do an mon hoc_tan duc

i

MC LC

CHNG 1. M U ..................................................................................................................................... 1

CHNG 2. TNG QUAN TI LIU ........................................................................................................... 3

2.1 Gii thiu v Nitrosomonas sp. ............................................................................................................. 3

2.1.1 Phn lp, duy tr v nui cy ............................................................................................................. 3

2.1.2 c im v ging ............................................................................................................................ 4

2.1.3 Nitrosomonas europaea .................................................................................................................... 7

2.1.4 Nitrosomonas eutropha ..................................................................................................................... 8

2.1.5 Nitrosomonas marina........................................................................................................................ 9

2.1.6 Nitrosomonas mobilis ..................................................................................................................... 10

2.2 Gii thiu v Nitrobacter sp. ............................................................................................................... 11

2.2.1 Nitrobacter winogradsky ................................................................................................................. 11

2.2.2 Nitrobacter alkalicus ...................................................................................................................... 12

2.2.3 Nitrobacter hamburgensis ............................................................................................................... 14

2.2.4 Nitrobacter vulgaris ........................................................................................................................ 15

2.3 Qu trnh nitrate ho ........................................................................................................................... 15

2.3.2 Cc yu t nh hng n qu trnh nitrate ho ............................................................................... 25

2.3.3 Kh nng bm dnh ......................................................................................................................... 27

2.4 Thu sinh khi Nitrosomonas europaea theo m (batch) v lin tc ...................................................... 28

2.4.1 B tr th nghim ............................................................................................................................. 29

2.4.2 Kt qu th nghim.......................................................................................................................... 30

2.5 Gii thiu v Acetobacter xylinum v mng bacterial cellulose ............................................................ 34

2.5.1 S lc vi khun Acetobacter xylinum ............................................................................................ 35

2.5.2 Cc c im cu trc ca BC ......................................................................................................... 36

2.5.3 Vai tr ca bacterial cellulose i vi Acetobacter xylinum ............................................................. 37

2.5.4 Cc yu t nh hng n qu trnh ln men to BC ....................................................................... 38

2.5.5 Cc ng dng ca bacterial cellulose ............................................................................................... 39

2.6 Nghin cu v h vi khun nitrate ho v ng dng ............................................................................. 40

CHNG 3. NGHIN CU TNH HUNG ................................................................................................ 43

3.1 Qui trnh thc hin .............................................................................................................................. 43

3.2 To cht mang BC .............................................................................................................................. 43

3.3 Ln men thu sinh khi h vi khun nitrate ho ..................................................................................... 46

3.4 Phng php xc nh ammonia, nitrite v nitrate ............................................................................... 47

3.4.1 Xc nh hm lng ammonia NH3 theo TCVN 2662-78 v 4563-88 .............................................. 48

3.4.2 Xc nh hm lng nitrite NO2- theo TCVN 2658-78 v 4561-88 .................................................. 50

3.4.3 Xc nh hm lng nitrite NO3- theo TCVN 4562-88 .................................................................... 52

3.5 Kt qu bc u ................................................................................................................................ 53

3.5.1 C nh vi khun ln BC ................................................................................................................. 53

3.5.2 Th nghim ch phm..................................................................................................................... 54

TI LIU THAM KHO ................................................................................................................................. 57

ii

DANH MC BNG

Bng 1. Cc mi trng khc nhau dng nui cy vi khun nitrite ho. Mi trng 1, 2 v 3 xut tng

ng bi Soriano v Walker (1968), Watson (1971) v Krinmel v Harms (1982) i vi vi khun phn lp

t t, mi trng 4 xut bi Watson (1965) i vi vi khun phn lp t bin v mi trng 5 xut

bi Koops v cng s (1976) i vi vi khun phn lp t nc l. Tt c cc mi trng u cn 5 g/l

CaCO3 hoc 4 g/l HEPES nh cht m pH [7] ............................................................................................... 4

Bng 2. Cc c im phn loi ging vi khun nitrite ho [7] ....................................................................... 5

Bng 3. Nng lng thu c t qu trnh oxi ho cc hp cht v c so vi t glucose (-686 kcal/mol)

[11] ..................................................................................................................................................................... 16

iii

DANH MC HNH

Hnh 1. Hnh thi t bo Nitrosomonas di knh hin vi quang hc [7] ....................................................... 5

Hnh 2. Hnh thi t bo Nitrosomonas di knh hin vi in t cho thy c s khc nhau v hnh dng

v kch thc t bo gia cc loi trong cng ging Nitrosomonas. IM l mng bao t bo cht

(intracytoplasmic membrane), C l carboxysome [7]....................................................................................... 5

Hnh 3. Cu trc t bo thuc ging Nitrosospira [7] ...................................................................................... 6

Hnh 4. Cu trc t bo thuc ging Nitrosovibrio [7] ..................................................................................... 6

Hnh 5. Cu trc t bo thuc ging Nitrosolobus [7] ...................................................................................... 7

Hnh 6. Cu trc t bo thuc ging Nitrosococcus [7] ................................................................................... 7

Hnh 7. Quan st t bo Nitrosomonas europaea di knh hin vi quang hc. Bar = 5 m [4] .................. 8

Hnh 8. Quan st t bo Nitrosomonas europaea di knh hin vi in t. Bar = 1 m [4] ........................ 8

Hnh 9. Quan st t bo Nitrosomonas eutropha di knh hin vi in t. Bar = 1 m [4]......................... 9

Hnh 10. Quan st t bo Nitrosomonas marina di knh hin vi in t qut. Bar = 1 m [4] ................. 9

Hnh 11. Quan st t bo Nitrosomonas marina di knh hin vi in t. Bar = 1 m [4]........................ 10

Hnh 12. Cu trc t bo Nitrosomonas mobilis di knh hin vi in t qut. Bar = 1m [4]................. 10

Hnh 13. Cu trc t bo Nitrosomonas mobilis di knh hin vi in t. Bar = 1 m [4] ........................ 11

Hnh 14. Vi khun Nitrobacter winogradsky c nhum Gram m, c dng quen ngn. Bar = 1 m [4]

............................................................................................................................................................................ 12

Hnh 15. Khun lc Nitrobacter alkalicus sau 2 thng nui cy pH 10. Bar = 0,1 cm [15] ..................... 12

Hnh 16. Tin mao (F) Nitrobacter alkalicus di knh hin vi. Bar = 1 m [15].................................... 13

Hnh 17. Cu trc t bo Nitrobacter alkalicus di knh hin vi in t. Lp v bao bn ngoi (S) c

cu to gm nhiu tiu n v protein ghp vi nhau. Bar = 1 m [15] ....................................................... 13

Hnh 18. Cu trc lp v bn ngoi t bo vi khun Nitrobacter alkalicus. Bar = 0,5 m [15].................. 13

Hnh 19. Cc bo quan Nitrobacter alkalicus (M) mng intracytoplasmic, (P) polyphosphate v (H) ht

polyhydroxybutyrate. Bar = 1 m [15] ............................................................................................................ 14

Hnh 20. Vi khun Nitrobacter hamburgensis c nhum Gram m, c dng qu l. Bar = 250nm. [4] 14

Hnh 21. Cu trc t bo Nitrobacter vulgaris gm lp mng introcytoplasmic v carboxysome. Bar =

250 nm. [4] ........................................................................................................................................................ 15

Hnh 22. Hng di chuyn electron da trn th kh ca cc cht trong t bo. Nhng cht kh lun l

cht cho in t, v cc cht oxi ho lun l cht nhn in t. [11] ............................................................ 17

Hnh 23. Hai giai on ca qu trnh nitrate ho ............................................................................................ 18

Hnh 24. Cu trc carboxysome v cc enzyme cu to lp v [18]. ........................................................... 20

Hnh 25. Con ng vn chuyn in t Nitrosomonas [2-3, 8-9, 11] ...................................................... 23

Hnh 26. Con ng vn chuyn in t Nitrobacter [3, 8-9, 11, 16] ....................................................... 24

Hnh 27. S to thnh nitrite trong qu trnh sinh trng ca N. europaea trn mi trng c cy 4%

dch ging v hm lng ammonia ban u l 7,8 mM [12] ......................................................................... 28

Hnh 28. Thit b fermenter dng nui cy theo m v lin tc xc nh cc yu t sinh trng ti u

cho Nitrosomonas [14] ..................................................................................................................................... 30

Hnh 29. Tc ng ca st n s pht trin ca Nitrosomonas trong nui cy theo m. (A): ng cong

sinh trng mu i chng, khng c st. (B): ng cong sinh trng mu th nghim c hm lng

st l 0,5ppm [14] ............................................................................................................................................. 32

Hnh 30. S sinh trng ca Nitrosomonas trong ch nui cy lin tc. ng cong pha trn l mt

t bo theo thi gian, ng cong pha di l hm lng (NH4)2SO4 [14] ........................................... 33

Hnh 31. Cu trc BC mt s loi vi khun ................................................................................................. 34

iv

Hnh 32. BC c to t mi trng tnh (a) v mi trng lc (b) ............................................................. 37

Hnh 33. (a) h vi si BC v (b) thc vt, phng i x5000 [5]......................................................... 40

Hnh 34. Hnh chp di knh hin vi in t nhng mu g A. altissima c loi lignin. A v B: kch

thc mu 300-500m. C v D: kch thc mu 500-710m [10] ............................................................... 41

Hnh 35. Ch phm sau khi c nh, kch thc 300-1500m. Thi gian c nh ln lt l A: 6h, B: 12h,

C: 24h v D: 72h [10] ....................................................................................................................................... 42

Hnh 36. Thit b c nh dng khuy o, dung tch 50L. Sau 4 ngy c nh, kh nng nitrate ho ca

ch phm t cao nht [10]............................................................................................................................... 42

Hnh 37. th th hin hot lc nitrate ho ca ch phm theo thi gian. Tin hnh th nghim lp li 3

ln [10] ............................................................................................................................................................... 42

Hnh 38. Quan st t bo Acetobacter xylinum di knh hin vi kim tra cc c im vi th ........... 44

Hnh 39. Khun lc Acetobacter xylinum ........................................................................................................ 44

Hnh 40. Sau khi m bo chc chn ging ang c l Acetobacter xylium th t ng ging gc (A) ta cy

chuyn qua ng thch nghing (B), nu thao tc khng ng s dn n tp nhim (C) ........................... 45

Hnh 41. Cng vi vic cy chuyn, ta s tin hnh nhn ging cp 1 trong ng nghim. (1+2) lp mng

BC to ra sau 5 ngy nhn ging, (3) cc t bo kt thnh di mu nu nht ............................................... 45

Hnh 42. Tin hnh nhn ging cp 2, sau khi cy ging vo erlen cha mi trng bnh (A), yn

erlen iu kin nhit phng trong 5 ngy s thy xut hin lp mng mng pha trn (B), nu n

1 tun th lp mng s cng dy (C) ................................................................................................................ 45

Hnh 43. Tin hnh ln men thu BC bng cch cy vo khay nha cha mi trng ln men (A), sau 7

ngy ta s thu c sn phm (B). ................................................................................................................... 46

Hnh 44. Trong qu trnh ln men, nu nguyn liu nc da gi b nhim bo t nm mc th kh

nng mc mc ln khi ln men l rt cao. Ngoi ra, nguyn nhn tp nhim cn do ra dng c khng

sch v khay b h np nn bo t nm lt vo .............................................................................................. 46

Hnh 45. (A) Dch ging Nitrobacter sp., (B) Dch ging Nitrosomonas sp. ............................................... 47

Hnh 46. Hnh thi t bo Nitrobacter (bn tri) v Nitrosomonas (bn phi) di knh hin vi. .............. 47

Hnh 47. ng chun NH3 ............................................................................................................................. 49

Hnh 48. ng chun NO2- ............................................................................................................................ 51

Hnh 49. ng chun NO3- ............................................................................................................................ 53

Hnh 50. BC sau khi x l v hp tit trng, pH trung tnh c s dng lm cht mang ....................... 54

Hnh 51. Ch phm BC thu c sau 24, 48, 72 gi c nh......................................................................... 54

Hnh 52. Ch phm BC cc ch by-hp ph khc nhau c em th nghim trn nc bin ....... 54

CHNG 1. M U

1

CHNG 1. M U

Trc tnh hnh bin i kh hu din ra ngy mt gay gt nh hin nay, vic hng

n nhng quy trnh chn nui, trng trt theo hng sinh thi, thn thin vi mi trng l

mt i hi nng bng ca x hi, c bit l Vit Nam, khi m nng nghip va l truyn

thng lu i va l n by kinh t a c nc thot ngho v thot khng hong nh lch

s minh chng. Thi im thng 4/2010, cc tnh khu vc ven bin v ng bng sng

Cu Long ang gnh chu s hn hn, nhim mn nghim trng gy nguy hi n an ninh

lng thc ca c nc v trn th gii. Bi ton cho mt nn nng nghip bn vng hin

ang t ra cho cc nh khoa hc, nh qun l Vit Nam phi gii quyt nhanh, mnh v kp

thi, nu khng nhng h lu tip theo t s suy gim nng nghip s rt kh lng.

Con tm m cy la hin l m hnh nng nghip sinh thi c nh gi cao v

hiu qu kinh t ln tc ng tch cc ln mi trng, khi lun canh gia v tm v v la th

cc cht dinh dng trong v tm s c la hp th v do gim bt nhiu chi ph ho

cht bn phn. Ngoi ra, nui tm tht trong iu kin qung canh nh vy s hn ch dch

bnh, m bo tm pht trin bnh thng v tn dng tt ngun nc ang b xm mn nh

hin nay.

Bn cnh m hnh nng nghip sinh thi nui trng qung canh, th m hnh nui tm

cng nghip vi mt cao nc ta vn l ngun cung cp chnh cho xut khu. Thi Lan

l nc c lng tm xut khu ln nht th gii, sn lng ln n 250.000 tn/nm, theo

sau l cc nc trong khu vc ng Nam vi h thng nui tm cng nghip quy m

ln, dn n 80% lng tm trn th gii c sn xut t khu vc ny. p ng nhu cu

v con ging, cc trang tri nhn ging tm p dng hnh thc nui vi mt rt dy c

c tm ging ln lng thc n, v nui trong nhng b c lp (trung bnh th tch 4 m3) nn

thc n tha tn ng trong b l rt ln, khi lng thc n ny b phn hu ra cc hp

cht th cp v cui cng v ammonia do cc nhm vi khun amon ho thc hin, chnh

lng ammonia ny s gy nh hng xu n cht lng tm ging v sau [13].

tm pht trin tt, cn kim sot lng ammonia (NH3) phi nh hn 0,1 ppm

(1,33-1,53 mg/L pH 8,0 v nhit 28-300C), nu lng ammonia tng cao s lm tm bt

mi km, sinh trng chm v d nhim bnh. Thng thng ngi ta s thng xuyn lun

chuyn n 40% lng nc trong b gim nng cc cht thi c hi, vic lm ny

gy tnh trng ph dng cho cc ngun nc ln cn, dn n tng nguy c xut hin to c

v vi sinh vt gy bnh. Do vy, vic qun l ngun nc trong cc trang tri nui tm (tm

ging v tm thng phm) l mt yu cu tin quyt trong vic nng cao sn lng v hn

ch thit hi n mi trng xung quanh [10, 13].

Hin c nhiu gii php k thut kim sot nng ammonia (TAN total

ammonia nitrogen) trong nc, phng php ph bin l cho nc nui tm chy qua mt lp

vt liu m, vi khun nitrate ho s c gi trn lp vt liu m ny s tin hnh oxy ho

ammonia trong nc thi, c th tin hnh sc kh tng hiu qu, cc nghin cu lin quan

CHNG 1. M U

2

c p dng nh phng php dng chy chm (submerged flow biofilter) (Abeysinghe et

al. 1996), nitrate ho hiu sut cao qua lp m (high-rate linear-path trickling nitrification

filter) (Twarowska et al. 1997) [17], bench-scale fluidized bed bioreactor (Ng et al. 1996), h

thng x l lin tc vi vt liu m l gel alginate c nh vi khun nitrate ho (Kim et al.

2000) cc nghiu cu ny tin hnh qui m pilot v t c nhng thnh cng nht

nh. C th, h thng x l nc thi dng phng php ny lm gim n 25% lng

ammonia theo nghin cu tin hnh bi Chin v Ong (1997) trang tri nui tm thng

phm. Tuy vy, nhc im ln ca cc phng php trn chnh l chi ph u t ban u ln,

tn km trong qu trnh bo tr-vn hnh, hn na, cng ngh s dng kh phc tp nn kh

ng dng i vi nhng h nui tm c quy va v nh vn l thnh phn ch lc trong sn

xut tm ging nhng nc ng Nam .

1. Phng php dng chy qua lp m

[17]

2. Phng php dng chy chm [17]

Gii php s dng trc tip ch phm vi sinh x l ammonia hin ang c ng

dng nhm khc phc nhc im ca phng php vt liu m. 2 h vi khun c s dng

trong ch phm vi sinh thuc nhm ho t dng, Nitrosomonas sp. v Nitrobacter sp., y l

nhng chng c tc sinh trng chm, thng c sn trong nc nui tm nhng v vic

thay nc thng xuyn lm ra tri h vi khun c li ny, kt qu l hot lc nitrate ho

t nhin trong b nui tm b gim ng k. Cc ch phm thng mi th hin trn th trng

rt nhiu nhng hiu qu tht s th vn cha r rng bi cc nh sn xut thng ni qu

hiu qu ca ch phm. Cc nguyn nhn dn n s km hiu qu khi s dng ch phm vi

sinh t do ny (dng lng, dng bt) l chng c kh nng thch nghi km vi mi trng

nc nui tm tng a phng, khu vc, s cnh tranh c cht vi chng vi sinh vt ni ti

trong b nui tm. Tr ngi ln nht l vic duy tr mt t bo vi khun nitrate ho phi

mc cao th mi c kh nng phn gii ammonia mt cch r rng, iu ny rt kh m bo

khi vi khun nitrate ho cng b ra tri trong qu trnh thay nc.

Chnh v th, mc tiu nghin cu ca ti l kt hp gia 2 phng php trn li vi

nhau, lm th no to ra mt ch phm c kh nng nitrate ho va r tin, thch hp cho cc

h nui trng va v nh, tit kim chi ph u t, bo dng trang thit b, va c hot lc

nitrate ho cao, h vi sinh vt t b ra tri v gim t l thay nc thng xuyn. Cht mang

BC (bacterial cellulose) c th l mt gii php trn vn cho cc tiu ch trn.

CHNG 2: TNG QUAN TI LIU

3

CHNG 2. TNG QUAN TI LIU

2.1 Gii thiu v Nitrosomonas sp.

Winogradsky l ngi t nn tng nghin cu v cc loi vi khun oxi ho ammonia

(vi khun nitrite ho) vo nm 1892. Tuy vy, vic phn lp v nghin cu su hn v cc

loi vi khun ny khng c tip ni nn trong mt thi gian di, ngi ta ch bit n

chng Nitrosomonas europaea. Bt u t nm 1960, Watson v cng s m ra mt k

nguyn mi trong vic phn lp v nui cy loi vi khun ny, h pht hin v t tn cho

hn 16 chng vi khun oxi ho ammonia khc. T n nay, kin thc v loi vi khun ny

lin tc c cp nhp, ngi ta dng k thut PCR xc nh on gen amoA (m ho

cho trung tm hot ng ca enzyme cha kho trong qu trnh nitrite ho l ammonia

monooxygenase-AOB) v phng php pht hin trc tip vi khun ny bng k thut FISH

(fluorescence in situ hybridization). Vi cc thnh tu ny, vic kim sot cc loi vi khun

oxi ho ammonia trong thc t tr nn d dng hn rt nhiu.

2.1.1 Phn lp, duy tr v nui cy

phn lp mt loi vi khun no , ta nm vng nguyn tc c cht no th s c

vi sinh vt loi phn hu, v vy cn ly mu nhng ni m vi khun nitrite ho tp

trung cao. Tip theo, s dng phng php MPN ng vi mt lot cc loi mi trng nui

cy khc nhau nhm phn nh tc ng ca yu t mi trng ln qu trnh nghin cu.

Chng hn, trong qu trnh tng sinh khi t mi trng acid, pH thp v thm vo urea nh

ngun c cht sinh nng lng duy nht, ngi ta c th phn lp c nhng chng tri ca

vi khun nitrite ho, c th thch nghi trong iu kin kht nghit. V tng qut, hai yu t

nh hng quan trng nht l ammonia (NH3) v nng mui, tip n l nhit v pH

mi trng.

Sau khi c c cng thc mi trng thch hp nht cho vi khun nitrite ho pht

trin, ta s dng mt trong hai cch sau phn lp vi khun nitrite ho l: phng php MPN

(s lng t bo chc chn c th) da trn phn phi Poisson khi phn tch mt lot mu pha

long theo nhiu t l khc nhau vo cc ng nghim cha mi trng nui cy thch hp nht

cho vi khun nitrite ho pht trin; phng php tri a petri t mu ban u. Cn lu l vi

khun nitrite ho pht trin cc k chm, nn i vi phng php MPN th c th xc nh

mu c cha t bo vi khun sau 2-3 tun nui cy (da trn s thay i mu sc cho cht ch

th pH gy ra), phng php tri a th cn ch n 6-8 tun mi thu c khun lc.

Cc mi trng chun nhn ging c lit k bn di, i vi mi trng gi

ging, th CaCO3 (5 g/l) hoc N-2-hydroxyethyl-piperarine-N-2ethanesulfonic acid

(HEPES, 4,77 g/l) c dng nh mt cht m pH. Khi nui cy, th pH lun c chnh

bng NaHCO3 10%. Gi tr pH ti u ph thuc vo nng cc mui ammonium thm vo.

10 mM mui ammonium thm vo pH 8.0 c xem l to iu kin rt tt cho qu trnh

pht trin ca vi khun. Nu nui cy th tch ln th cn phi khuy hoc sc oxy vo.

Nhit 300 l thch hp vi qu trnh pht trin ca vi khun. Thi gian cy chuyn trong


4

khong 3-4 thng, c th tr vi khun nitrite ho dng dch lng ch khng nht thit

dng thch nghing. Vic lu tr bng phng php lnh su v c bn t ra khng thnh

cng.

Bng 1. Cc mi trng khc nhau dng nui cy vi khun nitrite ho. Mi trng 1, 2 v 3

xut tng ng bi Soriano v Walker (1968), Watson (1971) v Krinmel v Harms (1982) i vi vi khun phn lp t t, mi trng 4 xut bi Watson (1965) i vi vi khun phn lp t bin v mi trng 5 xut bi Koops v cng s (1976) i vi vi khun phn

lp t nc l. Tt c cc mi trng u cn 5 g/l CaCO3 hoc 4 g/l HEPES nh cht m pH [7]

Thnh phn Mi trng

1 2 3 4 5

Nc ct (ml) 1000 1000 1000 600

Nc bin (ml) 1000 400

NH4Cl (mg) 535 1320 500

(NH4)2SO4 (mg) 500 130

MgSO4.7H2O (mg) 40 200 49,3 200

CaCl2.2H2O (mg) 40 20 147 20

KH2PO4 (mg) 200 54,4 50

K2HPO4 (mg) 87 114

KCl (mg) 74,4

NaCl (mg) 584

Chelated iron FeNaEDTA (mg) 1 1

FeSO4.7H2O (g) 973,1

Fe-EDTA (mg) 0,5

Na2MoO4.2H2O (g) 100 1

(NH4)6Mo7O24.4H2O (g) 37,1

MnCl2.4H2O (g) 200 2

MnSO4.4H2O (g) 44,6

CoCl2.6H2O (g) 2 2

CuSO4.5H2O (g) 20 25 20

ZnSO4.7H2O (g) 100 43,1 100

H3BO3 (g) 49,4

Phenol red 0,5% (ml) 0,1 1 1

Cresol red 0,5% (ml) 1 1

2.1.2 c im v ging

Vic phn loi vi khun ny v c bn da vo hnh dng ca t bo, sau l s sp

xp ca mng bao t bo cht (intracytoplasmic membrane). Vi cc tiu ch ny, ngi ta

phn loi vi khun nitrite ho thnh 5 ging chnh l Nitrosomonas, Nitrosococcus,

Nitrosospira, Nitrosolobus v Nitrosovibrio. Trong , hin c 16 loi vi khun c phn

bit da trn t l G+C trong DNA, hot ng ca carboxysome, hot tnh urease, tc

chuyn ho NH3, nng ti hn ammonia trong mi trng, yu cu v mn v nng

mui ti hn.


5

Bng 2. Cc c im phn loi ging vi khun nitrite ho [7]

c im Nitrosococcus Nitrosolobus Nitrosomonas Nitrosospira Nitrosovibrio

Hnh dng Dng trn n

ellipse

Dng thu a

hnh

Dng que

thng Cun xon c

Dng que mnh,

hi cong

Kch thc (m)

1,5-1,8x1,7-2,5 1,0-1,5x1,0-

2,5

0,7-1,5x1,0-

2,4

0,3-0,8x1,0-

8,0 0,3-0,4x1,1-3,0

Kiu tin mao Mc thnh chm Mc ri rc Mc cc Mc ri rc Mc cc

Cu trc mng C l xut hin

thnh cm

Chia thnh

nhiu ngn

C l xut

hin ri rc

Gp cun vo

bn trong

Gp cun vo

bn trong

Ging Nitrosomonas (Winogradsky, 1982): t bo v c bn l hnh que, mng bao t

bo cht c cc l nm ri rc, i khi lp mng ny gp cun vo bn trong t bo

cht.

Hnh 3. Hnh thi t bo Nitrosomonas di knh hin vi quang hc [7]

Hnh 4. Hnh thi t bo Nitrosomonas di knh hin vi in t cho thy c s khc nhau v hnh dng v kch thc t bo gia cc loi trong cng ging Nitrosomonas. IM l mng bao

t bo cht (intracytoplasmic membrane), C l carboxysome [7]

Ging Nitrosospira (Winogradsky, 1933): t bo l dng que xon, i khi c dng

du phy. Khng quan st thy mng bao t bo cht.


6

Hnh 5. Cu trc t bo thuc ging Nitrosospira [7]

Ging Nitrosovibrio (Harms v cng s, 1976): t bo dng du phy. Khng quan st

thy mng bao t bo cht. Mng trong t bo gp cun vo bn trong t bo cht

c ghi nhn.

Hnh 6. Cu trc t bo thuc ging Nitrosovibrio [7]

Ging Nitrosolobus (Watson v cng s, 1971): t bo c dng thu a hnh, chia

thnh ngn bi mng t bo cht.


7

Hnh 7. Cu trc t bo thuc ging Nitrosolobus [7]

Ging Nitrosococcus (Winogradsky, 1892): i din cho phn lp gamma-

proteobacterial ca vi khun nitrite ho. C hai loi tiu biu l N. oceani v N.

halophilus vi dng cu cho n ellipse, c ghi nhn c mng bao t bo cht vi

cc l tp trung thnh tng cm.

Hnh 8. Cu trc t bo thuc ging Nitrosococcus [7]

2.1.3 Nitrosomonas europaea

Vi khun ny c t tn l europaea v c phn lp ln u tin chu u

(1892), c dng hnh que, hai cc t bo c dng hnh trn, kch thc t 0,8-1,1 x 1,0-1,7

m, v c bn th tn ti tng t bo ring l, Gram m. Kh nng di ng th cha c ghi

nhn. Carboxysome cng khng c trong t bo cht. Khng c hot tnh urease. pH

khong 7,8 th t bo chu c nng mui ammonium ln n 400 mM. Hng s Ks oxi

ho NH3 nm trong khong 30-56 M. Tuy khng i hi phi c mui trong mi trng,


8

nhng t bo c th chu c nng mui NaCl ln n 500 mM. Vi khun phn b cc

h thng x l rc, ngun nc b ph dng ho v thnh thong tn ti trong t [4].

T l mol% G + C trong DNA l 50,6-51,4. Dng in hnh: ATCC 25978. GenBank

accesion number (16S rRNA): M96399.

Hnh 9. Quan st t bo Nitrosomonas europaea di knh hin vi quang hc. Bar = 5 m [4]

Hnh 10. Quan st t bo Nitrosomonas europaea di knh hin vi in t. Bar = 1 m [4]

2.1.4 Nitrosomonas eutropha

Vi khun ny c tn eutropha c ngha l sng ni c hm lng dinh dng cao, c

nhiu hnh dng t hnh que n hnh tri l, kch thc 1,0-1,3 x 1,6-2,3 m, thnh thong kt

thnh chui ngn. Cu trc thnh t bo Gram m. Hu ht cc dng u c th di ng c.

Carboxysome nm trong t bo cht. T bo khng c hot tnh urease. pH 7,8 th kh nng

chu nng mui ammonium ln n 600 mM v hng s Ks oxi ho NH3 l 35-36 M.

Khng i hi phi c mui trong mi trng, nhng t bo vn sng c khi nng NaCl

ln n 500 mM. Rt thng thy s lng nhiu trong cc cng rnh, ngun nc ph

dng v thnh thong trong t [4].


9

T l mol% G + C trong DNA l 47,9-48,5. Dng in hnh: C-91, Nm 57. GenBank

accesion number (16S rRNA): AJ298739, AY 23795, M96402.

Hnh 11. Quan st t bo Nitrosomonas eutropha di knh hin vi in t. Bar = 1 m [4]

2.1.5 Nitrosomonas marina

Vi khun ny c tn marina v thng gp bin. T bo dng que di, 2 u c

dng hnh trn, kch thc 0,7-0,9 x 1,4-2,3 m. Cu to thnh t bo Gram m, c thm mt

lp b sung bn ngoi c cu to bi mt dy cc i phn t. Kh nng di ng cha c

ghi nhn. Khng c carboxysome. Cn phi c mui trong mi trng nui cy, nng

NaCl ti u cho vi khun nm trong khong 400 mM, t bo c th chu n nng 800

mM. Ngoi ra, t bo c th chu c nng mui ammonium n 200 mM pH 7,8. Hot

tnh urease dng tnh. Phn b ph bin bin v cc khu vc ven bin [4].

T l mol% G + C trong DNA l 47,4-48. Dng in hnh: Nm 22. GenBank accesion

number (16S rRNA): Z46990.

Hnh 12. Quan st t bo Nitrosomonas marina di knh hin vi in t qut. Bar = 1 m [4]


10

Hnh 13. Quan st t bo Nitrosomonas marina di knh hin vi in t. Bar = 1 m [4]

2.1.6 Nitrosomonas mobilis

Vi khun c tn mobilis ngha l di ng c. V c bn, t bo c dng hnh cu,

ng knh t 1,5 n 1,7 m. Tuy nhin, mt s dng c dng hnh que, kch thc 1,5-1,7 x

1,7-2,5 m. T bo thng dng n hoc cp i, thnh thong kt thnh chui ngn. Cu

trc thnh t bo Gram m. T bo di ng nh mt b si gm t 1 n 22 tin mao, c

chiu ngang khong 12 nm v chiu di 3-5 m. Khng c carboxysome trong t bo cht. T

bo sng ni c mn va, vi nng NaCl ti u khong 100 mM, tuy nhin khi tng

nng ln n 600 mM th t bo vn sng c. pH 7,8 th t bo chu c nng cc

hp cht ammonium n 300 mM. Hot tnh urease m tnh. Dng vi khun ny c phn

lp khu vc nc l v cc h thng x l nc thi [4].

T l mol% G + C trong DNA l 49,3. Dng in hnh: Nc 2. GenBank accesion

number (16S rRNA): AF287287, AJ298728, M96403.

Hnh 14. Cu trc t bo Nitrosomonas mobilis di knh hin vi in t qut. Bar = 1m [4]


11

Hnh 15. Cu trc t bo Nitrosomonas mobilis di knh hin vi in t. Bar = 1 m [4]

2.2 Gii thiu v Nitrobacter sp.

2.2.1 Nitrobacter winogradsky

Tn dng vi khun ny c t theo tn nh sinh vt hc Winogradsky l ngi u

tin phn lp c n. Vi khun c dng que ngn, hnh qu l, thnh thong c dng cu,

kch thc 0,6-0,8 x 1,0-2,0 m. Carboxysome thng xut hin dng th vi trong nguyn

sinh cht. T bo c th di ng nh tin mao gn cc v bn hng. Nhiu dng vi khun

nitrite ho ny l loi ho t dng khng bt buc. Khi nui trn mi trng c nhiu loi

dinh dng, vi khun nitrite ho s u tin dng nitrite trc, sau mi oxi ho cc hp cht

hu c cn li. Vi khun cng c th s dng pyruvate, acetate v glycerol nh ngun sinh

nng lng, cng vi dch chit nm men, casamino acid, peptone, NH3 v nitrate nh ngun

cung cp nit. Di iu kin mi trng gm cc cht khong v c hay mi trng hn

hp nhiu loi dinh dng, thi gian th h dao ng t 8 n 14 gi. Trong khi mi trng

giu cht hu c th vi khun pht trin chm nn thi gian th h t 70 n 100 gi. Vi

khun c kh nng sng trong iu kin k kh vi nitrate l cht nhn in t v to ra cc

dng nitrite, nitric oxide (NO), nitrous oxide (N2O). T bo t nhy cm vi oxy khi c

chuyn t iu kin k kh sang iu kin hiu kh. NO c th l ngun c cht thay th nitrite

cho vi khun oxi ho trong iu kin hiu kh thnh nitrate. Nhng dng vi khun in hnh

ch yu c phn lp t t. Ngoi ra, vi khun ny cn phn b nc ngt, bin, cng

rnh, h thng x l nc thi v phn bn. [4]

T l mol% G + C trong DNA l 61,7. Dng in hnh: ATCC 25391.


12

Hnh 16. Vi khun Nitrobacter winogradsky c nhum Gram m, c dng quen ngn. Bar =

1 m [4]

2.2.2 Nitrobacter alkalicus

Vi khun ny c tn alkalicus ngha l alkaline, kim v kh nng sng trong pH

kim kh cao t 6,5 n 10,2 v pH ti thch l 9,5. Vi khun c dng qu l, kch thc t

0,6-0,8 x 1,2-1,8 m. Vch t bo c thm mt lp bc bn ngoi c cu trc bi cc tiu

n v protein theo mt trt t nht nh. Nitrite l ngun sinh nng lng cho vi khun iu

kin mi trng khong v c, trong khi mi trng hu c th ngi ta khng ghi nhn c

s pht trin ca vi khun. i lp vi cc loi vi khun nitrite ho khc, ngi ta khng pht

hin thy carboxysome trong vi khun ny, do sinh trng th vi khun bt buc phi

dng ngun carbon dng ion carbonate. Vi khun ny c phn lp lp bn trong cc h

nc khong c hm lng Na2CO3 cao (soda lake) hoc t vi, khong (soda soil)

Siberia v Kenya. [4]

T l mol% G + C trong DNA l 62. GenBank accesion number (16S rRNA): AF

069956.

Hnh 17. Khun lc Nitrobacter alkalicus sau 2 thng nui cy pH 10. Bar = 0,1 cm [15]


13

Hnh 18. Tin mao (F) Nitrobacter alkalicus di knh hin vi. Bar = 1 m [15]

Hnh 19. Cu trc t bo Nitrobacter alkalicus di knh hin vi in t. Lp v bao bn

ngoi (S) c cu to gm nhiu tiu n v protein ghp vi nhau. Bar = 1 m [15]

Hnh 20. Cu trc lp v bn ngoi t bo vi khun Nitrobacter alkalicus. Bar = 0,5 m [15]


14

Hnh 21. Cc bo quan Nitrobacter alkalicus (M) mng intracytoplasmic, (P) polyphosphate

v (H) ht polyhydroxybutyrate. Bar = 1 m [15]

2.2.3 Nitrobacter hamburgensis

Vi khun c phn lp u tin thnh ph Hamburg, c, v tng nh s kin

ngi ta t tn thnh ph cho dng vi khun ny. Hnh thi v cu trc t bo tng t

nh cc dng vi khun nitrite ho khc. Carboxysome hin din t bo cht. Kh nng di

ng nh tin mao ui v bn hng. Pht trin km trn mi trng khong v c nhng

li c kh nng pht trin tt mi trng hn hp. Khi mi trng c nitrite, pyruvate, dch

chit nm men v peptone th tc sinh trng t cao nht. y l dng duy nht c th

pht trin tt trong mi trng c cht hu c. C nirtite v cht hu c u c s dng

trong qu trnh bin dng ca t bo. T bo cn c th kh nitrate phc v qu trnh sinh

trng. Vi khun kh nhy cm vi oxy khi chuyn t iu kin k kh sang hiu kh. Dng vi

khun ny c phn lp t t Hamburg (c), Yucatan (Mexico) v Corse (Php). [4]

T l mol% G + C trong DNA l 61,6. Dng in hnh l X 14. GenBank accesion

number (16S rRNA): L11663.

Hnh 22. Vi khun Nitrobacter hamburgensis c nhum Gram m, c dng qu l. Bar = 250nm. [4]


15

2.2.4 Nitrobacter vulgaris

Vulgaris trong ting La tinh ngha l ph bin. c im hnh thi v cu trc tng t

nh cc dng nitrite ho khc. Carboxysome hin din trong t bo cht. Phn ln t bo

trong dng ny c 2 pha sinh trng, pha u tin s oxi ho nitrite thng, pha th hai oxi

ho cc hp cht hu c khc. Mc sinh trng trong pha u tin (ho t dng) thng

chm hn pha th hai (ho d dng). T bo dng Z c thi gian nhn i l 140 gi pha

u tin v 25 gi pha th hai. C nitrate v oxy u l cht nhn in t v acetate hoc

pyruvate l cht cho in t khi t bo s dng hp cht hu c. T bo sn sinh ra nhng

polymer ngoi bo gip lin kt nhng t bo xung quanh li to thnh cm, mng (biofilm).

im khc bit vi cc dng nitrite ho khc l vi khun Nitrobacter vulgaris khng nhy

cm vi oxy chuyn t mi trng k kh sang mi trng hiu kh. Trn mng nguyn sinh

cht Nitrobacter vulgaris th ch c duy nht mt loi cytochrome c 32 kDa trong khi N.

winogradsky v N. hamburgensis th c c loi cytochrome 30 kDa. Dng vi khun ny c

phn lp t t, nc ngm, nc ngt hoc nc l, cng rnh v c trong t mi. N.

vulgaris l loi ph bin nht trong loi Nitrobacter, thm ch n c xut hin trong b tng

v nhng to nh xy dng t , gch. [4]

T l mol% G + C trong DNA l 59,4. Dng in hnh l Z.

Hnh 23. Cu trc t bo Nitrobacter vulgaris gm lp mng introcytoplasmic v carboxysome. Bar = 250 nm. [4]

2.3 Qu trnh nitrate ho

Cc loi vi khun ho nng v c (chemolithotrophic) thng l loi t dng, u s

dng chu trnh Calvin c nh CO2 nh ngun carbon duy nht. vi khun nitrate ho,

c xp vo loi ho t dng (chemoautotrophic) s s dng nng lng thu c t qu

trnh oxi ho ammonia hay nitrite phc v cho vic kh CO2 thnh carbohydrate. Khi mt

phn t CO2 i vo chu trnh Calvin, n cn n 3 phn t ATP v 2 phn t NADPH. iu

kh khn l nng lng thu t qu trnh oxi ho cc phn t v c (ni chung) ca vi khun

nitrate ho li nh hn rt nhiu so vi vic oxi ho hon ton glucose loi vi khun thng


16

thng. T l P/O trong qu trnh oxi ho phosphoryl ho vi khun nitrate ho thng bng

1, chnh v t l qu thp nh vy, nn vi khun nitrate ho ni ring v cc loi vi khun ho

nng v c khc ni chung, cn oxi ho mt lng ln cc hp cht v c sinh trng v

sinh sn, iu ny ni ln tc ng ca h vi khun ny i vi mi trng rt su sc.

Bng 3. Nng lng thu c t qu trnh oxi ho cc hp cht v c so vi t glucose (-686

kcal/mol) [11]

Phn ng Go (kcal/mol)

H2 + 1/2 O2 H2O -56,6 NO2

- + 1/2 O2 NO3

- -17,4

NH4+ + 1 O2 NO2

- + H2O + 2H

+ -65,0

S0 + 1 O2 + H2O H2SO4 -118,5

S2O32-

+ 2O2 + H2O 2SO42-

+ 2H+ -223,7

2Fe2+

+ 2H+ + 1/2 O2 2Fe

3+ + H2O -11,2

vi khun nitrate ho, nng lng gii phng t qu trnh oxi ho ammonia ln nitrite

c s dng tng hp nn ATP (hot ho cc enzyme, c cht, l hp cht d tr nng

lng) v NAD(P)H (ngun cung cp electron trong chui vn chuyn in t, cung cp H+

dng kh cc hp cht khc, l hp cht cha nng lng kh). Chnh 2 hp cht quan

trng ny, m vi khun nitrate ho s dng kh CO2 v cc hp cht khc phc v cho qu

trnh bin dng ca t bo.

Qu trnh tng hp ATP vi khun nitrate ho v nguyn tc rt ging vi hot ng

tng hp ATP ca ti th cc vi sinh vt c nhn tht. Con ng chnh to ATP l thng

qua cc hot ng oxi ho ammonia v phn ly nc ( vi khun nitrite ho) v oxi ho nitrite

( vi khun nitrate ho), H+ c to ra v y vo periplasm (l khong khng gian gia

mng trong v mng ngoi ca t bo vi khun), t to nn gradient H+ lm cho H+ chy

qua phc ATPase vo trong cytoplasm (nguyn sinh cht) gip ti to ADP thnh ATP. Con

ng ph (din ra Nitrobacter sp., trong khi Nitrosomonas sp. th khng hin din)

to ATP l thng qua hot ng ca cht kh NADH v hot ng ca phc vn chuyn in

t trn mng, gip tng hp ATP.

Qu trnh tng hp NADH th kh khn hn nhiu v hin cha c l gii r hon

ton, v n lin quan n qu trnh vn chuyn electron theo chiu ngc li (reverse electron

transport RET). L do chnh v nhng phn t nh ammonia, nitrite th c th kh cao (E0

NO3-/NO2

- = 0,421 V) hn so vi NAD+ (E0 NAD

+/NADH = -0,32) nn VI KHUN nitrate

ho khng th cung cp electron trc tip t qu trnh oxi ho ammonia v nitrite tng hp

nn NAD(P)H qua h enzyme NADH-oxidoreductase. Bi v electron ch c th di chuyn t

cht cho (donor) c th kh thp hn n cht nhn (acceptor) c th kh cao hn. gii

quyt kh khn ny, vi khun nitrate ho s dng lc y proton (proton motive force

PMF) o li chiu di chuyn electron, qua cc cht vn chuyn in t, nhm kh NAD+

v NADH. Ngoi ra PMF cn dng cho s chuyn ng ca tin mao, cc hot ng trao i

cht trong t bo.


17

C th thy nu so snh vi qu trnh to nn NADH v ATP qua con ng ng

phn hay chu trnh Krebs cc vi sinh vt khc, th qu trnh to NADH v ATP vi khun

nitrate ho tiu tn nhiu nng lng hn v lng sn phm to ra t hn. Nhng v ngun c

cht ammonia, nitrite m vi khun nitrate ho s dng li khng b cnh tranh nh cc ngun

c cht khc (glucose, lipid, protein), nn vi khun nitrate ho vn thch nghi c vi con

ng bin dng bng cc hp cht ny.

Hnh 24. Hng di chuyn electron da trn th kh ca cc cht trong t bo. Nhng cht kh lun l cht cho in t, v cc cht oxi ho lun l cht nhn in t. [11]

Qu trnh nitrate ho l qu trnh chuyn ho ammonium (NH4+) thnh nitrate (NO3

-)

bi tc ng ca vi sinh vt trong iu kin hiu kh. Qu trnh ny c thc hin bi hai

nhm vi khun tun t ni tip nhau, gm 2 bc: giai on nitrite ho (nitrition) chuyn

NH4+ thnh nitrite (NO2

-) v giai on nitrate ho (nitration) t nitrite thnh nitrate (NO3-).

Chuyn NH4+ thnh NO2

-

Nhm vi khun chuyn ammonium thnh nitrite l Nitrosomonas (N. europaea, N. aligocarbogenes) oxi NH4

+ thnh NO2

- qua sn phm trung gian l hydroxylamine

(NH2OH)

2NH4+ + O2 2NH2OH + 2H

+

NH2OH + O2 NO2- + 2H

+ + H2O + 275 kJ


18

Chuyn NO2- thnh NO3

-

Nhm vi khun chuyn nitrite thnh nitrate l Nitrobacter (N. agilis, N. winogradsky) theo phn ng sau:

NO2- + 0,5O2 NO3

- + 73 kJ

Hnh 25. Hai giai on ca qu trnh nitrate ho

Trc khi i su vo cc qu trnh xy ra trn mng t bo cht ca vi khun nitrate

ho, cn im li kin thc lin quan n cc hp cht nit, cc phc vn chuyn in t.

2.3.1.1

-3). Ammonia NH3

ammonium NH4+

-

. Nitrite

NO2-

+5.

S tn ti ca ammoni trong nc c phn thnh 2 loi: NH3 (kh ho tan) v NH4+

(dng ion ho). S phn chia ny ph thuc vo pH, nhit v mn ca nc, nhng nh

hng ca pH l quan trng hn c. Ch c dng NH3 l gy c cho ng vt nui trng. Khi

hm lng NH3 trong nc qu mc cho php s lm nh hng n vt nui v d nhim

bnh t vi khun. Thc t trong ngnh thu sn, hm lng NH3 quy nh trong nc phi

nh hn 0,1ppm, khi hm lng ln n 0,45ppm s lm gim 50% tc sinh trng ca

tm.

ammonia-nitrogen

(NH3 -nitrogen (NH4+

-nitrogen hay to -

3

:


19

, 2-

:

2.3.1.2 M lin quan

o Cytochrome -

2+ hay Fe

3+

-

550nm.

Cy .

26 .

o Periplasm space

(-)

vi

khun - vi khun

(peptidoglyca

(x

-

.


20

27

Gram (-)

o Carboxysome: l nhng vi bo (microcompartment) c cha enzyme lin quan n

qu trnh c nh carbon. Cu to ca carboxysome do polyhedral protein bao bc bn

ngoi, c ng knh t 80 n 140 nm. Chc nng ca carboxysome c nhn nhn

l ni tp trung hm lng CO2 cao nhm tng cng hiu qu hot ng v t l

enzyme RuBisCo trong chu trnh Calvin, gip khi u chu trnh Calvin c thun

li. Nhng bo quan ny c tm thy cc loi vi khun s dng CO2 nh ngun

carbonhydrate nh vi khun lam (cyanobacteria) v rt nhiu loi vi khun ho dng

khc. Carboxysome l mt v d cho rt nhiu nhm protein tuy c cu to ging nhau

nhng thc hin chc nng khng tng ng, nguyn nhn do khc nhau da trn

cu to hnh thi ca lp v c hnh thnh bi protein .

Hnh 28. Cu trc carboxysome v cc enzyme cu to lp v [18].


21

2.3.1.3 Qu trnh nitrite ho Nitrosomonas sp.

Hai enzyme cha kho y l ammonia monooxygenase - AMO (oxi ho NH3 thnh

NH2OH v H2O) v hydroxylamine oxidoreductase (oxi ho NH2OH thnh NO2-). Trong t

bo vi khun nitrite ho, c ch chung nh sau: u tin, enzyme ammonia monooxygenase

xc tc chuyn NH3 ngoi mi trng thnh NH2OH theo phn ng:

NH3 + O2 + 2e- + 2H

+ NH2OH + H2O + 0,7 kcal/mol (1)

(1)

.

Hydroxylamine (NH2

a 1 proton (H+).

NH2OH + O2 + H2O NO2- + H2O + H

+ + 83,3 kcal/mol (2)

3 2

2-

(2)

.

2-

.

2 2- +

(OH)2 a

ion H+

.

phn ng (1) 3

NH2 (2)

2

O2, H+ -

NH2OH + H2O NO2- + H

+ + 4[H

+ + e

-] (3)

2OH ra NO2-

,

do phn ng (2) c th tch thnh 2 giai on nh sau:

NH2OH (HNO) + 2[H+ + e

-]

(HNO) + H2O NO2- + H

+ + 2[H

+ + e

-]

Nh vy, phn ng chung ca qu trnh nitrite ho nh sau:

NH3 + 1,5O2 NO2- + H

+ + H2O + 84 kcal/mol

iu cn lu l trong qu trnh nitrite ho, c cht ca ammonia monooxygenase l

NH3 ch khng phi NH4+, v vy qu trnh nitrite ho din ra mnh nht pH trung tnh hoc


22

kim (pH 7,5-8,5). Vi khun nitrite ho thuc loi hiu kh tu tin, trong iu kin thiu oxy

chng s oxi ho tip ra N2O v NO. [6]

Theo Hagopian v cng s (1998) th

2

-

n chuyn ngc in t

-

(3)

NADH2- theo co

NADH2 :

NH3 + O2 + NADH2 NH2OH + H2O + NAD+

chui vn chuyn in t ca cc VI KHUN thng thng th NADH s nh H+ ra

periplasm v nhng in t cho cc cht chuyn in t nhm tng hp ATP,

VI KHUN khng c h vn chuyn in t nh vy, nn

NADH2 c tng hp ra s

ammonia (AMO-catalyzed), cn li s dng cho chu trnh Calvin.

(v qu trnh phn ly nc din ra enzyme ny, cung cp cho t bo H+ v e-

2

2

).

vi khun (hay ammonia-

ox

. V khi cytochrome aa3 kh oxi v nc, n s dng 2 H+ kt hp vi

0,5O2 v ng thi bm 2 H+ khc ra periplasm. Lng H+ tch t periplasm to ra gradient

v quay tr vo cytoplasm thng qua phc ATPase. Do , c th kt lun chui vn chuyn

in t vi khun nitrite ho thc cht l chuyn e- t qu trnh oxi ho ammonia qua cc

cht chuyn in t v tng hp nn ATP.

23

Hnh 29. Con ng vn chuyn in t Nitrosomonas [2-3, 8-9, 11]

24

Hnh 30. Con ng vn chuyn in t Nitrobacter [3, 8-9, 11, 16]

CHNG 3: NGHIN CU TNH HUNG

25

2.3.1.4 Qu trnh nitrate ho Nitrobacter sp.

Enzyme cha kho y l nitritoxydase (nitrite oxydase, nitrite oxidoreductase, NO2-

OR), l mt enzyme c cha nhn Fe-S v trung tm hot ng cha Mo. Trong t bo vi

khun nitrate ho, c ch chung nh sau:

NO2- + H2O NO3

- + 2[H

+ + e

-] + 17,8 kcal/mol

Khc vi qu trnh nitrite ho, nguyn t oxy dng oxi ho NO2- c ly t H2O

thay v phn t O2. Hai electron sinh ra t qu trnh oxi ho NO3- c chuyn n

cytochrome c ri n cytochrome aa3 v kt hp vi oxy t phn t O2 to thnh nc

trong nguyn sinh cht. iu cn lu l cytochrome aa3 cn hot ng nh mt bm proton,

khi n thc hin kh O2 v nc th cng bm H+ ra periplasm.

Ngoi ra, t enzyme NO2- OR, 2 electron cn c chuyn qua cytochrome c550 v

n phc cytochrome bc1. Electron sau khi qua phc ny s kt hp vi ubiquinone to

thnh ubiquinol, v n lt mnh ubiquinol s nh in t v proton cho NAD+ chuyn

NAD+ thnh NADH. Con ng ny l ngc so vi chiu vn chuyn in t thng thng

ca cytochrome bc1, electron di chuyn c th cn phi c t l cyt c550 (dng kh)/cyt

c550 (dng oxi ho) v ubiquinol/ubiquinone cao y H+ chuyn t periplasm v

cytoplasm thng qua phc cytochrome bc1, nh s ht electron chuyn t cytochrome c550

v pha ubiquinone. Tng t nh vy, electron chuyn t ubiquinol n enzyme NADH

oxidoreductase (NADH dehydrogenase) th cn c t l ubiquinol/ubiquinone v

NAD+/NADH cao dn H+ t periplasm v to nn dng in t t ubiquinol chuyn qua

enzyme NADH dehydrogenase, kt qu l sn sinh ra NADH. T bo s s dng NADH sinh

ra cho cc hot ng oxi ho phosphoryl ho chui vn chuyn in t t FMN n

cytochrome o560 n ti to ATP, v dng NADH cho chu trnh Calvin nhm c nh CO2.

Mt khc, nu so snh vi chiu di chuyn thng thng in t cc vi khun ho

nng v c khc, th s chnh lch mc in th gia NADH/NAD+ v cyt c (dng kh -

Fe3+

)/ cyt c (dng oxi ho - Fe2+) t hn phi nh hn mc chnh lch thng thng n mc

th in ho H+ (~200 mV) khng cho s di chuyn ca H+ i vo periplasm nu electron c

di chuyn t NADH n cytochrome c. iu ny c ngha, vic di chuyn in t t

cytochrome c n NADH oxidoreductase nhm kh NAD+ thnh NADH tuy ngc vi

hng vn chuyn thng thng th vi khun nitrate ho iu ny vn xy ra nh cc bm

proton t trong cc phc vn chuyn in t gn trn mng.

Ngoi ra, th in ho H+ hai bn mng khng phi lc no cng n nh v H+

periplasm vn b thm vo bn trong qua cc l nh trn mng, iu ny lm gim gradient

nng H+ v tng mt H+ bn trong t bo cht, vi khun nitrate ho gii quyt kh khn

ny bng cch o li chiu tng hp ATP t bn trong ra bn ngoi nhm phc hi li

gradient H+ periplasm lun cao hn cytoplasm [9].

2.3.2 Cc yu t nh hng n qu trnh nitrate ho

Cc vi khun ho nng v c ni chung s hu nhng c ch ho sinh c bit gip

chng tn ti v phn b trong mt khong khng gian rt rng mt t v di nc.


26

Ngi ta cng phn lp c chng trong ct v nc ma. Do l vi sinh vt t dng, nn

chng c kh nng t tng hp sinh khi nh c c ch sinh ho cha h enzyme phn gii

cc cht v c, v d nh mt s loi c th ng thi oxi ho urea v methane cng lc vi d

ho rt nhiu loi hydrocarbon khc nhau. Hn na, cc t bo ny c kh nng duy tr s

sng trong iu kin thiu ngun c cht bng nh gim hot ng h hp ni bo v ng

ho xung mc thp nht. i vi, vi khun nitrate ho th chng b c ch bi nh sng v

c bit nhy cm vi s xo ng khi pH kim ti u v nhit m (~25-300C). Ta s

xem xt cc yu t nh hng c th nh sau:

Nng ammonia

Theo nghin cu ca Anthonisen v cng s (1976) th khi hm lng ammonia vt

qu t 10 n 150 mg/l th s c ch qu trnh nitrite ho. Qu trnh nitrate ho nhy

cm hn, nu ammonia vt hn mc 0,1 n 1 mg/l th vi khun nitrate ho b c

ch. i vi nng ion NO2- th t 0,22 n 2,8 mg/l s c ch qu trnh nitrate ho.

Hin cha c nghin cu no ghi nhn nng NO3- mc no s c ch qu trnh

nitrate ho.

Nng oxy ho tan (DO)

Tuy vi khun nitrate ho l loi hiu kh bt buc nhng chng c th sng trong thi

gian di iu kin thiu oxy di cc tng nc pha y h, trong cc b x l nc

thi v bn hot tnh. Ngi ta ghi nhn c mt 102 t bo/ml vi khun nitrite

ho trong mt b x l k kh c nng TAN 52 mg/l. cc b x l nc thi lin

tc th nng vi khun nitrite ho t ti u nng oxi ho tan khong 0,2 mg/l.

Qu trnh nitrite ho vn xy ra mc oxi ho tan thp 0,05 mg/l m vn phn gii

c mt lng ng k ammonia. i vi vi khun nitrate ho th c sc sng tng

t nh vy, trong mt s trng hp mt t bo cn nhiu hn vi khun nitrite ho

vi ln. Vic pht trin cc b x l hiu kh kn c mt vi khu vc k kh cc b

cho php tin hnh ng thi qu trnh nitrate ho v phn nitrate ho, chuyn t

ammonia ra n N2 (Davidson v cng s, 1986). Nng oxi ho tan ti u cho h vi

khun nitrate ho pht trin tt l 3-4 mg/l O2 (Prosser, 1989).

Nhit

Vi khun nitrate ho l loi vi khun a m, nhit ti thch cho chng pht trin

trong khong 25-300C. i vi qu trnh nitrite ho vn din ra nhit t 7-350C

trong khi qu trnh nitrate ho th nhit din ra rng hn t 5-420C.

pH dung dch

Hu ht cc vi khun nitrate ho c gi tr pH ti u dao ng trong khong 7,5-8,0.

Ti gi tr pH nh hn 6 hoc ln hn 10 th qu trnh nitrate ho din ra rt chm v

b c ch. Qu trnh oxi ho ammonia bi vi khun nitrite ho lm gim tnh kim ca

mi trng, v th m ca pH ca mi trng l rt quan trng, trong mt s

trng hp cn b sung km gi pH. Vi khun Nitrosomonas th hot ng trong

khong pH 6,7-9,2 trong khi vi khun Nitrobacter th hot ng trong khong pH 8-

9,2. S thch nghi ca VI KHUN i vi gi tr pH qu cao hoc qu thp u i

hi phi c mt khong thi gian. Trong nhiu nghin cu cho thy cc chng vi


27

khun t dng c th thch nghi c c gi tr pH rt thp nh 3-4 hoc gi tr pH

rt cao 11-12. Trong cc ngun nc thi thng c pH nm trong di trung tnh v

kim nh, v th qu trnh nitrate ho vn din ra thun li m t cn s tc ng chnh

pH v pH ti thch.

S c mt ca cc cht c

Nitrosomonas sp. v Nitrobacter sp. u nhy cm vi mt vi hp cht c thng

gp trong nc thi. Tuy nhin, mc nhy cm ca Nitrosmonas sp. thng cao

hn vi Nitrobacter sp., cc hp cht ny gy ngng qu trnh nitrate ho v c ch

cc h enzyme trn mng ca t bo. Hu ht cc hp cht dit khun u tc ng

nghim trng n hot ng sng ca vi khun nitrate ho (iu ny gii thch v sao

cc trang tri nui trng thu sn c ngun nc thi b nhim hu c qu cao v

trong qu trnh nui, ngi ta s dng cht dit khun tiu dit vi khun gy

bnh cho ng vt nui trng, ng thi dit lun h vi khun c li, lm cho kh

nng t lm sch t nhin ca ao nui b mt tc dng). C th lit k mt vi hp cht

gy c ch vi khun nitrate ho nh sau: nitrapyrin (2-chloro-6-trichloromethyl-

pyridine), allylthiourea (2-propenyl-thiourea), sodium azide (NaN3), DIECA (diethyl-

dithiocarbamate), Acetylen (C2H2), sodium chlorate (NaClO3).

2.3.3 Kh nng bm dnh

H vi khun nitrate ho c bit c khuynh hng tit ra nhng polymer ngoi bo

v dnh vo cht mang. Khi cht mang c lp lipopolysaccharide ph bn ngoi th s bm

dnh c tt hn. Nu b mt cht mang khng ph hp v nu c nc ra tri, nhng cm

t bo s hnh thnh, gi l zoogloeas. Mng sinh hc (biofilm) to bi vi khun nitrate ho c

khi lng ring t t 1,14 g/cm3 v tng khi lng ring kh l 0,03 g/cm3. Trong ngun

nc t nhin, vi khun nitrite ho thng kt vi nhau thnh tng chm v ni trn b mt

hn l dng t do. Diab v Shilo (1988) chng minh vi khun nitrate ho c th bm

dnh t 70-95% ln mt b mt mn, tr sau 30 pht k t khi cho tip xc.

Tc ng tch cc ca cht mang ln hot ng sinh l ca h vi khun nitrate ho

hin vn cn kh b n. Powell v Prosser (1986) ghi nhn l qu trnh nitrate ho trong t,

s t nhy cm hn 10 ln so vi qu trnh nitrate ho xy ra trong nc nu dng cng

nitrapyrin lm cht c ch. Abeliovich (1987) nhn thy s lng t bo ca Nitrosomonas sp.

gim nhanh trong iu kin k kh dch t bo t do so vi t bo nm trong lp bn, c

ghi nhn kh nng sng st trong hng thng. Diab v Shilo (1988) cng chng minh kh

nng tn ti mnh m ca vi khun nitrate ho trong iu kin c nh cc ch k kh v

thiu ngun dinh dng. Audic v cng s (1984) khm ph ra hot ng h hp nitrate ho

ca vi khun c nh trong ct s tng n 130% so vi bnh thng, v tc nitrate ho

tng rt mnh trong gi u tin. Bi v hot ng nitrate ho v kh nng sng st s rt thp

nu vi khun dng t do, v th yu cu cn phi c nh l cn thit, vic to ra nhng cht

mang nhn to s nhn hiu qu ca qu trnh nitrate ho t nhin ln rt cao.


28

2.4 Thu sinh khi Nitrosomonas europaea theo m (batch) v lin tc

Nm 1960, cc nghin cu ho sinh v cc phn ng oxi ho ammonia thnh nitrous

acid vi khun Nitrosomonas t ra yu cu l phi lun c sn sinh khi vi khun, nhng

v loi vi sinh vt ny c thi gian tng sinh khi rt lu nn l mt tr ngi trong vic m

rng quy m th nghim. V thi im y cc nghin cu v qu trnh nui cy ch mi dng

li ch phn lp v gi ging, ch cha i su vo vic ti u cc qu trnh ln men thu sinh

khi, chnh v vy Skinner v Walker bt tay vo thc hin nghin cu nh hng cc cu

t trong mi trng nui cy (Ca, Mg, K, Co, Mn, Zn) cng nh ch batch v lin tc s

tc ng nh th no n s sinh trng ca vi khun [14].

Cng cn lu l vo nm 1958, Engel v Alexander trong bi vit v nui cy vi

khun Nitrosomonas europaea a ra ng cong sinh trng ca vi khun ny, v sn

phm trao i cht NO2- o c th trng vi qu trnh pht trin n. Do , c mt cch

nh lng s sinh trng ca loi vi khun ny l thng qua sn phm trao i cht to

thnh.

Hnh 31. S to thnh nitrite trong qu trnh sinh trng ca N. europaea trn mi trng c cy 4% dch ging v hm lng ammonia ban u l 7,8 mM [12]


29

2.4.1 B tr th nghim

i vi ging vi sinh vt, Skinner s dng dng Nitrosomonas europaea c Jensen

phn lp an Mch. H xem ging vi khun ny l thun chng sau khi kim tra bng cc

phng php sau: (1) cy trn vi khun vo a pertri cha peptone v 300C trong iu

kin hiu kh, sau 6 ngy khng ghi nhn bt k khun lc no mc ln; (2) khi quan st hnh

thi tiu bn git treo, khng pht hin thy cc dng d hnh cng nh kim tra bng phng

php nhum Gram, thy l vi khun khng di ng, dng que, Gram m.

Nu trong ging b nhim tp khun hoc thm ch cc loi vi sinh vt ho sinh d

dng khc (thng l cc loi nm c kh nng dng NH3 nh ngun c cht), ta s phn lp

li qua hai bc: u tin dng phng php hp nh m khun lc, sau ly cy cc

khun lc vo erlen cha mi trng thch hp. Sau 3 tun, nhit 250C, erlen no c

lng ammonia b gim (nh lng bng phng php o ammonia) th s em ra th trn

mi trng thch cha peptone, nu khng pht hin b nhim tp khun th erlen cha

dng Nitrosomonas thun.

Nui cy trong erlen: thnh phn cc loi mui tinh khit trong 1 lt mi trng s

dng nc ct l: 0,5g (NH4)2SO4; 0,07g KH2PO4; 0,05g MgSO4.7H2O; 0,05g CaCl2.2H2O,

sau khi pha xong s tit trng mi trng. Ta cho 4ml dung dch Na2CO3 5% w/v tit trng

vo mi trng nhm iu chnh pH v 8,0. Kinh nghim cho thy l hm lng cc loi

mui c th khc nhau nhng khng lm nh hng n s pht trin ca vi khun, ch c

lng CaCl2 v MgSO4.7H2O cn phi cn chnh xc nhm trnh kt ta cc mui phosphate

khi mi trng c chnh v pH kim.

Nui cy trong fermenter: thnh phn cc loi mui tinh khit trong 1 lt mi trng

s dng nc ct l: 18 g (NH4)2SO4; 0,4 g KH2PO4; 0,05 g MgSO4.7H2O; 0,05 g

CaCl2.2H2O v khi cn thit s b sung 0,10,6 mg Fe (cho vo dng FeSO4 to phc vi

EDTA).

K thut m t bo: tc sinh trng ca Nitrosomonas c m trc tip di

knh hin vi bng bung m hng cu. Mt ng knh x20 lm t khong cht fluorite c

b tr thm nhm lm tng kh nng phng i hnh nh t 10 n 15 ln, gip mt c th

nhn trc tip c vi khun.

nh lng ammonia: nng ammonia trong canh trng c nh lng bng

cch em 2 ml mu i chng cht, sau to mi trng kim bng cch nh vi git NaOH

40% w/v, NH3 bay hi ra c ngm trong dung dch acid boric v chun trc tip bng

H2SO4 N/50.


30

Hnh 32. Thit b fermenter dng nui cy theo m v lin tc xc nh cc yu t sinh trng ti u cho Nitrosomonas [14]

2.4.2 Kt qu th nghim

2.4.2.1 Nui cy trong erlen

Mi trng s dng nui vi khun ging vi mi trng m Engel v Alexander

(1958) xut, ngoi tr vic s dng Na2CO3 thay v K2CO3 cng nh khng s dng

FeNaEDTA v cht ch th pH. Mi m nui cy khong 5 lt, nui trong erlen (loi 500ml,

cho vo erlen 100ml mi trng), dng my lc vng, nhit 270C, thu c 19mg sinh

khi kh/L v mt vi sinh vt l 200.106 t bo/ml. Engel v Alexander ghi nhn kt qu

n 72,4mg sinh khi kh/L vi mt t bo m c l 190.106 t bo/ml. Mt t bo

m c c th thp hn mt t bo thc t (CFU), y l l do v sao m kt qu thu

c li khc nhau.


31

nh hng ca calcium (Ca) v magnesium (Mg)

Erlen cha mi trng nui cy (nhng khng cho CaCl2v MgSO4 vo) s em i tit

trng, sau thm vo tng erlen dung dch CaCl2 v MgSO4 ( c tit trng) vi

hm lng khc nhau kim tra tc ng ln vi khun. Cy 1ml ging Nitrosomonas

v 300C. T l sinh trng ca vi khun c xc nh bng cch o nng NH3

nhng thi im khc nhau. Kt qu thu c cho thy vic thm 1,2 hay 5mg

CaCl2.2H2O hoc MgSO4.7H2O vo mi 100ml mi trng cng khng lm nh

hng n s pht trin ca vi khun. Nguyn nhn l do trong lng ging cy vo

c 0,05mg CaCl2 v 0,05mg MgSO4 p ng nhu cu v Ca v Mg ca vi khun

trong th nghim ny. Kingma Boltjes (1935) th cho rng vi khun pht trin ti u

th cn gp 6 ln lng calci ni trn.

nh hng ca potassium phosphate (KH2PO4)

Trong 1 th nghim tng t, vi cc nng KH2PO4 khc nhau cng khng nh

hng n s pht trin ca vi khun N. europaea, do ch cn thm khong 0,28mg

KH2PO4 cho mt ln cy ging.

nh hng ca nhng ion kim loi khc

St: vic thm st vi nng 2 ppm khng nh hng n s pht trin ca vi

khun.

Nickel, chromium: thm Ni (di dng nickel sulphate) hoc Cr (di dng

chrome alum) khng gy nh hng, nhng khi qu 0,25 ppm nhng kim loi

ny s lm kim hm s sinh trng.

Cobalt, manganese v km: vic thm vo Co (di dng nitrate), Mn (di

dng sulphate) hay Zn (di dng sulphate) vi nng 1 ppm khng gy nh

hng n s pht trin.

ng: c kh nng c ch vi khun hon ton khi nng l 0,5 v 0,2; cn

khi nng thp 0,1 ppm th s gy km hm nh hn. ng c cho vo

dng CuSO4.

nh hng ca thp khng r

Ngi ta kim tra xem s pht trin ca vi khun din ra nh th no nu nui trong

fermenter lm bng thp khng r. Tin hnh cho 1 mu thp khng r c tit

trng vo erlen kim tra, v erlen i chng th khng c. Cy vi khun

Nitrosomonas vo vi mt ban u l 2,5.106 t bo/ml, em 300C. Kt qu ghi

nhn cho thy trong 18 gi u erlen i chng v 24 gi u erlen kim tra, s

lng t bo gim, sau bt u tng theo hm log trong 20 n 30 gi nui cy tip

theo ri t n pha cn bng. Thi gian nhn i pha log l 8 gi c 2 erlen th

nghim, iu ny cho thy khng c nh hng bi thp khng r i vi s tng sinh

ca vi khun.

2.4.2.2 Nui cy trong fermenter

Fermenter c s dng trong th nghim c 2 loi, loi nh (ng knh 10cm, chiu

cao 30cm, cha 1,8 lt mi trng) dng xc nh iu kin sinh trng ti u, v loi ln

(ng knh 23cm, chiu cao 30cm, cha 7 lt mi trng) dng thu sinh khi bng


32

phng php ln men lin tc. Nhit c chnh trong khong 28320C, cm bin pH gi

mi trng trng thi kim, khuy o vi tc 600rpm.

S sinh trng ca Nitrosomonas trong nui cy theo m

Mi trng cha 0,5% (NH4)2SO4 c bm vo fermenter, chnh pH 7,4 bng dung

dch Na2CO3 5%, thm vo 100ml dch ging. Nhit nui cy gi 300C. Sau vi

gi, gi tr pH gim nh, iu chng t s sinh trng ca vi khun bt u, mu

vi sinh vt s c ly ra v nh lng t bo. Vic nui cy tip tc din ra trong 5

ngy, v mu s c kim tra nh k. Kt qu cho thy pha log din ra trong khong

t ngy th 3 n ngy th 4, mt t bo t ti a ln n 520.106 t bo/ml

ngy th 5 v sau th gim nh, s sinh trng i vo pha cn bng (Hnh 33).

Tc ng ca st chelate FeNaEDTA ln s sinh trng ca vi khun

Th nghim thm st chelate vo mi trng nng 0,5 ppm v pH mi trng gi

77,4. Kt qu cho thy khi thm st vo mi trng th mt t bo tng t 56.106

t bo/ml n 910.106 t bo/ml trong vng cha n 4 ngy, thi gian nhn i pha

log l 16 gi (Hnh 33). Trong 2 th nghim khc, st c thm vo mi trng vi

nng l 0,5 v 0,6 ppm, theo tng s t bo tng t 740.106 t bo/ml ln

850.106 t bo/ml. Do , vi mi trng c a ra trn th st l mt nhn t c

nh hng n s pht trin ca Nitrosomonas, c bit khi nng t bo t 500.106

t bo/ml tr ln.

Hnh 33. Tc ng ca st n s pht trin ca Nitrosomonas trong nui cy theo m. (A): ng cong sinh trng mu i chng, khng c st. (B): ng cong sinh trng mu

th nghim c hm lng st l 0,5ppm [14]


33

S sinh trng ca Nitrosomonas trong nui cy lin tc

Trong th nghim ny, phng php c s dng l nui cy vi sinh vt theo m cho

n khi t c mt t bo hoc nng NH3 mong mun. Sau thm lin tc

mi trng nui cy vo v xc nh nh hng ca cc tc chy khc nhau ln

mt t bo v hm lng c cht (NH4)2SO4. Kt qu thu c cho thy trong sut

giai on u ca th nghim, mt t bo tng t 11.106 t bo/ml ln 371.106 t

bo/ml trong vng 74 gi (Hnh 34) thi gian nhn i sinh khi trung bnh l 16 gi,

lc ny lng c cht NH3 gim xung mc thp nht. Sau , mt dng mi trng

chy vo fermenter vi tc 30ml/gi v duy tr trong 3 ngy tip theo, trong sut

thi gian ny, mt t bo vn duy tr mc khng i nhng nng NH3 li tng

rt chm mc d n vn duy tr mc thp. Sau , mi trng trong fermenter c

pha long bng cch cho vo mi trng mi lm mt t bo gim xung cn

125.106 t bo/ml, tip tc vi sinh vt pht trin theo m cho n khi t c mt

493.106 t bo/ml. Sau li chnh mi trng chy vo lin tc vi tc chm

trong 3 ngy tip theo. Trong sut giai on nui cy ln 2 cn duy tr mi trng

ging vi giai on nui cy ln 1. Trong th nghim y, khi cho mi trng chy

vo fermenter c dch nui cy 1,8 lt vi tc 30ml/ gi th t l pha long l 0,016

th tch/gi v thi gian lu trung bnh ca t bo l 60 gi ng vi t bo ang giai

on pha log iu kin nui cy theo m. Tuy nhin, v mt l do cha xc nh, t

bo thu c qu trnh ln men lin tc li c thi gian nhn i ln hn 16 gi so

vi t bo thu c qu trnh ln men theo m trong cng thi gian.

Hnh 34. S sinh trng ca Nitrosomonas trong ch nui cy lin tc. ng cong pha

trn l mt t bo theo thi gian, ng cong pha di l hm lng (NH4)2SO4 [14]

Tc ng ca hm lng (NH4)2SO4 ln s sinh trng ca vi khun

Trong th nghim u tin ca lot th nghim ny, mi trng nui cy c cha

(NH4)2SO4 vi hm lng 0,5 g/L. Canh trng bt u c nui cy theo m t

mt 60.106 t bo/ml sau s thm mi trng mi vo vi tc 30ml/gi. Duy


34

tr ch ny trong vi ngy, trng thi n nh c thit lp trong fermenter, mt

t bo c duy tr mc 120.106 t bo/ml v nng (NH4)2SO4 khong 0,2g/L.

Khi tng tc cp mi trng ln 78 ml/gi, th mt trng thi n nh mi c

thit lp, nng (NH4)2SO4 khong 0,61 g/L v mt t bo t 40.106 t bo/ml.

Kt qu ny cho thy, khi tng lu lng cp mi trng c th lm ra tri t bo

v khi nng (NH4)2SO4 tng th c th lm kim hm qu trnh tng sinh khi

vi khun.

Tc ng ca vic sc kh/khuy o ln s sinh trng ca vi khun

Tin hnh nui Nitrosomonas trong mt fermenter loi ln, nhit 290C, hm

lng (NH4)2SO4 l 6 g/L, khuy o tc 1250 rpm v sc kh vo fermenter vi

lu lng 1 lt khng kh/pht. Nui cy theo m n khi vi khun tiu th ht lng

ammonia sau mi chuyn sang nui cy lin tc. Mi trng cp vo vi tc

250ml/gi sao cho nng (NH4)2SO4 duy tr mc di 0,1 g/L. Mi trng chy

trn ra ngoi c thu li trong mt bnh cha v lm lnh xung di 100C. Sau 5

ngy nui cy thu c 30 lt canh trng vi sinh vt vi mt 600.106 t bo/ml,

tng ng 2,14 g sinh khi kh. Mt khc, khi dng mi trng c hm lng

(NH4)2SO4 l 8 g/L, vi cng ch nui cy nh trn th canh trng vi sinh vt thu

c c mt ln n 800.106 t bo/ml.

2.5 Gii thiu v Acetobacter xylinum v mng bacterial cellulose

Ngoi thc vt, nm v to c cellulose l thnh phn cu trc c bn hnh thnh nn

vch t bo, cellulose cn c tng hp bi mt s loi vi khun, c gi l cellulose vi

khun (bacterial cellulose BC). Con ng tng hp v c ch iu ha tng hp BC cc

loi ging nhau, nhng cu trc BC mi loi th khc nhau. C cc im khc bit ng k

v thuc tnh vt l ca cc sn phm cellulose, ch yu l chiu di ca chui glucan (c

c trng bi mc polymer ha), tnh kt tinh v trng thi kt tinh ca n. Ty thuc vo

mi loi m trng thi kt tinh ca cellulose khc nhau. T tnh cht vt l ca sn phm

nh bn, ha tan trong cc dung mi, tnh chu nh hng ca cc tc nhn bin tnh

cng khc nhau.

Hnh 35. Cu trc BC mt s loi vi khun


35

Acetobacter xylinum (A.aceti ssp. xylinum, A. xylinus), l vi sinh vt to cellulose hu

hiu nht. Gn y, n c xp vo ging mi Gluconacetobacter, bao gm cc loi l G.

xylinus, G. hansenii, G. europaeus, G. oboediens v G. intermedius. Nhng nghin cu v

cellulose vi khun tp trung vo i tng A. xylinum c v nghin cu c bn v cc ng

dng rng ri.

2.5.1 S lc vi khun Acetobacter xylinum

Phn loi Theo Bergey, A. xylinum thuc h thng phn loi sau:

Lp: Schizomycetes B: Pseudomonadales B ph: Pseudomonadieae

H: Pseudomonadaceae Loi: Acetobacter xylinum

c im hnh thi

A. xylinum c dng hnh que, thng hay hi cong, c th di ng hoc khng di ng

v khng sinh bo t. T bo A. xylinum ng ring l hoc kt thnh chui di, c kh nng

to vng dy trn mi trng nui cy.

c im sinh l v sinh ha A. xylinum l vi khun Gram m, nhng c im Gram ca chng c th b bin i

do t bo gi i hay do iu kin mi trng. A. xylinum thuc loi vi khun hiu kh bt

buc, nn chng tng trng b mt tip xc gia mi trng lng v mi trng kh.

Chng khng s dng mui NH4+

lm ngun N duy nht, ng thi trn b mt mi trng

dch th to thnh mng dy nhy, c cha cellulose. Ngoi ra, A. xylinum to: (1) phn ng

catalase dng tnh, (2) oxy ha ethanol thnh CO2 v H2O, (3) khng tng trng trn mi

trng Hoyer, (4) khng to sc t nu, (5) tng hp cellulose.

A. xylinum c th s dng nhiu ngun ng khc nhau v ty thuc vo chng m

ngun ng no c s dng tt nht. Chng c th chuyn glucose thnh acid gluconic,

iu ny lm cho pH mi trng gim t 1 n 2 n v. Nhit ti u A. xylinum pht

trin l t 25 n 300C v pH t 5,4 n 6,3. Theo Hestrin (1947), pH ti u ca A. xylinum l

5,5 v chng khng pht trin nhit 370C ngay c trong mi trng dinh dng ti u.

Khi nui trn mi trng c, lc t bo cn non, khun lc mc ring r, nhy v trong sut,


36

xut hin sau 3 n 5 ngy. Khi gi, t bo mc dnh nhau thnh tng cm v khun lc mc

theo ng nui cy.

2.5.2 Cc c im cu trc ca BC

Cellulose l mt polymer khng phn nhnh bao gm nhng gc glucopyranose ni

vi nhau bi ni -1,4. Cc nghin cu c bn cho thy BC c cu trc ha hc ging vi PC

(plant cellulose cellulose thc vt). Tuy nhin, cu trc a phn v thuc tnh ca BC khc

vi PC. Cc si mi sinh ra ca BC kt li vi nhau hnh thnh nn cc si s cp

(subfibril), c chiu rng khong 1,5 nm, l nhng si mnh nht c ngun gc t nhin. Cc

si c bn kt li thnh cc vi si (microfibril). Cc vi si nm trong cc b (bundle), v cui

cng hnh thnh cc di (ribbon). Trong khi chiu rng ca cc si cellulose c to ra t g

thng l 30.000-75.000 nm hay g bul (Betula) l 14.000- 40.000 nm. Nhng di vi si

cellulose mn c chiu di thay i t 1-9 m lm hnh thnh nn cu trc li dy c, c

n nh bi cc ni hydrogen. BC khc PC v ch s kt cht, v mc polymer ha. BC c

mc polymer ha t 2.000-6.000; mt vi trng hp t ti 16.000-20.000, trong khi mc

polymer ha thc vt l 13.000-14.000. So vi PC, BC c kt tinh cao hn, thm nc

tt hn, sc bn c hc trng thi m cao hn v d un nn hn. BC c hnh thnh qua

mt qu trnh phc tp gm nhiu giai on, cho sn phm c kch thc khc nhau theo s

sau:

Cu trc ca BC ph thuc cht ch vo iu kin nui cy. iu kin nui cy tnh.

Vi khun tng hp nhng ming cellulose trn b mt ca dch nui cy, ti ranh gii gia b

mt dch lng v khng kh giu oxy. Cc ming BC ny c gi l BC trn mi trng tnh

(S-BC: Static BC). Cc si cellulose s cp lin tc c y ra t nhng l c xp dc

trn b mt ca t bo vi khun, kt tinh li thnh cc vi si, v y xung su hn trong mi

trng dinh dng. Cc di cellulose t mi trng tnh to nn cc mt phng song song

nhng khng t chc, c vai tr chng cho qun th t bo A. xylinum. Cc si BC k nhau

c to ra t mi trng tnh ni vi nhau v b nhnh t hn cc si BC c to t mi

trng lc (A-BC: Agitated-BC). A-BC c to ra di dng cc ht nh, cc ht hnh sao

v cc si di, chng phn tn rt tt trong mi trng. Cc si an li vi nhau trong mi

trng lc ging nh m hnh k , c c hai hng song song v vung gc. S khc nhau

v cu trc khng gian ba chiu ca hai dng S-BC v A-BC c quan st r rng hn bng

knh hin vi in t. Nhng si S-BC ko di v chng trn cc si khc theo chiu an cho

nhau. Nhng si A-BC th ri rm v cong. Ngoi ra, b mt ct ngang ca si A-BC (0,1-

0,2m) ln hn si S-BC (0.05 0.10m). S khc nhau v hnh thi gia hai loi BC ny

lm mc kt tinh, kch c kt tinh ca chng khc nhau [1].


37

Hnh 36. BC c to t mi trng tnh (a) v mi trng lc (b)

Hai dng kt tinh ph bin ca cellulose trong t nhin l I v II, c phn bit bi

cc k thut phn tch bng tia X, quang ph, v tia hng ngoi. Cellulose I c th c

chuyn thnh cellulose II, nhng cellulose II th khng th chuyn thnh cellulose I.

Cellulose I c tng hp bi a s thc vt v A. xylinum mi trng tnh. Cc

chui -1,4-glucan cellulose I c sp xp song song vi nhau theo mt trc. Trong khi

, cc chui -1,4-glucan cellulose II th xp mt cch ngu nhin, hu nh khng song

song v ni vi nhau bi mt s lng ln ni hydrogen, lm cho cellulose II c bn v

nhit. Rt t t bo Eukaryote tng hp cellulose II trong khi A. xylinum th tng hp c c

2 loi cellulose I v II. Trong phn t cellulose I c s hin din ca I v I, do c cu to

bi glucose dng hay dng . Dng I bn v nhit ng hc hn. Glucose dng v

glucose dng l hai dng ng phn khng gian ca nhau. Hai dng ng phn ny ch khc

nhau s nh hng ca nhm hydroxyl. Hai dng ny c cc cellulose c ngun gc t

to, vi khun , thc vt. BC c dng I nhiu hn PC. S-BC c nhiu dng I hn A-BC. S

khc nhau v t l I gia hai loi S-BC v A-BC lm phng i cc ch s kt tinh, ch s I t

l thun vi kch c kt tinh. A-BC c ch s kt tinh thp hn v kch c kt tinh nh hn S-

BC. Mt phn ng k cellulose II c A-BC. Trong t nhin, cellulose II ch c tng hp

mt vi vi sinh vt (mt s to, mc, v vi khun nh Sarcina ventriculi, A. xylinum). Hin

nay, sn phm cng nghip ca cellulose II ch yu c c l da trn s bin i ha hc

cellulose thc vt.

2.5.3 Vai tr ca bacterial cellulose i vi Acetobacter xylinum

A. xylinum l vi khun khng c kh nng quang hp. Trong mi trng t nhin, a

s vi khun tng hp cc polysaccharide ngoi bo hnh thnh nn lp v bao quanh t

bo, mng BC l mt v d. Nhng t bo vi khun sn xut cellulose c by bn trong

mng li polymer. Mng li ny l vt chng cho qun th vi sinh vt lun b mt tip

gip gia mi trng lng v khng kh. H thng li polymer lm cho cc t bo c th

bm cht trn b mt mi trng v lm t bo thu nhn cht dinh dng mt cch d dng

hn so vi khi t bo trong mi trng lng khng c mng li cellulose. Mt vi tc gi


38

cho rng, cellulose c tng hp bi A. xylinum cn ng vai tr tch tr v c th c s

dng khi vi sinh vt ny b thiu ngun dinh dng. S phn hy cellulose c xc tc bi

enzyme exo-glucanase hay endo-glucanase, s hin din c hai loi enzyme ny c pht

hin trong dch nui cy mt vi chng A. xylinum sn xut cellulose. Nh vo tnh do v

tnh thm nc ca cc lp cellulose m cc t bo vi khun khng li c nhng thay i

bt li trong mi trng sng nh gim lng nc, thay i pH, xut hin cc cht c, v

cc vi sinh vt gy bnh. Cc vi khun A. xylinum c th tng trng v pht trin bn trong

lp v bao. Cellulose bao quanh t bo vi khun bo v chng khi tia cc tm. Khong 23%

t bo A. xylinum c bao BC sng st sau 1 gi x l tia cc tm. Tch BC khi t bo, kh

nng sng ca t bo gim ng k, ch cn 3%.

2.5.4 Cc yu t nh hng n qu trnh ln men to BC

Phng php ln men: Ngy nay, sn xut BC c thc hin bng nhiu phng

php ln men khc nhau nh nui cy lc, nui cy tnh trn khay Vic la chn

phng php no thc hin l ty thuc vo nh hng ng dng v sn phm BC

s c c tnh ha l v cu trc khc nhau ng vi tng phng php khc nhau. Khi

nui cy tnh hay b mt, mng BC (S-BC) c tch ly trn b mt mi trng nui.

S-BC thng mi thng dng nh Nata-de Coco, mng rung truyn m thanh, lm

mng tr bng Cn khi nui cy lc th BC (A-BC) s c dng si huyn ph, hay

dng khi khng ng nht (dng ht nh, hoc hnh cu, hnh elip). Kiu nui ny

thch hp hn cho sn xut BC cng nghip v cho cc ng dng khc ca A-BC nh:

m phm, vt liu, cht nh ha Kiu ln men BC ny cha c nghin cu v

tin hnh Vit Nam. Theo cc nh nghin cu th phng php nui cy chm cho

hiu sut sinh tng hp BC cao hn v cc thit b ln men chm c th cung cp tt

lng oxy cho t bo hot ng. ng thi BC ny cn c kh nng gi nc cao hn

t phng php tnh. Tuy nhin, tr ngi y l thng pht sinh cc chng t bin

Cel- mt cch ngu nhin sau thi gian ln men. Cc chng Cel- ny s khng cn kh

nng sn sinh BC na, v th lm hiu sut BC gim ng k.

Nhit : Nhit ti u cho sn sinh cellulose l 25-300C.

Gi tr pH: Nhn chung, khong pH ti u sn sinh BC l 4-7. Theo Hestrin v

Schramm (1954) cho rng pH di 7 l quan trng hn c. Cn Fiedler v cng s th

cho pH t 5-7 th ti u. Hu ht pH bng 5 hay 6 c dng trong cc nghin cu.

Nhng sn xut cellulose cng nghip th pH t 4-4,5 th cho kt qu u th hn do

gim s tp nhim. A. xylinum ng thi cng sn xut ra 2 loi: enzyme cellulase v

bacterial cellulose. Cc nghin cu gn y cho thy, tuy ng thi cng sn xut 2

loi: cellulose v cellulase nhng c th khng ch pH ca mi trng dinh dng

iu chnh mc sn sinh ra 1 trong 2 loi ny. Khi pH mc t 4 - 4,5 th lng

BC c sn sinh ra nhiu nht, cn pH mc t 5 5,5 th lng enzyme cellulase

c sn sinh cao hn. Theo S. Hestrin v M. Schramm (1954), HCl v NaOH thch

hp dng iu chnh pH mi trng.

Nng oxy: Nng O2 lin quan trc tip n vic sinh tng hp BC. Khi khng

kh giu O2 (duy tr nng ln hn 39%) c cho vo ti thi im 8h sau khi nui


39

cy th sn lng BC tng ln 1,5 ln v nng sut BC tng t 11% ln n 18%. Tuy

nhin, vi nng O2 nh th, nhng cho vo t khi bt u nui cy th s lng t

bo suy gim mt cch nhanh chng v hu nh vi khun khng sn sinh BC na.

iu ny tc gi gii thch v s lng t bo sng st ti thi im bt u nui cy

qu thp nn khng th tm thy BC trong iu kin nng O2 ha tan cao (v trong

iu kin ny lm c ch vi khun). Trong mi trng nui cy tnh, ti nng O2

10% v 15% mt vi chng A. xylinum cho sn lng BC cao hn nng O2 l

20%. Khi nng O2 tng n 30% s lm gim ng k sn lng BC trong khi s

tng trng ca t bo vn tip tc duy tr.

Ngun dinh dng carbon: Nhng c cht c nh gi l cho sn lng BC cao,

bao gm: glucose, sorbitol v manitol. Glycerol, galactose, lactose, sucrose v maltose

c xem l c cht thch hp. Nhng c cht m vi khun ny khng c kh nng s

dng to mng cellulose l sorbose, mannose, cellobiose, erythritol, ethanol v

acetate. Ngun carbon t glucose c cho l thch hp trong vic tng hiu sut kh

nng sn sinh BC.

Ngun dinh dng nitrogen: Mi trng c dng nghin cu l mi trng c

0,5% cao nm men v 0,5% pepton. i vi mt s dng A. xylinum li cho thm

tryptophan, dch chit ng vo trong mi trng. Tuy nhin dch chit ng cho hiu

qu hn c. Cc acid amin nh methionin, glutamate th lun cn phi c trong mi

trng v nhng acid amin ny c nh hng quan trng n s pht trin ca t bo

v s tng hp cellulose. Ngi ta tm ra ammonium dihydrogen phosphate c th

c s dng thay cho cao nm men nh mt ngun cung cp nit v thm vo mt

lng nh dch chit bt bp (t 0,125 ti 0,5 ml/l) th gia tng sn lng BC.

2.5.5 Cc ng dng ca bacterial cellulose

V bacterial cellulose c nhng c tnh nh mc tinh khit cao, kt tinh cao,

mt dy, kh nng gi cu trc tt, kh nng ht nc v cha nc mnh v din tch b

mt ring ln khi so snh vi cc loi cellulose thc vt, nn ngi ta p dng n trong

nhiu lnh vc khc nhau nh cng nghip dt nhum, giy, thc phm, y dc, x l cht

thi, truyn thng, hm m v tinh sch ho cht. Ta c th lit k mt s ng dng ph bin

nh sau:

Thc phm: v mt ho hc, cellulose c s dng trong cc quy trnh ch bin

thc phm vi chc nng lm ng c v n nh cu trc. ng dng u tin phi

k n l lm thch da (nata de coco). Do thch da c cu trc ging nh gel v

hon ton khng tiu trong ng tiu ho, nn n c tc dng tng t cc cht x,

rt hu ch cho h tiu ho. Philippine v nhiu nc khc, ngi ta cn ch bin

bacterial cellulose thnh dng bnh snack rt c a chung. K t nm 1970, khi

pht hin ra hp cht monacolin K (mevinolin) t loi nm Monascus c kh nng

km hm qu trnh sinh tng hp cholesterol, ngi ta kt hp gia thch da v

monacolin K thnh mt loi thc phm chc nng c tn l monascus-nata. Ngoi ra,

v c tnh vt tri v b mt mm mi v h si cao nn bacterial cellulose cn c

ng dng rng ri trong cc quy trnh ch bin thc phm khc. n nm 1992,


40

bacterial cellulose c a vo thc ung dnh cho ngi n king Nht Bn.

Acetobacter cng c ng dng cng vi nm men trong sn phm dch chit tr (tea

extract) c b sung ng, sn phm ny gi l kombucha hay l tr Manchurian, gip

ci thin sc kho ngi s dng [5].

Y dc: bacterial cellulose chu c lc ko cng cao, c xp cao v cu trc vi

si nn c ng dng trong y dc rt nhiu. i vi cc bnh kinh nin nh l lot

chn, nm lit ging v hoi t v bnh i tho ng th rt kh cha tr, nhng

bnh ny tr thnh nhng th thch ln cho c bnh nhn v ngnh chm sc sc

kho. Vic cha tr nhng bnh trn lin quan n nhiu gii php s dng cc cht

mang khc nhau (hydrocolloid, hydrogel, mng sinh hc hoc mng tng hp) nhm

cung cp mt trng thi mi trng c m cao gip cha tr vt thng t hiu

qu. Ngy nay i vi nhng vt liu bao vt thng ngi ta yu cu phi tng t

nh mt loi da nhn nhn to, c v cu trc ln chc nng. Vt liu ny khng gy

c, khng sinh nhit v tng hp vi c th, c kh nng chng nhim trng, gim

s r r bi dch vt thng, gim s au n trong qu trnh iu tr, d dng bao

kn vt thng, d chuyn hoc tim thuc vo vt thng, kh nng hp thu mnh

dch khi vt thng b vim, bn ko cao, d co gin v to cm gic thoi mi, d

to hnh ng vi cc vt thng khc nhau v khng gy au n khi bao vt thng.

Cellulose t vi khun do c cu trc xp nn n cho php chuyn nhng loi thuc

trc tip vo vt thng thng qua vic tim, ng thi n li l mt lp bo v trnh

s xm nhim vi sinh vt. Kh nng gi nc ca cellulose vi khun rt cao, cng vi

h si nh hn 100 ln so vi h si t cellulose thc vt. Cng ty Biofill (Brazil)

u t vo lnh vc ny v cho ra i 2 sn phm l Bioprocess v Gengiflex, dng

trong bao bc vt thng ngoi. Mt ch phm cellulose vi khun khc l Prima Cel,

c sn xut bi cng ty Xylos (M) cng p dng th nghim trn quy m ln.

Hnh 37. (a) h vi si BC v (b) thc vt, phng i x5000 [5]

2.6 Nghin cu v h vi khun nitrate ho v ng dng

Nm 2009, Manju v cng s trung tm quc gia v ng vt bin, n nghin

cu v a ra quy trnh c nh h vi khun nitrate ho ln bt g ca cy thn tho Ailantus

altissima (300-1500m) nhm chuyn ho lng ammonia trong nc nui tm sang nitrate.

Ch phm c tn thng mi l TANOX (Total Ammonia Nitrogen Oxidizer). Ging vi sinh

vt c phn lp t t nhin v t trong cc b nui tm, c Achuthan v cng s tuyn


41

chn nm trong mt cng trnh nghin cu vo nm 2006. Thi gian c nh theo phng

php th cng by-hp ph l 72h th ch phm c hot lc nitrate ho cao nht. Ngoi ra, cc

tc gi cng ch to mt thit b khuy o hot ng theo m gip nng cao nng sut v

hiu sut c nh.

Sau cc th nghim, nhm nghin cu tm ra cc thng s sau:

i vi cc chng vi khun nitrate ho: c nui thu sinh khi fermentor 2 L trong

7 ngy bng mi trng Watson (1965). Khi , hot lc nitrate ho ca 4 chng vi khun t

cao nht. Lng ammonia chuyn ho l 4,00,53 mg/L.ngy (vi khun nitrite ho, phn lp

t t nhin) v 6,20,78 mg/L.ngy (vi khun nitrite ho, phn lp t ao nui). Lng nitrite

chuyn ho l 5,371,2 mg/L.ngy (vi khun nitrate ho, phn lp t t nhin) v 6,890,98

mg/L.ngy (vi khun nitrate ho, phn lp t ao nui).

i vi cht mang: sau khi x l lignin theo phng php ca Wood v Saddler

(1988), s c nghin nh ra t kch thc 300-1500 m, c din tch b mt 1,87 m2/g.

Ch phm sau khi c c nh, nu bo qun nhit phng th kh nng nitrate

ho gim i phn na.

Phng php c nh: ngi ta nui thu sinh khi c 4 chng vi khun nitrate ho

chung mt thit b, sau tin hnh by-hp ph vi bt g. Kt qu l vi nng t bo

ban u 105 t bo/ml th 1 gam ch phm sau c nh trong 3 ngy s c hot lc nitrtate ho

cao nht.

S dng ch phm nh sau: dng 0,2 g ch phm ng vi 20 L nc bin cn x l,

mc chuyn ho ammonia t trung bnh 2,4 mg/L trong 3 ngy u tin. Trong cc th

nghim, sn phm nitrate ho to thnh (NO3-) khng pht hin c do qu trnh phn nitrate

ho bi cc chng vi sinh vt khc trong nc. iu ny cho thy ch phm TANOX c th

xem nh gip loi b nitrogen trong nc trit (thnh kh nit thot ra ngoi), mc d bn

cht ch l chuyn t ammonia sang nitrate.

Hnh 38. Hnh chp di knh hin vi in t nhng mu g A. altissima c loi lignin. A v B: kch thc mu 300-500m. C v D: kch thc mu 500-710m [10]


42

Hnh 39. Ch phm sau khi c nh, kch thc 300-1500m. Thi gian c nh ln lt l A: 6h, B: 12h, C: 24h v D: 72h [10]

Hnh 40. Thit b c nh dng khuy o, dung tch 50L. Sau 4 ngy c nh, kh nng nitrate ho ca ch phm t cao nht [10]

Hnh 41. th th hin hot lc nitrate ho ca ch phm theo thi gian. Tin hnh th

nghim lp li 3 ln [10]


43

CHNG 3. NGHIN CU TNH HUNG

3.1 Qui trnh thc hin

Nitrosomonas sp.

Nitrobacter sp.

Nhn ging cp 1

Nhn ging cp 2

Acetobacter xylinum

Nhn ging

To gi th BC

X l ph hpC nh VSV

Kim tra mu

Mu nc th nghim

Xc nh ch

tiu ho l

Xc nh hot lc nitrate ho

ca ch phm

Kt qu v bn lun

S 1. Quy trnh c nh h vi khun nitrate ha ln BC v th nghim ch phm.

3.2 To cht mang BC

Ging Acetobacter xylinum do B mn Cng ngh Sinh hc, Khoa k thut Ho hc, trng

i hc Bch Khoa TPHCM cung cp.

Mi trng gi ging (thch nghing) Glucose 20 g/l

Ammonium sulfate (SA) (NH4)2SO4 8 g/l

Diammonium phosphate (DAP) (NH4)2HPO4 2 g/l

Nc da gi 1 lt


44

Agar 20 g/l

Khng cho acid acetic

Kh trng 1210C trong 20 pht

Mi trng nhn ging cp 1, cp 2 Glucose 20 g/l



Nc da gi 1 lt Acid acetic m c 5 ml/l Kh trng 1210C trong 20 pht

Mi trng ln men thu BC Glucose 20 g/l



Nc da gi 1 lt Acid acetic m c 5 ml/l

Thanh trng 1000C trong 2 pht

Hnh 42. Quan st t bo Acetobacter xylinum di knh hin vi kim tra cc c im

vi th

Hnh 43. Khun lc Acetobacter xylinum


45

Hnh 44. Sau khi m bo chc chn ging ang c l Acetobacter xylium th t ng ging gc (A) ta cy chuyn qua ng thch nghing (B), nu thao tc khng ng s dn n

tp nhim (C)

Hnh 45. Cng vi vic cy chuyn, ta s tin hnh nhn ging cp 1 trong ng nghim. (1+2) lp mng BC to ra sau 5 ngy nhn ging, (3) cc t bo kt thnh di mu nu nht

Hnh 46. Tin hnh nhn ging cp 2, sau khi cy ging vo erlen cha mi trng bnh

(A), yn erlen iu kin nhit phng trong 5 ngy s thy xut hin lp mng mng pha trn (B), nu n 1 tun th lp mng s cng dy (C)


46

Hnh 47. Tin hnh ln men thu BC bng cch cy vo khay nha cha mi trng ln men

(A), sau 7 ngy ta s thu c sn phm (B).

Hnh 48. Trong qu trnh ln men, nu nguyn liu nc da gi b nhim bo t nm mc

th kh nng mc mc ln khi ln men l rt cao. Ngoi ra, nguyn nhn tp nhim cn do ra dng c khng sch v khay b h np nn bo t nm lt vo

3.3 Ln men thu sinh khi h vi khun nitrate ho

Ging vi khun c phn lp t ch phm vi sinh do vin Thu Sn II cung cp.

Mi trng nhn ging v ln men Nitrosomonas sp. (NH4)2SO4 3 g/l

K2HPO4 0,5 g/l

MgSO4 0,05 g/l

CaCl2 0,04 g/l

Phenol red 0,05 mg/l

FeSO4 0,05 mg/l

CaCO3 1 g/l


47

Nc ct 1 lt

Kh trng 1210C trong 20 pht

Mi trng nhn ging v ln men Nitrobacter sp. NaCl 0,3 g/l

KNO2 hoc NaNO2 0,5 g/l KH2PO4 0,25 g/l

MgSO4 1,4 g/l

CaCO3 1 g/l

Nc ct 1 lt Kh trng 1210C trong 20 pht Trong c 2 trng hp, khi lm mi trng thch nghing th agar thm vo khong 20g/L.

Hnh 49. (A) Dch ging Nitrobacter sp., (B) Dch ging Nitrosomonas sp.

Hnh 50. Hnh thi t bo Nitrobacter (bn tri) v Nitrosomonas (bn phi) di knh hin vi.

3.4 Phng php xc nh ammonia, nitrite v nitrate

Cc phng php s dng theo TCVN trong phn tch nc ung.


48

3.4.1 Xc nh hm lng ammonia NH3 theo TCVN 2662-78 v 4563-88

Tiu chun quy nh phng php so mu vi thuc th Nessler xc nh hm

lng nit di dng ammonia. NH3 s tc dng vi thuc th cho mu t vng n nu sm

trong mi trng kim. Phng php ny cho php xc nh hm lng ammonia t 0,01-

5mg trong 1 lt nc. Khi nng ammonia ln hn gii hn trn, phi pha long mu trc

khi xc nh.

Nguyn tc:

2(2KIHgI2) + NH3 + 3KOH (NH2)Hg-O-HgI + 7KI + 2H2O

(NH2)Hg-O-HgI l phc cht mu vng, c hp thu cc i bc sng 430nm.

C th xc nh ammonia. C th xc nh ammonia trc tip trong mu nc hoc xc nh

sau khi ct mu. Khi nc b bn, c mu th thng c ct trc khi xc nh.

Cht gy nhiu:

+ Mt s hp cht amine mch thng, hp cht cloramine hu c mch vng, acetone,

aldehyde, ru c th phn ng vi thuc th Nessler cho phc cht mu vng, gy

sai s kt qu o.

+ Ion sulfur gy kt ta vi Nessler nn cn loi b bng carbonate ch. kim tra

ion sulfur ta s cho vo mu nc mt lng nh acid sulfuric (c 5ml th cho 2ml

acid, t l 1:3). Sau khi cho acid, mu nc b c l c sulfur. Trng hp phi

cho vo 100ml mu th 10 git km acetate 25%. Khi kt ta lng xung, ly phn

nc trong xc nh.

+ Xc nh ammonia trong phng ring, trnh ammonia c trong khng kh xm

n

do an mon hoc_tan duc

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