Sinh trng v pht trin ca vi sinh vt

Vietsciences- Nguyn Ln Dng, Bi Th Vit H Chng trnh Vi sinh vt hc Sinh trng l biu th s tng trng cc thnh phn ca t bo. i vi cc vi sinh vt c hnh thc sinh sn bng ny chi hay phn i th sinh trng dn ti s gia tng s lng t bo. T bo tng trng n mt mc nht nh th s phn ct thnh hai t bo th h con c kch thc hu nh bng nhau. i vi cc vi sinh vt a nhn th s phn cch nhn khng ng hnh vi s phn ct t bo - s sinh trng lm tng kch thc t bo m khng lm tng s lng t bo. V vi sinh vt rt nh b cho nn l i tng rt khng thun tin nghin cu v sinh trng v pht trin. Chnh v vy m khi nghin cu v sinh trng, ngi ta thng xt n s bin i v s lng ca c qun th vi sinh vt. 14.1. NG CONG SINH TRNG S sinh trng qun th vi sinh vt c nghin cu bng cch phn tch ng cong sinh trng trong mt mi trng nui cy vi sinh vt theo phng php nui cy theo m (batch culture) hoc trong mt h thng kn. C ngha l vi sinh vt c nui cy trong mt thit b kn, trong qu trnh nui cy khng thay i mi trng v thi gian nui cy cng ko di th nng cht dinh dng cng gim st, cc cht ph thi ca trao i cht cng tng ln. Nu ly thi gian nui cy l trc honh v ly s logarit ca s lng t bo sng lm trc tung s c th v c ng cong sinh trng ca cc vi sinh vt sinh sn bng cch phn i. ng cong ny c 4 giai on (phases) khc nhau. 11/03/2009

Hnh 14.1: ng cong sinh trng trong h thng kn (Theo sch ca Prescott, Harley v Klein)

14.1.1. Giai on Tim pht (Lag phase) Khi cy vi sinh vt vo mt mi trng mi s lng thng khng tng ln ngay, l giai on Tim pht hay pha Lag. Trong giai on ny t bo cha phn ct nhng th tch v khi lng tng ln r rt do c s tng cc thnh phn mi ca t bo. Nguyn nhn l do t bo trng thi gi, thiu ht ATP, cc cofactor cn thit v ribosome. Thnh phn mi trng mi khng ging mi trng c cho nn t bo cn mt thi gian nht nh tng

hp cc enzyme mi nhm s dng c cc cht dinh dng mi. Cc t bo cng c th b thng tn v cn mt thi gian hi phc. Bt k v nguyn nhn g th kt qu vn l t bo phi t trang b li cc thnh phn ca mnh, ti to ADN v bt u tng khi lng. Giai on tim pht di hay ngn lin quan n bn thn tng loi vi sinh vt v tnh cht ca mi trng. Nu tnh cht ha hc ca mi trng mi sai khc nhiu vi mi trng c th giai on tim pht s ko di. Ngc li, nu cy t giai on logarit vo mt mi trng c thnh phn tng t th giai on tim pht s rt ngn li. Nu cy vi sinh vt t giai on tim pht hay t giai on t vong th giai on tim pht s ko di. 14.1.2. Giai on logarit (Log Phase) hay Pha Ch s (Exponential Phase) Trong giai on ny vi sinh vt sinh trng v phn ct vi nhp ti a so vi bn tnh di truyn ca chng nu gp mi trng v iu kin nui cy thch hp. Nhp sinh trng ca chng l khng thay i trong sut giai on ny, cc t bo phn i mt cch u n. Do cc t bo sinh ra ch khc nhau rt t cho nn ng cong sinh trng l mt ng trn nhn ch khng gp khc (hnh 14.1). Qun th t bo trong giai on ny c trng thi ha hc v sinh l hc c bn l nh nhau cho nn vic nui cy giai on ny thng c s dng nghin cu sinh ha hc v sinh l hc vi sinh vt. Sinh trng logarit l sinh trng ng u, tc l cc thnh phn t bo c tng hp vi tc tng i n nh. Nu cn bng dinh dng hay cc iu kin mi trng thay i s dn n s sinh trng khng ng u. S sinh trng khi nhp tng hp cc thnh phn ca t bo tng i bin ha s bin i theo cho n khi t ti mt s cn bng mi. Phn ng ny rt d quan st thy khi lm thc nghim chuyn t bo t mt mi trng ngho dinh dng sang mt mi trng giu hn. T bo trc ht phi to nn cc ribosome mi c th nng cao nng lc tng hp protein, sau l s tng cng tng hp protein v ADN. Cui cng tt yu dn n tc pht trin nhanh chng. Lc chuyn qun th t bo t mt mi trng giu dinh dng ti mt mi trng ngho th cng c kt qu v s sinh trng khng ng u nh vy. Vi sinh vt trc c th thu c t mi trng nhiu thnh phn ca t bo nhng khi chuyn sang mi trng ngho chng cn c thi gian to ra cc enzyme cn thit sinh tng hp cc thnh phn khng c sn trong mi trng. Sau t bo mi c th phn ct, ADN mi c th ti to, nhng vic tng hp protein v ARN l chm cho nn t bo nh li v t chc li s trao i cht ca chng cho n khi chng c th sinh trng tip. Sau s sinh trng cn bng s c hi phc v tr v li giai on logarit. Cc th nghim trn y cho thy s sinh trng ca vi sinh vt c kim sot mt cch chnh xc, phi hp v phn ng nhanh chng vi nhng s bin i ca mi trng. Khi s sinh trng ca vi sinh vt b hn ch bi nng thp ca cc cht dinh dng cn thit th sn lng t bo cui cng s tng ln cng vi s tng ln ca cc cht dinh dng b hn ch (hnh 14.2a). y chnh l c s s dng vi sinh vt trong vic nh lng vitamin v cc nhn t sinh trng khc. Tc sinh trng cng tng ln cng vi s tng nng cc cht dinh dng (hnh 14.2b). Hnh dng ca ng cong hu nh phn nh tc

 hp thu cht dinh dng nh s chuyn vn protein ca vi sinh vt. Lc nng cht dinh dng cao th h thng vn chuyn s bo ha v tc sinh trng khng tng ln cng vi s tng ln ca nng cht dinh dng.

Hnh 14.2: Nng cht dinh dng v sinh trng (a )- nh hng ca s hn ch cht dinh dng i vi sn lng chung ca vi sinh vt. Lc nng cao th sn lng chung s t ti n nh. (b)- nh hng ca s hn ch cht dinh dng ti tc sinh trng.

14.1.3. Giai on n nh (Stationary Phase) hay Pha Cn bng Qua giai on Logarit s sinh trng ca qun th cui cng s dng li, ng cong sinh trng i ngang (hnh 14.1). Nng vi khun trong giai on n nh thng vo khong 109/ml. Vi cc vi sinh vt khc thng khng t c n nng ny. Vi ng vt nguyn sinh v vi to thng ch t n nng 10 6/ml. ng nhin, s lng t bo cui cng quyt nh bi nh hng chung ca iu kin dinh ng, chng loi vi sinh vt v cc nhn t khc. Trong giai on ny s lng t bo sng l khng thay i, c th do s lng t bo mi sinh ra cn bng vi s lng t bo cht i, hoc l t bo ngng phn ct m vn gi nguyn hot tnh trao i cht. C nhiu nguyn nhn lm cho qun th vi sinh vt chuyn sang giai on n nh. Trong nguyn nhn ch yu l s hn ch ca cht dinh dng. Nu mt cht dinh dng thit yu b thiu ht nghim trng th s sinh trng s chm li. Vi sinh vt hiu kh thng b hn ch bi nng oxygen. Oxygen thng ha tan t trong nc, O2 trong ni b mi trng rt nhanh chng b tiu th ht, ch c cc vi sinh vt sinh trng b mt mi trng mi c nng O2 sinh trng. V vy khi nui cy vi sinh vt phi s dng ti my lc hay cc bin php thng kh khc. Qun th vi sinh vt cng c th b nh ch sinh trng khi gp s tch ly ca cc sn phm trao i cht c hi. Mt s vi sinh vt k kh (nh Streptococcus) c th ln men ng lm sn sinh mt lng ln acid lactic hay cc acid hu c khc, lm acid ha mi trng v c ch s sinh trng ca vi sinh vt. ng thi s tiu

hao ht ng cng lm cho t bo i vo giai on n nh. Sau na l, mt s chng c cho thy khi s lng vi sinh vt t n mt gii hn nht nh th s sinh trng c th b nh ch. S sinh trng ca vi sinh vt chuyn sang giai on n nh c th do kt qu chung ca rt nhiu nhn t khc nhau Nh chng ta. thy vi khun khi nui cy theo m s chuyn sang giai on n nh khi thiu thc n. Trong t nhin, do nhiu mi trng c nng ch dinh dng rt thp nn vi sinh vt thng chuyn sang giai on n nh. i vi vi khun vic chuyn sang giai on n nh c th l mt loi thch ng tt. Nhiu loi vi khun khng c s bin ha r rt v hnh thi (nh hnh thnh bo t ni sinh-endospore) nhng chng c th thu nh kch thc li, thng do cht nguyn sinh co li v nhn gi (nucleoid) m c li. Mt bin i quan trng hn l, khi thiu thc n vi khun s sinh ra mt loi protein i (starvation proteins) lm cho t bo khng nhiu hn vi cc thng tn bng nhiu con ng khc nhau. Chng lm tng cc lin kt peptidoglycan v s bn vng ca thnh t bo. Chng hn Dps (DNA-binding protein from starved cells), mt loi protein kt hp vi ADN ly t cc t bo i, c th bo v cho ADN. Phn t Chaperones cn tr s bin tnh ca protein v hi phc li c cc protein b tn thng. V nhng vic v nhiu c ch khc m cc t bo i c th kh b cht i v khng c vi tnh trng b i, vi s bin ha ca nhit , s tn thng v xy ha v s thm thu, cng nh tng sc khng vi cc ha cht c hi (nh chlorine chng hn). Nhng ci bin ny rt c hiu qu v lm cho mt s vi khun c th sng li sau vi nm b i. R rng vic hiu r nhng vn ny s c tm quan trng thc tin to ln i vi y hc v vi sinh vt hc cng nghip. Chng cn c th chng minh vi khun thng hn (Salmonella typhimurium) v nhiu vi khun gy bnh khc c th c kh nng gy bnh mnh hn khi b i. 14.1.4. Giai on t vong (Death Phase) Vic tiu hao cht dinh dng v vic tch ly cc cht thi c hi s lm tn tht n mi trng sng ca vi sinh vt, lm cho s lng t bo sng gim xung. l c im ca giai on t vong. Ging nh giai on logarit, s t vong ca qun th vi sinh vt cng c tnh logarit (t l t bo cht trong mi gi l khng i). Tng s t bo sng v t bo cht khng thay i v cc t bo cht cha b phn hy. Mun xc nh s lng t bo sng phi pha long ra ri cy ln thch a v a vo iu kin thch hp xc nh s khun lc xut hin. Mc du phn ln vi sinh vt t vong theo phng thc logarit nhng sau khi s lng t bo t nhin gim xung th tc cht ca t bo chm li. l do mt s c th sng li nh c tnh khng c bit mnh. V iu ny v nhng nguyn nhn khc lm cho ng cong ca giai on t vong c th kh phc tp. 14.1.5. Tnh ton v qu trnh sinh trng Khng t cc nh vi sinh vt hc tnh ton v tc sinh trng ca vi sinh vt trong giai on logarit. Tnh ton nhp sinh trng s lm c s cho cc nghin cu v sinh l hc, sinh thi hc vi sinh vt, v cn gii quyt mt s vn ng dng trong sn xut cng nghip.

Trong giai on logarit mi c th vi sinh vt tin hnh phn ct trong mt thi gian hng nh. S lng t bo tng theo phng thc 2n. Thi gian gia hai ln phn chia lin tip hay thi gian cn cho s tng i s t bo c gi l thi gian th h (generation time hay doubling time). V d a mt t bo vo mi trng nui cy, c 20 pht phn ct mt ln th sau 20 pht c 2 t bo, sau 40 pht c 4 t bo v tip tc nh vy (bng 14.1) Bng 14.1: Mt v d v sinh trng theo logarit Thi gian* 0 20 40 60 80 100 120 S ln phn ct 0 1 2 3 4 5 6 2n 20=1 21=2 22=4 23=8 24=16 25=32 26=64 S lng (N0 x 2n) 1 2 4 8 16 32 64 lg10 Nt 0,000 0,301 0,602 0,903 1,204 1,505 1,806

*Thi gian th h l 20 pht, gi thit l nui cy t 1 t bo

S lng logarit t bo l 2n, n l s th h. C th biu th cc s liu trong bng 14.1 bng cng thc sau y:

Trong : N0 l s lng t bo ban u; Nt l s lng t bo thi gian t; n l s th h. T cng thc trn c th bin i nh sau v s th h n c tnh bng logarit thp phn:

Khi nui cy phn m (batch culture) tc sinh trng trong giai on logarit c th biu th bng hng s tc sinh trng bnh qun k (mean growth rate constant k). l s th h sinh ra trong n v thi gian, thng biu th bng s th h trong 1 gi:

Thi gian cn thit tng gp i tng s t bo l thi gian th h bnh qun (mean generation time) hay thi gian tn gp i bnh qun (mean doubling time) v c biu th

bng g. Nu t=g th N t= 2N0. Thay vo cng thc trn ta c:

Thi gian th h bnh qun l o s ca hng s tc sinh trng bnh qun:

Thi gian th h bnh qun g c th cn c trc tip vo th bn logarit (semilogarithmic plot) v hng s tc sinh trng tnh ra (hnh 14.4). V d ,s lng vi khun ti gi th 10 l t 103 tng ln n 109 th :

(th h/h)

gi/th h hay 30 pht/th h

Hnh 14.3: Sinh trng th h ca vi sinh vt (biu th 6 th h)

Hnh 14.4; Xc nh thi gian th h.

(Theo sch ca Prescott,Harley v Klein).

Thi gian th h c th xc nh bng ng cong sinh trng ca vi sinh vt. Ly thi gian l trc honh v ly s lng t bo lm trc tung. Thi gian tng gp i s lng ca qun th (thi gian th h) c th c trc tip trn th

Thi gian th h thay i ty theo chng loi vi sinh vt, iu kin nui cy. Mt s vi khun thi gian th h khng qu 10 pht (0,17h) trong khi mt s vi sinh vt nhn thc (eucaryotic) li di ti vi ngy (Bng 14.2). Thi gian th h trong t nhin thng l di hn so vi khi nui cy. Bng 14.2: Thi gian th h ca mt s loi vi sinh vt Vi sinh vt Beneckea natriegens Escherichia coli Bacillus subtilis Staphylococcus aureus Pseudomonas aeruginossa Clostridium botulinum Rhodospirillum rubrum Anabaena cylindrica Mycobacterium tuberculosis Treponema pallidum Scenedesmus quadricauda Chlorella pyrenoidosa Asterionella formosa Euglena gracilis Ceratium tripos Tetrahymena geleii Leishmania donovani Paramecium caudatum Acanthamoeba castellanii Giardia lamblia Saccharomyces cerevisiae Monilinia fructicola Nhit (0C) Vi khun v Vi khun lam 37 40 40 37 37 37 25 25 37 37 To 25 25 20 25 20 ng vt nguyn sinh 24 26 26 30 37 Nm 30 25 Thi gian th h (gi) 0,16 0,35 0,43 0,47 0,58 0,58 4,6-5,3 10,6 Khong 12 33 5,9 7,75 9,6 10,9 82,8 2,2-4,2 10-12 10,4 11-12 18 2 30

14.2. XC NH S SINH TRNG CA VI SINH VT C nhiu cch thng qua vic xc nh s bin i s lng v cht lng vi sinh vt hiu c s sinh trng ca vi sinh vt, bit c tc sinh trng v thi gian th h. Di y s gii thiu cc phng php thng dng nht cng cc u, khuyt im ca cc phng php ny. Khng c phng php no l tt nht, la chn phng php no cn ph thuc vo tng trng hp c th. 14.2.1. Xc nh s lng t bo Phng php n gin nht xc nh s lng t bo l m trc tip di knh hin vi. Dng cc phng m m va nhanh chng, d dng, li r tin nht, li c th quan st thy kch c v hnh dng t bo. Thng dng phng m Petroff-Hausser m t bo ng vt nguyn sinh. Dng phng m hng cu c th m c cc t bo nhn nguyn thy cng nh t bo nhn tht. Vi t bo nhn nguyn thy cn nhum mu hoc l dng knh hin vi tng phn pha hay knh hin vi hunh quang (phase-constrast or fluoresence microscope) d quan st hn. Phng m c cu trc c mt su nht nh li c chia ra thnh cc nh (hnh 14.5). Khi m s lng ta a dch pha long vo phng m, y l knh (lamelle/ cover glass) ln trn, sau tin hnh m s lng di knh hin vi. Khuyt im ca phng php ny l khng xc nh c vi cc mu c s lng vi khun qu nh, chnh xc cng khng cao v khng phn bit c gia t bo sng v t bo cht. Hnh 14.5: Phng m Petroff-Hauser: (a)- Mt nhn nghing ca phng m- Phng m cha dch huyn ph vi khun l khong khng gian bn di l knh; (b)- Gia phin knh c phng m vi cc nh; (c) phng i khong x 400-500 tin hnh m s lng vi khun trong cc nh. Ly s lng bnh qun tnh ra mt vi khun trong mu vt. Trong phm vi 1mm2 c 225 nh , do s lng vi khun trn 1mm2 l (s vi khun/mm) x 25; v phng m c chiu dy l 0,02mm do nng vi khun trong phng m l: (s vi khun)/m2 x 25 (tng s nh) x 50= s vi khun/mm 3. V 1 cm3=1 mm3 x 103cho nn gi th s lng vi khun bnh qun trong mi nh l 28 th trong 1 cm3 c nng vi khun l 28 x 25 x 50 x103= 3,5 x 107vi khun. Nhn vi pha long ban u (nu c) s bit c nng vi khun trong mu kim tra.

Vi ng vt nguyn sinh, vi to v nm men c th dng my m in t nh loi my Coulter Counter xc nh s lng. Nguyn l l hai bn mi l nh c in cc v ni in. Khi t bo trong dch huyn ph i qua l nh th c mi t bo i qua th in tr li tng ln (hoc tnh dn in gim xung) v sinh ra mt tn hiu in, my m s t ng ghi s. Kt qu xc nh ca loi my ny kh chnh xc, c th ng dng rng ri xc nh s lng hng cu v bch cu, nhng phng php ny khng thch hp xc nh s lng vi khun v d b can thip bi cc ht nh v cc vt cht dng si trong mu vt. C hai phng php ni trn u khng phn bit c t bo sng v t bo cht. xc nh s lng t bo sng ngi ta thng dng phng php cy dch pha long ln b mt mi trng thch a. Sau khi nui cy mi vi khun s to thnh 1 khun lc. V d pha long 1 x 10-6 m c 150 khun lc th c ngha l mt vi khun trong mu l 1,5 x 108. Dng dng c m khun lc cng thm thun tin. Phng php ny cho bit s lng cc t bo sng ca vi sinh vt. Phng php ny n gin, nhy cm v thch hp ng dng rng ri xc nh s lng vi sinh vt sng khi phn tch cc mu thc phm, nc, t...Tuy nhin kt qu cng chu nh hng ca mt s nhn t. Nu vi khun dnh thnh khi khng tch ri nhau ra th kt qu thu c l thp hn thc t., v mi khun lc khng pht trin t mt t bo ring r. V vy kt qu thu c t phng php ny c coi l s n v hnh thnh khun lc (CFU-colony forming unit). CFU khng hon ton ph hp vi s t bo sng trong mu vt. Trong qu trnh s dng phng php ny nn s dng pha long no cho s khun lc xut hin trn a ch nm trong phm vi khong 30-300 m thi. ng nhin mi trng dinh dng khng th p ng chung cho mi loi vi sinh vt, do kt qu thu c bao gi cng thp hn thc t. Khi trn thch vi dch pha long th thch ngui khng lm cht vi khun hay lm thng tn vi mt s loi mn cm vi nhit . Vic cy cy dch pha long trn b mt ri dn u bng que gt thy tinh thng cho kt qu cao hn v s lng vi sinh vt so vi phng php trn vi mi trng thch cha ng.

Hnh 14.6: Tch khun lc v phng php kim tra s lng vi sinh vt thng qua m khun lc mc trn mi trng thch a. (a) (b)- Cch ria cy tch khun lc ring r (khng dng m s lng) (c)(d)- Cch pha long ri trn vi mi trng thch cha ng (e)(f)- Cch dn dch pha long bng que gt trn mt thch (cho s lng khun lc nhiu hn).Theo sch ca K.P.Talaro,2005.

xc nh s lng vi sinh vt cn c th nui cy giy lc lc dch pha long mu vt. Phng php ny gi l phng php mng lc (membrane filter). Dng mt thit b lc c bit t va mt giy lc hnh trn c cc l nh hn kch thc vi khun v cc vi sinh vt khc. Sau khi lc t giy lc ln mi trng thch thch hp hoc thm t mng lc bng dch mi trng thch hp ri nui cy 24 gi. m s khun lc mc trn giy lc tnh ra mt vi khun sng c mt trong mu vt (hnh 14.7)

Hnh 14.7: Phng php lc mng xc nh s lng vi sinh vt

Phng php ny thch hp s dng phn tch vi sinh vt trong nc. C th dng cc mi trng khc nhau thch hp vi cc nhm vi sinh vt khc nhau (hnh 14.8)

Hnh 14.8: Cc loi khun lc mc trn mng lc. Theo sch ca Prescott,Harley v Klein (2005) (a)- Tng s vi khun mc trn mi trng tiu chun, Dng ch th mu nhum khun lc cho d im; (b)- Dng mi trng thch hp kim tra nhm vi khun coliform c ngun gc t phn (khun lc bt mu xanh); (c)- Dng mi trng thch m-Endo xc nh vi khun E.coli v cc Coliform khc- khun lc c mu lc; (d)- Nm si v nm men mc trn mi trng Thch - Mch nha.

Phng php mng lc cn dng m trc tip vi khun. Dch mu vt c lc qua mt mng polycarbonate mu en. Vi khun trn mng lc c nhum mu hunh quang bng thuc nhum acridine da cam hoc DAPI (diamidino-2-phenylindole). Quan st di knh

hin vi hunh quang c th thy cc t bo vi sinh vt hin ln mu da cam hay mu lc trn mt nn en. Hin c nhng kit thng mi cho php phn bit t bo sng v t bo cht khi kim tra. 14.2.2. Xc nh khi lng t bo S sinh trng ca vi sinh vt khng ch biu hin s lng t bo m cn c s tng trng ca tng khi lng t bo. Phng php trc tip nht l xc nh trng lng kh ca t bo. Trc ht cn ly tm thu nhn sinh khi t bo. Sau ra t bo ri lm kh trong l sy ri cn trng lng kh. Phng php ny thch hp xc nh s sinh trng ca nm. Phng php ny tn thi gian v khng tht mn cm. i vi vi khun v trng lng tng c th l rt nh, thm ch phi ly tm ti vi trm ml mi s lng xc nh trng lng sinh khi kh. Phng php nhanh hn, mn cm hn l dng phng php o c nh tn x nh sng. Mc tn x nh sng t l thun vi nng t bo. Lc nng vi khun t n 107 t bo/ml th dch nui cy s vn c, nng cng tng th c cng tng theo v lm cn tr nh sng i qua dch nui. C th o tn x nh sng bng quang ph k (spectrophotometer). mt mc hp th nh sng thp, gia nng t bo v gi tr hp th nh sng c quan h tuyn tnh (hnh 14.9). Ch cn nng vi sinh vt t ti nng c th o c l u c th dng phng php o c trn quang ph k xc nh s sinh trng ca vi sinh vt. Nu hm lng mt s vt cht trong mi t bo l ging nhau th tng lng cht trong t bo c tng quan trc tip vi tng sinh khi vi sinh vt. Chng hn, thu t bo trong mt th tch nht nh ca dch nui cy, ra sch i ri o tng lng protein hay tng lng nitrogen, c th thy s tng qun th vi sinh vt l ph hp vi s tng tng lng protein (hay N). Cng tng t nh vy, vic xc nh tng lng chlorophyll c th dng o sinh khi to; o hm lng ATP c th bit c sinh khi ca cc vi sinh vt sng.

Hnh 14.9: o s lng vi sinh vt bng phng php o c.

Thng qua vic o hp th nh sng c th xc nh c sinh khi vi sinh vt. Khi s lng t bo tng ln s dn n vic tng c, mc tn x nh sng nhiu hn v quang ph k s o c mc tng ln ca tr s hp th nh sng. Trn quang ph k c hai thang chia : pha di l tr s hp th nh sng, pha trn l mc thu quang. Khi tr s hp th nh sng tng ln th mc thu quang h xung. 14.3. NUI CY LIN TC VI SINH VT Trong cc phn trn chng ta xem xt vic nui cy phn m (batch cultures) trong cc h thng kn, tc l khng c chuyn b sung cht dinh dng, cng khng thi loi cc sn phm c hi sinh ra trong qu trinh sng. Giai on logarit ch duy tr qua vi th h sau chuyn vo giai on n nh. Nu nui cy vi sinh vt trong mt h thng h, trong qu trnh nui cy thng xuyn b sung cht dinh dng v thi loi cc cht cn b th c th lm cho mi trng lun gi trng thi n nh. l h thng nui cy lin tc (continuous culture system). Trong h thng ny s sinh trng ca vi sinh vt lun gi c trng thi logarit, nng sinh khi vi sinh vt lun gi c n nh trong mt thi gian tng i di. Gi th ta c mt bnh nui cy trong vi khun ang sinh trng, pht trin. Ta cho chy lin tc vo bnh mt mi trng mi c thnh phn khng thay i. Th tch bnh nui cy gi n nh. Dng mi trng i vo b p cho dng mi trng i ra vi cng mt tc . Ta gi th tch ca bnh l v (lit), tc dng mi trng i vo l f (lt/ gi). Tc (hay H s) pha long c gi l D (f/v). i lng D biu th s thay i th tch sau 1 gi. Nu vi khun khng sinh trng v pht trin th chng s b rt dn ra khi bnh nui cy theo tc :

dX/dt = D.X

X l sinh khi t bo Ngi ta thng dng hai loi thit b n nui cy lin tc vi sinh vt. l Chemostat v Turbidostat. 14.3.1. Chemostat Khi s dng Chemostat nui cy vi sinh vt ngi ta a mi trng v khun vo bnh nui cy vi lng tng ng vi tc a mi trng cha vi khun ra khi bnh nui cy (xem hnh 14.10). Trong mi trng mt s cht dinh dng thit yu ( nh mt vi acid amin) cn khng ch nng trong mt phm vi nht nh. V vy tc sinh trng ca vi sinh vt trong h thng quyt nh bi tc mi trng mi c a vo h thng v nng t bo ph thuc vo nng cc cht dinh dng c hn ch. Nhp i mi cht dinh dng biu th bi nhp pha long D (dilution rate). Tc lu thng ca cht dinh dng (ml/h) c biu th bng f v th tch bnh nui cy l V (ml): D= f/V Chng hn nu f l 30ml/h v V l 100ml th nhp pha long D l 0,30h-1. C s lng vi sinh vt v thi gian th h u c lin quan n nhp pha long (hnh 14.11). Trong mt phm vi nhp pha long tng i rng th mt vi sinh vt trong h thng l khng thay i.. Khi nhp pha long tng ln, thi gian th h h xung (tc sinh trng tng ln), khi cht dinh dng hn ch b tiu hao ht. Nu nhp pha long qu cao th vi sinh vt b loi ra khi bnh nui cy trc khi kp sinh si ny n bi v lc nhp pha long cao hn tc sinh trng ca vi sinh vt. Nng cc cht dinh dng hn ch tng ln khi nhp pha long tng cao vi c t vi sinh vt s dng chng.

Hnh 14.10: Nui cy lin tc trong Chemostat v Turbidostat

Hnh14.11: H thng nui cy lin tc (Chemostat)

Khi nhp pha long rt thp th nu tng nhp pha long s lm cho c mt t bo v tc sinh trng u tng ln. l do hiu ng ca nng cht dinh dng i vi nhp sinh trng (growth rate). Quan h ny c lc c gi l quan h Monod (Monod relationship). Trong iu kin nhp pha long thp , ch c t i cht dinh dng c cung cp th t bo phi dng phn ln nng lng duy tr s sng ch khng dng sinh trng, pht trin. Lc nhp pha long tng ln, cht dinh dng tng ln, t bo c nhiu nng lng c cung cp, khng nhng duy tr s sng m cn c th dng sinh trng, pht trin, lm tng cao mt t bo. Ni cch khc, khi t bo c th s dng nng lng vt qu nng lng duy tr (maintenance energy) th nhp sinh trng s bt u tng ln.

Hnh 14.12: T l pha long trong chemostat v sinh trng ca vi sinh vt

14.3.2. Turbidostat Turbidostat l loi h thng nui cy lin tc th hai. Thng qua t bo quang in (photocell) o hp th nh sng hay c trong bnh nui cy t ng iu chnh lu lng mi trng dinh dng, lm cho c hay mt t bo gi mc nh d kin. Turbidostat v Chemostat c nhiu im khc nhau. Trong h thng Turbidostat mi trng khng cha cc cht dinh dng hn ch, nhp pha long khng c nh. Turbidostat hot ng tt nht khi nhp pha long cao trong khi Chemostat li n nh nht v hiu qu nht khi nhp pha long tng i thp. H thng nui cy lin tc l rt c li v cc t bo lun trng thi sinh trng thuc giai on logarit. Hn na c th dng lm m hnh nghin cu s sinh trng ca vi sinh vt trong iu kin nng cht dinh dng thp tng t nh mi trng t nhin. H thng nui cy lin tc rt c ch trong vic nghin cu nhiu lnh vc khc. Chng hn, nghin cu tc dng tng h gia cc loi vi sinh vt trong iu kin mi trng tng t nh mi trng nc ao h nc ngt. H thng nui cy lin tc c s dng trong cc ngnh

vi sinh vt hc cng nghip v thc phm. 14.4. NH HNG CA CC NHN T MI TRNG N S SINH TRNG CA VI SINH VT Nh chng ta bit vi sinh vt c kh nng p ng vi s bin ha ca nng cht dinh dng, nht l cc cht dinh dng hn ch. S sinh trng ca vi sinh vt chu nh hng rt ln i vi cc nhn t vt l, ha hc ca mi trng sng. Hiu bit v nh hng ca cc nhn t mi trng i vi s sinh trng ca vi sinh vt gip ch rt nhiu cho vic khng ch vi sinh vt cng nh i vi vic nghin cu s phn b sinh thi ca vi sinh vt. ng ch l mt s vi sinh vt c th sng c trong nhng iu kin cc oan (extreme) v kh sng (inhospitable). Cc vi sinh vt nhn nguyn thy (Procaryotes) c th sinh tn ti mi ni c th sinh sng. Nhiu ni cc vi sinh vt khc khng th tn ti c nhng vi sinh vt nhn nguyn thy vn c th sinh trng rt tt. Chng hn vi khun Bacillus infernus c th sng su 1,5 dm di mt t, ni khng c xy v c nhit cao n 600C. Nhng vi sinh vt c th sinh trng c trong nhng hon cnh h khc nh vy c gi l cc vi sinh vt a cc oan. Trong phn ny chng ta s xem xt nh hng ca mt s nhn t ch yu ca mi trng i vi s sinh trng ca vi sinh vt (bng 14.3) Bng 14.3: Phn ng ca vi sinh vt vi cc nhn t mi trng nh ngha Vi sinh vt i din Hot tnh ca nc v dung cht Vi sinh vt a p C th sinh trng trong Staphylococcus aureus, (Osmotolerant) mt phm vi rng v Saccharomyces hot tnh ca nc v nng thm thu. Vi sinh vt a mn (Halophile) Cn sinh trng nng Halobacterium,Dunaliella, NaCl cao, thng l Ectothiorhodospira t 0,2 mol/L tr ln. pH a acid (Acidophile) Sinh trng tt nht Sulfolobus,Picrofilus, Ferroplasma, trong phm vi pH 0-5,5 Acontium, Cyanidum caldarium. a trung tnh (Neutrophile) Sinh trng tt nht Escherichia, Euglena, Paramecium trong phm vi pH 5,58,0 a kim(Alkalophile) Sinh trng tt nht Bacillus alcalophilus, trong phm vi pH 8,5- Natronobacterium 11,5 Nhit a lnh(Psychrophyle) Sinh trng tt nht Bacillus psychrophilus, Thut ng

Chu lnh(Psychrotroph)

a m(Mesophile)

150C hay thp hn. C th sinh trng 070C nhng sinh trng tt nht 20-300C, cn c th sinh trng c khong 350C Sinh trng tt nht 25-450C.

Chlamydomonas nivalis Listeria monocytogenes, Pseudomonas fluorescens

a nhit(Thermophile)

a nhit cao (Hyperthermophile)

Hiu kh bt buc (Obligate aerobe) K kh khng bt buc (Facultative anaerobe)

K kh chu Oxy (Aetolerant anaerobe) K kh bt buc (Obligate anaerobe) Vi hiu kh (Microaerophile)

a p (Barophile)

Escherichia coli, Neisseria, Gonorrhoeae, Trichomona vaginalis. C th sinh trng Bacillus stearothermophilus, nhit 550C hoc cao Thermus aquaticus, Cyanidium hn, nhit thch hp caldarium, Chaetomium nht thng l gia 55 thermophile v 650C Thch hp pht trin Sulfolobus, Pyrodictium, nhit gia 80 v Pyrococcus. 0 khong 113 C Nng xy Hon ton da vo O2 Micrococcus luteus, Pseudomonas, ca khng kh sinh Mycobacteriun, phn ln To, Nm trng v V nguyn sinh Khng cn O2 sinh Escherrichia, Enterococcus, trng nhng sinh Saccharomyces cerevisiae trng tt hn khi c mt O2. Sinh trng nh nhau Streptococcus pyogenes khi c mt hay khng c xy B cht khi c mt O2 Clostridium, Bacteroides, Methanobacterium, Trepomonas agilis. Cn O2 mc thp Campylobacter, Spirillum volutans, hn2-10% sinh Treponema pallidum trng v b tn hi trong khng kh (20%) p sut Sinh trng nhanh hn Photobacterium profindum, khi p sut thy tnh cao Shewanella benthica, Methanococcus jannaschii

14.4.1. Hot tnh ca nc v dung cht V t bo vi sinh vt tch vi mi trng ca chng bng loi mng sinh cht c tnh thm thu chn lc cho nn vi sinh vt chu nh hng ca s bin i nng thm thu trong

mi trng. Nu mt vi sinh vt c a vo dung dch c nng thm thu thp th nc s xm nhp vo t bo, nu khng c s khng ch hu hiu th t bo s b trng ln v v ra. Nh c cc th ni hm m c th gim thp nng thm thu ca t bo cht. Lc tnh thm thu ca mi trng thp hn tnh thm thu ca t bo cht, cc vi sinh vt nhn nguyn thy cng c th ph v cc knh mn cm vi p sut lm cho dung cht thm ra. Phn ln vi khun, to v nm thng c thnh t bo vng chc, c th duy tr hnh thi v tnh hon chnh ca t bo. Lc a t bo cc vi sinh vt c thnh t bo vng chc vo mi trng p sut thm thu cao th nc s thot ra, mng sinh cht s tch ra khi thnh t bo v to ra tnh trng co nguyn sinh. S mt nc ny ca t bo s tn hi ti mng t bo cht, t bo s mt kh nng trao i cht v ngng sinh trng. iu ny lin quan n vic bo qun thc phm nh mui mn hoc tm ng (lm mt, ngm mt ong...). Nhiu vi sinh vt s dng dung cht hn hp (compatible solute) lm cho nng thm thu ca nguyn sinh cht cao hn mi trng chung quanh, lm cho mng sinh cht vn gn c vi thnh t bo. S d gi l dung cht hn hp l v dung cht c th thch hp t bo sinh trng v pht trin ngay khi c nng cao trong t bo. Phn ln vi sinh vt nhn nguyn thy c th nng cao nng thm thu trong t bo mi trng p sut thm thu cao l nh tng hp hoc hp thu choline, betaine, proline, acid glutamic v cc acid amin khc. Vic nng cao nng K+ cng c th mt mc no gip nng cao nng thm thu trong t bo. To v nm th s dng saccharose v cc polyol, v d nh arabitol, glycerol, mannitol,... t c mc ch nh vy. Polyol v acid amin l nhng dung cht l tng nng cao nng thm thu trong t bo bi v chng khng ph hy cu trc v chc nng ca enzym. Nhiu vi sinh vt nhn nguyn thy nh Halobacterium salianarium s dng K+ nng cao nng thm thu trong t bo, Na+ cng c tc dng ny nhng khng s dng c cao nh K+. Cc enzyme ca Halobacterium cn nng mui cao duy tr hot tnh. ng vt nguyn sinh do khng c thnh t bo nn phi s dng cc khng bo (vacuoles) bi xut phn nc d tha khi sng trong mi trng c nng thm thu thp. Tc dng tng h gia phn t nc v phn t dung cht c gi l hiu ng thm thu (osmotic effect) cn hiu ng c cht (matric effect) l biu th cc phn t nc bi hp ph (adsorption) trn b mt cc cht rn. Hai hiu ng ny dn n vic gim st phn nc c th dc vi sinh vt s dng. V nng thm thu trong mi trng c nh hng su sc i vi vi sinh vt cho nn nh lng kh nng s dng nc cc nh vi sinh vt hc thng dng khi nim hot nc aw (water activity aw) biu th tnh hu hiu ca nc. Cng c th dng th nng nc (water potential) tng quan vi aw biu th tnh hu hiu ca nc. Hot nc ca mt dung dch l 1/100 ca m tng i ca dung dch ny (tnh theo %), cng l tng ng vi t l gia p sut bay hi ca dung dch ny (Psoln) v p sut bay hi ca nc tinh khit (Pwater):

Hot nc ca mt dung dch hay mt cht rn c th xc nh bng cch a vo mt

vt cha kn v o m tng i sau khi h thng t ti trng thi cn bng (equilibrium). V d, mt mu vt sau khi t ti trng thi cn bng trong h thng ny m khng kh t ti 95% bo ha, th cng tc l khng kh cha 95% m khi t ti cn bng cng nhit vi mt mu nc thun khit, hot nc ca mu vt ny l 0,95%. Hot nc t l nghch vi p sut thm thu, nu p sut thm thu cao th tr s aw l thp. Cc vi sinh vt khc nhau c nng lc thch ng khc nhau rt ln i vi mi trng c hot nc thp (bng 14.4) Bng 14.4: Tr s tng i v hot nc (aw) thp nht i vi s sinh trng ca vi sinh vt (theo A.D. Brown, 1976) Hot nc 1,00 0,95 Mi trng Nc thun khit Bnh m Vi khun, Nm, To Phn ln VK Gram (-) khng a mn Phn ln trc khun Gram (-) Basidiomycetes Fusarium Phn ln Mucor,Rhizopus, Bacillus. i gia sc Phn ln cu khun, Nm men c bo t ti. Salami Staphylococcus Thc phm mui Saccharomyces rouxii (trong mui), Penicillium H mui Halobacterium, Aspergillus, C mui Dunaliella , Actinospora Ng cc, ko, qu kh Aspergillus Scla, mt ong, sa bt Saccharomyces rouxii (trong ng), Xeromyces bisporus ADN b ph hy

0,90 0,85 0,80 0,75 0,70 0,60 0,55

C th k thm vi v d khc v tr s aw: mu ngi- 0,995; qu ti- 0,97-0,98; nc bin0,98; tht gia sc ti- 0,97; sir- 0,90; gim bng- 0,90; lp xng- 0,85; mt qu- 0,80; nc ng bo ha- 0,76; bt m- 0,65... Vi sinh vt mun gi lng nc bng cch duy tr dung cht ni bo nng cao khi sinh trng trong mi trng c hot nc thp s gp kh khn kh ln. Nhng vi sinh vt c th tn ti trong nhng iu kin nh vy c gi l cc vi sinh vt chu p (osmotolerant). Chng c th sinh trng c trong mt phm vi nng thm thu hoc hot nc kh rng. V d , vi khun Staphylococcus aureus c th nui cy trn mi trng c nng NaCl cao ti 3 mol/L. Chng cng c th thch ng sinh trng trn da ngi. Nm men Saccharomyces rouxii c th sinh trng trn dung dch ng c hot nc thp n 0,6. To lc Dunaliella viridis c th chu c nng NaCl cao n 1,7mol/L hoc nng bo ha.

Mc du mt s t vi sinh vt c th thc s chu p nhng phn ln vi sinh vt ch c th sinh trng tt hot nc khong 0,98 (tng ng vi aw ca nc bin) hoc cao hn na. Li dng iu ny ngi ta s dng phng php sy kh hay dng mui, dng ng bo qun thc phm, phng tp nhim bi vi sinh vt. Tuy nhin nhiu nm chu p vn c th lm h hng cc thc phm sy kh hoc p mui, tm ng. Vi sinh vt a mn (Halophile) hon ton thch ng vi mi trng cao p (hypertonic), cn nng NaCl cao sinh trng. Phm vi nng mui cn thit sinh trng i vi nhm vi khun a mn cc oan (extreme halophilic bacteria) l 2,8-6,2 mol/L (nng mui bo ha). Ti Bin Cht (Dead Sea)- mt h thp nht th gii nm gia Israel v Jordan, v ti h i Dim (Great Salt Lake) bang Utah (Hoa K) v ti cc mi trng khc c nng mui gn vi bo ha, c th phn lp c cc C khun (archeon) thuc chi Halobacterium. Chng cng cc vi khun a mn cc oan khc khng ging vi phn ln cc vi sinh vt chu p (osmotolerant) ch khng phi l n gin thng qua vic nng cao nng dung cht ni bo, m ch yu l sa i cu trc protein v mng ca mnh thch ng vi nng mui cao. Nhng vi khun a mn cc oan ny duy tr nng kali ni bo sao cho p sut thm thu cao hn mi trng sng; nng K+ ni bo c th ti 47 mol/L. Cc enzym, ribosom v protein vn chuyn ca cc vi khun ny cn nng K+ cao duy tr tnh n nh v hot tnh. Ngoi ra nng Na+ cao cng gip cho s n nh ca t bo v mng sinh cht ca vi khun Halobacterium. Nu nng Na + qu thp th thnh t bo v mng sinh cht s hon ton b ph hy. Vi khun a mn cc oan thch ng thnh cng vi iu kin mi trng mui cao, ni c th tiu dit hu ht cc sinh vt khc. Tuy nhin chng cng bit ha (specialized), mt i tnh linh hot (flexibility) sinh thi v ch c th sinh trng trong mt t mi trng cc oan. 14.4.2. pH pH l s o hot tnh ion hydrogen ca mt dung dch v l s logarit m ca nng ion hydrogen (biu th bng nng phn t): pH= -log[H+]= log (1/[H+ ]) Thang pH t pH 0,0 (1,0 mol H+) n pH 14,0 (1,0 x 10 -14mol H+). Mi n v pH i biu cho s bin i 10 ln v nng ion hydrogen. Hnh 14.13 cho thy ni c tr m vi sinh vt c th sinh trng l rt rng, t pH rt acid (pH 1-2) n nhng h hay t rt kim vi pH gia 2 v 10. pH c nh hng r rt i vi s sinh trng ca vi sinh vt. Mi vi sinh vt u c mt phm vi pH sinh trng nht nh v pH sinh trng tt nht. Vi sinh vt a acid (acidophile) c pH sinh trng tt nht l pH 0-5,5 ; i vi vi sinh vt a trung tnh l pH 5,5-8,0 ; i vi vi sinh vt a kim (alkalophile) l pH 8,5-11,5. Vi sinh vt a kim cc oan c mc sinh trng ti u pH 10 hay cao hn na. Ni chung, cc nhm vi sinh vt khc nhau u c phm vi sinh trng ring ca mnh. Phn ln vi khun v ng vt nguyn sinh l a trung tnh. Phn ln nm l a hi acid (pH 4-6). Cng c nhiu trng hp ngoi l. V d, to Cyanidium caldarium v c khun Sulfolobus acidocaldarius thng

sng trong cc sui nc nng acid, chng sinh trng tt nhit cao v pH t 1 n 3. C khun Ferroplasma acidarmanus v Picrophilus oshimae c th sinh trng pH=0 hay rt gn vi 0. Bng14.5: Thang pH

Mc du vi sinh vt thng c th sinh trng trong mt phm vi pH kh rng, v xa vi pH tt nht ca chng, nhng tnh chu ng (tolerance) ca chng cng c gii hn nht nh. Khi pH trong t bo cht c s bin ha t ngt s lm ph v mng sinh cht hoc lm c ch hot tnh ca enzyme hay proteine chuyn mng, do lm tn thng n vi sinh vt. Vi sinh vt nhn nguyn thy b cht khi pH ni bo gim xung thp hn 5,0-5,5. S bin i pH ca mi trng s lm thay i trng thi in ly ca phn t cc cht dinh dng, lm h thp kh nng s dng chng ca vi sinh vt. Khi pH trong mi trng c s bin ha tng i ln th pH ni bo ca phn ln vi sinh vt vn gn trung tnh. Nguyn nhn c th l do tnh thm ca H+ qua mng sinh cht l tng i thp. Vi sinh vt a trung tnh thng qua h thng vn chuyn s dng K+ thay cho H+. Vi sinh vt a kim cc oan nh Bacillus alcalophilus dng Na+ ni bo thay th cho H+ ca mi trng bn ngoi, gi cho pH ni bo gn vi trung tnh. Ngoi ra h thng cht m ni bo (intering buffering) cng c vai tr quan trng trong vic duy tr pH n

nh. Vi sinh vt phi c nng lc thch ng vi s bin i pH ca mi trng th mi c th sinh tn. i vi vi khun, h thng vn chuyn ngc K+/H+ v Na+/H +c th dng khc phc nhng bin i nh v pH. Nu pH qu acid cc c ch s pht huy tc dng. Lc pH gim xung ti pH 5,5-6,0 vi khun thng hn (Salmonella typimurium) v Escherichia coli c th tng hp ra mt lot cc protein mi v c gi l mt phn ca p ng chng chu acid. ATPase chuyn v proton c dng sn sinh ra nhiu ATP hoc bm proton Ra ngoi t bo. Nu pH bn ngoi gim xung cn 4,5 hay thp hn na vi khun s tng hp ra cc phn t i km, chng hn nh cc protein gy sc acid (acid shock proteins) hay cc protein gy sc nhit (heat shock proteins). Chng c dng phng nga s bin tnh acid ca cc proten khc v gip sa cha li cc protedins b bin tnh. Vi sinh vt thng sinh ra cc cht thi trao i cht c tnh acid hay kim lm thay i pH mi trng sng. Vi sinh vt ln men s dng ngun carbonhydrat to ra cc acid hu c. Cc vi sinh vt dinh dng ha nng v c (chemolithotrophs) nh Thiobacillus c th xy ha cc hp cht lu hunh dng kh sinh ra acid sulfuric. Mt s vi khun khc thng qua vic phn gii cc acid amin lm sinh ra NH3 v lm kim ha mi trng. Ngi ta thng b sung cc cht m (buffers) vo mi trng nui cy phng nga s c ch qu trnh sinh trng ca vi sinh vt khi pH bin ha qu ln. Phosphat l cht m thng c s dng, in hnh l mui H2PO4- acid yu v mui HPO42- kim yu :

H+ + HPO42- H 2PO4OH- + H2PO4 - HPO42- + HOH

Nu b sung H+ vo h thng m n s kt hp vi HPO42- to ra acid yu. Nu b sung OH- vo h thng m n s kt hp vi H2PO 4- to thnh nc. Nh vy l pH mi trng khng b bin ha qu ln. Trong cc mi trng phc tp th peptid v cc acid amin cng c nng lc m (buffering effect) rt mnh. 14.4.3. Nhit Cng ging nh cc sinh vt khc, nhit ca mi trng cng c nh hng rt ln i vi vi sinh vt. Trn thc t, do vi sinh vt thng l cc sinh vt n bo cho nn chng rt mn cm vi s bin ha ca nhit , v thng b bin ha cng vi s bin ha v nhit ca mi trng xung quanh. Chnh v vy, nhit ca t bo vi sinh vt cng phn nh trc tip nhit ca mi trng xung quanh. Mt nhn t quyt nh nh hng ca nhit i vi s sinh trng ca vi sinh vt l tnh mn cm vi nhit ca cc phn ng xc tc nh enzym. Trong phm vi nhit thp, khi nhit tng ln s lm tng tc sinh trng ca vi sinh vt, v phn ng xc tc nh enzyme cng ging nh cc phn ng ha hc ni chung, khi nhit tng ln 100C tc phn ng s tng gp i. V cc phn ng trong t bo u tng cho nn ton b hot ng trao i cht s tng ln khi nhit

cao hn, v vi sinh vt s sinh trng nhanh hn. Lc nhit tng ln n mt mc nht nh th nhit cng tng tc sinh trng cng gim. Khi nhit tng qu cao vi sinh vt s cht. Khi nhit qu cao s gy ra s bin tnh ca enzym, ca cc th vn chuyn (transport carriers) v cc protein khc. Mng sinh cht s b tn thng v hai lp lipid s b ha tan. Do mc du nhit cng cao cc phn ng xc tc tin hnh cng nhanh nhng do cc nguyn nhn ni trn m t bo b tn thng n mc kh hi phc v dn n vic c ch sinh trng. Ti iu kin nhit rt thp mng sinh cht b kt ng li, enzyme cng ngng hot ng. Ni chung, nu vt qu nhit tt nht i vi vi sinh vt, chc nng v kt cu t bo u b nh hng. Nu nhit rt thp, tuy chc nng chu nh hng nhng thnh phn ha hc v kt cu khng nht thit chu nh hng. Do nh hng hai mt, va c li va c hi ca nhit i vi vi sinh vt m c th xc nh cc loi nhit c bn (cardinal temperaturre) i vi s sinh trng ca vi sinh vt. l nhit thp nht (minimum), nhit tt nht (optimum) v nhit cao nht (maximum) i vi s sinh trng.

Hnh 14.13: nh hng ca nhit i vi tc sinh trng ca vi sinh vt (Theo sch ca Prescott, Harley v Klein)

Mc du ng biu din nh hng ca nhit i vi sinh trng ca vi sinh vt l ph thuc vo tng vi sinh vt, tng iu kin khc nhau nhng nhit tt nht thng gn vi nhit cao nht hn l so vi nhit thp nht. Ba nhit c bn ca cng mt loi vi sinh vt khng phi l c nh m thng ph thuc vo pH, thc n v cc nhn t khc. Chng hn, mt loi ng vt nguyn sinh c tin mao l Crithidia fasciculate sng trong ng tiu ha ca mui c th sinh trng trn mi trng n gin nhit 22-270C nhng nhit 33-340C th li khng sinh trng c nu khng b sung vo mi trng ion kim loi, acid amin, vitamin v lipid. Nhit c bn ca cc vi sinh vt khc nhau l khc nhau rt nhiu. Nhit tt nht c th thp t 00C n cao ti 750C. Nhit thp nht sinh trng c th n -20 0C. Nhit cao nht c th vt qu 1000C. Nhn t ch yu quyt nh phm vi sinh trng ny c th l nc. Ngay trong iu kin ti cc oan th vi sinh vt cng cn c nc trng thi dch th mi c th sinh trng. i vi s ng vi sinh vt th phm vi nhit sinh trng thng trong khong 30 0C. Mt s vi sinh vt (nh Cu khun lu - Nesseria gonorrhoeae)

c phm vi nhit sinh trng rt hp. Trong khi cng c nhng vi sinh vt (nh Enterococcus facalis) li c phm vi nhit sinh trng rt rng. Nhit sinh trng cao nht l khc nhau gia cc nhm ln vi sinh vt. Nhit sinh trng cao nht i vi ng vt nguyn sinh (protozoa) l 500C. Mt s to v nm c th sinh trng nhit cao ti 55-60 0C. Mt s vi sinh vt nhn nguyn thy c th sinh trng 1000C (nhit nc si) hay gn nh vy. Gn y ngi ta cn pht hin thy c nhng vi sinh vt sinh trng c c nhng iu kin nhit cao hn 1000C. c cc thng bo cho bit pht hin thy cc vi sinh vt nhn nguyn thy ti dch phun giu sulphid (black smoker) vt nt di y bin - ni nhit nc cao ti 3500C. Nhng vi sinh vt ny sinh trng, pht trin rt tt nhit 113 0C v cn c th sinh trng, pht trin nhit cao hn na. p sut cao ming ni la di y bin lm cho nc nhiu siu cao vn tn ti trng thi dch th ( p sut 265 atm nc bin s si 4600C). Pht hin ny cho thy protein, mng, acid nucleic ca cc vi sinh vt ny c tnh khng nhit rt cao. y l nhng vt liu rt tt gip cho vic nghin cu c ch n nh ca mng v cc cao phn t sinh hc. Tng lai c th ngh n kh nng thit k cc enzyme c th pht huy tc dng trong nhng iu kin rt cao. Nhng enzyme bn nhit t cc vi sinh vt ny s c nhng ng dng rt quan trng trong cng nghip v trong nghin cu khoa hc. Chng hn men Taq polymerase nhn c t c khun Thermus aquaticus c ng dng rng ri trong phn ng chui polymerase. R rng l vi sinh vt nhn nguyn thy c th sinh trng c nhng nhit cao hn vi sinh vt nhn tht. l v vi sinh vt nhn tht khng c th to ra c cc mng c quan t c chc nng tng ng iu kin nhit cao hn 600C. Ngoi ra cc c quan quang hp cng hu nh khng n nh nh vy v do khng pht hin thy c s sinh trng ca cc vi sinh vt quang hp trong mi trng nhit rt cao. Cn c vo phm vi nhit sinh trng c th chia vi sinh vt thnh 5 nhm. Bng 14.6: Phm vi nhit (N) i vi s sinh trng ca vi sinh vt Vi sinh vt Bacillus psychrophilus Micrococcus cryophilus Psedomonas fluorescens Staphylococcus aureus Enterococcus faecalis Escherichia coli Neisseria gonorrhoeae Thermoplasna acidophilum Bacillus stearothermophilus Thermus aquaticus Sulfolobus acidocaldarius Pyrococcus abyssi Pyrodictium occultum N thp nht N tt nht VSV khng quang hp -10 23-34 -4 10 4 25-30 6,5 30-37 0 37 10 37 30 35-36 45 59 30 60-65 40 70-72 60 80 67 96 82 105 N cao nht 28-30 24 40 46 44 45 38 62 75 79 85 102 110

Pyrolobus fumarii

90 106 Vi khun quang hp v vi khun lam Rhodospirillum rubrum 30-35 Anabaena variabilis 35 Osillatoria tenuis Synechococcus eximius 70 79 To nhn tht Chlamydomonas nivalis -36 0 Fragilaria sublinearis -2 5-6 Chlorella pyrenoidosa 25-26 Euglena gracilis 23 Skeletonema costatum 6 16-26 Cyanidium caldarium 30-34 45-50 Nm Candida scottii 0 4-15 Saccharomyces cerevisiae 1-3 28 Mucor pusillus 21-23 45-50 ng vt nguyn sinh Amoeba proteus 4-6 22 Naegleria fowleri 20-25 35 Trichomonas vaginalis 25 32-39 Paramecium caudatum 25 Tetrahymena pyriformis 6-7 20-25 Cyclidium citrullus 18 43

113 45-47 84 4 8-9 29 >28 56 15 40 50-58 35 40 42 28-30 33 47

Vi sinh vt a lnh (Psychrophile): l cc vi sinh vt c th sinh trng 00C, sinh trng tt nht 150 C hay thp hn, nhit cao nht ch l khong 20 0C. Ti Nam cc v Bc cc d dng phn lp cc vi sinh vt thuc nhm ny. V c ti 90% nc bin thp hn hay bng 50C nn ti c lng ln cc vi sinh vt a lnh. Chlamydomonas nivalis l mt loi to a lnh, chng sinh bo t mu ti lm cho khi bng tuyt c mu phn hng (pink). Phn ln vi khun a lnh thuc v cc chi Pseudomonas, Vibrio, Alcaligenes, Bacillus, Arthrobacter, Moritella, Photobacterium, v Shewanella. C khun Methanogenum a lnh gn y c phn lp ti h Ace Chu Nam cc.Vi sinh vt a lnh thng qua nhiu loi phng thc thch ng c vi mi trng lnh. Chng pht huy c ch rt tt tng hp protein, enzym, cc h thng vn chuyn. Mng t bo ca vi sinh vt a lnh c cha nhiu cc acid bo khng bo ha, c th gi c trng thi cht bn lu (semifluid) khi gp lnh. Tuy nhin nhiu vi sinh vt a lnh nhit cao hn 200C mng t bo s b ph hi. Nhiu vi sinh vt sinh trng tt nht nhit 20-300 C, nhit cao nht l cao hn 350C, nhng chng vn c th sinh trng trong iu kin 0-70C.Chng thuc v nhm a lnh khng bt buc (Psychrotrophs hay facultative psychrophiles). Nhng vi khun v nm

thuc nhm ny l nguyn nhn chnh lm h hng thc phm gi lnh. Vi sinh vt a m (Mesophile): l cc vi sinh vt sinh trng tt nht 20-450C, nhit sinh trng thp nht l 15-200C. Nhit sinh trng cao nht l khong 45 0C hoc thp hn. Phn ln vi sinh vt l thuc v nhm ny. Hu nh mi vi khun gy bnh cho ngi u l vi sinh vt a m, bi v thn nhit ca ngi l 37 0C.

Hnh 14.14 :Phm vi nhit sinh trng ca vi sinh vt (Theo sch ca Prescott, Harley v Klein).

Vi sinh vt a nhit (Thermophile): l cc vi sinh vt sinh trng c nhit 550C hay cao hn na. Nhit sinh trng tt nht i vi chng l 33-650C. Thnh phn ch yu ca nhm ny l vi khun (ch yu l x khun), mt t to v nm (bng 14.6). Chng pht trin trong ng phn chung , di y cc ct rm r hay c kh, trong ng dn nc nng, trong cc sui nc nng...Vi sinh vt a nng khc vi vi sinh vt a m ch chng c h thng tng hp enzyme v protein bn nhit (heat-stable) v c th hot ng nhit cao. Mng sinh hc ca chng c lipid bo ha mc cao, c im si cao hn v v vy vn gi c nguyn vn nhit cao. C mt s t cc vi sinh vt a nhitc th sinh trng nhit 900C hay cao hn. Nhit sinh trng cao nht l 100 0C. Ngi ta xp cc vi sinh vt c nhit sinh trng tt nht 80-1130C vo nhm Vi sinh vt a siu nng (Hyperthermophiles). Chng thng khng th sinh trng bnh thng nhit thp hn 550C. Vi khun Pyrococcus abyssi v Pyrodictium occultum l v d v nhng vi sinh vt a siu nhit c tm thy nhng y bin nng. 14.4.4. Nng oxygen Cc vi sinh vt sinh trng trong iu kin c oxygen c gi l vi sinh vt hiu kh (aerobe), cn cc vi sinh vt sinh trng trong iu kin khng c oxygen c hi l cc vi sinh vt k kh (anaerobe). Hu ht cc c th a bo u phi cn sinh trng trong iu

kin c oxygen, chng l cc sinh vt hiu kh bt buc (obligate aerobes). Oxygen lcht nhn in t cui cng trong chui vn chuyn in t khi h hp hiu kh. Ngoi ra, cc vi sinh vt nhn tht (eucaryotes) hiu kh cn dng oxygen tng hp sterol v cc acid bo khng bo ha. Cc vi sinh vt k kh khng bt buc (facultative anaerobes) khng cn oxygen sinh trng nhng khi c oxygen th sinh trng tt hn. Khi c oxygen chng s dng phng thc h hp hiu kh. Cc vi sinh vt k kh chu oxygen (aerotolerant anaerobes) nh vi khun Enterococcus faecalis c th sinh trng nh nhau trtong iu kin c oxygen cng nh khng c oxygen. Ngc khun Bacteroides, Fusobacterium, Clostridiun pasteurianum, Methanococcus.... s b cht khi c oxygen.

Hnh 14.15: Oxygen v s sinh trng ca vi khun Ch thch: Cc nhm vi sinh vt xem trong bi Mi chm biu hin khun lc ca vi khun trong hay trn b mt mi trng. SOD v catalase l biu th vi khun c tn ti enzyme superoxide dismutase v catalase hay khng?(Theo sch ca Prescott,Harley v Klein)

Vi sinh vt k kh chu oxygen v vi sinh vt k kh bt buc khng sinh nng lng thng qua qu trnh h hp, chng thu c nng lng thng qua qu trnh ln men hay h hp k kh (anaerobic respiration). Sau cng, phi k n nhm vi sinh vt vi hiu kh (microaerophiles), chng khng sinh trng c trong iu kin khng kh bnh thng (20% O2) v cn sinh trng trong iu kin nng O2 khong 2-10% Quan h gia vi sinh vt v oxygen c th xc nh bng mt th nghim n gin nha sau: nui cy vi sinh vt trong ng nghim cha mi trng c hoc mi trng c bit nh mi trng cha thioglycollate (l cht kh lm gim nng oxygen trong mi trng). Cng mt nhm vi sinh vt c th c nhiu loi quan h khc nhau vi O2. C 5 loi hnh u c th thy vi sinh vt nhn nguyn thy (prpcaryotes) v ng vt nguyn sinh. Nm thng l hiu kh, ch tr mt s loi c bit, nht l nm men, thuc loi k kh khng bt buc. To hu nh u thuc loi hiu kh bt buc. ng ch l nng lc c th sinh trng c trong mi trnghiu kh ln mi trng k kh lm cho vi sinh vt c tnh linh hot cao v chnh l mt loi u th sinh thi hc.

Mc du O2 c th lm cht cc vi sinh vt k kh bt buc, nhng trong mi trng hiu kh vn c th phn lp c chng. l do chng thng sng chung vi loi k kh khng bt buc v b ny tiu th ht O2 , to nn mi trng k kh cc b gip cho vi sinh vt k kh bt buc c th sinh trng c. V d trong khoang ming vi khun k kh bt buc Bacteroides gingivalis c th sinh trng c trong cc khe k kh quanh rng. S khc bit trong quan h ca vi sinh vt vi O2 do nhiu nguyn nhn khc nhau, bao gm vic bt hot ca protein v tc dng c hi ca O2 trong iu kin hiu kh. Cc enzyme c th b bt hot khi cc nhm mn cm nh sulfhydryls b oxy ha. Chng hn nh enzyme c nh m nitrogenase l loi rt mn cm vi O 2 . V hai in t bn ngoi ca oxygen khng thnh cp do rt d tip nhn in t v b kh. Flavoprotein, mt s thnh phn t bo khc v s bc x u c th thc y vic kh oxy, to thnh cc sn phm kh nh gc t do superoxide, hydrogen peroxide, gc hydroxyl. O2 + e- O2- (gc t do superoxide) O2 + e- + 2H+ H 2O2 (hydrogen peroxide) O2 + e- + H+ H 2O + OH-(gc hydroxyl) Cc sn phm kh oxy ny l cc k c hi v chng l cc cht oxy ha mnh v ph hy nhanh chng cc thnh phn t bo. Vi sinh vt no phi c nng lc t chng li c cc sn phm kh ny mi trnh khi b tiu dit. Bch cu trung tnh (neutrophils) v i thc bo (macrophage) li dng cc sn phm c hi ny tiu dit cc vi sinh vt gy bnh xm nhp c th. Nhiu vi sinh vt sinh ra cc enzyme chng li cc sn phm kh c hi ny. Vi khun hiu kh bt buc v vi khun k kh khng bt buc thng cha cc enzyme nh superoxide dismutase (SOD) v catalase, chng phn bit xc tc vic ph hy gc superoxide v hydrogen peroxide. Peroxidase cng c th dng ph hy hydrogen peroxide:

2O2- + 2H+ superoxide dismutase O2 + H 2O 2H2O2catalase 2H 2O + O 2 H2O2 + NADH + H+peroxidase 2H2O + NAD +
Vi sinh vt k kh chu oxygen c th thiu catalase nhng hu ht lun c superoxide dismutase. Vi khun k kh chu oxygen Lactobacillus plantarum dng ion Mn2+ thay th SOD phn gii gc t do ca superoxide. Tt c cc vi sinh vt k kh bt buc u khng c hai loi enzyme ni trn hoc c vi nng rt thp v do khng c nng lc

chng chu c vi oxygen. V vi sinh vt hiu kh cn O2, trong khi vi sinh vt k kh li b cht v O2, cho nn vic nui cy hai nhm ny pha bng cc phng php hon ton khc nhau. Lc nui cy khi lng ln vi sinh vt hiu kh phi nui cy trn my lc hay phi thi khng kh v khun vo bnh (hay ni) nui cy . Cn khi nui cy vi khun k kh th phi loi b ht O2. C th dng cc phng php sau y: - S dng mi trng k kh c bit, c cha cc cht kh nh thioglycolate hay cysteine. Khi ch to mi trng cn un ln lm tan cc thnh phn v cng ng thi loi tr ht O 2 ha tan trong mi trng. Khi vi sinh vt k kh c th mc c ln trn b mt mi trng. - Nui cy trong cc t nui cy k kh (anaerobic work chamber) ht chn khng v b sung bng kh nitrogen. Thng cn cn b sung c kh CO2 bi v nhiu vi khun k kh sinh trng tt khi c tn ti mt lng nh kh CO2 . - Mt phng php rt ph bin khi nui cy mt lng nh vi sinh vt k kh l dng bnh k kh (Gas Pak jar). Trong h thng ny li dng H2 v cht xc tc palladium lm cho O2 kt hp vi H2 to thnh nc khng c O 2 trong mi trng. Cc cht kh a vo mi trng thch cng c th gip loi b O2. - C th dng ti nha to ra cc mi trng k kh khi nui cy mt lng nh cc vi sinh vt k kh. Trong ti nha cha CaCO3 v cht xc tc to ra iu kin k kh giu CO 2. Mt dung dch c bit c a vo ti nha sau a hp lng (hp Petri) hay cc dng c nui cy khc vo v hn kn ti nha li. Ty tng trng hp c th m s dng cc phng php nui cy k kh khc nhau.

Hnh 14.17: Nui cy vi sinh vt k kh trong cc bnh k kh. (Theo sch ca Prescott,Harley v Klein).

14.4.5 p sut (Pressure) Phn ln vi sinh vt c th sng trn lc a hay trn b mt nc, l nhng ni c p sut khng kh l 1 atm (atmosphere) v khng chu nh hng r rt g ca p sut ny.Nhng y bin (ni c su 1000m tr ln) li chim n 75% th tch i dng. nhng ni p p sut cao n 600-1000m, nhit lnh ti 2-3 0C. Trong mi trng cc oan (extreme) nh vy vn c mt s vi sinh vt thch ng tn ti. Phn ln thuc v nhm chu p (barotolerant). Tuy p sut tng ln cao s c nh hng n chng, nhng nh hng bt li ny nh hn nhiu khi so vi cc vi sinh vt khng chu p (nontolerant). Mt s vi khun sng trong ng tiu ha ca nhng ng vt khng xng sng di bin su (nh amphipods v holothurians) l nhng vi khun a p (barophilic). Chng sinh trng cng nhanh trong iu kin p sut cao. Chng c vai tr quan trng trong vng tun hon cc cht dinh dng di y bin. Ti khe bin Mariana gn Philippine (su khong 10 500m) ngi ta phn lp c nhng vi khun a p c th sinh trng trong iu lin 20C vi p sut khong 400-500atm. Nhng vi khun ny hin bit l thuc v cc chi Photobacterium, Shewanella, Colwellia...Mt s thuc v C khun va a p va a nhit (thermobarophiles), chng hn nh Pyrococcus spp., Methanococcus jannasschi... 14.4.6. Bc x (Radiation) Th gii m chng ta ang sng y cc loi bc x in t trng (electromagnetic radiation). Cc bc x ny hnh thnh nh sng trn mt nc v lan truyn trong khng kh. C ly gia hai nh sng hay cui sng c gi l di sng (wavelength). Khi di sng gim i th th nng lng bc x tng ln. Tia gamma hay tia X c nng lng cao hn bc x ca nh sng nhn thy (nh sng kh kin) hay tia hng ngoi. Bc x in t trng cn ging nh mt dng nng lng hp bi cc photon (quang t). Mi photon u c nng lng nht nh, nng lng cao hay thp quyt nh bi di sng ca bc x.

nh sng mt tri l ngun bc x ch yu trn tri t, bao gm nh sng kh kin (visible light), tia t ngoi (ultraviolet), tia hng ngoi (infraded rays) v sng radio (v tuyn in). nh sng kh kin l loi thng thy v quan trng nht trong mi trng chung quanh chng ta: mi s sng u ph thuc vo cc c th c kh nng quang hp da vo nng lng ca mt tri. Bc x mt tri c 60% nm vng tia hng ngoi ch khng phi vng nh sng kh kin. Tia hng ngoi l ngun nhit lng ch yu ca tri t. tm mt bin ch thy c rt t bc x t ngoi 290-300nm (nanometre). Tia t ngoi c bc sng thp hn 287nm hp th bi oxygen trong khng kh v to ra tng ozone (O3) cao cch mt t khong 25-50km. Tng ozone hp th cc tia t ngoi bc sng tng i di v gii phng ra O2 . Bi v tia t ngoi rt c hi cho sinh vt nn vic tng ozone tiu tr bt tia t ngoi l c tc dng rt quan trng i vi s sng trn tri t. V cc loi bc sng trong nh sng mt tri phn b ng u trong phm vi nh sng kh kin cho nn ta thy nh sng mt tri c bn c mu trng. Nhiu bc x in t trng l rt c hi i vi vi sinh vt, nht l cc bc x c bc sng ngn, cao nng lng l cc bc x ion ha (ionizing radiation), chng lm nguyn t mti in t (electron) hoc ion ha (ionize). C hai loi bc x ion ha. Mt l, tia X to ra bi con ngi, hai l,tia gamma ( tia ) sinh ra trong qu trnh tan r cc ng v phng x (radioisotope). Bc x ion ha mc thp s lm sn sinh cc t bin (mutations) v gin tip lm cht vi sinh vt. Bc x ion ha cao s trc tip git cht vi sinh vt. Mc du vi sinh vt c tnh khng cao hn v cc bc x ion ha so vi cc sinh vt khc, nhng vi liu lng cao chng s git ht vi sinh vt. Chnh v vy c th dng bc x ion ha dit khun. Tuy vy, mt s sinh vt nhn nguyn nthy (nh vi khun Deinococcus radiodurans v cc vi khun sinh vt sinh bo t) c th vn tn ti c ngay c cc mc bc x ion ha kh cao. Bc x ion ha gy cho t bo rt nhiu bin ha, c th ph v lin kt hudrogen, oxy ha lin kt i, ph hy cu trc vng, cao phn t ha mt s phn t.Oxygen c th lm tng cc hiu ng ny, c th l do vic sn sinh gc t do hydroxyl (OH-)... Mc du c rt nhiu thnh phn t bo chu nh hng, nhng nguyn nhn quan trng nht gy cht l s ph hy ADN. V bc sng ngn (10-400nm) c nng lng cao cho nn bc x t ngoi (Ultraviolet radiation) c th tiu dit cc loi vi sinh vt. Bc x t ngoi (UV) mnh nht bc sng 260nm. Chng d b ADN hp th, lm cho trn 1 si n ADN hnh thnh nhng song phn t (dimers) thymine, chng lm c ch qu trnh ti to (replication) v cng nng ca ADN. Thit hi ny c th c sa cha qua mt s con ng. Theo con ng quang hot ha mt loi enzyme quang hot ha s dng nh sng xanh lam tch song phn t thymine. Trong hot ha ti mt on ngn ADN c cha cc song phn t thymine c th b ct ri v i ch thnh mt on ADN bnh thng. Thit hi ny cng c th sa cha nh cc protein recA trong qu trnh ti t hp (recombination) hoc qu trnh SOS. Thit hi khng c th c khc phc nu nh liu lng UV qu ln, to nn nhng tn tht qu nng. Mc du bc x UV qu nh (thp hn 290 v 300nm) kh c th lt xung b mt tri t, bc x UV nhng bc sng 325-400nm cng c th gy hi cho vi sinh vt. Chng phn ct

tryptophan thnh nhng quang sn phm (photoproducts) c hi. Nhng sn phm ny tc dng ng thi vi cc bc x gn t ngoi lm ph v si ADN. C ch c th ca tc dng ny khng ging vi c ch tc dng ca UV vi bc sng 260nm. nh sng kh kin l ngun nng lng ch yu ca qu trnh quang hp. (photosynthesis) v vy rt cn cho cc sinh vt. Nhng nu nh sng kh kin qu mnh s c th gy hi hay lm cht vi sinh vt. Tham gia vo qu trnh ny c mt loi sc t gi l cht quang mn (photosensitizers) v oxygen. Cc sc t vi sinh vt nh chlorophyll, bacteriochlorophyll, cytochromes,v flavin c th hp th nh sng mt tri v b kch hot to ra cc cht quang mn. Cc cht quang mn (P) khi b kch hot c th chuyn nng lng cho O2 lm ra oxygene n - singlet oxygen ( O2).

nh sng P P (hot tnh) P hot tnh + O2 P + O 2

Oxygenn l cht c hot tnh rt mnh, l cht oxy ha mnh c th ph hy nhanh chng t bo, chng cng l nhn t ch yu c cc i thc bo (phagocytes) dng dit khun.

Hnh 14.18: Phm vi bc sng ca cc bc x in t trng - Phn nh sng kh kin c trnh by pha di. (Theo sch ca Prescott,Harley v Klein).

Nhiu vi sinh vt trong khng kh hoc sng trn b mt cc vt tip sc vi khng kh s

dng sc t carotenoid bo v chng li vi quang oxy ha (photooxidation). Carotenoid c th lm ph hy cc oxygen n, hp thu nng lng ca oxygen n v bin thnh trng thi phi hot tnh. C cc vi sinh vt quang hp ln vi sinh vt khng quang hp u s dng sc t vo mc ch ny. 14.5. S SINH TRNG CA VI SINH VT TRONG MI TRNG T NHIN 14.5.1. Cc nhn t ca mi trng lm hn ch s sinh trng Mi trng sinh sng ca vi sinh vt l phc tp v thng xuyn bin i.Vi sinh vt c trng cho mi mi trng c th b bao bc bi s bin i ca cc cht dinh dng v cc nhn t mi trng khc. ng l vi sinh vt sinh trng trong mt mng sinh hc (biofilm). Vi sinh vt sinh trng trong mt vi mi trng (microenvironments) cho n khi mi trng hay cc nhn t dinh dng t ti s sinh trng gii hn. Nguyn tc lng ti thiu ca Liebig xc nh rng: tng sinh khi ca mt c th quyt nh bi s c mt ca cht dinh dng vi nng thp nht theo nhu cu ca c th. Nguyn tc ny c th ph hp cho iu kin phng th nghim cng nh trong mi trng t v nc. S tng ln ca mt nhn t dinh dng cn thit (chng hn nh phosphate) s lm tng ln qun th vi sinh vt cho n khi mt s nhn t dinh dng khc tr thnh nhn t gii hn. Nu mt cht dinh dng no l gii hn th tng cc nhn t dinh dng khc khng c ch li g. Tnh hnh thc t cn phc tp hn th na. Nhiu nhn t gii hn c th nh hng thng xuyn ln qun th vi sinh vt, chng hn nh nhit , pH, nh sng, nng mui... nh lut chng chu ca Shelford (Shelfords law of tolerance) xc nh: vi sinh vt c mt yu cu nht nh i vi cc nhn t mi trng, Thp hay cao hn yu cu ny th vi sinh vt khng th tn ti v sinh trng mc du vn c y cc cht dinh dng. Chng hn mi vi sinh vt c mt phm vi nhit sinh trng nht nh. Cng tng t nh vy i vi pH, nng oxygen, nng mui... S sinh trng ca vi sinh vt ph thuc vo c s cung cp cht dinh dng ln kh nng chng chu vi cc iu kin ca mi trng. Khi vi sinh vt c iu kin dinh dng sinh trng mnh m th cng ng thi sinh ra cc cht thi c hi v lm hn ch s sinh trng ca chng. p ng vi mc dinh dng thp (mi trng ngho - oligotrophic environments) v c s cnh tranh, nhiu vi sinh vt chim ot thc n v khai thc chng lm ngun cnh tranh. Vi sinh vt thng bin i hnh thi lm tng b mt v nng lc hp thu cht dinh dng. Cc vi sinh vt nhn nguyn thy hnh que bin thnh dng mini hay ultramini (siu nh) hoc mc ra cc ci cung (prosthecate) p ng vi tnh trng thiu thc n. S thiu ht cht dinh dng s dn n nhiu bin i vi sinh vt, chng hn chng c th tng bc khp li hot ng ca cc gen lin quan n trao i cht , tr vic duy tr gen qun gia (housekeeping gene). Nhiu nhn t c th lm ci bin mc dinh dng trong mi trng ngho. Chng hn vi sinh vt c th tch cc cht dinh dng hn ch ra (nh l st) khin khng cn st cnh tranh na. Khng kh cng c th cung cp cht dinh dng gip cho s sinh trng ca vi

sinh vt. iu ny c th thy c trong phng th nghim cng nh ngoi thin nhin. pht hin thy cht hu c trong khng kh c th xc tin s sinh trng ca vi sinh vt trn mi trng pha long (dilute media). Mi trng sinh trng c lm giu bng cht hu c trong khng kh cng c th lm tng r rt s pht trin ca qun th vi sinh vt. Ngay trong nc ct- thng cha du vt cht hu c, cng c th hp thu nhng hp cht 1 carbon t khng kh gip cho s sinh trng va vi sinh vt. S tn ti cc cht dinh dng trong khng kh v tnh trng sinh trng ca vi sinh vt, nu khng xem xt n s c th nh hng n cc thc nghim v sinh ha hc hay sinh hc phn t, cng nh cc nghin cu v s sinh trng ca vi sinh vt trong cc mi trng dinh dng ngho (oligotrophic). Cc cht t nhin (natural substances) cng c th c ch trc tip ti s sinh trng v pht trin ca vi sinh vt trong cc mi trng dinh dng thp. Cc cht bao gm phenol, tannin, ammonia, ethylene, v cc hp cht lu hunh bay hi. y c th l mt phng thc gip vi sinh vt trnh tn dng cc nng lng gii hn trc khi c cung cp cc cht dinh dng cn thit. Cc ha cht ny l rt quan trng trong bnh l hc thc vt v c th gip khng ch cc bnh vi sinh vt trong t. 14.5.2. Kim tra s lng vi sinh vt nhn nguyn thy tuy sng nhng khng nui cy c Khi nghin cu s sinh trng ca qun th vi sinh vt nhn nguyn thy (procariotic) trong thin nhin bn ngoi phng th nghim cn phi xc nh s lng vi sinh vt sng. Trong lch s vi sinh vt hc thng ngi ta nh ngha vi sinh vt sng l c th sinh trng, c th hnh thnh khun lc (colony) hoc to ra c r rt trn mi trng dch th. John R.Postgate i hc Sussex (nc Anh) l mt trong nhng hc gi u tin xc nh vi sinh vt chu c ch (stressed) khi sng trong mi trng thin nhin, hoc trong nhiu mi trng phng th nghim c bit, l c bit mn cm vi cc c ch th sinh (secondary stresses). Cc c ch ny lm cho vi sinh vt tuy sng nhng khng c th to thnh khun lc trn cc mi trng c bnh thng vn dng nui cy chng. xc nh c s sinh trng ca cc vi sinh vt ny Postgate a ra mt phng php thc nghim gi l th nghim vi hot tnh Postgate (Postgate Microviability Assay). Trong th nghim ny em vi sinh vt nui cy trn lp mt thch mng di l knh (coverslip), lm cho chng khng qua c giai on sinh trng n bo m ch bin i hnh thi t bo, coi l biu hin tn hiu sng (life sign) T , nhiu nh nghin cu pht trin cc phng php hin vi mn cm v phng php cht ng v xc nh s tn ti v ngha ca dng vi khun sng nhng khng nui cy c. Chng hn dng khng th hunh quang v thuc nhum acridine orange dnh gi mc s lng; hoc l dng phng php s lng kh nng ti a (MPN-most probable number)...Vic s dng ch s gii phng hot tnh phng x ca vt cht t bo cng c dng xc nh nh hng ca hiu ng c ch i vi vi sinh vt. Cc phng php do Postgate ra l rt quan trng. Nhiu nghin cu cho thy c khi mt s vi khun nh Escherichia coli, Vibrio cholerae, Klebsiella pneumoniae, Enterobacter aerogenes, Enterococcus faecalis mt nng lc sinh trng trn cc mi trng phng th nghim theo

cc k thut nui cy tiu chun, nhng chng vn gi vai tr gy bnh truyn nhim. Tnh trng mt qun th hn hp trong mi trng thin nhin l rt phc tp. Thng thng ch c khong 1-10% cc t bo l c th hnh thnh khun lc. Trong tng lai c th phi tm ra cc mi trng nui cy thch hp hn i vi cc vi sinh vt cn cha c bit n. Hin nay ngi ta s dng k thut PCR v phn tch ARN ca tiu th ribosome nh gi tnh a dng ca qun th cc vi sinh vt cha nui cy c. 14.5.3. Cm ng mt v cc qun th vi sinh vt T nhiu thp k nay, cc nh vi sinh vt hc vn ngh rng qun th vi sinh vt l tp hp ca cc c th ring bit, sinh trng v hot ng c lp vi nhau. Tuy nhin gn y ngi ta pht hin thy nhiu vi khun c kh nng giao tip v hot ng hp tc vi nhau. Mt trong nhng cch c bn vi sinh vt hp tc c vi nhau l cm ng mt hay cn li l s t cm ng. l hin tng trong vi sinh vt t iu chnh mt thng qua qu trnh cm nhn hm lng cc phn t tn hiu, i khi gi l cc cht t cm ng (autoinducer) bi v chng c th kch thch t bo tit ra chng. Nng cc phn t tn hiu tng ln cng vi s tng ln ca s lng vi khun trong qun th cho n khi t n ngng c trng (i vi qun th ) v ra tn hiu cho vi khun rng mt qun th n mc ti hn hay cn gi l quorum. Vi khun lc s bt u biu hin cc gen ph thuc mt t bo ti hn nhm iu chnh mt t bo. Cm ng mt c pht hin c vi khun Gram m v Gram dng. Cm ng mt c ngha quan trng vi vi sinh vt. C th ly v d v s sinh tng hp v gii phng cc enzyme ngoi bo. Nu cc enzyme ny ch c gii phng nh mt s t vi khun, chng s b khuch tn v khng pht huy c tc dng do b pha long. Vi cch iu khin bng cm ng mt , vi khun s t n mt qun th ln trc khi chng gii phng enzyme v kt qu l hm lng enzyme ln pht huy tc dng. chnh l li th ca vi sinh vt trong c th vt ch cng nh trong cc mi trng t, nc. Nu vi sinh vt gy bnh c th t c n nng ln ti mt im no trn c th vt ch trc khi sn sinh cc nhn t c lc v xm nhp c vo cc m vt ch, chng s c c hi ln hn trong vic lm mt tc dng kh nng t v ca vt ch v do c th lan ra ton b c th vt ch. iu ny gii thch mt kiu khc ca cm ng mt . Dng nh cm ng mt rt quan trng i vi nhiu vi sinh vt trong vic thit lp mi quan h cng sinh hay k sinh i vi vt ch. Cm ng mt c pht hin u tin v tm hiu r nht vi khun Gram m. Cc tn hiu thng gp nht vi khun Gram m l acyl homoserine lactones (HSLs). l cc phn t nh c cu to bi chui acyl t 4 n 14 C lin kt vi homoserine lactone (hnh 14.19). Chui acyl ny c th c nhm keto hay nhm v tr C th 3. Cc phn t Acyl HSLs khuch tn vo cc t bo ch (target cell) (hnh 14.19). Khi t n hm lng ln, cc phn t Acyl HSLs s bm vo cc protein th th c bit (R) v gy ra s thay i cu trc protein. Thng thng khi phc h HSLs-protein c hot ha, chng c tc dng nh cht cm ng, chng bm vo cc im ch trn ADN v kch thch s phin m ca cc gen nhy cm vi nng t bo ti hn. Cc gen cn thit tng hp HSL cng c

to ra thng xuyn, do nhiu cht t cm ng c tng hp v gii phng.

Hnh 14.19: Cm ng mt vi khun Gram m. (Theo sch ca Prescott,Harley v Klein). (a) Cu trc chung ca acyl homoserine lactone, cht c bit n nh l tn hiu cm ng mt (iu chnh mt t bo) hay cn gi l cht t cm ng. (b) Lc minh ha cch hot ng ca cm ng mt nhu vi khun gram m. Cc protein th th hot ng vi vai tr cht cm ng (inducer) c k hiu bng ch R. ng k gy nt biu th acyl HSL synthase khng phi lc no cng c to ra tng ng vi cc cht t cm ng.

vi khun Gram m, rt nhiu qu trnh nhy cm vi cc tn hiu acyl HSL v cm ng mt . Mt s v d c nghin cu k l (1) s sn sinh cht pht quang sinh hc (bioluminescence) bi Vibrio fischeri, (2) Pseudomonas aeruginosa tng hp v gii phng cc yu t gy bnh, (3) S chuyn cc yu t di truyn nh qu trnh tip hp Agrobacterium tumefaciens, v (4) S sn xut khng sinh Erwinia carotovora v Pseudomonas aureofaciens. Vi khun Gram dng cng iu ha hot ng bng cm ng mt , thng thng bng tn hiu l cc oligopeptide. Cc v d in hnh l s tip hp Enterococcus faecalis, kch thch kh nng ti np ADN (competence induction) Streptococcus pneumoniae, s kch thch to bo t Bacillus subtilis, v s sn sinh rt nhiu c t v cc yu t gy bnh Staphylococcus aureus. Cm ng mt thm ch kch thch s pht trin khun ty kh sinh v to streptomycin Streptomyces griseus. i vi trng hp ca Streptomyces griseus th c l tn hiu l g-butyrolaton ch khng phi l oligopeptide.

Chc nng quan trng na ng quan tm ca cm ng mt l s y mnh vic to mng sinh hc (biofilm) bi vi khun gy bnh Pseudomonas aeruginosa, v c th n ng vai tr quan trng trong bnh x ha u nang (crystic fibrosis). S to mng sinh hc rt c ngha i vi vi sinh vt gy bnh v n bo v vi khun khi s tn cng ca khng sinh v cc cht ty ra. S iu chnh mt t bo c th rt hiu qu bn trong mng sinh hc do hm lng acyl HSL t b pha long v c th tng ln nhanh chng. Trong iu kin ny, hai loi vi khun khc nhau c th kch thch nhau bng cch sn xut ra cc tn hiu tng t nhau, l trng hp mng sinh hc c cha cc vi khun gy bnh P. aeruginosa v Burkhoderia cepacia. Cm ng mt l mt v d v s hot ng a t bo trong cc c th giao tip v hp tc hot ng nh l mt n v thng nht. Cc v d khc v hot ng phc hp nh trn l s hnh thnh cc dng khun lc v s hnh thnh th qu Nim vi khun (Myxobacteria).

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