Vi sinh vt Dinh dng ca vi sinh vt 13.1. YU CU DINH DNG CA VI SINH VT 13.1.1. Thnh phn ho hc ca t bo vi sinh vt C s vt cht cu to nn t bo vi sinh vt l cc nguyn t ho hc. Cn c vo mc yu cu ca vi sinh vt i vi cc nguyn t ny m ngi ta chia ra thnh cc nguyn t a lng v cc nguyn t vi lng. Cc nguyn t ch yu bao gm: C, H, O, N, P, S, K, Mg, Ca v Fe. Trong s ny c 6 loi ch yu (chim n 97% trng lng kh ca t bo vi sinh vt), l C, H, O, N, P v S. Cc nguyn t vi lng thng l Zn, Mn, Na, Cl, Mo, Se, Co, Cu, W, Br v B. T l cc nguyn t ho hc tham gia cu to t bo vi sinh vt l khng ging nhau cc nhm vi sinh vt khc nhau. V d nm men, nm si v vi khun c lng cha trung bnh ca 6 nguyn t ch yu l khng ging nhau (bng 13.1): Bng 13.1: Lng cha trung bnh cc loi nguyn t ch yu trong t bo mt s nhm vi sinh vt (% trng lng kh) Nguyn t C H O N P S Vi khun ~50 ~8 ~20 ~15 ~3 ~1 Nm men ~50 ~7 ~31 ~12 Nm si ~48 ~7 ~40 ~5 -

Theo cc ti liu ca Tempest (1969), Pirt (1975) v Herbert (1976) th thnh phn trung bnh ca cc nguyn t to nn t bo vi sinh vt ni chung l nh sau:

Bng 13.2: Thnh phn cc nguyn t cu to nn sinh khi t bo

Nguyn t

% trng lng kh* Trung bnh Bin 50 45-58 21 12 8 3 1 1 0.5 1 0.5 0.5 1 18-31 5-17 6-8 1.2-10 0.3-1.3 0.2-5 0.1-1.1 0.02-2.0 0.01-5.0

Cc ngun dinh dng in hnh c s dng cho sinh trng VSV trong mi trng CO2, hp cht hu c H20, 02, cc hp cht hu c NH3, NO3-, cc hp cht hu c cha N Nc, cc hp cht hu c. Phosphate v cc hp cht cha P. SO4-2, H2S, v cc hp cht cha S. K+ (c th thay th bng Rb+) Mg2+ Ca2+ ClFe3+, Fe2+ v phc cht ca Fe Na+ Ly t cc ion v c khc

C O N H P S K Mg Ca Cl Fe Na

Nhng 0.5 nguyn t khc,Mo, Ni, Co, Mn, Zn, ..

*Cc t bo bao gm 70% trng lng l nc v 30% l cc nguyn liu kh khc. Mc trung bnh ny c tnh theo sinh trng ca vi khun Gr(-) trong iu kin d tha cht dinh dng nui cy theo m. Vi khun lu hunh (sulfur bacteria), vi khun st (iron bacteria) v vi khun i dng (marine bacteria) c lng cha cc nguyn t S, Fe, Na, Cl nhiu hn so vi cc nhm vi khun khc. To Silic (diatom) c cha lng SiO2 kh cao trong thnh t bo. Thnh phn cc nguyn t ho hc cn thay i trong mt phm vi nht nh tu thuc vo tui nui cy v iu kin nui cy. Khi nui cy trn cc mi trng c ngun N

phong ph th lng cha N trong t bo s cao hn so vi khi nui cy trn cc mi trng ngho ngun N. Cc nguyn t ho hc ch yu tn ti trong t bo vi sinh vt di dng cht hu c, cht v c v nc. Cht hu c thng bao gm protein, carbon hydrat, lipid, acid nucleic, vitamin v cc sn phm phn gii ca chng cng nh cc cht trao i cht. phn tch cc thnh phn hu c trong t bo thng s dng hai phng php: mt l, dng phng php ho hc trc tip chit rt tng thnh phn hu c trong t bo, sau tin hnh phn tch nh tnh v nh lng. Hai l, ph thnh t bo, thu nhn cc thnh phn kt cu hin vi ri phn tch thnh phn ho hc ca tng kt cu . Cht v c thng ng ring r di dng mui v c hoc kt hp vi cht hu c. Khi phn tch thnh phn v c trong t bo ngi ta thng phn tch tro sau khi nung t bo nhit 5500 C, cht v c thu c di dng cc oxit v c c gi l thnh phn tro. Dng phng php phn tch v c c th nh tnh hay nh lng tng nguyn t v c. Bng 13.3:Thnh phn ha hc ca t bo vi khun (theo F.C.Neidhardt et al.,1996) Phn t kh (1) / t bo - Nc - Cc i phn t +Protein +Polysaccharide +Lipid +ADN +ARN - Cc n phn t +Aminoacid v tin th +ng v tin th +Nucleotid v tin th - Cc ion v c Tng cng % khi lng 96 55 5 9,1 3,1 20,5 3,0 0,5 2 0,5 1 100 24 609 802 2 350 000 4 300 22 000 000 2,1 255 500 S phn t S loi phn t 1 khong 2500 khong 1850 2 (2) 4 (3) 1 khong 660 khong khong khong khong khong 350 100 50 200 18

Ch thch: (1) -Khi lng kh ca t bo vi khun Escherichia coli ang sinh trng l khong 2.8 x 10-13g. (2) - Gi thit Peptidoglycan v Glycogen l 2 thnh phn ch yu. (3) - T bo cha vi loi phospholipid, do tnh a dng ca thnh phn acid bo gia cc chi vi khun khc nhau v do nh hng ca iu kin sinh trng m c nhiu hnh thc tn ti ca mi loi phospholipid. Nc l thnh phn khng th thiu duy tr hot ng sng bnh thng ca t bo. Nc thng chim n 70-90% trng lng t bo. chnh lch gia trng lng ti v trng lng kh chnh l lng nc trong t bo, thng biu th bng t l % tnh theo cng thc sau y: (Trng lng ti - Trng lng kh) / Trng lng ti x 100%. n v trng lng t bo trong dch nui cy thng c biu th bng n v g/l hay mg/ml. Phng php nung kh t bo nhit 5500C thng lm phn gii mt s hp cht ca t bo v vy khi tnh trng lng kh ca t bo nn dng phng php sy kh 1050C hay lm kh nhit khng cao trong chn khng, hoc lm kh nhanh nh tia hng ngoi... 13.1.2. Cc cht dinh dng v chc nng sinh l Vi sinh vt ch yu thu nhn c cht dinh dng t mi trng bn ngoi. Cn c vo chc nng sinh l khc nhau trong t bo m ngi ta thng chia cc cht dinh dng thnh 5 nhm ln: 1) Ngun carbon (source of carbon) L ngun vt cht cung cp C trong qu trnh sinh trng ca vi sinh vt. Trong t bo ngun C tri qua mt lot qu trnh bin ho ho hc phc tp s bin thnh vt cht ca bn thn t bo v cc sn phm trao i cht. C c th chim n khong mt na trng lng kh ca t bo. ng thi hu ht cc ngun C trong cc qu trnh phn ng sinh ho cn sinh ra trong t bo ngun nng lng cn thit cho hot ng sng ca vi sinh vt. Mt s vi sinh vt dng CO2 lm ngun C duy nht hay ch yu sinh trng, khi ngun C khng phi l ngun sinh nng lng. Vi sinh vt s dng mt cch chn lc cc ngun C. ng ni chung l ngun C v ngun nng lng tt cho vi sinh vt. Nhng tu tng loi ng m vi sinh vt c nhng kh nng s dng khc nhau. V d trong mi trng cha glucose v galactose th vi khun Escherichia coli s dng trc glucose (gi l ngun C tc hiu) cn galactose

c s dng sau (gi l ngun C tr hiu). Hin nay trong cc c s ln men cng nghip ngi ta s dng ngun C ch yu l glucose, saccharose, r ng (ph phm ca nh my ng) tinh bt (bt ng, bt khoai sn...), cm go, cc ngun cellulose t nhin hay dch thu phn cellulose. Nng lc ng ho cc ngun C cc vi sinh vt khc nhau l khng ging nhau. C loi c kh nng s dng rng ri nhiu ngun C khc nhau, nhng c loi kh nng ny rt chn lc. Chng hn vi khun Pseudomonas c th ng ho c ti trn 90 loi hp cht C, nhng cc vi khun thuc nhm dinh dng methyl (methylotrophs) th ch ng ho c cc hp cht 1C nh methanol, methane... Ngun C ch yu c vi sinh vt s dng gm c ng, acid hu c, ru, lipid, hydrocarbon, CO2, carbonat... (Bng 13.4) Bng 13.4: Ngun C c vi sinh vt s dng Ngun C ng Acid hu c Ru Lipid Hydrocarbon Carbonate Cc ngun C khc Cc dng hp cht glucose, fructose, maltose, saccharose, tinh bt, galactose, lactose, mannite, cellobiose, cellulose, hemicellulose, chitin... acid lactic, acid citric, acid fumaric, acid bo bc cao, acid bo bc thp, aminoacid... ethanol lipid, phospholipid kh thin nhin, du th, du paraffin NaHCO3, CaCO3, phn Hp cht nhm thm, cyanide, protein, pepton, acid nucleic...

Hnh 13.1: Sn lng sinh trng ti u khi vi sinh vt d dng s dng cc ngun C khc nhau Ngun carbon thng c s dng trong cng nghip ln men l r ng (molasses). S khc nhau gia r ng ma v r ng c ci c thy r trong bng 13.5 Bng 13.5: Thnh phn ha hc ca r ng c ci v r ng ma Thnh phn ng tng s Cht hu c kh ng Protein (N x 6,25) K Ca Mg P Biotin Acid pantoteic Inositol Tiamin T l % % % % % % % mg/kg mg/kg mg/kg mg/kg R ng c ci 48-52 2-17 6-10 2-7 0,1-0,5 khong 0,09 0,02-0,07 0,02-0,15 50-110 5000-8000 khong 1,3 R ng ma 48-56 9-12 2-4 1,5-5,0 0,4-0,8 khong 0,06 0,6-2,0 1,0-3,0 15-55 2500-6000 khong 1,8

T l cc nguyn t trong cc hp cht cao phn t vi sinh vt c th thy r trong bng sau y:

Bng 13.6: T l cc nguyn t trong cc cao phn t t bo vi sinh vt

Thnh phn Protein RNAd DNAd peptidoglycan Phospholipit Lipopolysaccharide Lipit trung tnh Acid Teichoic Glycogen PHB PHA (C8)m Polyphosphatd Cyanophycinoa.

% trng lng kh Trung bnh Bin dao ng 55 15c-75 21 5c 30e 3 1c 5f 3 0g 20h 9 0i-15 3 0h-4j 0-45k 0l-5d 3 0-50k 0-80k 0-60k 0-20n 0-10

%C %H %O %N %S %P 53 36 36 47 67 55 77 28 28 45 56 68 7 4 4 6 7 10 12 5 6 7 9 15 23 34 34 40 19 30 11 52 49 37 23 61 25 16 17 17 7 2 2 27 1 10 10 5 3 15 39 -

b.

c. d. e. f. g. h. i. j. k. l. m. n.

Theo Herbert (1976). Cc thng s c thu nhn t cc vi sinh vt khc nhau, khng in hnh cho mt nhm no. E. coli (trong pha sinh trng log). Theo Neidhardt et al. (1990). Cc t bo c ngun d tr C. Bao gm cc cao phn t nh ARN, ADN, polyphosphate hoc mt s thnh phn ca thnh t bo. Ti mc c t l sinh trng cao. Cc t bo sinh trng chm. Cc loi k sinh khng c thnh t bo. Vi khun Gram(+). Cc chng thay th ngun phospholipid bng cc cht tng t cha P t do, trong iu kin hn ch ngun P Vi khun Gram(-) Cc t bo trong iu kin hn ch ngun N. Hn ch ngun P. PHA (polyhydroxyaldehyde) cha 3-hydroxyoctanoic acid. Mt s nm men v vi khun.

o.

Mt s vi khun lam c ngun d tr N cyanophycin [(asp-arg)].n

*PHB= Poly- - hydroxy butyrate 2) Ngun N (source of nitrogen) Ngun N l ngun cung cp N cho vi sinh vt tng hp nn cc hp cht cha N trong t bo. Thng khng l ngun nng lng, ch mt s t vi sinh vt t dng (thuc nhm ammon ho-ammonification, nhm nitrate ho- nitrification) dng mui ammone, mui nitrate lm ngun nng lng. Trong iu kin thiu ngun C mt s vi sinh vt k kh trong iu kin khng c oxy c th s dng mt s aminoacid lm ngun nng lng. Ngun N thng c vi sinh vt s dng l protein v cc sn phm phn hu ca protein ( peptone, peptide, aminoacid...), mui ammone, nitrate, N phn t (N2), purine, pyrimidine, urea, amine, amide, cyanide...(bng 13.7) Bng 13.7: Ngun N c vi sinh vt s dng Ngun N Protein v cc sn phm phn gii ca protein Ammone v mui ammone Nitrate N phn t Cc ngun N khc Cc dng hp cht peptone, peptide, aminoacid... (mt s vi sinh vt tit men proteinase phn gii protein thnh cc hp cht phn t nh hn ri mi hp thu c vo t bo) NH3, (NH4)2SO4,... (d c hp thu) KNO3 (d c hp thu) N2 (vi vi sinh vt c nh N) purine, pyrimidine, urea, amine, amide, cyanide (ch mt s nhm vi sinh vt mi c th ng ho c)

Ngun N thng c s dng nui cy vi sinh vt gm c pepton, bt c, bt nhng tm, bt u tng, bt kh lc, cao ng, cao tht, cao nm men... Vi sinh vt s dng chn lc i vi ngun N. Chng hn x khun sn sinh terramycin s dng cao ng vi tc nhanh hn so vi s dng kh u tng hay kh lc, bi v ngun N trong cao ng l cc sn phm phn gii d hp thu ca protein. Cao ng c coi l ngun N tc hiu, cn kh du c coi l ngun N tr hiu. Loi N tc hiu l c li cho s sinh trng ca vi sinh vt, cn loi tr hiu li c li cho s hnh thnh cc sn phm trao i cht. Khi sn xut terramycin chng hn, ngi ta phi hp s dng cao ng v kh du theo mt t l nht nh phi hp gia giai on sinh trng to sinh khi v giai on sinh tng hp cc sn phm trao i cht, nhm mc tiu l nng cao sn lng terramycin.

Nng lc hp thu mui ammone v nitrate vi sinh vt l kh mnh. Ion NH4+ sau khi c t bo hp thu c th c trc tip s dng, do cc ngun mui ammone c coi l ngun N tc hiu. Cn nitrate sau khi c hp th cn kh thnh NH4+ ri mi c vi sinh vt s dng. a s cc vi khun hoi sinh (saprophyte), vi khun ng rut, vi sinh vt gy bnh ngi, ng vt, thc vt...u c th dng mui ammone, mui nitrate lm ngun N. Chng hn cc vi khun Escherichia coli, Enterobacter aerogenes, Bacillus subtilis, Pseudomonas aeruginosa...u c th s dng ngun (NH4)2SO4 v NH4NO3 lm ngun N; x khun c th s dng KNO3 lm ngun N; nm si c th s dng KNO3 lm ngun N. Lc dng cc mui nh (NH4)2SO4 lm ngun N nui cy vi sinh vt cn ch l sau khi vi sinh vt hp thu NH4+ th s lm h thp pH ca mi trng. Ngi ta gi l nhng mui c tnh sinh l acid. Ngc li khi dng cc mui nitrate (nh KNO3) sau khi vi sinh vt hp thu NO3- th s lm nng cao pH ca mi trng. Ngi ta gi l cc mui c tnh sinh l kim. lm cho pH trong cc mi trng nui cy vi sinh vt t b bin ng ngi ta b sung thm cc cht c tnh m (buffer substance). 3) Ngun mui v c (source of inorganic salt) Cc mui v c l ngun cht dinh dng khng th thiu i vi s sinh trng ca vi sinh vt. Chng c cc chc nng sinh l ch yu l: tham gia vo thnh phn ca cc trung tm hot tnh cc enzyme ca vi sinh vt, duy tr tnh n nh ca kt cu c i phn t v t bo, iu tit v duy tr cn bng p sut thm thu ca t bo, khng ch in th oxy ho kh ca t bo v l ngun vt cht sinh nng lng i vi mt s loi vi sinh vt (bng 13.8). Bng 13.8: Mui v c v chc nng sinh l ca chng Nguyn t Hp cht s dng P KH2PO4, K2HPO4 (NH4)2SO4, MgSO4 Chc nng sinh l L thnh phn ca acid nucleic, nucleoprotein, phospholipid, coenzyme, ATP... Lm nn h thng m gip iu chnh pH mi trng. L thnh phn ca cc aminoacid cha S, mt s vitamin; glutathione c tc dng iu chnh in th oxy ho kh trong t bo. L thnh phn trung tm hot tnh ca enzyme phosphoryl ho hexose, dehydrogenase ca acid isocitric, polymerase ca acid nucleic, thnh phn ca chlorophyll v bacterio-chlorophyll.

S

Mg

MgSO4

Ca

CaCl2, Ca(NO3)2 NaCl

To tnh n nh ca mt s cofactor, enzyme duy tr, cn cho s dng trng thi cm th ca t bo. Thnh phn ca h thng chuyn vn ca t bo, duy tr p sut thm thu, duy tr tnh n nh ca mt s enzyme. L cofactor ca mt s enzyme, duy tr p sut thm thu ca t bo, l nhn t n nh ca ribosome mt s vi khun a mn. Thnh phn ca sc t vi khun v mt s enzyme, l vt cht ngun nng lng ca mt s vi khun st, cn thit tng hp chlorophyll v c t vi khun bch hu.

Na

K

KH2PO4, KH2PO4

Fe

FeS04

Trong qu trnh sinh trng vi sinh vt cn cn ti mt s nguyn t vi lng. Nhng nguyn t ny cng c vai tr quan trng mc du ch cn vi s lng rt nh, khong 10-8-10-6 mol/ L mi trng nui cy. Nguyn t vi lng tham gia vo thnh phn enzyme v lm hot ho enzyme. (Bng 13.9) Bng 13.9: Tc dng sinh l ca nguyn t vi lng Nguyn t Zn Mn Mo Se Co Cu W Br Tc dng sinh l C mt trong alcohol dehydrogenase, lactodehydrogenase, phosphatase kim, ARNpolymerase, ADNpolymerase... C mt trong peroxyd dismutase, carboxylase ciitric synthetase C mt trong reductase nitrate, nitrogenase, dehydrogenase formic. C mt trong reductase glycin, reductase formic. C mt trong mutase glutamic. C mt trong cytochrome oxydase. C mt trong dehydrogenase formic. C mt trong urease, cn cho s sinh trng ca vi khun hydrogen.

Nu thiu nguyn t vi lng trong qu trnh sinh trng th hot tnh sinh l ca vi sinh vt b gim st, thm ch ngng sinh trng. Do nhu cu dinh dng ca vi sinh vt l khng ging nhau cho nn khi nim v nguyn t vi lng chi c ngha tng i. Vi sinh vt thng tip nhn nguyn t vi lng t cc cht dinh dng hu c thin nhin, cc ho cht v c, nc my hay ngay t trong cc dng c nui cy bng thu

tinh. Ch trong nhng trng hp c bit mi cn b sung nguyn t vi lng vo mi trng nui cy vi sinh vt. V nhiu nguyn t vi lng l kim loi nng cho nn nu d tha s gy hi cho vi sinh vt. Khi cn b sung thm nguyn t vi lng vo mi trng cn lu khng ch chnh xc liu lng. 4) Nhn t sinh trng Nhn t sinh trng (growth factor) l nhng hp cht hu c m c nhng vi sinh vt cn thit sinh trng tuy vi s lng rt nh v khng t tng hp so vi nhu cu. Cc vi sinh vt khc nhau c nhng yu cu khng ging nhau v chng loi v liu lng ca cc nhn t sinh trng. Sau y l mt s v d (bng 13.10). Bng 13.10: Cc nhn t sinh trng cn thit di vi mt s loi vi sinh vt Vi sinh vt Acetobacter suboxydans Clostridium acetobutylicum Streptococcus pneumonia Leuconostoc mesenteroides Staphylococcus aureus Corynebacterium diphtheria Clostridium tetani Lactobacillus arabinosus Cht sinh trng APAB, Acid nicotinic APAB choline pyridoxal thiamin b-alanin uracil acid nicotinic acid pantothenic Streptococcus faecalis methionine acid folic Lactobacillus delbruckii arginine Nhu cu / ml 0-10 ng 3 mg 0,15 ng 6 mg 0,025 mg 0,5ng 1,5 mg 0~4 mg 0,1 mg 0,02 mg 1,0 mg 0,02 mg

Lactobacillus casei

tyrosine thymonucleoside biotin ephedrin

50 mg 8 mg 0-2 mg 1 ng

Ch thch: 1 mg= 10-6g; 1ng= 10-9g Vi sinh vt t dng v mt s vi sinh vt d dng (nh Escherichia coli) thm ch c th sinh trng m khng cn bt k nhn t sinh trng no. Mt khc, cng mt loi vi sinh vt nhng nhu cu i vi nhn t sinh trng cng thay i tu theo iu kin mi trng. V d Mucor rouxii khi sinh trng trong iu kin k kh th cn thiamin (B1) v biotin (H), nhng trong iu kin hiu kh th li t tng hp c cc vitamin ny. C trng hp cha gii thch c bn cht ca nhu cu v nhn t sinh trng mt s loi vi sinh vt. Thng thng b sung vo mi trng cc cht hu c nh cao nm men, cao tht, dch un ng thc vt (nhng, gi ) l c th p ng c nhu cu v nhn t sinh trng. Cn c vo s khc nhau v cu trc ho hc v chc nng sinh l ca cc nhn t sinh trng ngi ta chia nhn t sinh trng thnh cc nhm vitamin, aminoacid, purine v pyrimidine. Vitamin l nhn t sinh trng c tm thy bn cht ho hc sm nht. Hin nay ngi ta pht hin c nhiu loi vitamin c tc dng l nhn t sinh trng. Mt s vi sinh vt c th t tng hp c vitamin, nhng nhiu loi khc li cn c cung cp vitamin trong mi trng dinh dng th mi sinh trng c. Vitamin ch yu l coenzyme hay cofactor ca cc enzyme tham gia vo qu trnh trao i cht. Mt s vi sinh vt khng t tng hp c nhng aminoacid no , cn b sung vo mi trng cc aminoacid hay b sung peptide chui ngn. Chng hn vi khun Leuconostoc mesenteroides cn ti 17 loi aminoacid mi sinh trng c. Mt s vi khun cn cung cp D-alanin tng hp thnh t bo. Purine v pyrimidine ch yu c dng lm coenzyme hay cofactor ca cc enzyme cn thit cho qu trnh tng hp nucleoside, nucleotide v acid nucleic. Bng 13.11: Chc nng ca mt s vitamin thng thng i vi vi sinh vt Vitamin Chc nng -Carboxyl ha (c nh CO2) Biotin (H) -Trao i cht mt carbon V d v cc vi sinh vt cn cung cp Leuconostoc mesenteroides (B)

Saccharomyces cerevisiae (F) Ochromonas malhamensis (A) Acanthammoeba castellanii (P) Lactobacillus spp. (B)

-Sp xp li phn t Vitamin B12

-Nhm mang methyl trong trao i Euglena gracilis (A) cht mt carbon To silic v nhiu vi to khc (A) Acanthammoeba castellanii (P) Enterococcus faecalis (B) Tetrahymena pyriformis (P) Lactobacillus casei (B) Tetrahymena spp. (P) Proteus morganii (B) Hanseniaspora spp. (F) Paramecium spp. (P) Lactobacillus spp. (B) Tetrahymena pyriformis (P) Brucella abortus (B) Haemophilus influenza (B) Blastocladia pringsheimii (F) Crithidia fasciculata (P) Caulobacter vibrioides (B) Dictyostelium spp. (F) Tetrahymena pyriformis (P) Bacillus anthracis (B)

Acid folic

-Trao i cht mt carbon

Acid lipoic

-Chuyn nhm acyl

Acid pantotenic

-Tin th ca CoA (oxy ha pyruvat, trao i axit bo)

Pyridoxin (B6)

-Trao i acid amin

-Tin th ca NAD, NADP Niacin

-Tin th ca FAD, FMN Riboflavin (B2)

-Chuyn nhm aldehyd (kh Thiamin (B1) carboxyl pyruvat, oxy ha acid keto)

Phycomyces blakesleeanus (F) Ochromonas malhamensis (A) Colpidium campylum (P)

Ch thch: B-Vi khun; F-Vi nm; A-Vi to; P-ng vt nguyn sinh 5) Nc Nc l thnh phn khng th thiu vi sinh vt c th sinh trng. Chc nng sinh l ca nc trong t bo l: - Ho tan v chuyn vn cc cht, h tr cho vic hp thu cht dinh dng, gii phng cc sn phm trao i cht. - Tham gia vo hng lot cc phn ng ha hc trong t bo. - Duy tr cu hnh thin nhin n nh ca cc i phn t nh protein, acid nucleic... - L th dn nhit tt, hp thu tt nhit lng sinh ra trong qu trnh trao i cht v khuch tn kp thi ra bn ngoi duy tr s n nh ca nhit bn trong t bo. - Duy tr hnh thi bnh thng ca t bo. - Thng qua qu trnh thu phn hay kh nc khng ch kt cu ca t bo (enzyme, vi ng, tin mao...) v s tho lp virt. Tnh hu hiu ca nc i vi s sinh trng ca vi sinh vt thng c biu th bng hot ng (hot ) ca nc (water activity, aw). l t l gia p lc hi nc ca dung dch trong nhng iu kin nhit v p lc nht nh vi p lc ca hi nc thun khit trong cng nhng iu kin nh vy: aw = p w / pw0 y Pw l p lc hi nc ca dung dch, cn aw0 l p lc ca hi nc thun khit. Pw0 ca nc thun khit l 1.0. Dung dch cng cha nhiu dung cht (cht ho tan) th aw cng nh. Vi sinh vt thng sinh trng trong iu kin c aw trong khong 0,6-0,99. i vi mt s loi vi sinh vt khi aw qu thp th tc sinh trng v tng sinh khi gim. Cc vi sinh vt khc nhau c aw thch hp khng ging nhau (bng 13.12) Bng 13.12: aw thch hp nht cho sinh trng mt s nhm vi sinh vt

Vi sinh vt Vi khun ni chung Nm men Nm si Vi khun a mn Vi nm a mn Nm men a p sut thm thu cao

aw 0,91 0,88 0,80 0,76 0,65 0,60

Nhn chung aw thch hp nht cho s sinh trng ca vi khun cao hn ca nm men v nm si. Vi sinh vt a mn c aw thch hp nht cho s sinh trng l kh thp. Phn nc c th tham gia vo cc qu trnh trao i cht ca vi sinh vt c gi l nc t do. Phn ln nc tn ti trong t bo vi sinh vt l nc t do. Phn nc lin kt vi cc hp cht hu c cao phn t trong t bo c gi l nc lin kt. Nc lin kt mt i kh nng ho tan v lu ng. 13.1.4. Khi nim v s sinh trng trong iu kin hn ch cc cht dinh dng mi trng nui cy lc trong phng th nghim, khi tt c cc cht dinh dng c cung cp cho s sinh trng ca vi sinh vt c thit k ti u th s d tha xy ra vo lc u v cc t bo sinh trng theo logarit vi tc sinh trng l ln nht. Tuy nhin, trong mi h thng mi trng v k thut nui cy, s sinh trng ca vi sinh vt khng th tip din mi m khng b gii hn trong mt khong thi gian di. Mt tnh ton n gin chng minh nhn nh ny l: sau 2 ngy sinh trng theo logarit, mt t bo vi sinh vt c 20 pht li nhn i mt ln s to ra xp x 2 x 1043 t bo. Gi s khi lng trung bnh ca mi t bo l 10-12 g th ton sinh khi t bo trn s c khi lng gp gn 400 ln khi lng ca qu t. V vy, trong mi mt th tch nui cy, s sinh trng lun lun sm b gii hn do s cn kit ca mt hoc vi cht dinh dng. Thut ng cc cht dinh dng hn ch c s dng vi rt nhiu ngha, v thng vn b nhm ln. Cc cht dinh dng hn ch c kh nng nh hng n s sinh trng trong cc mi trng nui cy vi sinh vt theo hai cch ring bit: ha hc v v ng hc. S hn ch ha hc c nh ngha l khi lng ln nht sinh khi c th c to ra trong iu kin gii hn cc cht dinh dng. Nguyn l Liebig bt ngun t cc nghin cu v s mu m trong nng nghip ca Justus von Liebig vo nm 1840. Trong nghin cu ny ng tm ra rng hm lng ca mt cht dinh dng no s quyt nh n nng sut ma mng, min l tt c cc cht dinh dng khc c mt

mt cch d tha (phng trnh 1). Gii hn ng hc xut hin khi nng cc cht dinh dng l thp (trong phm vi t miligram ti microgram trong mi lit), s hn ch cc cht dinh dng s iu khin tc sinh trng ring ca t bo (). iu khin ng hc v tc sinh trng thng ko theo cc ng lc bo ha v phng trnh Monod (phng trnh 2) c s dng m t mi quan h gia nng ca cc cht dinh dng i vi tc sinh trng ring ca t bo (). X = X0 + ( S0 - S) x YX/S (1) = max x x s / (KS + S) (2) Trong S0 l nng ban u v s l nng cui cng ca cc cht dinh dng b hn ch S; X(X0) l nng sinh khi (ban u); l sn lng sinh khi thu c i vi cht dinh dng S, max l tc sinh trng ring ln nht, v KS l hng s i lc c cht Monod. iu ny th hin r trong hnh 13.2 i vi s sinh trng trong h thng nui cy kn. Cc t bo ban u sinh trng khng gii hn cho n khi s tiu th cc cht dinh dng hn ch b ht dn, dn n tc sinh trng suy gim dn, sau tc sinh trng ngng hn. l lc t n nng cui cng ca sinh khi. Trong nui cy lin tc, ngi b sung mi trng mt cch lin tc v mt lng mi trng d tha c loi b. Tc b sung thm vo ca cc cht dinh dng b hn ch s iu khin ng thi c v nng sinh khi trong mi trng nui cy (Pirt, 1975; Kovarova v Egli, 1998).

Hnh 13.2: ng hc ca s gii hn sinh trng ca vi sinh vt trong nui cy ng do gii hn nng ca cht dinh dng (c cht) S. S0 l nng c cht ban u, s l nng thc ca c cht, X l nng sinh khi; X0: nng sinh khi ban u; Y: sn lng sinh khi thu c i vi c cht S.

Trong thc nghim, ngi ta c th nui cy cc t bo trong cc iu kin c bit r, nh cc cht dinh dng hn ch s c xc nh. i vi vic nui cy cc vi sinh vt d dng nghin cu v to ra cc sn phm sinh khi, mi trng c thit k ph bin vi ngun carbon v nng lng gii hn, tt c cc cht dinh dng khc c cung cp d tha. Tuy nhin, trong qu trnh cng ngh sinh hc, s gii hn bi cc cht dinh dng ch khng phi ngun carbon gi chc nng iu khin cc trng thi sinh l v qu trnh trao i cht ca vi sinh vt. S hn ch cc cht dinh dng no thng kch thch hoc tng cng s to thnh rt nhiu cc sn phm trao i cht v cc enzyme ca vi sinh vt. V d, nng sut s c tng ln trong qu trnh ln men to cht khng sinh do sinh trng trong mi trng hn ch photphat, s sn xut acid citric trong mi trng c s hn ch Fe-, Mn-, hoc Zn. Cn s sinh tng hp ca NAD l c thc hin trong iu kin hn ch Zn-Mn. Vic tch ly cc nguyn liu d tr ni bo PHB hoc PHA (cht do sinh hc-bioplastic) s b gii hn bi ngun cung cp hp cht giu nitrogen. R rng l s sinh trng ca vi sinh vt c iu khin thng xuyn khng phi ch bi mt cht dinh dng m bi s kt hp ca hai hay nhiu cht dinh dng ng thi (Kovarova v Egli, 1998). 13.1.5. Thit k v phn tch mi trng sinh trng ti thiu sinh trng v tng hp cc nguyn liu t bo cho bn thn mnh, vi sinh vt phi thu nhn cc thnh phn cu trc (hay cc tin cht ca chng) v nng lng cn thit t mi trng sng. Do , nui cy vi sinh vt trong phng th nghim th cc cht dinh dng phi c cung cp y vo mi trng v cc cht dinh dng phi dng m cc vi sinh vt ny c th s dng c. Do c s a dng sinh l ca th gii vi sinh vt m c v s cc mi trng vi thnh phn dinh dng khc nhau c a ra, vi mc ch hoc l lm giu mt cch chn lc hoc l nui cy mt nhm v sinh vt c th no (LaPage v cs, 1970; Balows v cs 1992; Atlas, 1997). Tt c cc mi trng ny u cha cc thnh phn vi cc chc nng dinh dng r rng, c bit l cn nhc v chc nng cu trc hoc sinh nng lng. Tuy nhin, hu ht cc nghin cu v cht dinh dng c tin hnh nh tnh ch khng phi nh lng v cc cht dinh dng khc nhau c thm vo nhiu hn hay t hn mt cch ty . Ngoi ra, rt nhiu cc mi trng nui cy c cha cc thnh phn khng c bit r rng bi v s dng cc nguyn liu hu c nh ng, khoai ty, Trong cng nhng iu kin nh: nhit hoc pH, tc sinh trng ring ln nht ca vi sinh vt b nh hng bi s a dng ca cc cht dinh dng trong mi trng. iu ny c minh ha mt cch c th i vi s sinh trng ca Salmonella typhimurium (th nghim bi Schaechter v cs, 1958). H s dng 22 mi trng c thnh phn khc nhau v nhn thy cc tc sinh trng khc nhau cc mi trng

trong cc iu kin d tha cc cht dinh dng. Kt qu cho thy cht lng cc tin cht a vo mi trng khong cho php iu chnh tc sinh trng mt cch r rng nht. Thit k mi trng v kim tra cc cht dinh dng gii hn Thit k mi trng sinh trng Trong thit k mi trng sinh trng, quyt nh u tin c a ra l chn la nng cao nht cho php to ra sinh khi (Xmax), v xc nh cc cht dinh dng gii hn (theo nguyn l Liebig). in hnh, mi trng sinh trng cho cc vi sinh vt d dng c thit k vi ngun nng lng - carbon ring bit s gii hn lng sinh khi c to ra, nhng ngc li tt c cc cht dinh dng khc (c thm vo di dng cc hp cht n) c cung cp d tha. Da vo gi tr X max, c th tnh ton c nng ti thiu ca cc nguyn t khc nhau cn thit trong mi trng nui cy. m bo s d tha ca tt c cht dinh dng khng gii hn trong mi trng th nng ca chng c nhn vi nhn t d (FE). Bng cch ny, nng ca cht dinh dng i hi trong mi trng tng trng (Ereq) gp x ln theo l thuyt i vi ngun carbon. Ereq = X max / YX/E x FE (3) YX/E (the individual average elemental growth yield) l sn lng tng trng trung bnh da trn tng nguyn t. Mt v d cho vic thit k mi trng khi gii hn ngun carbon, cho php to sn lng sinh khi kh t 10g/l sinh (bng 13.13). Cn ch rng, trong mi trng ny cc thnh phn c la chn sao cho c th thay i nng ca mi nguyn t (v d c th thay th MgCl2 v NaHSO4 bng MgSO4). Hn na, mi trng ny ch c tnh cht m yu (weakly buffered), do cn thit phi khng ch pH trong sut qu trnh sinh trng. Cch thc ny c s dng cho vic thit k mi trng nui cy cc vi sinh vt hiu kh vi mt sinh khi thp v trung bnh. Phc tp hn l thit k ca mi trng cho nui cy vi sinh vt k kh, trong rt nhiu thnh phn ca mi trng d dng kt ta ti th oxy ha kh cn thit, hoc mt t bo cao trong c cha cc cht ha tan hoc vn c tnh ca mt s mi trng.

Bng 13.13: Thit k mi trng ti thiu b gii hn bi ngun C cho php sn lng sinh khi kh t 10g/l a,b

Thnh phn mi trng

Ngun

Glucose NH4Cl NaH2 PO4 KCl NaH2SO4 MgCl2 CaCl2 FeCl2 MnCl2 ZnCl2 CuCl2 CoCl2

Cc nhn t Khi lng d tha vi cc nguyn t ngun (g/l) (g sinh khi carbon tng kh/g nguyn ng t) C, nng 1 1 10 lng N 8 3 3.75 P 33 5 1.52 K 100 5 0.5 Na 100 5 0.5 Mg 200 5 0.25 Ca 100 10 1.0 Fe 200 10 0.5 4 Mn 10 20 0.02 Zn 104 20 0.02 Cu 105 20 0.002 5 Co 10 20 0.002

Nng sut sinh trng

Khi lng cc thnh phn cu to (g/l)

25.0 14.33 5.88 0.95 1.87 0.98 2.77 1.13 0.046 0.042 0.0042 0.0044

a. Da vo sn lng tng trng ca cc nguyn t trong sinh khi kh. b. Theo Pirt (1975), Egli v Fiechter (1981). Sn lng tng trng ca C v cc nguyn t vt Zn, Cu, Mo, Mn Nhn t YX/E c phn tch t sinh khi kh khi nui cy trong iu kin khng gii hn tng trng ca h thng ng. i vi carbon, oxy, v hydro, YX/E khng th c tnh ton chnh xc trc tip t cc thnh phn c bn ca t bo do nhng thnh phn ny khng ch to nn sinh khi, m cn c cc chc nng trao i cht khc.Ngoi ra, trong bng khng ni n mt s lng ln cc cht nhn in t cn thit phi c m bo cho qu trnh sinh trng. Tnh cht ha hc ca cc thnh phn trong mi trng sinh trng phi c tnh n khi chn FE. V d, phn ln cc nguyn t vi lng d dng kt ta trong mi trng sinh trng pH trung tnh hoc kim v do gim bt kh nng hp th sinh hc (kh khn xc nh). Do , chng c thm vo nhiu gp 10 ti 20 ln (Bridson v Brecker, 1970). Trong cng ngh sinh hc, qu trnh nui cy theo m (batch) v nui cy theo m c b sung (fed-batch) c nghin cu t lu v rt c li khi thit k cc mi trng cha

tt c nguyn t vi lng chnh xc lng cn thit, sao cho tt c cc nguyn t phi c tiu th ht ti cui k tng trng. Tuy nhin, rt kh khn c th t c iu ny do tnh a dng ca cc nguyn t v s ph thuc ca chng vo iu kin nui cy.Tt nhin, trong cng ngh sinh hc, vic ti u ha mi trng l rt quan trng tnh ton s tiu th cht dinh dng v hn ch ti a s hao ph nguyn liu, ha cht. Bng 13.14: Cc nhn t tng trng sn lng ca cc cht cho v nhn in t Cc cht cho in t H2 S2O3 Fe2+ NH4+ - NO3NO2_ - NO3 Cht nhn in t O2 NO3- - N2 NO2- - N2 N2O- - N2

YX/H2 = 12g/mol YX/S2O3 = 4g/mol YX/Fe2+ = 0.35g/mol YX/NH4 = 1.3-2.6/mol YX/NO2 = 0.9-1.8g/mol YX/O2 = 10a-42bg/mol YX/NO3 = 27g/molc YX/NO2 = 17g/molc YX/N2O = 9g/molc

a. i vi cc c cht kh l methane hoc n-alkanes. b. i vi cc cht oxy ha l glucose. c. i vi Paracoccus denitrificans vi ngun carbon l glutamate 13.2. CC LOI HNH DINH DNG CA VI SINH VT Vi sinh vt c tnh a dng rt cao cho nn cc loi hnh dinh dng (nutritional types) l kh phc tp. Cn c vo ngun C, ngun nng lng, ngun in t, c th chia thnh cc loi sau y (bng 13.15) Bng 13.15: Cc loi hnh dinh dng ca vi sinh vt (I)

-Ngun C (Carbon sources) +T dng (autotroph) +D dng (heterotroph) CO2 l ngun C duy nht hay ch yu Ngun C l cht hu c

-Ngun nng lng (Energy sources) +Dinh dng quang nng (phototroph) +Dinh dng ho nng (chemotroph) Ngun in t (Electron sources) + Dinh dng v c (lithotroph) + Dinh dng hu c (organotroph) Dng cc phn t v c dng kh cung cp in t Dng cc phn t hu c cung cp in t Ngun nng lng l nh sng Ngun nng lng l nng lng ha hc gii phng ra t s oxy ho hp

C th m hnh ha chc nng sinh l ca cc cht dinh dng i vi s sinh trng ca vi sinh vt qua hnh 13.3 sau y:

Hnh 13.3: M hnh s lc v chc nng sinh l ca cc cht dinh dng i vi

s sinh trng ca vi sinh vt. C th em phn ln vi sinh vt phn thnh bn nhm chnh (bng 13.16) Bng 13.16: Cc loi hnh dinh dng ca vi sinh vt (II) Loi hnh dinh dng Ngun nng lng; i din Hydrogen; in t; Carbon -T dng quang nng v c Quang nng; H2, H2S, Vi khun lu hunh, mu ta,mu (photolithoautotrophy) lc; Vi khun lam. S hoc H2O; CO2 -D dng quang nng Quang nng; Cht Vi khun phi lu humh mu ta, hu c mu lc. hu c (photoorganoheterotrophy) -T dng ho nng v c (chemolithoautotrophy) -D dng ho nng

Ho nng (v c); H2, Vi khun oxy ho S, vi khun H2S, Fe2+, NH3, hoc hydrogen, vi khun nitrt ho, vi NO2-, CO2 khun oxy ho st.

Ho nng (hu c);

hu c (chemoorganohetero- Cht hu c trophy)

ng vt nguyn sinh, nm, phn ln cc vi khun khng quang hp (bao gm c cc vi khun gy bnh).

Loi T dng quang nng v c cn c gi l Photolithotrophic autotrophy; loi D dng quang nng hu c cn c gi l Photoorganotrophic heterotrophy; loi T dng ha nng v c cn c gi l Chemolithotrophic autotrophy; loi D dng ha nng hu c cn c gi l Chemoorganotrophic heterotrophy. Chng ta s xem xt k hn cc qu trnh trao i cht ca tng nhm vi sinh vt ny trong chng Trao i cht. Cc vi sinh vt thuc loi hnh T dng quang nng v c v D dng quang nng v c c th li dng nh sng sinh trng. Chng c vai tr quan trng trong qu trnh din bin ca mi trng sinh thi trong giai on c xa ca Tri t. Vi sinh vt T

dng ho nng v c phn b rng ri trong t v trong nc, chng tham gia tch cc vo cc vng tun hon vt cht trn Tri t. Vi sinh vt D dng ho nng hu c dng cht hu c va lm ngun carbon va lm ngun nng lng. Hu ht cc loi vi khun, nm, ng vt nguyn sinh bit u thuc loi hnh D dng ho nng hu c. Tt c cc vi sinh vt gy bnh bit u thuc loi ny. Trong loi hnh d dng ho nng hu c li chia thnh hai nhm: Nhm Hoi sinh (metatrophy) dng cht hu c cht (xc ng thc vt) lm ngun carbon. Nhm K sinh (paratrophy) k sinh trn c th thc vt, ngi v ng vt hp thu cht dinh dng. Chng khng th sng c khi tch ri khi vt ch. Tuy nhin gia hai nhm ny cn c nhng loi hnh trung gian l Hoi sinh khng bt buc (facultive metatrophy) v K sinh khng bt buc (facultive paratrophy). Mt s chng vi sinh vt pht sinh t bin (t bin t nhin hay t bin nhn to) mt i nng lc tng hp mt (hoc mt s) cht cn thit cho sinh trng (thng l nhn t sinh trng nh aminoacid, vitamin), chng ch sinh trng c khi b sung vo mi trng cc cht ny. Ngi ta gi chng l loi hnh Khuyt dng (auxotroph). Cc chng hoang di tng ng c gi l loi hnh Nguyn dng (prototroph). Ngi ta thng s dng cc chng vi sinh vt khuyt dng trong nghin cu Di truyn hc vi sinh vt. Khng c ranh gii tuyt i gia cc loi hnh dinh dng ca vi sinh vt. Vi sinh vt d dng khng phi tuyt i khng s dng c CO2 m ch l khng th dng CO2 lm ngun carbon duy nht hay ch yu sinh trng. Trong iu kin tn ti cht hu c, chng vn c th ng ha CO2 to ra t bo cht. Tng t nh vy, vi sinh vt t dng khng phi l khng c th s dng cht hu c sinh trng. Ngoi ra, mt s vi sinh vt c th thay i loi hnh dinh dng khi sinh trng trong nhng iu kin khc nhau. V d vi khun phi lu hunh mu ta (purple nonsulfur bacteria) khi khng c cht hu c c th ng ha CO2 v thuc loi vi sinh vt t dng; nhng khi c cht hu c tn ti th chng li c th s dng cht hu c sinh trng v lc chng l cc vi sinh vt d dng. Hn na, vi khun phi lu hunh mu ta trong iu kin k kh v c chiu sng c th sinh trng nh nng lng ca nh sng v thuc loi dinh dng quang nng; nhng trong iu kin hiu kh v khng chiu sng th chng li sinh trng nh nng lng sinh ra t qu trnh oxy ha cht hu c v thuc loi dinh dng ha nng. Tnh bin i loi hnh dinh dng vi sinh vt r rng l c li cho vic nng cao nng lc thch ng ca chng i vi s bin i ca iu kin mi trng. 13.3. MI TRNG NUI CY (Culture medium) Mi trng nui cy l cc c cht dinh dng c pha ch nhn to nhm p ng cho yu cu sinh trng, pht trin v sn sinh cc sn phm trao i cht ca vi sinh vt. Mi trng dinh dng dng trong nghin cu vi sinh vt v trong qu trnh sn xut cc sn phm ca vi sinh vt. Mi trng dinh dng l yu t quan trng trong cng nghip ln men, cng nghip sinh tng hp nh vi sinh vt.

13.3.1. Nguyn tc pha ch mi trng nui cy 1) Chn cc cht dinh dng thch hp: Ni chung mi trng dinh dng cn p ng cc nhu cu ca vi sinh vt v ngun C, ngun N, ngun mui khong, ngun nhn t sinh trng v nc. V loi hnh dinh dng ca vi sinh vt l phc tp, cc vi sinh vt khc nhau c nhng yu cu khng ging nhau v cc cht dinh dng cho nn c rt nhiu cng thc pha ch mi trng nui cy. Sch Danh lc mi trng nui cy (A Compilation of Culture Media) xut bn t nm 1930 cng ghi ti trn 2500 loi mi trng nui cy khc nhau. Mi trng nui cy vi sinh vt t dng hon ton pha ch t cc hp cht v c. V d nui cy vi khun Thiobacillus thiooxidans gm c cc thnh phn nh sau (g/l): (NH4)2SO4 -0.4; MgSO4.7H2O - 0,5; FeSO4 - 0,01; KH2PO4 - 4; CaCl2 - 0,25; S- 10; pH: 7,0, kh trng 121 C trong 20 pht. Cc vi khun ny s dng CO2 trong khng kh (hay ha tan trong nc) cung cp ngun carbon. Vi cc vi sinh vt t dng quang nng ngoi vic cung cp cc cht dinh dng cn tit cn cn chiu sng cung cp nng lng cho chng. i vi vi sinh vt d dng cn cung cp cht hu c v nhu cu dinh dng ca cc nhm khc nhau l rt khc nhau. nui cy vi khun Escherichia coli c th dng mi trng kh n gin sau y (g/l): Glucose - 5; NH4H2PO4- 1; MgSO4.7H2O - 0,2; K2HPO4 - 1; NaCl - 5; pH: 7,0-7,2, kh trng 112 C trong 30 pht. Nhng cngc nhng vi khun d dng yu cu nhng mi trng nui cy rt phc tp. V d vi khun Lactobacillus bifidus cn mi trng sau y (trong 1 lt mi trng m c gp i) : K2HPO4 - 5g; Na-Acetat - 50g; NZ Case Peptone- 10g; Lactose- 70g; Alanin, Cystin, Tryptophan- mi loi 0,4g; Asparagin- 0,2g; Xanthin, Adenin, Guanin, Uracin - mi loi 0,02g; Dung dch Mui B- 10ml; Pyridoxin-HCl - 2,4mg ; Tiamin-HCl 0,4mg; Riboflavin- 0,4mg; Acid nicotinic- 1,2mg; Ca-Pentotenat - 0,8mg ; Biotin- 8,0 mcg (microgram); Acid folic- 20 mcg; Acid p-aminobenzoic- 20 mcg; Tween 80 - 1g. iu chnh pH n 6,8. Thm bng thao tc v trng 100ml dung dch Acid ascorbic 1% lc qua nn lc vi khun. iu chnh n pH 6,5. Li thm bng thao tc v trng Sa ngi loi kem sao cho nng t c l 2%. Dung dch Mui B c thnh phn nh sau (g/l): MgSO4.7H2O-10; FeSO4.7H2O-0,5; NaCl-0,5; MnSO4.2H2O- 0,337. Thng thng thay cho cc nhn t sinh trng ngi ta thng dng Peptone (thay cho tng aminoacid) v cao nm men (thay cho cc nhn t sinh trng). Mi trng thng dng nui cy cc vi khun d dng l Mi trng Cao tht-Pepton vi thnh phn nh sau (g/l): Cao tht (Beef extract) - 5; Peptone- 10; NaCl- 5; pH: 7,0-7,2; kh trng 121 C trong 20 pht. Mi trng nui cy vi khun Brevibacterium spp. c thnh phn nh sau (g/l): Cao nm men (Yeast extract)-10; Glucose- 20; CaCO3 - 20. Ngi ta chia mi trng nui cy thnh nhiu loi khc nhau.o

Cn c vo thnh phn mi trng ta c: mi trng thin nhin, mi trng tng hp.

Mi trng thin nhin (complex medium): y l loi mi trng cha cc cht hu c thin nhin khng bit r thnh phn ha hc hoc thnh phn ha hc khng n nh, v vy cn c gi l mi trng khng xc nh v ha hc (chemically undefined medium). Cc mi trng Cao tht-Pepton, mi trng Mch nha, mi trng LB (LuriaBertani) l cc v d ca loi mi trng ny. Thnh phn ca mi trng LB l nh sau (g/l): Peptone - 10; Cao nm men - 5; NaCl -10; pH: 7,0; kh trng 1210C trong 21 pht. Cao tht l nc chit tht c c c li. Cao tht cha cc cht m hu c, ng, vitamin, mui khong- tt c u d tan trong nc. Peptone l dng thy phn bng protease hay bng acid i vi tht, casein, gelatin sau lm kh li thnh dng bt. Peptone cha phong ph cc cht m hu c, cng c mt s vitamin v ng. Cao nm men l dch t phn (autolysate) t bo nm men c c c li. Cao nm men cha phong ph vitamin nhm B, cng c cha cc cht m hu c v ng. Ngoi cc loi ni trn mi trng thin nhin cn c ch to t cc nguyn liu khc nh nc chit khoai ty, nc chit gi u, nc chit t, nc chit rm r, nc chit lng v bt ng, cm go, sa, huyt thanh, nc p c rt, nc da. Vi sinh vt a phn (coprophilous microorganisms) c th dng nc phn lm cht dinh dng. Gi thnh ca mi trng thin nhin thng thp, cho nn khng ch c s dng trong phng th nghim m cn c th c s dng trong cc x nghip ln men cng nghip. Bng 13.17: Thnh phn mt s loi peptone v dch thy phn protein Ch phm(CP) Peptone Pharmacon Peptone Vitte Peptone Canbaum Peptone Gee Peptone Roche CP thy phn nh pepsin CP thy phn nh trypsin Dch thy phn gluten Dch thy phn gelatin Ch thch: (1)- Cc polypeptid cao phn t c kt ta bng tannin khi c mt 2% H2SO4. (2)- Cc polypeptid c kt ta bng tannin khi mi trng c phn ng trung tnh. Phn ng Cc thnh phn (% trong protein) Biure (1) (2) (3) + 63.5 9.7 27.8 + 44.1 26.7 29.2 + 40.2 27.4 32.4 + 44.5 23.2 32.3 + 25.3 11.7 63.0 + 42.1 25.2 32.7 + 2.0 14.9 82.1 0 0 100 0 0 100

(3)- Cc aminoacid t do v cc peptid khng b t ta bi tannin. Mi trng tng hp (synthetic medium): y l loi mi trng c thnh phn ha hc c bit r cho nn cn c gi l mi trng xc nh v ha hc (chemically defined medium). V d mi trng Gause thch hp cho X khun vi thnh phn nh sau (g/l): Tinh bt tan - 20; KNO3 - 1; NaCl - 0,5; K2HPO4 .3H2O - 0,5; K2HPO4 .3H2O 0,5; FeSO4 .7H2O- 0,01, pH: 7,2-7,4; kh trng 1210C trong 21 pht. Mi trng tng hp c gi thnh cao v trn loi mi trng ny vi sinh vt pht trin tng i chm, ni chung thch hp s dng trong phm vi phng th nghim. C nhng vi khun i hi cc mi trng tng hp kh n gin, chng hn nh vi khun Escherichia coli vi mi trng sau y: K2HPO4-7,0g; KH2PO4-2,0g; (NH4)2SO41,0g; MgSO4-0,1g; CaCl2-0,02g; Glucose-4-10g; Nguyn t vi lng (Fe,Co,Mn,Zn,Cu,Ni,Mo)-mi loi 2-10g; Nc ct- 1000ml.

Escherichia coli C nhng vi sinh vt i hi cc mi trng tng hp rt phc tp (v t tin). Sau y l v d v mi trng tng hp dng nui cy vi to Euglena: acid glutamic-6g; acid aspartic-4g; Glycine-5g; Sacchaose-30g; Acid malic-1,04g; Acid boric-1,14mg; Thiamine HCl-12mg; KH2PO4- 0,6g; MgSO4-0,8g; CaCO3-0,16g; (NH4)2CO3- 0,72g; FeCl3-60mg; ZnSO4- 40mg; MnSO4-6mg; CuSO4- 0,62mg; CoSO4- 5mg ; (NH4)2MoO41,34mg; Nc 1000ml. Mt v d khc v mi trng tng hp nui cy vi khun Leuconostoc mesenteroides: K2HPO4- 0,6g; KH2PO4- 0,6g; NH4Cl- 3g; MgSO4- 0,1g; Glucose- 25g; Na acetate- 20g; Cc amino acid- mi loi 100-200g (gm c alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophane,

tyrosine, valine); Purinevaf Pyrimidine mi loi 10mg (gm c adenine, guanine, uracil,xanthine); Vitamin- mi loi 0,01-1mg (gm c biotin, folate, nicotinic acid, pyridoxal, pyridoxamine, pyridoxine, ribolavine, thiamine, pantothenate, paraaminobenzoic acid). Nguyn t vi lng - mi loi 2-10g; Nc ct- 1000ml.

Cn c vo trng thi ca mi trng ngi ta chia ra thnh mi trng c, mi trng bn c v mi trng dch th.

Mi trng c (solid medium): y l loi mi trng c lm ng c li nh c b sung thm thch (agar-agar), gelatin hay silica gel. Mi trng c phi m bo c cc yu cu sau y: - Khng b vi sinh vt nui cy s dng.

Rau cu ch vng -Gi c trng thi c trong nhit nui cy vi sinh vt. D ha tan khi un nng (thng c iu chnh bng lng cha cht lm c trong mi trng) -Nhit lm c mi trng khng qu thp. -Cht lm c mi trng khng c hi i vi vi sinh vt -Cht lm c khng b ph hy khi kh trng mi trng -Gi c trng thi trong sut ca mi trng. -Gi thnh khng qu cao, pha ch mi trng d dng.

Thch l sn phm ch to t Rau cu ch vng (Gracilaria verucosa) hay cc to bin khc thuc chi Gracilaria hay Gelidium. Thch c cha khong 70% agarose v khong 30% agaropectin. lm tan thch cn un mi trng n 1000 C v lm gi mi trng thch trng thi lng cn gi nhit khong 50-600C (trong ni cch thy). lm cho mi trng c li cn h nhit xung 40-450C. Ty cht lng ca thch m khi lm mi trng ngi ta cho vo vi t l 15-20g/l. Khi cn nui cy vi sinh vt trn cc mi trng thch c pH t 6 tr xung th cn iu chnh mi trng ti pH trung tnh trc khi kh trng, sau mi iu chnh li n pH thch hp (nu khng thch c b thy phn trong iu kin pH thp v nhit cao).

Hnh 13.4: Robert Koch (1843-1910)

Hnh 13.5: Fannie Eilshemius (1850-1934) v Walther Hesse (1846-1911)

Ngi u tin ngh n s dng mi trng c trong nghin cu vi sinh vt l Robert Koch khi tnh c thy cc khun lc ca vi khun trn c khoai ty v ng dng cc lt khoai ty lm mi trng phn lp vi khun vo nm 1881. Ngi u tin dng gelatin ch to mi trng c cng vo nm ny l mt tr l ca Koch, ng Frederick Loeffler. Vic dng thch lm cht ng c l do c Minora Tarazaemon pht hin; khi nu thc n vi to bin v khi ngui c thy thc n ng c li (1882). Ngi u tin dng thch thay th gelatin trong mi trng nui cy l v ca Walther Hess (mt tr l khc ca Koch) - b Fannie Eilshemius Hess. Sau y l vi c im ch yu ca Thch v Gelatin: c im Nng thng dng Thch 1,5-2,0 Gelatin 5-12

(%) Nhit ha tan (0C) Nhit ng (0C) pH Cht khong (%) CaO (%) MgO (%) N (%) Vi sinh vt c th s dng lm cht dinh dng

96 40 Hi acid 16 1,15 0,77 0,4 Tuyt i a s khng s dng

25 20 Acid 14-15 0 0 18,3 Nhiu vi sinh vt c th s dng

Mi trng bn c (semisolid medium): Mi trng bn c l mi trng ch cha 0,2-0,7% thch v thng c s dng quan st kh nng di ng ca vi sinh vt, quan st hiu gi thc khun th (phage)... Mi trng dch th (liquid medium): Mi trng dch th hay mi trng lng l cc mi trng khng b sung cc chy lm ng c mi trng. thng kh phi dng ti my lc hay cc ni ln men c h thng thi kh v trng (v khun) v h thng khuy o lm tan u bt kh. Mi trng dch th ngoi vic s dng trong nghin cu ti phng th nghim cn c s dng rng ri trong sn xut ln ti cc nh my ln men cng nghip. - Cn c vo mc ch s dng ngi ta chia mi trng nui cy thnh nhiu loi khc nhau Mi trng c s (minimum medium): Cc vi sinh vt tuy c yu cu dinh dng khng ging nhau nhng ni chung v c bn th cc cht dinh dng l ging nhau. Mi trng c s l mi trng c cha cc cht dinh dng c bn cn thit cho s sinh trng, pht trin ca a s vi sinh vt. Mi trng c s thng dng l Mi trng cao tht - peptone. Mi trng c s c dng lm thnh phn c bn cho nhng mi trng c bit, ty theo yu cu ca tng nhm vi sinh vt m b sung thm cc cht dinh dng cn thit. Mi trng lm giu hay cn gi l mi trng gia ph (enrichment medium): Trn mi trng c s cho thm mt s cht dinh dng c bit thch hp vi vic nui

cy mt s nhm vi sinh vt. Cc cht b sung thm c th l mu, huyt thanh, cao nm men, m ng vt hay thc vt. V d nui cy vi khun Bordetella pertussis ngi ta dng mi trng c s Difco 0048 nhng b sung bng thao tc v trng mu th ( lc qua nn lc) sau khi kh trng mi trng 15 pht 1210C, sao cho nng cui l 15%.

Bordetella pertussis Mi trng gim bit (differential medium) Mi trng gim bit dng trong vic gim nh cc loi vi sinh vt khc nhau xc nh v tr phn loi ca chng. Cc mi trng gim bit v phng php s dng c trnh by trong Tp I (Th gii vi sinh vt). Chng hn khi xc nh kh nng sinh protease th b sung casein hay gelatin, kh nng sinh amylase th thm tinh bt tan, kh nng sinh lipase th thm du n v ch th mu, kh nng sinh H2S th thm Pb acetat, ..Ngi ta thng dng mi trng EMB (Eosin Methylene Blue) gim bit vi khun ng rut. Mi trng ny c thnh phn nh sau: Peptone-10g; Lactose-5g; Saccharose-5g K2HPO4- 2g; EosinY-0,4g; Methylene Blue-0,065g; Nc ct-1000ml; pH=7,2. Mi trng ny c ch vi khun Gram (+) v mt s vi khun Gram (-). T mi trng ny kim tra thm mt vi th nghim vi cc khun lc xut hin c th phn lp c nhiu loi vi khun ng rut Gram (-) theo s sau y: a- Ln men lactic, sinh acid. b- Sinh acid mnh,khun lc chiu sng thy c mu ta, phn quang c mu lc nh kim ...Escherichia coli bb-Sinh acid yu, khun lc c mu nu g ...Enterobacter,Serratia, Klebsiella, Hafnia

aa- Khng ln men lactic, khng sinh acid, khun lc trong v mu... Proteus, Salmonella, Shigella. Mi trng chn lc (Selective medium) Dng mi trng chn lc phn lp tng nhm vi sinh vt ring bit t mt qun th vi sinh vt trong t nhin. Da vo yu cu dinh dng c bit ca tng nhm vi sinh vt hoc tnh mn cm khc nhau i vi ha cht, vi cht khng sinh m a thm vo mi trng nhng cht tng thch, nhm c ch s sinh trng ca cc nhm vi sinh vt khc v gip cho phn lp c nhm vi sinh vt cn nghin cu. C nhng mi trng chn lc c thit k da trn nhu cu dinh ng c bit ca tng nhm vi sinh vt nht nh. V d dng cellulose hay du parafin lm ngun carbon duy nht khi phn lp nhm vi sinh vt phn hy celluose hay phn hy parafin, dng protein lm ngun nitrogen duy nht phn lp vi sinh vt sn sinh proteinase, dng mi trng khng cha nitrogen phn lp vi sinh vt c nh nitrogen. V d mi trng v m Ashby dng phn lp vi khun Azotobacter c thnh phn nh sau: Mannit-1%; KH2PO40,025%, MgSO4.7H2O-0,02%; NaCl-0,02%; CaSO4.2H2O-0,01%; CaCO3-0,5%. Cng c loi mi trng chn lc thm 10% phenol s lm c ch s sinh trng ca vi khun v vi nm nhng li c th phn lp c x khun. Nu thm vo mi trng Bi sulphat th c th c ch c cc vi khun Gram (+)v phn ln cc vi khun Gram (-), nhng li phn lp c vi khun thng hn (Salmonella typhi). Thm vo mi trng Brilliant green hay Crystal violet th c ch c vi khun Gram (+) nhng li phn lp c vi khun Gram (-). Trm vo mi trng Streptomycin th c th c ch c nhiu loi vi khun nhng li phn lp c vi nm. Thm vo mi trng Na propionat c th c ch c nm si nhng li phn lp c nm men. Trong K thut di truyn (Genetic engineering) ngi ta thng xuyn s dng cc mi trng chn lc cha cc khng sinh xc nh tch ra cc chng mang gen ti t hp. Trong thc t c nhng mi trng va l mi trng chn lc, va l mi trng gim bit. V d phn lp t cu khun vng (Staphylococcus aureus) ngi ta thm vo mi trng 7,5% NaCl, Mannit v ch th mu acid-kim. Vi khun ny va chu c nng NaCl cao , va chuyn ha mannit thnh acid.

Staphylococcus aureus Sau y l mt s cht c b sung vo mi trng (MT) chn lc khi cn thit phn lp mt s nhm vi sinh vt nht nh: Potassium tellurite (MT Mueller tellurite) phn lp Corynebacterium diphtheriae; Tellurite v Crystal violet (MT Mitis-salivarius) phn lp Streptococcus; Na azide (MTAzide glucose) phn lp Streptococcus; Phenylethanol (MT Phenylethanol) phn lp Staphylococcus v Streptococcus; Nc p c chua (MT nc p c chua) phn lp vi khun lactic t nc bt; Desoxycholate, citrate (MT Desoxycholate citrate) phn lp vi khun ng rut Gram(-); Mt(bile),citrate, brilliant green (MT SS) phn lp Salmonella v Shigella; Malachite green dye (MT Lowenstein-Jensen) phn lp Mycobacterium; Chloramphenicol (MT Emmon) phn lp nm; Rose Bengal v Streptomycin (MT Martin) phn lp nm...

Corynebacterium diphtheriae Ngoi cc loi mi trng k trn cn c cc loi mi trng c bit khc. l Mi trng phn tch (assay medium) dng nh lng vitamin, cht khng sinh... l Mi trng kh (reduced medium) dng nui cy cc vi sinh vt k kh. l Mi trng nui cy m (Tissue-culture medium) chuyn phc v cho vic nui cy t bo v m ng, thc vt, hoc dng nui cy trn t bo cc nhm vi sinh vt chuyn k sinh nh virt, Chlamydia, Rickettsia, Spirochete. Mt s virt v Rickettsia khng pht trin c trn cc mi trng nhn to m phi nui cy trn phi g, trn t bo thn kh, trn c th ng vt thc nghim. Di y l mt vi gi quan trng khi chun b mi trng nui cy. Rt nhiu ng d b phn gii trong qu trnh kh trng pH kim (c bit vi s c mt ca photsphate v peptone), lm cho mu mi trng chuyn thnh mu nu v cc sn phm to thnh c th c ch s sinh trng ca vi sinh vt. trnh tnh trng , ngi ta kh trng mi trng pH acid nh hoc kh trng ring bit i vi ng. Tt c cc kim loi vi lng d dng to nn mui photphat khng tan v kt ta trong mi trng nui cy. iu ny c th c trnh bng cch b sung thm cc nhn t c i lc vi kim loi (metal-chelating agents) nh l EDTA (Ethylene Diamine Tetraacetic Acid) hay NTA (Nitrilotriacetic acid) hay i khi l cc axit cacboxylic nh citrate hay tartrate. Vic thm cc nhn t ny c hiu qu hai mt. Mt mt, n ngn chn s kt ta ca cc kim loi vi lng, mt khc n hot ng ging nh mt b cha cc kim loi , bng cch ny c th lm gim tnh c nh gim nng t do ca chng (ti mc m cc vi sinh vt c th s dng c). mi trng pH >7, cc kim loi kim th Ca v Mg (di dng vi lng) d dng kt ta vi s hin din ca photphate (hay s c mt ca ion carbonate khi s dng mi trng m l bicarbonat, hay c sn trong nc cng) to nn hm lng mui khng tan cao. Nhng kt ta ny i khi kh thy bng mt thng, c bit trong cc bnh nui cy lc do th tch nh ca mi trng. trnh iu ny, mi trng c th c kh trng pH hi axit (pH c iu chnh sau), hay mui photphat c kh trng ring r vi mi trng v kt hp sau khi lm ngui. Cn ch rng a s cc mi trng c in s dng trc nhng nm 60 ca th k trc thng khng bao gm cc nguyn t vi lng. S thm vo thng l khng cn thit bi v cc nguyn t vi lng c cha sn trong cc mui khng tinh sch c s dng chun b cho mi trng. Mi trng hin nay c chun b vi cc mui tinh sch cao nn khng ng ngc nhin l s tht bi trong vic to ra nhiu sinh khi sn phm nu khng c b sung cc nguyn t vi lng vo trong mi trng. Mt v d in hnh l mi trng c in M9 c s dng rt rng ri cho s sinh trng ca E.coli trong cc nghin cu di truyn. Mi trng ny khng cung cp thun li cc

nguyn t vi lng cho s pht trin ca E.coli trong mt vi th h, sau chng sinh trung chm li v cui cng l ngng li.

S HP THU CC CHT DINH DNG VI SINH VT tn ti, sinh trng v pht trin, t bo vi sinh vt phi thng xuyn trao i vt cht v nng lng vi mi trng bn ngoi. Mt mt chng tip nhn cc cht dinh dng t mi trng, mt khc chng thi ra mi trng mt s sn phm trao i cht. T bo vi sinh vt s dng cc cht dinh dng bt u t vic hp thu chng. C ch ca s hp thu ny c tnh chuyn ha, ni cch khc chng ch hp thu cc cht cn thit, vic hp thu cc cht khng s dng c l bt li i vi t bo. Vi sinh vt thng sng trong cc mi trng ngho cht dinh dng, do chng phi c nng lc vn chuyn cht dinh dng t mi trng c nng thp vo mi trng c nng cao bn trong t bo, tc l ngc li vi gradient nng . Nh th l gia trong v ngoi t bo c mt hng ro thm thu, l mng sinh cht c tnh thm thu chn lc. Chng cho php cc cht dinh dng xm nhp vo t bo v cn tr cc cht khc. Do tnh a dng v phc tp ca cc cht dinh dng nn vi sinh vt c nhiu phng thc khc nhau vn chuyn cc cht dinh dng. Quan trng nht l cch Khuch tn xc tin (Facilitated diffusion), cch Vn chuyn ch ng (Active transport) v cch Chuyn v nhm (Group translocation). cc vi sinh vt c nhn tht khng thy c cch Chuyn v nhm nhng c cch s dng qu trnh Nhp bo (Endocytosis).Cu to ca mng sinh cht c biu th qua hnh 13.6 sau y:

Hnh 13.6: Cu trc ca mng sinh cht (Theo sch ca Prescott, Harley v Klein).

13.4.1. S khuch tn xc tin (Facilitated Diffusion)

Mt s t cc cht, nh glycerol, c th i qua mng t bo cht theo phng thc Khuych tn b ng (Passive diffusion). Khuych tn b ng cn c gi tt l Khuych tn, l vic cc cht dinh ng chuyn t ch c nng cao n ch c nng thp. Khuych tn b ng mun lm cho t bo hp th c hiu qu mt s cht dinh dng cn c nng cht ny bn ngoi t bo cao hn bn trong. Tc hp thu ty theo lc t bo tng lng hp thu cht ny m gim xung. Tr phi loi cht dinh dng ny sau khi xm nhp t bo lp tc c s dng v khng lm nng cao nng cht trong t bo. Ch c nc (H2O), O2 v CO2, l nhng phn t rt nh mi thng c vn chuyn qua mng bng phng thc khuch tn b ng. Cc phn t tng i ln hn, cc ion v cc cht c tnh cc (polar substances) kh c th i qua mng sinh cht bng phng thc khuch tn b ng.

Hnh 13.7: Khuch tn b ng (ng thng) v khuch tn xc tin (ng cong) (Theo sch ca Prescott, Harley v Klein). Protein mang (carrier protein) cn gi l enzim permease l mt loi protein gn trn mng. Vi s h tr ca permease c th nng cao rt nhiu tc khuch tn qua mng c tnh thm thu chn lc. Phng thc vn chuyn qua mng vi s h tr ca permease c gi l s khuch tn xc tin (facilitated diffusion). Tc ca qu trnh khuch tn xc tin tng ln khi s chnh lch nng cht dinh dng gia trong v ngoi t bo tng ln. Khi nng cc cht dinh dng tng i thp th khun kh tng ln cao hn so vi phng thc khuch tn b ng. Lc gradient nng t ti mt tr s nht nh th dn n hiu ng bo ha. S tham gia ca Permease lm dn n hiu ng bo ha (hnh 13.7) ng ch l, lc permease b bo ha, s khuch tn xc tin khng tng ln do s tng mc chnh lch cht dinh dng trong v ngoi t bo. Quan h gia tc khuch tn xc tin v gradient nng cht dinh dng tong t nh mi quan h gia enzyme

v c cht, v khc hn vi ng biu din thng phn nh s khuch tn b ng. Ngoi ra s ging nhau gia permease v enzyme cn ch c tnh chuyn nht i vi cht vn chuyn, mi loi permease ch c th vn chuyn mt cch chn lc i vi mt s cht tng thch. D c s tham gia ca permease nhng khuch tn xc tin vn ng l phng thc vn chuyn khuch tn. Vic vn chuyn vn phi da vo s chnh lch nng cht dinh dng gia trong v ngoi mng. Khi mt i s chnh lch nng s vn chuyn s dng li. Qu trnh ny khng cn ti nng lng trao i cht (metabolic energy) ca t bo. Gradient nng c th duy tr khi t bo chuyn bin cht dinh dng c vn chuyn thnh mt hp cht khc hoc chuyn cht dinh dng ti mt v tr khc ca mng ( sinh vt c nhn tht). Tht th v khi thy mt s permease ny lin quan n protein ch cht ca thu knh mt ng vt c v, l cc protein thuc h MIP. Trong vi khun 2 loi knh MIP phn b rng ri nht l aquaporins vn chuyn nc v glycerol facilitators (cc nhn t xc tin glycerol) vn chuyn glycerol. Mc du c rt nhiu nghin cu i vi c ch khuch tn xc tin nhng qu trnh ny vn cha c hiu bit mt cch y . Hnh nh phc hp permease xuyn ngang qua mng t bo. Sau khi cht dinh dng c kt gn bn ngoi mng, cu hnh ca permease pht sinh bin ha phng thch c cht dinh dng vo bn trong mng. Permease sau li hi phc li cu hnh ban u v sn sng n nhn phn t dinh dng khc bn ngoi mng. Kt qu ca qu trnh ny l mt phn t khng tan trong lipid c th i vo t bo p li gradient nng ca n. Nn nh rng, c ch ny c th o ngc bi gradient nng , nu nng mt s vt cht trong t bo cao hn bn ngoi th cng c th thng qua phng thc ny m chuyn vn ra ngoi t bo. V thng qua hot ng trao i cht m t bo tiu hao rt nhanh cc cht dinh dng a vo t bo nn khng c chuyn cht dinh dng b a ngc ra ngoi (hnh 13.8). cc sinh vt nhn nguyn thy qu trnh khuch tn xc tin khng phi l phng thc vn chuyn ch yu v nng cht dinh dng bn ngoi t bo thng rt thp cho nn khng th thc hin c qu trnh khuch tn xc tin hp th cht dinh dng. Glycerol c vn chuyn bi qu trnh khuch tn xc tin E.coli, Salmonella typhimurum, Pseudomonas, Bacillus v nhiu vi khun khc. S khuch tn xc tin thng gp t bo sinh vt nhn thc, chng dng phng thc vn chuyn ny chuyn vn cc loi ng v amino acid vo t bo.

Hnh 13.8. Mt kiu Khuch tn xc tin (Theo sch ca Prescott, Harley v Klein) 13.4.2. S vn chuyn ch ng (Active Transport) Mc du s khuch tn xc tin gip chuyn vn c hiu qu cht dinh dng vo bn trong t bo khi nng cht ha tan bn ngoi cao hn bn trong t bo, nhng khng th vn chuyn c cht dinh dng khi nng cht ha tan trong t bo cao hn bn ngoi. Vi sinh vt thng sng trong cc mi trng c nng cht dinh dng rt thp, c th sinh trng v pht trin chng phi c th vn chuyn v hp thu c t mi trng cc cht dinh dng c nng thp. Khi khuch tn xc tin khng cn l phng thc vn chuyn hu hiu na m phi c nhng phng thc vn chuyn khc, trong quan trng nht l phng thc vn chuyn ch ng (active transpore) v phng thc chuyn v nhm (group translocation); c hai phng thc ny u cn ti nng lng. S vn chuyn ch ng l loi phng thc vn chuyn cc phn t cht ha tan ti ni c nng cao hn, tc l ngc li vi gradient nng v cn phi tiu hao nng lng. V s vn chuyn ch ng cn ti cc protein mang (permease) nn tng t vi s khuch tn xc tin trong mt s phng din. Permease c tnh chuyn nht cao i vi cc phn t c vn chuyn. Cc phn t cht ha tan c tnh cht tng t c th ln kt vi permease trong c hai trng hp - khuch tn xc tin v vn chuyn ch ng. Trong trng hp nng cc cht dinh dng kh cao s vn chuyn ch ng cng c hiu ng bo ha (hnh 13.9). Tuy nhin, s khc nhau ln nht gia hai loi ny l vn chuyn ch ng c th vn chuyn ngc nng nhng cn tiu hao nng lng trao i cht. Cc cht c ch trao i cht c th lm tr ngi vic sn sinh nng lng do lm c ch s vn chuyn ch ng, nhng khng lm nh hng n qu trnh khuch tn xc tin (ngay c trong thi gian ngn). Vi khun, c khun v cc vi sinh vt nhn tht c cc h thng vn chuyn protein kt hp (Binding protein transport systems) hoc protein vn chuyn hnh hp kt hp vi ATP (ATP-binding cassette transporters) hay cn gi l protein vn chuyn ABC (ABC transporter). Loi protein vn chuyn ny thng c to thnh mt phc th nh s kt

hp gia hai vng xuyn mng a nc (hydrophobic membrane - spanning domain) trn b mt t bo cht v hai vng gn vi nucleotide (hnh 13.9).

Hnh 13.9: Cng nng ca protein vn chuyn hnh hp c kh nng kt hp vi ATP (Theo sch ca Prescott, Harley v Klein) (1)=Protein mang cht ha tan c gn vi c cht vn chuyn v hng n phc cht protein vn chuyn ABC (2)=Protein mang cht ha tan gn vo protein vn chuyn v phng thch c cht, chuyn qua mng nh nng lng ca s thy phn ATP Vng xuyn mng hnh thnh mt l nh trong mng v vng kt hp nucleotide s gn vi ATP ri thy phn ATP hp th cht ha tan. Protein vn chuyn ABC tn dng protein lin kt c cht chuyn bit nm trn khe chu cht ca vi khun Gram m hoc bm trn mng lipid ti mt ngoi ca mng sinh cht vi khun Gram dng. Cc protein lin kt ny (cng tham gia vo qu trnh ha hng ng-chemotaxis) s gn vi phn t c vn chuyn, ri tng tc vi protein vn chuyn mng chuyn phn t ha tan vo trong t bo. Vi khun E.coli dng c ch ny vn chuyn nhiu loi ng (arabinose, maltose, galactose, ribose) v aminoacid (glutamate, histidine, leucine). Cc cht a vo vi khun Gram (+) phi i qua mng ngoi trc khi pht huy tc dng ca protein vn chuyn ABC v cc h thng vn chuyn ch ng khc. Cc phn t ng c th s dng mt protein l ph bin nh OmpF. Cc phn t ln hn phi dng ti cc protein l mng chuyn bit. Trong mt s trng hp, v d vic hp thu st v vitamin B12 phi dng ti cc protein vn chuyn v protein tip nhn mng ngoi c i lc cao chuyn bit. ng ch l protein vn chuyn ABC sinh vt nhn tht nhiu khi c tm quan trng ln trong y hc. Mt s t bo ung th s dng cc protein vn chuyn ny bm

thuc ra. Vic x ha nang l kt qu ca mt t bin lm bt hot mt protein vn chuyn ABC i vi chui chuyn ion chloride trong phi. Vi khun cng dng gradient proton pht sinh ra khi chuyn vn in t thc y s vn chuyn ch ng. Cc protein vn chuyn mng chu trch nhimi vi qu trnh ny thiu ht cc protein lin kt chu cht chuyn bit kt hp vi cc cht dinh dng. Lactose permease vi khun E.coli l mt v d in hnh. Permease ny l mt protein n c phn t lng khong 30 000. N vn chuyn phn t lactose khi c mt proton xm nhp t bo (nng proton cao bn ngoi t bo l do hot ng ca chui chuyn vn in t). S vn chuyn lin kt ca hai c cht theo cng mt hng c gi l vn chuyn ng hng (symport). Trong qu trnh ny nng lng tch t trong gradient proton c huy ng vn chuyn vt cht. Mc du c ch ca phng thc vn chuyn ny cn cha c hiu bit y nhng ni chung c cho rng proton v permease sau khi kt hp s ci bin hnh dng v i lc hp th cht dinh dng. Vi khun E.coli cng dng s vn chuyn ng hng vi proton vn chuyn aminoacid v cc acid hu c nh succinate v malate. Mt gradient proton cng c th thng qua vic hnh thnh mt gradient ion natri gin tip tc ng ln s vn chuyn ch ng (hnh 13.10). Cc cht vn chuyn c chuyn xuyn mng theo phng hng tng phn nh vy c gi l vn chuyn ngc hng (antiport). Gradient natri sinh ra trong ngoi t bo do h thng vn chuyn proton ngc hng ny s c th dn n vic hp thu ng v acid amin vo t bo. Mt ion natri c th lin kt vi mt protein mang v gy ra s bin i hnh dng. Protein mang s kt hp mt thit vi ng hay acid amin v nh hng chng chuyn vo bn trong t bo. Do nng natri trong t bo thp, ion natri c th tch ri ra khi protein mang v cht dinh dng c vn chuyn cng c tch ra theo. Cng vi vic ion natri di ng vo t bo vi khun E.coli th protein mang cng s chuyn ng melibiose v acid amin glutamate vo t bo ny.

Hnh 13.10: Tc dng ca gradient proton v natri trong vn chuyn ch ng (Theo sch ca Prescott, Harley v Klein). 1- Proton bm ra ngoi mng sinh cht khi vn chuyn in t 2-Gradient proton thng qua c ch vn chuyn ngc hng (antiport mechanism) y ion natri ra ngoi 3- Ion natri lin kt vi phc hp protein mang (carrier protein complex) 4-im kt hp vi cht ha tan (dung cht) bin i hnh dng v gn vi dung cht (ng hoc aminoacid) 5-Cu hnh ca protein mang (carrier) thay i, ion natri c chuyn vo trong t bo v sau dung cht cng ri khi protein mang S vn chuyn ng hng (symport) hay vn chuyn hip ng (cotransport) natri cng l mt qu trnh quan trng cc t bo nhn tht (eucaryotic) khi hp thu ng v acid amin. Nhng gradient ion natri thng sinh ra do vic thy phn ATP ch khng phi do lc chuyn ng ca proton.

Vi sinh vt thng thng qua nhiu h thng vn chuyn hp thu mt cht dinh dng. V d nh vi khun E.coli, t nht cng c ti 5 h thng vn chuyn hp thu ng galactose, 3 h thng vn chuyn hp thu glutamate v leucin, 2 h thng vn chuyn hp thu ion kali. Khi c nhiu h thng vn chuyn nh vy i vi cng mt c cht, cc h thng c s khc nhau v ngun nng lng tiu hao, v i lc i vi cht dinh dng v v phng thc iu tit h thng vn chuyn. Tnh a dng v cc phng thc vn chuyn nh vy gip cho vi sinh vt cng c u th cnh tranh mnh m trong iu kin mi trng d bin i. 13.4.3. S chuyn v nhm (Group Translocation) Trong vic vn chuyn ch ng, cc phn t ha tan vn chuyn qua mng m khng cn ci bin. Nhiu vi sinh vt nhn s cn c th thng qua vic chuyn v nhm hp thu cht dinh dng. Trong qu trnh ny vt cht c vn chuyn s c pht sinh bin ha ha hc. Nhm ny c th xp vo loi vn chuyn ph thuc nng lng v cn s dng nng lng trao i cht. H thng chuyn v nhm quen bit nht l Phosphoenolpyruvate: h thng phosphotransferase ng (PTS). Nhiu loi ng thng qua phng thc vn chuyn ny chuyn vo t bo vi sinh vt nhn s khi b phosphoryl ha v s dng phosphoenolpyruvate (PEP) lm th cho phosphate: PEP + ng (ngoi bo) Pyruvate + ng + P (ni bo) PET rt phc tp, vi khun E.coli v Salmonella typhimurium PTS to thnh bi 2 enzym (EI v EII) v 1 protein n nh nhit c phn t lng thp (HPr). HPr v Enzym EI l tn ti trong t bo cht. Enzym EII c nhiu bin ha trong cu trc, thng do hp thnh bi 3 tiu n v (subunits) hay vng kt cu (domains), trong EIIA (trc y gi l EIII) l protein t bo cht c th ha tan, EIIB cng l mt protein a nc (hydrophilic), EIIC l protein k nc nm trn mng t bo, hai loi sau thng kt hp li vi nhau. Trong qu trnh vn chuyn, di tc dng ca EI v HPr mt phosphate cao nng t PEP chuyn n enzym EII vi s gip ca Enzym I v HPr. Sau EII a 1 phn t ng qua mng vo t bo v c phosphoryl ha. EII ch vn chuyn chuyn ha tng loi ng v trong cc h thng PTS khc nhau c cc EII khc nhau, cn EI v HPr l ging nhau trong mi h thng PTS.

Hnh 13.11: Chuyn v nhm (Theo sch Microbiology ca Prescott, Harley v Klein) H thng PTS phn b rng ri cc vi sinh vt nhn s. Cc chi vi khun Escherichia, Salmonella, Staphylococcus v cc vi khun k kh khng bt buc (facultatively anaerobic) khc u c h thng PTS. Mt s vi khun k kh bt buc, nh thuc chi Clostridium, cng c h thng PTS. Mt s vi khun thuc chi Bacillus c c hai h thng ng phn (Glycolyse) v PTS. Nhng cc vi khun hiu kh hu nh khng c h thng PTS. Nhiu carbohydrate c vn chuyn bi h thng ny. Vi khun E.coli hp thu glucose, fructose, mannitol, saccharose, N-acetylglucosamine, cellobiose v nhiu carbohydrate nng phng thc chuyn v nhm. Ngoi vai tr dng vn chuyn, protein PTS cn l c quan cm th ha hc - cm th kh trong qu trnh ha hng ng. 13.4.4. S hp th St (Iron Uptake)

Hnh 13.12. Siderophore Hu nh tt c vi sinh vt u cn s dng st (Fe) cu to nn cc Cytochrome v nhiu enzym. St rt kh hp th v ion st (Fe3+) v cc dn xut ca chng rt kh ha tan, trong mi trng thng c rt t cc hp cht st d ha tan c th vcn chuyn vo t bo. Vic hp thu st ca vi sinh vt l ht sc kh khn. Nhiu vi khun v nm phi khc phc kh khn ny bng cch thng qua th mang st (siderophore). l nhng phn t c phn t lng thp li lin kt vi st v chuyn vn c vo t bo, thng l cc mui hydroxamates hoc phenolates-catecholates. Ferrichrome l mt loi hydroxamate c sinh ra bi nhiu nm; enterobactin l loi catecholate c sinh ra bi E.coli... Trong hnh bn ta thy 3 loi th mang st di cc dng khc nhau. Khi mi trng c cha vi hm lng thp cc st c th s dng vi sinh vt s tit ra cc th mang st (siderophones) kt hp vi st v chuyn n mng t bo. Lc s kt hp tip vi protein th th (receptor-protein) chuyn st vo trong t bo, hoc ton b phc th St-siderophone c chuyn vo trong nh mt protein vn chuyn ABC (ATP-binding cassette transporter). vi khun E.coli th th siderophone nm mng ngoi (outer membrane), sau khi Fe3+ c chuyn n khe chu cht (periplasmic space) th vi s h tr ca protein vn chuyn st s c chuyn qua mng sinh cht

(plasma membrane), sau Fe3+ s c kh thnh Fe2+. V st rt cn cho qu trnh sinh trng, pht trin ca vi sinh vt cho nn vi sinh vt cn s dng nhiu phng thc hp thu khc nhau p ng nhu cu ny.