu 3oC. Trong sut qu trnh t l acid to ra trung bnh l vo khonh 0,5 kg CAM/m3h (CAM c ngha l citric acid monohydrate, FW 210.14 c bn rng ri Chu u, CAA l acid citric anhydrate, FW 192.13 c bn M. Nhn t chuyn i l 1.094). Cng thi gian ny, lng nhit thot ra ngoi khng kh nm trong khong t 600-1.100 kg/m2h. V kh nng hp thu nhit ca khng kh km nn t l thng kh it nht l 4V khng kh /1V mi trng trong mi pht. V vy, cn phi tnh ton mc thng kh trong qu trnh ln men b mt bo m yu cu v lm lnh. Mt khc, lng nhit to ra trong qu trnh ny bao gm c lng nhit lm bc hi nc ca c cht trong mi trng 30-40%. Nh vy, qu trnh ln men acid citric hiu sut 75% cn nng ng l 160kg/m3, qu trnh ln men lng c th thu c nng acid citric 200-250kg/m3 Khi kt thc qu trnh ln men, cc khay c ly ra khi cc phng ln men, chuyn sang giai on tinh sch v lp li qu trnh sn xut. Cc khay trng c th c thay th xoay vng c th chun b cho qu trnh ln men sp ti. 3.6 Ln men chm: Sn xut acid citric vi quy m ln s dng phng php ln men chm tip tc c gia tng mc d n i hi nhiu k thut tinh vi phc tp. Ngi ta nh gi khong 80% lng acid citric sn xut trn ton th gii l do phng php ln men chm. u im ca phng php ny c th k n nh l s lng v kh nng sn xut cao cng nh gim chi ph thu nhn cng c dng bo cha cho s tht l c rt nhiu thng s hot ng c nghin cu li b sung vo k thut ny. Cc sinh vt thng rt nhy cm vi s chuyn i cc kim loi vt, c bit l Fe3+ v Mn2+ (Clark v cs., 1966). Nng ca Fe3+ l 1mg/l v ca Mn2+ l 2mg/l (Snell v Schweiger, 1949). Cn nhn thc r iu ny khi chn vt liu cho vo fermenter v

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sp t h thng ngoi vi tng t nh chun b ngun cung cp nc cho nh my. X l ph hp c cht C l iu khng th thiu v s c din t phn di. Nng O2 phi c duy tr trn 25% trng thi bo ho v s cung cp gin on O 2 s gy hi cho ln men. Ln men chm c th c tin hnh trong cc l khuy trn truyn thng nh nhng fermenter dng thp. Sau ny, bioreactor c la chn s dng nhiu hn bi v n c nhng u im vt tri so vi l phn ng khuy trn: gi thnh r hn, c th xy dng vi kch thc ln, hot ng m khng cn h thng khuy ln t gim kh nng tp nhim v sn xut t nhit hn vi huyn ph rn. Fermenter khuy trn c kh nng cha 50 n 100m3 trong khi fermenter dng thp c th cha th tch ln n 1000m3. V s nhy cm ca A. niger i vi hm lng nh cc kim loi nng c th c ho tan t vt liu xy dng v s n mn ca acid citric, v vy vic s dng cc kim loi khng b n mn trong tt c cc phn ca h thng ln men l iu cn thit. Vt liu tt nht l AISI 316 Ti khng r (1.4571, Din X10CrNiMoTi 1810) hoc cc loi vt liu chuyn bit tng t. Thm vo , fermenter v cc ng ngoi vi nn c chng r bi cc qu trnh bnh thng nh lm ph y vi hn hp dung dch acid sulfuric v acid nitric v thng gi trong khong 20h. Fermenter cho qu trnh ln men acid citric khng c lm khi tn ti p sut ng v s kh trng thng c thc hin bng hi nc sau khi l phn ng c ra sch m khng chu tc ng ca p lc (ch khong 0,2 bar). Qu trnh lnh c th c thc hin bi cc hi nc ngoi vi bm trn b mt ngoi ca fermenter.

Hnh 6: Quy trnh ln men chm sn xut acid citric t mt ng 20

S thng kh c thc hin bi cc phng php xua kh bnh thng ca cng nghip ln men nm mc. Fermenter thp thng c t l gia chiu cao v ng knh l 4:1 n 6:1. V vy, s gia tng p sut thu tnh lm gia tng kh nng ho tan ca O 2. Thng thng vic duy tr nng O2 cao hn 25-50% trong sut qu trnh l tng i d dng. S khuy trn c th gim hiu qu trong cc termenter dng thm nhng vn ny him khi gy nguy him. Cc ng thng gi c th c xy dng bn trong nhng tc ng ca chng b gii hn khi tin hnh nui cy theo hnh thc fed-batch. Cc fermenter c th c cy chng ging theo nhu cch khc nhau. Dung dch bo t trn ln vi Tween lm cho s phn tn d dng nng bo t cui cng l 2-25 x 106 bo t/L. S bo t trc t 6 - 8h c th lm bc I ca qu trnh ln men ngn li. Hay theo cch khc, h nm si c th c pht trin trc trong cc ging ht (Tveit, 1963). Theo cch ny, h nm si c sn xut trong cc pellet hoc flock c kch thc khng i. Nh ngh trn, loi vt liu th c s dng nh l ngun C quyt nh thao tc ca quy trnh. 3.6.1 Hn hp nhng ng v ln men fed-batch Mc d nhng th nghim u tin c tin hnh theo phng php ln men chm s dng nguyn liu ng tinh khit, cc quy trnh s dng mt ng nh mt ngun c cht th, r tin c nghin cu ng dng hu ht trong vic sn xut acid citric. Hn ch trong trng hp ny l tn ti nhiu cc hp cht khng phi l ng. Ging nh ln men ethanol, tc ln men acid citric b gim t ngt khi gia tng nng ng. V vy, phng php ln men theo m hin ti ch c s dng trong trng hp nng nguyn liu ban u thp. Do , phng php ln men bn lin tc c a chung hn hin nay. Trong ln men theo m, nng nguyn liu th ban u c th l 250kg/m 3, trong nng ng vo khong 120kg/m3. i vi phng php ln men bn lin tc nng ng trung bnh vo khong 150kg/m3. Ban u ch tin hnh vi m dch ln men, trong nng ng vo khong 100 kg/m3. Chnh v vy lng r ng ban u phi cha tt c l 200kg/m3. Kt qu l mt lng 130 kg/m3 acid citric (loi CAM) c thu nhn trong 6 ngy, vi nng sut ln men l 0,9 kg CAM/m3.h. Qu trnh trn c th dao ng trong mt gii hn nht nh. Theo HUSTEDE v RUDY (1976) mt lng HCF cn thit c th c thm vo qu trnh ln men c thun li hn, c th l vo giai on x l nguyn liu r Hnh 7: S ln men citric

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ng. Ngoi ra, vic b sung cht c ch HCF c th cn thit trong mt s giai on pht trin ca nm mc. 3.6.2 Ln men theo m s dng nguyn liu ng tinh - Quy trnh ln men s dng ng tinh khit c bit n u tin nhng him khi c s dng v gi nguyn liu th cao. Do vic gia tng tim kim, pht hin nhng ngun nguyn liu r tin ,c gi tr kinh t cao l rt quan trng hin nay. - Vi mt ng cng l mt ngun c cht (nguyn liu) quan trng hin nay Mt ng trc tin c x l thnh dng dung dch chun b cho ln men. Mt ng cng nh cc nguyn liu ng tinh khit khc (nh ng ma, c ci ng)c th trnh c tc hi ca cc ion kim loi bng cch x l vi nha trao i cation trong cng nghip. Theo ni dung trnh by trn, khng cn b sung cht c ch nu nng ion st di 1 mgL-1. Ch cn mt lng nh cht c ch HCF (1-10mg L-1) l gy ra s chuyn i t s sinh trng ca nm mc thnh s sn xut. - Trong 1 quy trnh ln men in hnh mt lng 300kg.m -3 dung dch ng c cht s c chun b 400C, pH: 1,6-1,8 (s dng axit sunfuric) dung dch ng ny s thc hin trao i cation mnh nhm lm gim ion st v mangan ln lt xung di 200ugL-1 v 5ugL-1. Sau , dung dch (ln men) ng c lc, thanh trng v chuyn vo ni ln men. Lng ng sucrose tng cng trong dung dch ln men c th dao ng trong khong 160 - 250kg.m-3, trong khi vi ng glucose trng hp con s ny 120 - 160kg.m-3. Do trong qu trnh ln men ng glucose b bin i, dn n ko di thi gian ln men. Sau khi b sung mui dng cht v HCF, pH ca dung dch tng ln 2,5 - 3,0. to iu kin qu trnh ln men thun li ta c th b sung kh amoniac (NH3) hay amoni hydroxide (NH4OH) nh mt phn dng cht. - Lc ny, cc bo t nm mc hay h si nm tng trng trc c cy vo h thng ln men, lng bo t nm mc c th dao ng trong khong 5 - 25.10 6 bo t/lit. Cho n khi bo t ny mm, s thng kh phi c duy tr mc khong 0,1 vvm.Trong sut giai on sinh trng ca h si nm c c trng bi cc cu ni ngn phn nhnh cng vi b mt li ca cc t bo . - y cng l iu kin tin quyt gii hn s tng trng ca nm mc v m rng sn sut axit citric. - Hnh thi nyc c do s hot ng ca cht c ch HCF v s gim nng 3+ Fe v Mn2+. Nu nguyn liu kh ban u khng c x l hay cc cht dinh dng a vo khng sch s dn n nng cao ca cc ion Fe 3+, Mn2+ c trong dung dch ln men v vic gii hn tng trng ca nm mc s khng c thit lp, hin tng ny c trng bi s xut hin nhng cu ni di, khng phn nhnh cng s gn kt cc cu ni li vi nhau nh t nhn v mt hnh thi ca nm mc. Ch khi du hiu hnh thi u tin c pht hin (hnh thnh cc cu ni di, khng phn nhnh) nh quan st di knh hin vi th hin tng ny c th c khc phc bng cch b sung cht c ch. - Vic to cc mu li trn dng nm mc c bit ny i hi 2 - 3 ngy cng vi s gim gi tr pH xung 1,6-1,8. Trong nhiu h thng, pH c duy tr gi tr ln hn 2 trong sut qu trnh ln men trnh cn tr ca pH thp. Sau thi gian 2 ngy, s to acid citric c bt u v nn c kim tra thng xuyn bng phng php chun . Nh cp trn, s thng kh c ngha rt quan trng trong ln men to acid

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citric. Vic dng thng kh, d ch trong mt thi gian ngn, cng lm gim ng k qu trnh ln men. - Tu thuc vo nng ng ban u, vic sn xut acid citric kt thc vo gia ngy th 9 v ngy th 12 ca qu trnh ln men. Nng sut ln men thung ln hn 1kg.m-3h-1 vi sn lng CAM trn lng ng tiu th vt qu 90%. 3.6.3 Ln men dng hn hp (kt hp) - Qu trnh ln men s dng nguyn liu ng tinh sch rt nhy cm vi ngun c cht ban u cng nh chu nhiu nh hng bi mt lng nh cc cht c ch. Chnh v vy lm bng n cuc cch mng tm kim mt quy trnh sn xut khc thun li hn. - S vic ny dn n hnh thnh phng php ln men bn lin tc. u tin mi trng mt ng c x l vi nng trung bnh (60-100kg.m-3) cng vi lng cao cht c ch HCF. Sau dung dch ng tinh sch nng cao (300kg.m-3) c a vo ni ln men. Cc bc tip theo tng t vi quy trnh m t trn. Sn lng v tc ln men ca phng php ln men bn lin tc tng ng vi phng php ln men theo m s dng ng tinh sch. 3.6.4 S ln men t cc nguyn liu khng tinh - Vic tn dng c cc sn phm ph thi t nhiu lnh vc khc nhau nh mt ngun c cht ln men nh du mt bc tin tiu biu cho ngnh cng nghip sn xut acid citric. - Vo nm 1961, trong khi c gng ngn chn s xm nhim, nh khoa hc Schweiger quan st thy nh hng c hi ca ion st trong dch ln men c th b hn ch bng cch s dng ion ng vi mt t l nht nh. Nu ion st khng c loi b xung mc cho php th vic s dng mt lng cao ion ng sau s gy hi cho qu trnh ln men, lm gim nng sut. Bn cnh , mt s nh khoa hc cho rng mc tiu ch yu ca hot tnh ion ng l mangan kim loi. - Vic s dng t nguyn liu th qua tinh sch v b sung ion Cu 2+ kim tra hot tnh c hi ca ion st/mangan, c trng cho cng ngh Miles ni ting, c th c xem nh mt cng ngh u vt nht hin nay. - Nhng th nghim nhm a cc cng ngh truyn thng nh s dng cc t bo c nh, ln men lin tc vo trong quy m cng nghip cho n nay vn cha thnh cng. Mi trng ln men lin tc vi cc t bo nm mc khng bt ng, s dng nguyn liu ng glucose, thu c nng acid citric hai gi tr 2 v 6g.L -1 vi nng sut vo khong 0.4gL-1h-1 ti tc pha long 0.075 h-1.. Tuy nhin nng ng cn li sau ln men o c l 40gL-1. Mt vi nhm nghin cu a ra cc phng php khc nhau nh: t bo nm mc bt ng bm trn vt mang thu tinh hay gn vo calcium alginate Cho n nay, hn ch ch yu ca cc phng php ny chnh l:vic lm gim tc pha long c c lng ng d ph hp vn lm gim nng sut v nng ng sn phm trong sn xut quy m cng nghip. 4. S THU HI V TINH SCH SN PHM - H khun ty t qu trnh nui cy b mt d dng c tch ra khi dung dch ln men, sau c lm tan r mt cch cn thn ri a thng vo trong thit b ty ra. Vic ra sinh khi nm mc l rt cn thit v nguyn liu thm nc gi li c

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khong 15% sn phm acid citric to ra trong qu trnh ln men. Nc ra ny s c b sung vo dch ln men. K n, lng sinh khi nm mc ny c lc kh nc, sy kh lm thnh thc n gia sc giu protein. - Trong quy trnh nui y chm, vic tch ri mng mc ra khi dung dch ln men kh hn nhiu v c thc hin bi mt thit b lc tch in vnh ai. Giai on lc ny thc s rt kh khn do cc cht nhy h si nm c tit ra trong sut qu trnh ln men. Trong nhng trng hp nh vy, cc cht h tr lc s c b sung. Nu h si nm c s dng lm thc n gia sc th cc cht h tr lc phi tiu ha c. Ngoi ra c th nhc n gypsum - mt cht h tr lc trung gian c tch ly ch yu trong giai on thu hi v tinh sch sn phm acid citric. Cc bc lc thng c kt hp vi vic gy kt ta oxalate - mt cht phi c loi b bi c tnh ca n. Gypsum c s dng s phn ng vi oxalate to thnh calcium oxalate. Gypsum c th c cho vo vt mc m vn khng s ri ro i vi vic kt ta oxalate. - Ngoi ra, dch ln men cng cha hn hp cc cht khng mong mun c trong nguyn liu th ban u cng nh cc hp cht lm gim sinh khi nm men. Chnh v vy vic tinh th ho trc tip acid citric i hi hn na cc bc tinh sch trong iu kin th nghim. Mt khc, mt s quy trnh tin hnh thu hi mui kim ca acid citric bng cch tinh th ho trc tip t dch ln men, sau trung ho ln lt cc hp cht kim loi kim thu nhn mui n, i hoc mui ba citrate. Cui cng cho bc hi cc mui trn cn khong 400gL-1, c tnh nh acid citric. - Nhn chung qu trnh thu hi v tinh sch acid citric c th c hon thnh bi ba phng php sau: kt ta, tinh ch v hp ph (ch yu s dng nha trao i ion). - Gy kt ta, phng php c in, c thc hin bng cch b sung calcium oxide to ra tricalcium citrate tetrahydrate ho tan. Thnh cng ca vic gy kt ta ph thuc vo mt vi nhn t sau: nng acid citric, nhit v pH trong sut qu trnh gy kt ta. Nu kt ta hiu qu th lng ln cc cht bn s nm li phn dung dch v c th c loi b bng cch lc kt ta. - Sau khi lc v ra, calcium citrate thu c tip tc cho phn ng 50 0C vi acid sulfuric nng trung bnh (60-70%). Tip theo, calcium sulfate (gypsum) v acid citric t do c tch ring nh lc. Phn nc lc sau c x l vi than hot tnh loi b tp cht mu v c cho qua nha trao i ion loi calcium sulfate d cng cc ion kim loi. Vic x l dch lc th vi hydrogen peroxide trc khi cho qua ct trao i ion cho hiu qu lc cao, c bit loi b hon ton cc hp cht carbon. Sau tin hnh bc hi dung dch acid citric tinh khit nhit di 40 0C trnh s caramen ho. - Citric acid monohydrate (CAM), sn phm ch yu c bn Chu u, c hnh thnh nhit di 36,5oC. Trn nhit ny, citric acid anhydrate (CCA) c to ra. Sn phm CAA c thng mi ho trn khp nc M v ng Nam . - Hn ch ng k ca vic thu hi acid citric chnh l mt lng ln gypsum c to ra (khong 1tn/tn CAM) b vt b, tr thnh rc thi. Bn cnh , vic s dng mt lng ng k nha trao i ion c th to ra cc ngun gy nhim mi trng. Tt c u lm gia tng gnh nng cho cng tc bo v mi trng vn rt nan gii.

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5. SN XUT ACID CITRIC T NHNG NGUN C CHT V SINH VT KHC Cho ti tn nhng nm 1970, Asp. niger hu nh vn l sinh vt duy nht c s dng trong sn xut acid citric, mc d khng c ngha l ch c loi sinh vt ny v nhng loi h hng gn gi ca n mi c kh nng sn xut acid citric. l A. awamori, A. carbonarius, A. fonsecaeus, A. foetidus, A. phoenicis thuc nhm Nigri Gams v al. (A. niger nhm Thom v Church, 1926). Nhng cng c mt ngoi l l A. wentii thuc nhm Wentii Gams v al. (A. wentii nhm Thom v Raper, 1945). A. wentii c pht hin v c cp patent bi kh nng sn xut acid tt (Waksman v Karow, 1946; Karow v Waksman, 1947) nhng khng c ng dng trong cng nghip. Perlman v Sim (1960) b sung thm vo danh sch cc ging A. clavalus, A. fumaricus, A. luchensis, A. saitoi, A. usamii. Cng tng t nh vy, nhng loi nm si khng c tn dng mc d mt vi patent lin quan c cp ti: Kinoshita v cs. (1961a) nghin cu k cng dng ca Penicillium janthinellum var. kuensanii (ATCC 13154) v P. restrictum var. kuensanii (ATCC 13155). Nng sut t trn 80% trn c cht ng tiu th (r ng) sau 7d ln men. Trong cng trnh ny, tc gi cng c tham kho nhng Pencillia khc trong Manual of the Penicillia ca Raper v Thom (1949) v kh nng sn xut acid citric. Trng hp khc na l trong patent ca Kinoshita v cs. (1961a) nu ra mt vi ging khc thuc Trichoderma viride, nh ging ATCC 13233 cng c th cho ra acid citric. Trong mi trng c hm lng c cht carbon 100g/L (khoai ty th hay khoai lang), nng sut t 75% trn tinh bt thm vo v 85% trn tinh bt tiu th, trong 7d ln men. Li th ca nhng quy trnh ny l s c sn amylase v cellulase trong c th sinh vt, khng nhy cm vi kim loi nng, khng c sn phm ph c to ra. Tuy nhin, nhng quy trnh nh th vn khng i vo hot ng cng nghip. Nhng sinh vt y ha hn khc cng c tm ra thuc nhm nm men, nh ging Candida v nhng ging tng t. Chng c kh nng pht trin trn ngun carbon n-alkan ln men to acid citric. Theo Yamada (1977), vic sn xut acid citric t nparaffin mch thng tng c nghin cu bi nn cng nghip Nht Bn t nhng nm 1960. Cc phn on paraffin mch thng khc nhau ( khong t C9 n C2O) l c cht thch hp. Cc nhn t quan trng khc c cp ti l pH v nng ion st. Gi tr pH nn c gi trn 5, pH thp hn s dn n vic hnh thnh cc polyol nh erythritol v arabitol (Tabuchi v Hara, 1973). Nng ion st vt mc lm gim ng k nng sut ln men ca Saccharomycopsis lipolytica, c l l do tng kh nng hot ng ca aconitate hydratase (Tabuchi v cs., 1973). Marchal v cs. (1977b), Behrens v cs. (1978) cho l qu trnh ln men acid citric c bt u tch lu sau khi s dng ht hon ton ngun N trong mi trng. Nhiu nghin cu cho thy vic duy tr mt t l P : C nht nh nh 0,1.10-3 2,0.10-3 : 1 trong sut qu trnh ln men s bo m nng sut cao n nh ca acid citric (Fukuda v cs., 1982). Nhng chng nm men c vai tr quan trng trong sn xut acid citric gm c: - Candida brumptii (C. catenulata) - Candida guilliermondii - Candida intermedia

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Candida lipolytica (Yarrowia lipolytica) Candida oleophila Candida parapsilosis Candida tropicalis Candida zeylanoides Rhodotorula Saccharomycopsis lipolytica (Yarrowia lipolytica) Mt bt li ca vic s dng nm men l s hnh thnh mt lng ln acid isocitric, ln ti hn 50% lng acid to thnh. Vn ny c th c khc phc bng cch chn lc cc chng t bin, ch yu l gim hot ng ca aconitate hydratase. Mt khc, s hnh thnh acid isocitric c th c kim ch bng cch b sung cht hot ng b mt (Matsumoto v cs., 1983) hay a kh oxy vo (Matsumoto v Ichikawa, 1986). Cuc khng hong du m 1974/73 kt thc hu nh ton b vic s dng n alkan nh mt ngun nguyn liu cho sn xt acid citric. Tuy nhin chnh trong thi im ny, nm men c nh gi cao bi kh nng s dng carbohydrate nh mt ngun carbon (Tabuchi v Abe, 1968; Tabuchi v cs., 1969; Lizuka v cs., 1969; Shimizu v cs., 1970; Abe v cs., 1970). c bit k t khi tm ra c nhng chng c kh nng ng ho fructose chm, nhng c cht nh mt r ng hay hn hp ng c dng cho vic sn xut ng thi acid citric v fructose (Uchio v cs., 1979). T nhng hng nghin cu m rng ny, ngi ta cng nhn thy ln men glucose cho nng sut acid citric tng t nh s dng alkan (Behrens v cs., 1986). Mt s hng ci tin khc nh: thay i ngun cung cp oxygen (Stottmieister v cs., 1986), to s thch nghi ban u cho t bo trn mi trng methionine cao (Barth v Krebs, 1985) hay tin nui cy t bo trn mi trng alkan ri sau chuyn sang mi trng glucose (Weissbrodt v cs., 1987a). Sau ny, ngoi glucose, ethanol cng c ngh lm ngun nguyn liu bi v cho c sn phm tinh hn. Vi A. niger, nhng ci tin khc c a ra c th tip tc s dng t hn, l ng dng nhng k thut mi nh c nh t bo hoc l nui cy bn lin tc. 6. SINH THI LN MEN - Vic thu hi acid citric t dch ln men thng i km vi vic tch lu mt lng ln cc sn phm trung gian. Khong 2,5 tn ph liu (tnh trn khi lng kh) c to ra trong qu trnh sn xut CAM, ch yu l gypsum (khong 60% tng s), theo sau l cc cht tan trong dch ln men v lng si nm ph thi. Vic p ng cc yu cu bt buc v sinh thi i hi nhiu bc tin hnh nhm lm gim thiu gnh nng mi trng v to kh nng tn dng cc ph liu. - Gypsum: nh cp trn, trong nhiu trng hp vic tch t gypsum ph thi khng th ko di. V vy, mt s quy trnh tinh lc tng c th nghim v thnh cng trong vic loi b gypsum cng mt s sn phm trung gian i km nh h si nm ph thi. Bn cnh , gypsum tinh khit c th c s dng nh mt loi vt liu xy dng in hnh hay nh mt loi thuc nhum ph ngoi giy. Ngoi ra, gypsum cn c th c chuyn i thnh dng cht hn gn gypsum hay thnh dng xi-mng khong sulfol.

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- Hin nay c nhiu kin cho rng qu trnh sn xut gypsum tinh khit t cc ngun ph thi khc nhau s mang li li nhun. iu ny ngc li s tc ng n kinh t ca cc c s to ra gypsum ph thi. Ngoi ra, mt vi nh nghin cu tng ngh xem xt phn ng ni bt gia calcium sulfate vi carbondioxid v ammonia nhm to ra calcium carbonate v amonium sulfate. C hai sn phm ny u c nhiu ng dng khc nhau. Tng t phn ng vi sodium carbonate cng to ra calcium carbonate v sodium sulfate. - H si nm c thi ra t qu trnh nui cy b mt v nui cy chm c th c sy kh v s dng nh mt loi thc n gia sc hay c bin i lm phn bn. - Dch thi t qu trnh gy kt ta calcium citrate c th cho bc hi to sn phm vinasse- c s dng nh mt loi thc n cho gia sc. Tuy nhin do hm lng cao cc hp cht v c c trong sn phm m ng dng ny c phn b hn ch. Ngoi ra, do nhng vn k thut cng nh yu cu nng lng ban u cao nn qu trnh x l ny t c chp nhn. Hin nay, trong cc nh my ln, hn hp dch thi ra c x l thng qua mt nh my x l nc thi ln. Vic b sung cc bc k kh ngy cng tr nn ph bin vi h thng x l nc thi s dng bn hot tnh. 7. NG DNG - Acid citric, vi v chua d chu cng c tnh ho tan nhiu trong nc, l mt acid quan trng trong cng nghip thc phm nh dng trong sn xut bnh ko, nc gii kht, hp rau qu, tht c, mt tri cy. Ngoi ra acid citric cn c s dng trong cc ngnh cng nghip m phm, dc phm cng nh trong sn xut cc cht ty ra. - Mui ca acid citric cng c s dng trong mt s cc ngnh cng nghip thc phm v dc phm. Trisodium citrate v acid citric c th trn ln vi nhau to dch mui m vi mt gi tr pH bt k. Aluminum citrate tng c xem nh mt tc nhn gn kt hiu qu i vi CM-cellulose hoc vi phn no nha polyarylamid b thy phn nhm nng cao quy trnh thu hi du m. - Trong nhng nm 1970, cc nh khoa hc nhn thy c rt nhiu cc acid polycarboxylic khng bn c sinh ra t qu trnh phn hu nhit ca calcium citrate. Cc hp cht ny c sulfon ho to ra acid sulphopolycarboxylic (SPC). Ester ca SPC l mt cht ty ra tuyt vi. Vic sn xut cc ester ca acid citric cng c ng dng trong cng nghip nha. Ngoi ra acid citric cn c s dng nh mt cht thm ht trong vic loi b SO2 t kh thi. Ngoi ra, do nhng vn k thut cng nh yu cu nng lng ban u cao nn qu trnh x l ny t c chp nhn. Hin nay, trong cc nh my ln, hn hp dch thi ra c x l thng qua mt nh my x l nc thi ln. Vic b sung cc bc k kh ngy cng tr nn ph bin vi h thng x l nc thi s dng bn hot tnh.

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1.

GII THIU: Nhng nguyn tc ln men acid lactic cng nghip c l c a ra t nhng nm 1857, khi Louis Pasteur cp ti trong tc phm ni ting: Mmoire sur la fermentation appele lactique. K t , c bit cng vi vic c lp c nhiu chng vi khun sinh acid lactic khc nhau, cng dng ca nhng chng vi sinh vt ngy cng tr nn quan trng cng vi s Hnh 8:Cng thc cu to pht trin ca nhn acid lactic loi. Nhng sn phm ln men t sa, rau, ng cc, tht cng nh trong vic bo qun mt s nguyn liu l nhng ng dng thc t trong ln men acid lactic. Vi khun lactic ng mt vai tr quan trng trong cc ngnh cng nghip, c bit cng nghip ch bin sa. Ni n vi khun lactic l ni n mt khi nim chung, bao qut. Chng l nhng vi sinh vt thuc nhm vi khun k kh ty gram dng. Nhng c tnh sinh hc v sinh ha c cp rt nhiu trong cc nghin cu khc nhau v d ca Balow v cs.,(1991). Trong phn ny chng ta ch ni n c tnh lm cho vi khun lactic c ng dng quan trng trong cc qu trnh cng nghip. Hnh 9: Cu trc acid lactic 2. C TNH SINH HC V SINH HA CA LN MEN ACID LACTIC CNG NGHIP

- Vi khun ln men ng hnh v ln men d hnh: C hai nhm vi khun ny u tn dng c tt con ng bin dng EMP ca chui chuyn ha glucose to sn phm cui acid lactic. Chng tham gia c vo con ng chuyn ha ng pentose thu c acid lactic cng vi cc sn phm khc nh acid acetic, ethanol, CO2.

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29

Hnh 10: Qu trnh ln men acid lactic d hnh

1919, Jensen a gi tn ln ng hnh men d ng cng phn bit chng vi khc nhau, xp chng nhm ny Vi ln men hnh l chnh sn acid lactic mi. Tuy

Nm Orlara cch men v ln hnh, cc khun sp vo hai khun ng ngun xut thng nhin,

Hnh 11: Qu trnh ln men acid lactic ng hnh

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phng thc ln men ng hnh cng c th thay i thnh ln men d hnh nu nng ca ngun C chnh gim xung di mc gii hn cho php (De Vries v cs., 1970). - Vi khun a nhit (Mesophilic bacteria) v chu nhit (Thermophilic bacteria) Mesophilic lactic acid tng trng ti u trong khong 28-45oC. Cn thermophilic lactic acid l 45-62oC, ty thuc tng ging. Trong ch bin sa, da vo nhit tng trng ngi ta phn bit ra: Thermobacterium, Streptobacterium v Betabacterium. Vic s dng nhng chng vi sinh vt chu nhit ny thun li im gim thiu nhng s c c th dn n s gy nhim trong sut qu trnh ln men. - Vi khun sinh acid lactic Mc d c kh nng sn sinh ra c acid vi vai tr l sn phm trao i cht chnh, nhng chng li rt nhy cm vi acid. V vy, quy trnh hng ti s dng lng ln ngun C, ng thi trung ha lng nng cao acid sinh ra, gi pH trong khong t 5,5-6. Vi khun sinh acid lactic l nhng vi sinh vt k kh khng bt buc. Chng cn mt ngun dinh dng phong ph, gm cc vitamin, amino acid v c nhng peptide nh. V vy nu khng kh trng k lng cc thnh phn mi trng ban u s rt d gy nhim cho mi trng nui cy. Thng thng vi khun acid lactic s dng ngun C thng thng nh glucose, fructose, lactose, maltose, sucrose. Chng khng s dng trc tip c tinh bt, tuy nhin cng c mt vi chng vi khun lactic phn hy c tinh bt trong qu trnh sn xut acid lactic. 3. GING SN XUT Cho n nay nhng c tnh di truyn hc ca vi khun lactic c tm hiu r, cng nh nhng p dng k thut gen trong lnh vc ny. Nhng phng thc ny c pht trin theo Devos v Vaughan (1944), Frizsimons v cs. (1994), Wei v cs. (1995). Nhng trn thc t, mc d ng dng trong vic s dng h thng ti t hp DNA c nghin cu hon chnh, vic sn xut acid lactic trong cng nghip vn cn tin hnh trn nhng phng php truyn thng, l s dng cc chng vi sinh vt c chn lc, kim tra bng thc nghim. Ngi ta s cn c vo nng sut to sn phm ca vi sinh vt trn nhng ngun vt liu th nht nh no c th tm ra nhng nhng chng ti u cho p ng cho nhng nhu cu trong cng nghip. i vi ngun nguyn liu th l glucose, mt s loi vi sinh vt c th s dng c thuc nhm Lactobacillus, nh Lactobacillus delbrueckii, c th l Lactobacillus delbrueckii ssp. delbrueckii (Teuber, 1993; Hammes v cs., 1991). Nhng loi vi sinh vt ny cng c th s dng lun c sucrose v th c th tin hnh ln men trn ng sucrose hay r ng, nhng chng khng s dng c lactose. Mt mt khc, Lactobacillus delbrueckii ssp. bulgaricus li c th dng c lactose m khng dng c sucrose, v vy c ng dng trong vic chuyn i nhng c cht cha lactose v d whey (nc sa). C nhng ging khc cng thuc loi vi sinh vt ny li khng c kh nng phn ct trn c cht galactose. Lactobacillus helveticus c th phn ct c lactose v c galactose, khng phn ct c sucrose, v vy cng c xem l mt vi sinh vt hu ch tiu biu khc trong

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vic chuyn i trn c cht whey. Lactobacillus delbrueckii ssp. lactic th c th s dng c trn c c cht cha glucose, sucrose v lactose. i vi c cht tinh bt, iu ng ni y l cc loi vi khun lactic khng c kh nng phn hy trc tip c tinh bt, cn c s thy phn tinh bt trc khi a thnh c cht cho qu trnh ln men. Vic chn la s dng loi vi sinh vt no trong trng hp ny cn ph thuc vo phng thc thy phn tinh bt. Nu s dng alpha hay beta amylase (t i mch, hay malt), nh cht xc tc trong chui ng ha thng thng (v d sn xut ethanol), ngun C chnh phi l maltose. Nhng loi vi sinh vt c kh nng trong ln men maltose nh Lactobacillus delbrueckii ssp. lactis, mt vi ging Lactobacillus delbrueckii v mt s chng khc (Teuber, 1993). Nu thc hin qu trnh ng ha vi enzyme alpha amylase hay glucoamylase, sn phm ch yu ca qu trnh thy phn l glucose v glucose oligosaccharides, nhng chng vi sinh vt nu trn cng c th p dng trong trng hp ny. c bit vi s pht hin ra chng vi sinh vt c tn l Lactobacillus amylophilus (Nakamura v Crowell, 1979) v Lactobacillus mylovorus (Nakamura, 1981), c hot tnh mnh trong qu trnh ln men vi c cht tinh bt, v tr thnh mt gii php thay th ca qu trnh ln men acid lactic trong cng nghip (Cheng v cs.,1991; Zhang v Cheryan, 1994). Nhng thnh vin ca nhm lactococci th him khi c s dng trong ln men acid lactic thng mi. Ngi ta c nhng c s da trn ngun c cht (nguyn liu th ban u) tm ra c chng vi sinh vt thch hp. Tuy nhin, cho d s dng ngun nguyn liu th no, th chng vi sinh vt c la chn phi cho nng sut cao. Nh cp trn, s la chn c thc hin bng mt s thc nghim n gin v c lng sai s ca phng php. V sn lng cn ph thuc nhiu vo nng H+ trong dch ln men, nn c th iu chnh tng nng acid lactic tng kh nng chuyn ha thc hin ca cc chng vi sinh vt. Vic p dng nhng hp cht ha hc c kh nng gy t bin (v d ethyl methane sulfonate) cng c a ra to ra cc bin th (th t bin) ca chng Lactobacillus delbrueckii (ATCC 9649) c kh nng chu c nng acid cao hn chng bnh thng v cho nng sut ln men cao hn (Demirci v Pmett, 1992). Mt vn na thng bt gp trong cng ngh sa l s c gy nhim ca nhiu loi bacteriophage khc nhau. V tt nhin ngi ta xc nh nhiu phage gy nhim trong qu trnh ln men acid lactic. C nhng du hiu cho rng nhng phage c c ngun gc t giai on prophase cn li v tn ti trong sut mt giai on di (ShimizuKadota v cs., 1983; Shimizu-Kadota v Tsuchida, 1984). Bng nhiu th nghim khc nhau, ngi ta a ra kt lun s b v tm cch loi b phage c th khc phc c prophage trong ging ban u. Tuy nhin, cng c nhiu ngi khc (nh Teuber v Lemke, 1983) cho rng khng phi nhng phage n ha li ng vai tr quan trng trong vic gy nhim. Trong cng nghip sa, ngi ta ngh ra cc phng php gii quyt nh trung ha cc phage gy nhim hoc l la chn ca ging c tnh khng phage (MataGilsinger, 1985; Teuber, 1993; Reed v Nagodawithana, 1995).

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Theo quy lut, c th to ra nhng iu kin c th cho sinh si ny n ca cc chng vi sinh vt to ging, i hi phi bo qun m vi khun nui cy trn nhng mi trng dinh dng nht nh theo tiu chun, v d mi trng nui cy MRS. 4. SN XUT TRN QUY M CNG NGHIP

Hnh 12: S qu trnh ln men acid lactic

Nhng th nghim hon thin quy trnh sn xut acid lactic thng mi bt u t nm 1881, c a ra bi Avery Littleton, USA. Mc ch nhm thay th cho ngun tartrates nhp khu, ri c s dng trong lm bt n bng calcicum lactate. Mc d khng c thun li cho lm, s thay th u tin c thit lp trong lnh vc cng nghip bng cng ngh sinh hc. Nm 1895, mt nh my sn xut acid lactic bng phng php ln men u tin c xy dng ln bi A. Boehringer Ingelheim, c (Buchta, 1983). Phng thc sn xut acid lactic bng ln men cho ra nhng sn phm cnh tranh c vi phng php tng hp ha hc. Trong phng php tng hp ha hc: ngi ta tin hnh bng phn ng oxi ha propene xc tc bi acid nitric v/hoc nitrogen peroxide trong s c mt ca oxygen, sinh ra alpha nitratopropinioic acid t c th c thy phn cho ra acid lactic v acid nitric (Platz v cs.,1972; Boichard v cs.,1973). Mt phng php khc ca tng hp acid lactic bng ha hc l thy phn lactic acid nitrile (Vogt v cs., 1966), t c bng phn ng ca acetaldehyde vi HCN. C hai phng php tng hp ha hc v phng php ln men acid lactic u gp phi vn trong qu trnh tinh ch t cht lng theo nhng tiu chun t ra trong nhng ng dng khc nhau. Giai on ny ng vai tr quan trng trong vic quyt nh gi thnh sn phm sau ny. 4.1 Nguyn liu th

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Ngoi kh nng gi thnh chp nhn c hay khng, nguyn liu th ban u cng ng mt phn quyt nh nh hng n quy trnh tinh ch sn phm cn thit sau ny. S la chn nguyn vt liu ph thuc vo ng dng ca sn phm. Gi thnh sn phm acid lactic ph thuc vo nguyn liu th v quy trnh tinh ch. Nhng ngun nguyn liu c cht mono hay disaccharide truyn thng thng s dng trong sn xut acid lactic l: - Glucose (dextrose) v glucose syrup, nhng sn phm cui ca qu trnh chuyn ha tinh bt s dng alpha amylase v glucoamylase - Maltose, sn phm ca qu trnh chuyn ha tinh bt s dng alpha amylase v beta amylase t malt i mch hoc l nhng ngun khc. - Sucrose, sn phm cui, sn phm trung gian (t syrup, tri cy) v sn phm ph (mt r ng) t c ci ng v ng ma. - Lactose thnh phn ca whey, y l ngun c cht t nhin ca hu ht vi khun lactic. Nh vy, tinh bt khng c s dng bi nhng vi khun lactic thng thng, tr Lactobacillus amylophilus v Lactobacillus amylovorus. Qu trnh thy phn tinh bt cn c tin hnh trc khi tin hnh ln men, phng php thy phn c th thc hin bng xc tc ha hc hoc bng enzyme. V maltose l sn phm chnh ca qu trnh ny nn ch mt s lng nh vi sinh vt c th s dng trong qu trnh ln men. Tng t nh sn xut ethanol trong cng nghip, s ha lng tinh bt v ng ha t c bng cch kt hp alpha amylase ca vi khun gc v glucoamylase t nm. V vy cc ch phm ng thu c t nguyn liu tinh bt th khc nhau khng khc nhau. Hydrol (dung dch thu c sau khi chuyn tinh bt thnh ng), sn phm ph ca sn xut dextrose, cng c dng nh ngun nguyn liu th. Sucrose - ly t nguyn liu th cng ph hp lm c cht cho ln men acid lactic v r rng chng l ngun c cht u tin c s dng trong cng nghip ln men. Hu ht cc chng Lactobacillus ln men ng hnh c th s dng ngun sucrose, vi Lactobacillus bulgaricus v Lactobacillus helveticus l cc ngoi l ng k. Vic s dng ngun nguyn liu r tin mt r ng cng l mt iu ng quan tm ln. C th ni rng, mt r ng, sau khi c x l ph hp, l mt ngun cung cp carbon ng k, tuy nhin cho nng sut thp hn v cng kh tinh ch sn phm hn sau ny. Lactose, ly t whey cng l mt ngun nguyn liu quan trng cho cc chng vi sinh vt. Tuy nhin, cng ging nh mt r ng, n cn nhng cng ngh tn km cho vic tinh ch sn phm. 4.2 Qu trnh ln men 4.2.1 Phng php calcium lactate c in Nguon C va E + Nguon N + O2 + Cac yeu to khac Sinh khoi + San pham + CO2 + H2O + Nhiet Ngi ta s dng nguyn liu th c cha ng (glucose, sucrose) vi nng khong 120-180g/L. B sung ngun nitrogen, c th l cc hp cht v c c cha N nh

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ammonia, dung dch ammonia, ammonium sulfate hoc ammonium phosphate cng vi cc hp cht hu c c cha N nh dch bp ngm, dch chit nm men, peptone v mt s digest protein khc, malt ny mm, cho hm lng N t 1-10 g/L. Nu khng k c thnh phn ca hp cht dinh dng khc, thnh phn khong (khong a lng) cng c cung cp bng cch thm vo phosphate v mui (nh mui sulfate ca magnesium, manganese hay st). Thng thng, nhng thnh phn phc hp (nh dch chit nm men, peptone,) c tnh quyt nh n kt qu nng t bo thu c (Monteagudo v cs., 1993). S tit trng, kh nhim cc thnh phn ca mi trng l rt cn thit trong qu trnh chun b nui cy, ln men. Qu trnh ln men c thc hin trong cc l phn ng vi th tch l thng l ln hn 100m3. Mt yu t quan trng l trong qu trnh ln men sn xut acid lactic thng sinh ra nhng cht c th gy n mn. Cn phi c nhng cch thc chng s n mn trn va l gi gn bo v l phn ng, va ngn chn s nhim vi sinh vt l cho dch ln men. Trc y, i vi nhng l phn ng c th tch nh, ngi ta thng lm cc b bng b-tng. Ngy nay, cng lm bng b-tng nhng vi sn lp ph ngoi thch hp, ngi ta cng dng thp khng r trnh c s n mn nh nu trn. Nhit phn ng ln men c iu khin trong khong hn 45oC, cung cp nhit phn ln trong giai on u, lm ngui ngay khi khi qu trnh ln men to nhit. Calcium carbonate dng khan, nghin vn c thm vo lc bt u hoc sau trong sut qu trnh ln men gi cho nng ca acid lactic dng t do thp n mc c th. Nh cp trn, gi tr pH duy tr khong 5,5-6. Cy ging c thc hin bng cch thm nhng th tch khong 10% mi trng nui cy duy tr c thnh phn nh mi trng ban u (RAUCH v cs., 1960). S bt u ln men (khong 6h sau khi fetch) c kim sot bi s thot ra ca kh CO 2 t calcium carbonate thm vo, ngn chn khng kh vo h thng. Do , s pht ra ca kh CO2 t qu trnh h hp c ngn chn mc va phi. Vic p dng nc vi sa trung ha khng cung cp nhng iu kin thun li. Duy tr tng i mt nhit cao (50oC) cho Lactobacillus delbrueckii hay nhng chng cng loi khc to chiu hng gim bt kh nng nhim khun. Hot ng ln men hon chnh sau 2-6 ngy, ty thuc nng ngun carbon cung cp. Trong ln men calcium lactate, mc gii hn nng ng cao hn c cho rng c tnh quyt nh bi tnh tan c ca calcium lactate. Ngi ta khng nh rng, nu dng mt nng ng cao hn (khong 260 g/L) c th kh thi trong ln men vi liu lng CaO tng ng nhm iu chnh pH khong 6,3 gy nn vic lng xung tip tc ca calcium lactate. 99,6% chuyn i trong 3 ngy c thc hin bng phng thc ny (Maslowski, 1988). Thng thng, v mt l thuyt, t l chuyn i t c t 85-95%. Khong 2% sn phm ph l acid acetic v acid propionic. 4.2.2 Qu trnh thu hi v tinh sch Qu trnh miu t s tinh ch v thu sn phm bng phng php calcium lactate c a ra bi Rauch v cs.(1960), n ph thuc vo tinh sch ca nguyn liu th s dng ln men. S dng ng tinh (dextrose, ng t c ci ng hay ng ma), dch ln men c gia nhit lm tan tt c calcium lactate v tip theo l x l vi acid sulfuric

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(78%). Calcium sulfate to ra c loi b qua qu trnh lc. Dung dch thu c cho bay hi (chn khng). Lng thch cao cn d kt ta trong dch acid lactic c lc loi b cng vi cc cht c mu m c th b hp ph bng carbon hot tnh. S khng tinh khit cng c th b gy ra bi cc ion kim loi nng, trong trng hp ny s c x l bng hexacyanoferrate hoc cho dung dch qua ct trao i ion v x l bng hydrogen peroxide hay potassium permanganate. tinh khit ca dung dch sn phm thu c c th t ti 80%. Dch ln men thu c t ln men trn nhng nguyn liu th c cht lng km i hi nhng bc tinh ch nghim ngt. Bao gm giai on tin tinh ch (tinh ch s b) bng cch lc, sau l lm kt tinh calcium lactate, c acid sulfuric gip phn hy cng thm x l bng nha resin trao i ion. Mun t c tinh khit cao hn, c th tin hnh lp li nhiu ln qu trnh lm kt ta v s dng acid sulfuric phn hy calcium lactate. Nhng ci tin khc cng c a ra nh s dng km lactate hay magnesium lactate thay th. c bit trong trng hp ln men s dng ngun nguyn liu th ban u l carbohydrate nh r ng, th s dng magnesium lactate t c mt hiu qu cao bi v n ci tin c c tnh kt tinh (Bode, 1969). Theo tnh ton, lng calcium sulfate to ra v loi b xp x lng acid lactic sn xut. V th, vn loi b thch cao tha ngy cng tr nn quan trng cng vi vic gia tng hiu sut ln men. Mt phng php khc tinh sch acid lactic l kt hp s ester ha, (vi methanol) v chng ct nhng hp cht ester d bay hi c xem l bc phn tch c hiu qu nht to ra nhng sn phm c tinh khit cao (Schopmeyer, 1943; Bowmans Chem, Ltd., 1962; Sepitka v Gaertner, 1962). S ester ha l qu trnh ngc dng ng thi phn tch ester d bay hi v k sau ester ha li bng nc trong giai on th hai to ra acid lactic t do v methanol c a vo li h thng. Phng php ny c cho rng sn sinh ra c acid lactic vi sch tt nht khng km theo mt sn phm thi no. Tuy nhin nhc im ca phng php ny l cn c trang thit b t tin, k thut hin i. Cn phng thc dng ion trao i, cho n nay ch mi s dng trong bc tinh ch, ch yu l tch acid lactic khi dch ln men (Kanazawa, 1977; Obara, 1989; Kulprathipania v Oroskar, 1991; Mantovani v cs., 1992; Mantovani v cs., 1992; Matsuda v Yoshida, 1992; Evangelista v cs., 1994). Cc quy trnh khc 4.2.3.1 Quy trnh ammonium lactate Trong sut qu trnh ln men, pH ca dch c duy tr trong khong 5,5-6. bng cch b sung lng sodium carbonate hoc sodium hydroxide, nhng cng cn phi c lp sodium lactate to ra hin din trong dch, y cng l mt vn ng lu . Ngi ta th trung ha acid lactic to ra bng ammonia (Poznanski v cs., 1976; Stieber v cs., 1977; Jarry v Lamonerie, 1988). kin ban u c th to ra ammonium lactate. C th thy r rng lng dung dch ammonium lactate c th b ester ha rt thun li v t thu li d dng acid lactic t lactic ester, gii phng ammonia hoc nhng hp cht mui ammonium v c hu ch khc (Walkep v cs., 1991; Sepitka v Sepitkova, 1992; Cockrem v Johnson, 1993; Kumagai v cs., 1994). Theo s khng nh r rng ca Walkup v cs. (1991), dng ammonium lactate khi phn ng vi mt alcohol s to ra 4.2.3

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ester tng ng, c th c tch ra v s dng to ra acid lactic sch, gi r, hoc bin i thnh acid acrylic sch, gi r. Cng trnh ca Cockrem v Johnson (1993) l a ra c mt danh sch cc alcohol c th ng dng. Cn vi quy trnh ca Kumagal v cs. (1994), ng cho rng s ester ha ammonium lactate c thc hin ti nhit cao s nh hng n s gii phng v thu hi li ammonia. Quy trnh ca Sepitka v Sepitkova (1992) bao gm ln men vi nng ng 200g/L bi Lactobacillus delbrueckii. Gi tr pH gi trong khong di 6,0 bng cch thm dung dch ammonia. Ru ln men c cho bay hi acid lactic t c nng khong 50-60%, bng cch x l vi mt lng acid sulfuric ph hp gii phng acid lactic v ha ln mt th tch tng i methanol. Nhit ca ton b qu trnh khng c vt qu 100oC. Sn lng acid lactic trong ton b qu trnh khong 95%, ngha l 90% ph thuc vo ngun ng ban u. 4.2.3.2 Quy trnh ln men lin tc Qa trnh ln men lin tc cho nng sut sn phm cao hn v v vy chng c thc hin trong nhiu dng khc nhau. Trong nhng th nghim trc y, ngi ta s dng huyn ph t bo (Childs v Welsby, 1966) v cui cng thu hi t bo (Vick v cs., 1983). H thng ln men nu s dng vi tc dng chy cao, nng sut tng ln khong 50g/L.h (Richter v cs., 1987). Khng ch cho nng sut cao, phng php ny c bit thch hp i vi c cht whey. Mt vi tc gi c nhng nguyn cu v ln men whey bng phng php ln men lin tc ny nh Boyaval v cs., 1987; Mehia v Cheryan v cs., 1987a; Aeschlimann v Von Stockar, 1990; Krischke v cs., 1991; Kulozik v cs., 1992; Boergardt v cs., 1994). Bn cnh ngi ta cng a ra nhng phng php khc s dng cc quy trnh kt hp (Chmiel v Paulsen, 1991). 4.2.3.3 ng dng ca c nh t bo Cc nh nghin cu trn th gii cng b ra khng t thi gian nghin cu phng php sn xut acid lactic bng h thng c nh t bo (Linko v cs., 1984; Mehaia v Cheryan, 1987b; Boyaval v Goulet, 1988; Bassi v cs., 1991) nhng cha ng dng c trong sn xut cng nghip. 4.3 Nhng vi sinh vt khc ngoi vi khun lactic Khng ch c vi khun lactic, ngi ta tm ra mt s lng khng nh cc chng vi sinh vt khc cng c kh nng sn xut ra mt lng ln acid lactic t nhng ngun carbon thng thng. V d nh chng Rhizopus. Trong lun n ni ting ca Foster (1949) Chemical activities of Fungi, ng ngh s dng Rhizopus cho sn xut acid lactic thng mi. Loi ny c th chuyn ha c nhng ngun ng khc nhau trong mi trng ln men tng hp. Mc d kin ny c a ra t lu(Snell v lowery, 1964), vic a vo s dng k thut ny n nay vn cha hon chnh. Gn y c nghin cu khc v qu trnh chuyn ha tinh bt bng Rhizopus oryzae bng ln men mt giai on (Hang, 1990). Mt s trng hp khc ngi ta s dng dng bo t vi khun ln men sn xut acid lactic. Nh:

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4.4

Sporolactobacillus inulinus: to ra c D-(-) acid lactic Bacillus coagulans

Cht lng sn phm v ng dng Acid lactic c rt nhiu ng dng trong cng nghip v i sng - Technical-grade lactic acid (acid lactic dng trong k thut) c mu t vng nht n mu nu ty theo nng ca n (20-80%): s dng trong cng nghip dt, sn xut ester lm dung mi v cht lm mm do. - Hu nh 80% acid lactic sn xut ra c dng trong cng ngh thc phm, lm cc cht c v chua v lm cht bo qun. chng c gi chung l food-grade lactic acid. Ty theo mi quc gia, s c nhng quy nh v hm lng v cht lng acid lactic s dng. Acid lactic s dng trong trng hp ny phi khng mu, cha ti thiu 80% acid lactic. Nh c v chua d chu, acid lactic cng c b sung vo cc loi ung ng chai, ng hp, nc hoa, nc p tri cy, trong mt, bnh ko, trong cc sn phm hp, trong lm bnh lm bt chua, bt nho. - Pharmacopocia-grade lactic acid: cha hm lng acid lactic 90%, cht khc khong 0,1%, phi khng mu, khng mi, c dng lm cc cht pha ch sn xut dc phm hoc nhng dn xut khc ca acid lactic (v d calcium lactate). - Plastic-grade lactic acid: cn phi nguyn cht, khng mu v hm lng cc cht khc di 0,01%. c s dng trong sn xut sn, vc ni, cc cht thm nh polymer. Tiu chun cht lng quan trng ca acid lactic l kh nng bn nhit. Trong nhng thp nn gn y, sn lng sn xut acid lactic lun duy tr n nh, Hnh 13: Mc tiu th acid lactic c th c lng lng acid sn xut c trn th gii nm 2005 khong 50000 t/a, chim khong 2/3 lng sn phm ca cng ngh ln men.

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