Khả năng phân hủy 2,4,5-T của một số chủng vi khuẩn

S ha bi Trung tm Hc liu i hc Thi Nguyn http://www.lrc-tnu.edu.vn

I HC THI NGUYN TRNG I HC S PHM

----------------///----------------

Phm Ngc Long

NGHIN CU KH NNG PHN HY 2,4,5-T V C IM PHN

LOI CA CHNG VI KHUN PHN LP T CC BIOREACTOR

X L T NHIM CHT DIT C/DIOXIN

LUN VN THC S SINH HC

Thi Nguyn - 2009


I HC THI NGUYN TRNG I HC S PHM

----------------///----------------

Phm Ngc Long

NGHIN CU KH NNG PHN HY 2,4,5-T V C IM PHN

LOI CA CHNG VI KHUN PHN LP T CC BIOREACTOR

X L T NHIM CHT DIT C/DIOXIN

Chuyn ngnh : Sinh hc thc nghim M s : 60.42.30

LUN VN THC S SINH HC

NGI HNG DN KHOA HC: TS. Nghim Ngc Minh

Thi Nguyn - 2009


Li cm n

Trc ht, ti xin by t lng bit n chn thnh v su sc ti TS

Nghim Ngc Minh. Trng Phng Cng ngh sinh hc Mi trng Vin

Cng Ngh sinh Hc tn tnh hng dn v du dt ti trong qu trnh

nghin cu v hon thnh lun n.

Trong qu trnh nghin cu va qua, ti nhn c s gip v ch

bo tn tnh ca PGS.TS. ng Th Cm H v cc anh ch Phng Cng

ngh sinh hc Mi trng, c bit l Ths. Nguyn B Hu, CN. Nguyn

Vn Bc, KS. Cung Th Ngc Mai, nhng ngi gip ti trong qu

trnh thc hin lun n ca mnh.

Ti cng xin by t lng bit n su sc ti Khoa sau i hc, khoa

Sinh-K thut nng nghip Trng i hc S phm i Hc Thi

Nguyn v lnh o Vin Cng ngh sinh hc, Vin Khoa hc v Cng ngh

Vit Nam to mi iu kin cho ti hon thnh kha lun ny.

Bn cnh , ti xin cm n nhng ngi thn trong gia nh v bn b

to iu kin ng vin gip ti c v vt cht v tinh thn ti c th

hon thnh bn lun vn ny.

Mt ln na ti xin chn thnh cm n!

H Ni, ngy 1 thng 10 nm 2009

Phm Ngc Long

BNG CH VIT TT

1,2,3,7,8-PeCDD 1,2,3,7,8-Pentaclorodibenzo-p-dioxin

2,3,7,8-TCDD 2,3,7,8-Tetraclorodibenzo-p-dioxin

2,4,5-T 2,4,5-trichlorophenoxyacetic acid

2,4-D 2,4,- dichlorophenoxyacetic acid

bp Base pair

DNA Deoxyribonucleic acid

LB Luria - Bertani

PAH Polycyclic aromatic hydrocarbon

PCDDs Polychlorinated dibenzo-p-dioxins

PCDFs Polychlorinated dibenzofurans

PCR Polymerase Chain Reaction

RNA Ribonucleic acid

rRNA Ribosomal ribonucleic acid

X-gal 5-bromo-4-chloro-3-indodyl- galactosidase

Lun vn thc s sinh hc Phm Ngc Long

S ha bi Trung tm Hc liu i hc Thi Nguyn http://www.lrc-tnu.edu.vn 1

MC LC

M U 1

PHN 1 TNG QUAN 3

1. S nhim ca 2,4,5-T v 2,4-D 3

2. c im v tnh cht ca 2,4,5-T v 2,4-D 5

2.1 Cht dit c 2,4,5-T 5

2.2 Cht dit c 2,4-D 7

3. nh hng ca 2,4,5-T, 2,4-D n mi trng v con ngi 8

3.1 nh hng ca 2,4,5-T v 2,4-D ti mi trng 8

3.2 nh hng ca 2,4,5-T, 2,4-D n con ngi 9

4. Mt s phng php x l cht c ha hc

trong c 2,4,5-T v 2,4-D 9

4.1 Phng php x l cht c ha hc bng ha hc, l hc, c hc 9

4.2 Phng php phn hy sinh hc 10

5. Kh nng phn hy 2,4,5-T v 2,4-D ca mt s vi sinh vt 15

6. Phn loi vi sinh vt 21

6.1. Phn loi theo phng php c in 21

6.2 Phng php phn loi bng sinh hc phn t 22

PHN 2 VT LIU V PHNG PHP 25

1. Vt liu, ha cht, cc thit b s dng trong nghin cu 25

1.1 Vt liu 25

1.2 Ha cht 25

1.3 Thit b, my mc 25

2. Phng php nghin cu 26

2.1 Mi trng nui cy 26



2.1.1 Mi trng SH1 dch (g/l) 26

2.1.2 Mi trng SH1 thch 26

2.1.3 Mi trng mui khong 26

2.1.4 Mi trng LB dch 27

2.1.5 Mi trng LB thch 27

2.1.6 Nc mui sinh l 27

2.2 Phng php nui cy v phn lp vi khun t mu t nhim

cht dit c/dioxin trong bioreactor hiu kh 27

2.2.1 Nui cy lm giu vi sinh vt 27

2.2.2 Phng php phn lp vi khun 27

2.3 Nghin cu hnh thi t bo ca chng vi khun 28

2.3.1 Nhum Gram 28

2.3.2 Quan st hnh thi t bo di knh hin vi in t qut 28

2.4 Phng php phn tch kh nng phn hy 2,4,5-T 29

2.5 Phn loi vi khun da trn so snh trnh t gen m ha 16S rRNA 29

2.5.1 Phng php tch DNA tng s t vi sinh vt 29

2.5.2 Nhn on gen 16S rRNA bng phng php PCR 30

2.5.3 in di kim tra trn gel agarose 31

2.5.4 Tch dng on gen m ha 16S rRNA 31

2.5.5 Bin np DNA ti t hp vo t bo E.coli 31

2.5.6 PCR trc tip t khun lc (colonyPCR) 32

2.5.7 Tch DNA plasmid theo Kit ca hng Fermentas 33

2.5.8 Xc nh trnh t on gen m ha 16S rRNA 34

2.5.9 Xy dng cy pht sinh chng loi 34



PHN 3 KT QU V THO LUN 35

1. Nui cy, phn lp chng vi sinh vt t mu t nhim cht

dit c/dioxin trong bioreactor hiu kh 35

1.1 Nui cy, lm giu tp on vi sinh vt 35

1.2 Phn lp chng vi khun 37

2. c im phn loi ca chng HR5.1 38

2.1 Hnh thi t bo 38

2.2 Phn loi da trn trnh t gen m ha 16S rRNA 39

2.2.1 Tch chit DNA tng s 39

2.2.2 Nhn on gen 16S rRNA ca chng HR5.1 bng k thut PCR 40

2.2.3 Tch dng gen 16S rRNA trong vector pBT 41

2.2.4 Xc nh trnh t gen 16S rRNA ca chng HR5.1 43

3 Nghin cu mt s c im ca chng HR5.1 47

3.1 Kh nng pht trin ca chng HR5.1 trn PAH 47

3.2 Kh nng phn hy 2,4,5-T ca chng HR5.1 49

3.2.1 nh hng ca mi trng nui cy c cha 2,4,5-T

ln s pht trin ca chng HR5.1 49

3.2.2 nh hng ca nng 2,4,5-T ln s pht trin

ca chng HR5.1 50

3.2.3 Kh nng phn hy 2,4,5-T ca chng vi khun HR5.1 54

KT LUN V KIN NGH 56

TI LIU THAM KHO 57



M U

Trong cuc chin tranh xm lc ca M tin hnh Vit Nam, hn 100

triu lt cht dit c cha 2,4,5-T, 2,4-D v 2,3,7,8 TCDD c ri xung hn

20% din tch ca min Nam. Theo cng b ca Stellman v cng s trn tp

ch Nature nm 2003 th 20 cht dit c khc nhau c s dng. Chu k bn

hy ca dioxin v cc cht tng t dioxin rt di, c khi n vi chc nm hoc

hng trm nm [15],[42]. Qua cc iu tra nghin cu ca nhiu c quan khoa

hc v cng ngh Vit Nam v quc t cho thy, t ca sn bay Nng

v Bin Ha tn lu ca PCDD, PCDF, 2,4,5-T v 2,4-D vn cn cao.

2,4,5-T, 2,4-D c hm lng ln ti hng vi trm nghn n vi triu g/kg

t. Ngoi ra mt lng khng nh cc cht DCP, TCP v PAH cng

c xc nh trong cc mu t ti khu vc b nhim c. Nghin cu p

dng phng php sinh hc kh c ti im nng Nng thu c

kt qu rt kh quan.

Tuy nhin x l cc im nhim cc b cht dit c/dioxin vi thi

gian ngn cn c cc cng ngh phn hy sinh hc ph hp. Hin nay, phng

Cng ngh sinh hc mi trng, Vin Cng ngh sinh hc ang tin hnh x

l t nhim cht dit c cha dioxin bng cng ngh tng cng sinh hc

trong cc bioreactor hiu kh v k kh. Trong qu trnh x l, ngoi s iu

khin v iu kin mi trng nh m, nhit th vai tr ca cc vi sinh

vt c trong bioreactor l rt quan trng. tng hiu qu v hon thin cng

ngh cn tng thm hiu bit v c im vi sinh vt c trong bioreactor, cng

nh vai tr ca cc vi sinh vt phn hy cht c c b sung vo

bioreactor. Nhm p ng yu cu ca thc tin , ti Nghin cu kh

nng phn hy 2,4,5-T v c im phn loi ca chng vi khun phn

lp t cc bioreactor x l t nhim cht dit c/dioxin c thc

hin.



Lun n thc hin c cc ni dung nghin cu sau y

Lm giu vi sinh vt t cc mu t trong bioreactor x l t

nhim cht dit c/dioxin.

Phn lp cc chng vi khun c kh nng pht trin trn 2,4,5-T v

2,4-D.

Nghin cu mt s c im ca chng vi khun phn lp c.

Phn loi nh tn chng vi khun c chn la.

Xc nh kh nng s dng 2,4,5-T ca chng vi khun nghin cu.

Lun n ny c thc hin ti phng Cng ngh sinh hc mi trng,

Vin Cng ngh sinh hc v l mt phn cp Vin Khoa hc v cng ngh

Vit Nam: Nghin cu x l ty c mt s hp cht hu c cha clo bng

cc phng php ha hc v sinh hc tin tin do PGS.TS. ng Th Cm

H ch tr.



PHN 1 TNG QUAN

1. S nhim ca 2,4,5-T v 2,4-D

T nm 1961 n nm 1971 qun i M ri hn 100 triu lt cht dit

c xung nhiu vng min Trung v Nam Vit Nam [42]. Cc cht dit c

c s dng bao gm: cht da cam, cht trng, cht xanh lc, cht xanh

lam, cht tm, cht hng, cc cht ny c gi tn theo mu nh du trn

cc thng phuy cha chng, mi thng khong 250l [42]. Cc cht dit c

thng l hn hp ca hai cht 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) v

2,4-D (2,4-dichlorophenoxyacetic acid) vi t l 50:50 (Bng 1). Dioxin l

tp cht c sinh ra trong qu trnh sn xut 2,4,5-T. Hm lng dioxin

trong cc cht dit c rt khc nhau, c tnh s lng dioxin cha trong cht

dit c m M dng trong chin tranh Vit Nam t 170 - 1000 kg [42], [15].

Bng 1.1. Thnh phn ha hc ca cc cht dit c qun i M s

dng trong chin tranh Vit Nam [42].

Tn

cht Thnh phn ho

cht m c tng ng

S dng trong

trong nm

S lng c tnh

ri (lt)

Cht hng

60% - 40% n-Butyl:

isobutylester ca 2,4,5-T

961-1081 g/l

acid tng ng

1961-1965 503.121;

413,852

Cht xanh

l cy

n-Butylester ca 2,4,5-T

Ging nh cht hng

Cha r, ri cng thi gian vi cht hng

31.026

Cht tm

50%n-Butylester

2,4,D

30% Butylester

1033 g/l acid

tng ng 1962-1965 1.892.773



2,4,5-T

20% isobutyl ester

2,4,5-T

Cht da

cam

(1)

50% n-Butyl ester

2,4-D

50% n-Butyl ester

2,4,5-T

1033 g/l acid

tng ng 1965-1970 45.677.937

(c th bao gm c cht da cam)

Cht da

cam

(2)

50% n-Butyl ester

2,4-D

50% n-Butyl ester

2,4,5-T

910 g/l acid

tng ng Sau 1968

(?)

Cha r nhng gi sang Vit Nam t nht l: 3.591.000

Cht trng

Khi lng acid c bn: 21,1% tri-isopropanolamine

mui ca 2,4-D v 5,7% picloram

Khi lng acid: 240,2 g/l

2,4-D v 64,9

g/l picloram

1966-1971 20.556.525

Cht xanh

(dng bt)

Acid dimethylarsinic

v Natri cacodylat

Acid: 65%

tng ng

Mui: 70% tng ng

1962-1964 25.650

Cht xanh

(dng dch)

21% Natri

cacodylat+ acid

cacodylic t nht chim 26% tng lng acid tng ng

Khi lng acid: 360,3 g/l

1964-1971 4.715.731

Ti cc cn c qun s c ca M trc y l ni tng tr v np cht dit

c ln my bay nh sn bay Bin Ha, Nng, Ph Ct c tn lu cc

cht c mc cao v rt cao, ln ti hng trm nghn ppt [15]. c bit l

cc sn bay Bin Ha, Nng hm lng 2,3,7,8-TCDD chim 90% tng

c, nhiu mu t 2,3,7,8-TCDD > 99% tt c c ca PCDD v



PCDF. Cc kt qu phn tch cn pht hin mt lng ln 2,4,5-T, 2,4-D,

dichlorphenol, trichlorophenol v mt s lng nh hydrocarbon thm a

nhn trong cc mu t ti khu vc nhim c [15]. Ngoi ra, nhim 2,4,5-T

v 2,4-D Vit Nam cn t cc ngun khc. Tuy nhin, trong khun kh lun

vn ny chng ti ch nghin cu v vi sinh vt c ngun gc t ngun

nhim cht dit c do chin tranh.

2. c im v tnh cht ca 2,4,5-T v 2,4-D

2.1 Cht dit c 2,4,5-T

2,4,5-T l tn gi tt ca acid 2,4,5-trichlorophenoxyaxetic. Cng thc ha

hc l C8H5O3Cl3, khi lng phn t 255,49 g/mol. Cng thc cu to c

th hin hnh 1.1.

Hnh 1.1 Cng thc cu to 2,4,5-T

2,4,5-T tinh khit c dng tinh th rn, khng mi, t khng mu n vng

nu nht, tan t trong nc, ha tan trong nc 30oC l 238 mg/kg, tan

tt trong dung mi hu c. T trng l 1,8 g/cm3 20oC. Nhit nng chy

trong khong 154oC -158oC [43].

2,4,5-T c s dng nh mt cht dit c c tc dng lm rng l cy,

c pht trin vo cui thp nin 40 ca th k XX v s dng trong nng

nghip. 2,4,5-T l cht c c tnh mnh, gy ung th, d thai, ri lon ni



tit, nhim c tuyn sinh dc v nhiu bnh nghim trng khc. S tng

qut qu trnh tng hp 2,4,5-T c trnh by hnh 1.2.

i ii

Hnh 1.2 S tng hp 2,4,5-T

i : Nhit vi NaOH trong CH3OH di p sut hi nc

ii: ClCH2COOH trong NaOH 140oC

Trong qu trnh tng hp 2,4,5-T i t nguyn liu ban u l 1,2,4,5-

tetrachlorobenzene, cn phi c nhit t 225 n 3000C v p sut dao

ng trong khong t 400 n 1500 psi. Tuy nhin, iu kin nh vy sn

phm ph l 2,3,7,8-TCDD c to ra v theo cc tc gi, hm lng

2,3,7,8-TCDD c trong 2,4,5-T vo khong t 0,07 ti 6,2 ppm (hnh 1.3) [40].



Hnh 1.3 C ch to ra sn phm ph 2,3,7,8-TCDD trong qu trnh tng

hp cht dit c 2,4,5-T

2.2 Cht dit c 2,4-D

Dichlorophenoxyacetic acid (2,4-D) c cng thc ha hc l C8H6Cl2O3

cng thc cu to c th hin hnh 1.4.

Hnh 1.4 Cu trc ca 2,4-D.

2,4-D c khi lng phn t 221,04g/mol, dng tinh khit 2,4-D

dng bt, c mu trng n mu vng. Nhit d nng chy l 140,5oC v nhit



bay hi l 160oC. nhit 25oC, 2,4-D c th c ha tan ci hm

lng 900mg/l.

2,4-D l thuc dit c c tng hp t cc auxin, l thuc dit c tn rng

Hin nay ch yu 2,4-D c s dng trong nhng hn hp pha trn vi cc

loi thuc dit c khc, c vai tr nh mt cht tng cng tc dng. N ang

c s dng rng ri trn khp th gii.

3. nh hng ca 2,4,5-T, 2,4-D n mi trng v con ngi

3.1 nh hng ca 2,4,5-T v 2,4-D ti mi trng

Qun i M ri cht dit c cha 2,4,5-T, 2,4-D v tp cht dioxin ln

khong 27% tng din tch Vit Nam. Khong hn 2 triu ha rng b tc

ng ca cht dit c [17]. Tc dng tc thi ca cht dit c l lm cho cc

loi cy rng b tri ht l, rt nhiu loi cy b cht, mi trng v sinh cnh

b thay i nhanh chng [17]. Ti cc vng rng b ri lp i lp li nhiu ln,

h sinh thi rng b ph hy hon ton v cho n nay ti nhng ni ny cha

c cy mc t nhin nh khu rng M (ng Nai), thung lng A Li

(Tha Thin Hu) v.v.[17]

Cht dit c sau khi c phun xung c th tch t khng nhng trong t

m cn phn tn trong lp nc mt, nc ngm, khng kh, tch t trong

thc vt, gy nhiu s c v him ha cho mi trng v t tc ng dy

chuyn n con ngi, ng thc vt v cc vi sinh vt. Hu qu l lm suy

thoi h sinh thi t nhin. Cc cht ny git cht cc ng vt, thc vt, vi

sinh vt v nhiu loi sinh vt khc lm cho chng khng th phc hi li

c, lm thay i hon ton cu trc qun x v chng loi ng vt, thc

vt [4], [22].

Cht c ha hc ngm vo trong t, tch t li trong c th thc vt nn

t b phn hy bi mt s yu t nh nh sng mt tri, tia cc tm, nhit .



Cc cht ny tn ti di dng hn hp v cc yu t mi trng nhiu khi

cha thun li cho cc qu trnh phn hy sinh hc t nhin. Chin tranh kt

thc hn 30 nm, lng cht c ha hc cn li trong t rt ln, c bit

l 2,4,5-T, 2,4-D, dioxin ti cc im nng. Ti cc cn c qun s c ca M

sn bay Nng, Bin ha v Ph Ct b nhim 2,4,5-T, 2,4-D, dioxin

.v.v. mc cao. Nghin cu chn la v p dng cc phng php thch

hp ty c ngay cc im nng l nhim v rt cn thit t ra cho cc

nh khoa hc v cng ngh cn quan tm gii quyt.

3.2 nh hng ca 2,4,5-T, 2,4-D n con ngi

c tnh c 3181 ngi lng Vit Nam b ri trc tip, vi khong 4,8 triu

ngi tip xc vi cht c ha hc do M ri xung [42].

Theo bo co mi nht ca Vin Y khoa Hoa K nm 2002, c 37 bnh

ngi lin quan n dioxin cc cp khc nhau nh ban clo, ung th m

mm, ung th dng Hodkin, ung th dng khng Hodkin, mt s bnh thn

kinh cp tnh, gai t ct sng, sy thai, d tt bm sinh v.v. [32].

Trong s nhng ngi tip xc vi cc cht c ha hc ny. Nhiu

ngi b phi nhim mc phi nhng cn bnh nguy him v mt s bnh

di truyn c sang cc th h sau [16].

4. Mt s phng php x l cht c ha hc trong c 2,4,5-

T v 2,4-D

4.1 Phng php x l cht c ha hc bng ha hc, l hc, c hc

Phng php chn lp hay c p dng cho cht thi nguy hi, rc thi, k

c cc cht c ha hc. u im ca phng php l gi thnh r nhng cht



c vn nm trong cc h chn lp khng c phn hy nn cc cht c ha

hc ny c th l ngun nhim tim tng cho mi trng v con ngi.

Cc phng php vt l nh quang ha, s dng cc tia cc tm, hay dng

p sut cao cng c hiu qu. Theo cc kt qu cng b cho thy rng s dng

phng php quang ha, 80% cht c b phn hy di tc ng ca chm

tia cc tm cng 20W/cm3 nhit 20oC trong 3 ngy. Tuy nhin

phng php ny ch p dng cho nhng lp t mng b mt dy di vi

milimet [14], [41].

Phng php thiu t cng c nhiu nc la chn x l dioxin.

Nguyn l ca phng php l dng nhit cao phn hy dioxin t hiu

qu n 99,99%, nhng phng php ny c nhc im l gi thnh x l

cao, l cha k n kinh ph o, vn chuyn t n l t v c th to ra

cc sn phm ph gy nhim th cp [18].

Phng php declo ha v oxy ha cng c nghin cu p dng vi cc

cht cha clo v c 2,3,7,8-TCDD. Phng php ny cng cho kt qu kh

tt, thng to ra nhng hp cht t clo v t c hn [19], [24],[14]. Nhc

im ca phng php ha hc l khng kim sot c sn phm to thnh,

cc sn phm ny thng gy nhim th cp.

Cc phng php x l c hc, vt l, ha hc ni chung u c nhc

im l tn km v khng trit , d gy nhim th cp cho mi trng.

Phng php x l nhim bng cng ngh sinh hc ang c c bit ch

do tnh an ton v kinh t ca n. Phng php ny ang c nhiu phng

th nghim trn th gii nghin cu, pht trin.

4.2 Phng php phn hy sinh hc

Phng php x l bng cng ngh sinh hc tuy mi m nhng c

c bit ch bi gi thnh h v thn thin vi mi trng. Phng php



phn hy sinh hc khng i hi cc iu kin phc tp nh nhit cao, p

sut, qu trnh xc tc v.v. Phng php ny tun theo qui lut chuyn ha

thuc chu trnh cacbon, nit, photpho v.v. khng gy ra nhim th cp, an

ton, thn thin vi mi trng v h sinh thi, chi ph thp do rt ph hp

vi iu hin kinh t nc ta. Mt khc, din tch t b nhim c Vit

Nam rt ln nn vic ng dng cc phng php ty c khc nh ha hc v

l hc kh c kh nng thc hin [2]. Tuy nhin nhc im ca phng

php ny l i hi thi gian di.

Qu trnh lm sch sinh hc c th thc hin quy m ln nh khc nhau

v iu kin hiu kh hoc k kh. Vic ty c bng phn hy sinh hc c

th c tin hnh ring r hoc kt hp vi cc phng php khc. Sau vi

thng hoc vi nm cc cht nhim c th c hon ton loi b bng

phng php phn hy sinh hc [9].

X l cht nhim theo phng php phn hy sinh hc c th i theo hai

hng chnh l lm giu sinh hc v kch thch sinh hc [9]. Lm giu sinh

hc (Bioaugmentation) l phng php s dng tp on vi sinh vt bn a

c lm giu hoc vi sinh vt s dng cc cht c t ni khc, thm ch

vi sinh vt c ci bin v mt di truyn b sung vo cc a im

nhim. Kch thch sinh hc (Biostimulation) l qu trnh thc y s pht trin

v hot ng trao i cht ca tp on vi sinh vt bn a c kh nng s

dng cc cht c hi thng qua vic thay i cc yu t mi trng nh pH,

m, nng O2, cht dinh dng, cc c cht, cc cht xc tc v.v.

Vic b sung vi sinh vt vo cc a im nhim i hi chi ph cao v

nhiu khi khng mang li hiu qu cao do nhiu nguyn nhn nh s cnh

tranh ca vi sinh vt, c ca mi trng, s thiu ht ngun dinh dng,

cc cht a lng v vi lng cn cho hot ng phn hy ca vi sinh vt [15].



Vit Nam, bin php chn lp tch cc phn hy cht dit c/dioxin

c nghin cu v p dng thnh cng trn quy m pilot hin trng.

Chn lp tch cc l s kt hp ca phn hy sinh hc, c lp, hp ph v

chn lp. ng Th Cm H v cng s tin hnh nghin cu th nghim

phn hy sinh hc i vi cc l x l 0,5m3; 1,5m3; 10m3 v 100m3 t

nhim cht dit c cha dioxin ti Nng [2], nm 2009 nhm nghin cu

x l hn 3000 m2 t nhim cht c ha hc ti sn bay Bin Ha bng

cng ngh phn hy sinh hc. Cc cng thc x l phn hy sinh hc c b

sung cc dng ch phm khc nhau cung cp cht dinh dng, cc cht cn

thit cho qu trnh oxy ha, kh loi b clo cc cht vi lng v cc cht thm

cho tp on vi sinh vt tham gia vo qu trnh ty c iu kin k kh v

hiu kh. S lng cc nhm vi sinh vt trc, trong sut qu trnh x l

c theo di. Cc chng nm, vi khun hiu kh, k kh, x khun c s

dng nghin cu kh nng phn hy 2,3,7,8-TCDD. Phng php nghin

cu vi sinh vt truyn thng v k thut sinh hc phn t im ch nh DGGE

v cc k thut sinh hc phn t khc c tin hnh nghin cu tp

on vi sinh vt ng thi phn lp cc chng vi sinh vt, nh tn loi vi sinh

vt s dng dioxin, dibenzofuran, hydrocabon thm a nhn phn lp t

ngun nhim k trn. tn lu ca dioxin v cc nhim khc c xc

nh bng phng php min dch v sc k khi ph. Phng php min

dch phn tch dioxin ca EPA Hoa K c tin hnh theo EnviroGrardTM

kt [2].

Sau tm nm nghin cu, cc nh khoa hc Vit Nam thu c nhng

kt qu rt kh quan. S lng vi sinh vt d dng t nhim c trc khi

x l khng cao, dao ng t 102 - 105 MPN/g hay CFU/g [2]. Nhng nhm

vi sinh vt khc cng tn ti trong loi t ny vi s lng v a dng thp.

Trong qu trnh x l qui m khc nhau, s lng vi sinh vt tng ng



k t 1.000-10.000 ln [2]. Sau hn hai nm x l bng cch b sung ch

phm Slow-D, DHS1, DHS2 v cc hp cht, vi lng, thnh phn xc tc,

cc cht hot ng b mt sinh hc, phi hp vi s thay i hm lng oxy

v thay i m, hiu qu ca qu trnh x l chn lp tch cc (kt hp

c lp, hp ph, chn lp v phn hy sinh hc) rt r rt. Trong tt c cc l

x l, sau 8 n 24 thng, t 50 n 70% tng c b gim [2].

Trong qu trnh x l cc quy m, hnh thc khc nhau, trong nhng

nm qua, cc cn b nghin cu thuc phng Cng ngh sinh hc mi trng

Vin Cng ngh sinh hc phn lp c mt s chng vi sinh c kh nng

vt s dng dibenzofuran, dioxin, cht dit c 2,4,5-T, 2,4-D v PAH t t

nhim cht c ha hc ti sn bay Nng. Cc vi khun c phn lp

gm Bacillus sp. BU3, Pseudomonas sp. BDN15, Pseudomonas sp. SETDN1,

Terrabacter sp. DMA, Rhodoccocus sp. HDN3 v.v. Mt s chng nm si

ch yu thuc chi Aspergillus nh Aspergillus sp. FDN30, Aspergillus sp.

FDN22, Aspergillus sp. FDN20. X khun cng c phn lp tuy khng

nhiu nhng cng , phn lp c mt s chng thuc chi Streptomyces

nh cc chng Streptomyces sp. XKDN11, Streptomyces sp. XKDN12 [1],

[2], [5], [8], [10], [11]. Vic nh tn v xc nh kh nng phn hy cht

nhim ca cc chng vi sinh vt ny gp phn lm sng t c ch v hiu qu

ca s phn hy sinh hc din ra trong qu trnh x l bng cng ngh phn

hy sinh hc c trin khai x l kh c qui m 3.000 m3 ti im

nng Bin Ha, ng Nai.

Kt qu phn tch vi sinh vt v ha hc cho thy cc ch phm s dng ti

hin trng thnh cng trong vic kch thch qu trnh phn hy sinh hc

ti ch. Tt c cc ch phm, cc hp cht trn u c th tm c v sn

xut ti Vit Nam v vy ch ng v gim gi thnh x l t nhim dioxin

trong cc cn c qun s c. Hn na, phng php chn lp tch cc c tnh



an ton cao v kh thi i vi khi lng b nhim cht dit c/dioxin n

vi ha v su trung bnh t mt n hai mt.

y nhanh tc phn hy sinh hc cht dit c/dioxin, cc nghin cu

x l kh c trong cc bioreactor c nhm nghin cu cng ngh x l

POP, Vin Cng ngh sinh hc tin hnh. y l phng php x l kiu tng

cng sinh hc (bioaugmentation) trong h thng c kh nng iu khin cc

thng s quan trng lin quan n qu trnh chuyn ha sinh hc nh nng

oxy, m, pH, nhit , nng cc cht b sung v thnh phn cc

nhm vi sinh vt lin quan n tng giai on chuyn ha sinh hc cht

nhim. y l kiu x l ex situ v tc chuyn ha c ci thin so vi

bin php x l chn lp tch cc do cc iu kin u c kim sot. t

nhim cht dit c/dioxin ti sn bay Nng c x l trong cc

bioreactor vi quy m 50 kg [3]. Cc chng vi sinh vt c hot tnh phn hy

tt cc cht nhim chnh c phn lp ti t nhim cht dit c/dioxin ti

chnh sn bay Nng v tp on vi sinh vt nui cy lm giu trn mi

trng dinh dng cha cc thnh phn cht nhim trong phng th nghim

c b sung trong qu trnh x l. Cc cht b sung trong qu trnh x l

c tnh ton v b sung theo tng giai on trong qu trnh kt hp vi

kim sot cht ch cc yu t v m v thng kh. Ngoi ra, cn kt hp

x l theo kiu kt hp hiu kh k kh c th chuyn ha hiu qu trong

c hai iu kin kh loi chlo v oxi ha ct vng 2,3,7,8-TCDD. Thnh phn

vi sinh vt trong thi gian x l tng ng k, t 106 ln n 1012 MPN/g,

trong trong cc bioreactor hiu kh, vai tr ca nm si c khng

nh l rt quan trng vi h si pht trin mnh trn t x l v kh nng

sinh cc loi enzyme ngoi bo nh laccase, v cc peroxidase vi hot tnh

cao. Sau thi gian 17 tun, tng c gim 44,1%[3].



5. Kh nng phn hy 2,4,5-T v 2,4-D ca mt s vi sinh vt

2,4-D v 2,4,5-T l hai thnh phn ch yu ca cc cht dit c m qun

i M s dng trong chin tranh Vit Nam [42]. Hin ti t ti cc khu

nhim cht c ha hc cc cn c qun s c ca M min Trung v

Nam Vit Nam hai hp cht trn vn nng cao. Trn th gii do 2,4-D

v 2,4,5-T c s dng t rt lu nn c nhiu cng b v kh nng phn

hy sinh hc hai hp cht ny bi vi sinh vt phn lp t ngun khc nhau

nh t nng nghip, t cng nghip v trm tch.

Khc vi 2,4-D, 2,4,5-T kh phn hy hn, hin khng c nhiu cng b

v phn hy sinh hc hp cht ny. Tuy nhin cng c mt s nghin cu

trn th gii v Vit Nam cng b v vi khun c kh nng phn hy 2,4,5-T

nh Burkholderia cepacia AC1100, Phanerochaete chrysosporium, Pleurotus

cornucopiae, Stenotrophomonas maltophilia PM, .v.v. Mt vi chng nm

Aspergillus terreus FDN41, Penicillium, Phanerochaete, Pleurotus.v.v.

[12],[21],[36]

Chng Pseudomonas pseudoalcaligenes NRRL B-18087 v Pseudomonas

pseudoalcaligenes NRRL B-18086 do Roy phn lp c c kh nng pht

trin trn c hai c cht 2,4,5-T, 2,4-D. Hai chng ny u c kh nng s

dng 2,4-D v 2,4,5-T l ngun nng lng v carbon duy nht cho chng

[23]. Theo Haugland v cng s, chng Pseudomonas cepacia AC1100 c

kh nng phn hy rt mnh 2,4,5-T, chng ny phn hy c 960g/ml

trong 24h 29oC [26]. Mt chng mi c ngun gc t chng Pseudomonas

cepacia AC1100 c nhn thm plasmid pJP4 quy nh kh nng phn hy

2,4-D t chng Alcalugenes eutrophus JMP134, chng ny c t tn l

RHJ1. Chng ny c kh nng phn hy 1000 g/ml 2,4-D v 2,4,5-T g/ml

trong 24h 29oC trn mi trng c cha hai c cht ny. Trong khi kh

nng phn hy 2,4,5-T v 2,4-D ca chng Pseudomonas cepacia AC1100



gim hn khi nui trn mi cha cng mt lc hai c cht, chng ny phn

hy c 26 g/ml 2,4,5-T v 24 g/ml 2,4-D cung trong 24h [26]. chng

RHJ1 2,4,5-T c chuyn ha thng qua con ng chlorohydroquinon, cn

2,4-D c phn hy thng qua con ng chloroatechol [26].

Vi khun s dng 2,4,5-T nh ngun cacbon v nng lng duy nht c

nghin cu y nht hin nay l Burkholderia phenoliruptrix AC110 (tn

c l Burkholderia cepacia AC1100) [20]. Cc gen tftA v tftB m ha hai

di n v (subunit) ca enzyme 2,4,5-T oxygenase tham gia chuyn ha

2,4,5-T sang 2,4,5-TCP (hnh 1.5). Tip theo 2,4,5-TCP chuyn thnh DCHQ

nh gen tftC m ha enzyme monooxynase cha putative flavin. Sau

DCHQ chuyn ha thnh CHQ, maleylaxetat, oxoadipat, succinat v axetat

bi cc gen tftCDEF [21].

Hnh 1.5 Con ng phn hu 2,4,5-T bi Burkholderia cepacia AC1100 [20]



Mt vi khun khc c kh nng khong ha hon ton 2,4-D v 2,4,5-T

l Nocardioides simplex 3E c phn lp bng cch lm giu vi 2,4,5-T, vi

khun ny c th phn hy c hai hp cht trn thnh trihydroxylat benzen

[45]. Nghin cu khc ca Mai v cng s cho thy qu trnh ng trao i

cht ca vi khun Stenotrophomonas maltophilia PM. Vi khun ny cn

mecoprop ng trao i cht 2,4,5-T [36].

La Th Thanh Phng v cng s cng b nghin cu phn lp chng

Pseudomonas sp. BDN15, chng ny c kh nng pht trin trn mi trng

c b sung 2,4,5-T. Sau 90 ngy iu kin nui tnh v nhit phng

chng BDN15 phn hy 39,37% vi nng ban u l 1000ppm 2,4,5-T

y cng l ngun nng lng v carbon duy nht trong mi trng nui cy

[6]. Nguyn Thanh Thy v cng s cng b chng nm si FDN41 c

kh nng phn hy 43,46% 2,4,5-T trong 20 ngy vi hm lng 2,4,5-T ban

u 905,34 g/ml. Ngoi ra chng ny cn c kh nng s dng mt s PAH

nh anthrancen, phenanthren .v.v. l ngun nng lng v carbon duy nht [12].

Khc vi s lng t ca cc vi khun phn hy 2,4,5-T, rt nhiu vi khun

phn hy 2,4-D c phn lp t nhiu v tr nhim khc nhau nh t

nng nghip, trm tch, khu vc x l rc thi v t nguyn thy. Cc vi

khun phn hy 2,4-D c xp thnh 3 nhm da vo enzyme phn hy v

cc c tnh l ha ca chng. Nhm th nht nm trong lp v -

Proteobacteria nh Achromobacter, Burkholderia, Delftia, Halomonas,

Pseudomonas. Nhng vi khun ny c cha gen tfd thng nm cc

plasmid v c th chuyn c t c th vi sinh vt n sang c th vi sinh vt

kia. Nhm th hai gm nhng vi khun nm trong lp - Proteobacteria

thuc chi Sphingomonas. Chng c phn lp t mi trng c cha clo.

Nhm th ba thuc chi Bradyrhizobium trong lp - Proteobacteria. Cc vi



khun ny c phn lp t t nguyn thy Canada, Hawaii, Chile v mt

s khu vc khc [28],[ 44].

Trong s cc vi khun tham gia phn hy 2,4-D, chng Alcaligenes

eutrophus JMP134 l chng c nghin cu kh k v con ng chuyn ha

cng nh cc gen m ha cho nhng enzyme tham gia phn hy 2,4-D (Hnh

1.6). H gen tfd gm c 6 gen tfdA, tfdB, tfdC, tfdD, tfdE, tfdF m ha cho cc

enzyme tham gia vo qu trnh phn hy 2,4-D to thnh axit succinic [44].

Hnh 1.6 Con ng phn hy 2,4-D ca chng Alcaligenes eutrophus

JMP134 [44]



Maltseva v cng s cng b chng Halomonadaceae strain 1-18 c

kh nng phn hy 3000 mg 2,4-D/l trong 3 ngy trn mi trng c b sung

50 mg cao men [37], khi nghin cu su hn, chng Halomonadaceae strain

1-18 nhm tc gi pht hin ra con ng phn hy 2,4-D ca chng ny

ging vi con ng phn hy 2,4-D ca chng Alcaligenes eutrophus

JMP134, ngoi ra Maltseva cng tm thy s c mt ca gen tfdA chng

ny. Nhm tc gi ny cng b 5 chng thuc cc chi Acinetobacter,

Serratiamarcescens, Stenothrophomonas, Flavobacterium v Penicillium

phn lp c t t nhim 2,4-D Brazin cng c kh nng phn hy 2,4-

D [37]. Nm 2002, Kitagawa tm thy mt s gen mi tham gia vo qu

trnh phn hy 2,4-D. Cc gen cadABC, cadK c tch dng t chng

Bradyrhizobium sp. strain HW13. Khi so snh trnh t nucleotide ca cc gen

cadABC vi trnh t gen TftA, TftB ca chng Burkholderia cepacia

AC1100 cho thy cc gen ny c tng ng 46%, 44% v 37% vi nhau.

Trnh t gen cadK cng c so snh vi trnh t gen TfdK ca chng

Ralstonia eutropha JMP134, kt qu cho thy 2 gen ny c tng ng

60%. [33]

Trong mt nghin cu ca Nguyn B Hu v cng s trn mu t nhim

c cht dit c cha dioxin sn bay Nng, nhm tc gi phn lp

c 3 chng vi khun phn hy 2,4-D u thuc chi Athrobacter c tn ln

lt l Athrobacter sp. DNB19, Athrobacter sp. DNB20 v Athrobacter sp.

DNB21. Hai gen cadA v tfdA m ha cho cc enzyme tham gia phn hy

2,4-D ca chng Athrobacter sp. DNB19 c xc nh vi mc tng

ng 93 n 94% vi cc trnh t gen tng ng c cng b trn

GenBank [8]. Theo Hong Th M Hnh v cng s cng b chng nm

FDN20 cng c ngun gc t mu t nhim cht c ha hc, c kh nng



loi b 72,92 g/ml tng ng vi 44,18% hm lng 2,4-D a vo ban

u [4].

Ngoi 2,4,5-T, 2,4-D l thnh phn chnh ca cht dit c cn c sn phm

ph ca cht dit c l dioxin. Dioxin l thnh phn rt c vi con ngi v

mi trng v vy trn th gii cng nh Vit Nam c nhiu cng trnh

nghin cu v kh nng phn hy dioxin bi vi sinh vt. Lyama v cng s

s dng Bacillus midousuji phn hy dioxin, sau 24h nui cy, Bacillus

midousuji phn hy 34% 2,3,7,8-TCDD v sau 48h khong 70% dioxin

b loi b [35]. Theo Hong v cng s chng Sphingomonas sp. RW1 c kh

nng chuyn ha 2,7-DCDD v 1,2,3,4-TCDD thnh 4-chlorocatechol v

3,4,5,6-tetrachlorocatechol. Sau chng RW1 tip tc chuyn ha 3,4,5,6-

tetrachlorocatechol thnh 2-methyoxy-3,4,5,6-tetrachlorophenol [27]. Habe

v cng s cng b hai chng Pseudonomas sp. CA10 v Terrabacter sp.

DBF63 u c kh nng s dng t 10-35% 2,7-DCDD v 1,2,3-TrCDD vi

nng ban u l 10 ppm [25].

Ngoi vi khun hiu kh c kh nng phn hy dioxin th vi khun k kh

bt buc, vi khun k kh khng bt buc cng c kh nng chuyn ha

dioxin. Vi khun k kh c vai tr quan trng trong qu trnh phn hy cc

hp cht hu c kh phn hy cha clo trong c dioxin. Rt nhiu nhm vi

khun c kh nng kh clo ca cc hp cht hu c cha clo trong mi

trng hot ng ca vi khun kh st, vi khun kh sunpht, v nhm kh

halogen v.v. Mt s chng vi khun k kh c kh nng kh clo c

nghin cu nh: Desulfitobacterium dehalogenans JW/IU-DC1 [34],

Dehalococcoides ethenogens 195, Dehalococcoides sp. CBDB1 [30],

Desulfitobacterium sp. PCE-1, Dehalococcoides sp. FL2 v.v. Trong s cc vi

sinh vt k kh nghin cu trong hai thp k qua th cc vi khun thuc chi

Dehalococcoides c quan tm hn c bi chng c kh nng loi kh clo



ca rt nhiu hp cht cha clo nh Trichlorethylene , Vinyl Chloride,

Trichlorodibenzo-p-dioxins, Polychlorinated dibenzofurans v.v. [30]. Theo

Bunge v cng s, chng Dehalococcoides sp. 195, Dehalococcoides sp.

CBDB1 c chng minh l c kh nng phn hy dioxin [19], chng

CBDB1 kh clo 1,2,3,4-TeCDD (46 M ) trong 84 ngy nui cy thnh

2,3-DiCDD v 2-MCDD [19]. Chng CBDB1 s dng clo trong dioxin l

cht nhn in t, t loi b clo ra khi phn t dioxin. Kt qu to ra sn

phm t c hn l 2,7; 2,8-DiCDD v 2-MCDD. Cc hp cht ny s d b phn

hy sinh hc bi cc vi sinh vt hiu kh. Chng CBDB1 cn c th s dng

1,2,3,7,8-PCDD l cht c c l 1 tng ng vi 2,3,7,8-TCDD [19].

Ti Vit Nam, ng Th Cm H v cng s cng b chng FDN30 c

kh nng phn hy hiu kh 2,3,7,8-TCDD. y l chng vi nm c phn

lp t t nhim cht c ho hc ti Nng. Sau hai tun, chng ny

phn hu 59% dioxin cha trong mi trng nui cy [20]. Ngoi ra, trong

qu trnh nghin cu x l cht dit c/dioxin cc quy m, hnh thc khc

nhau, trong nhng nm qua, nhm tc gi ny phn lp c mt s chng

vi sinh vt s dng dibenzofuran, dioxin, t t nhim cht c ha hc ti

sn bay Nng. Cc vi khun c phn lp l Bacillus sp. BU3,

Pseudomonas sp. BDN15, Pseudomonas sp. SETDN1, Streptomyces sp.

XKDN11, Streptomyces sp. XKDN12 [1], [2], [10], [13].

6. Phn loi vi sinh vt

6.1. Phn loi theo phng php c in

Phng php phn loi c in da trn cc c im v hnh thi, sinh l-

sinh ha.

Cc c im hnh thi nh kch thc, hnh dng, mu sc ca khun lc,

hnh dng, kch c ca t bo vi sinh vt; cch sp xp ca t bo (n, kp



hay dng chm, dng chui v.v. kh nng bt mu khi nhum Gram; cc c

im vi cu trc nh c tin mao, tim mao hay khng?, s lng ca tin

mao, tim mao, cch thc di chuyn ca t bo; hnh dng v v tr ca cc c

quan trong t bo v.v.

Cc c im v sinh l v trao i cht nh ngun nng lng, ngun

cacbon v nit m sinh vt s dng, kiu dinh dng, cc sn phm ln men,

gii hn v nhit v nhit ti thch cho sinh trng v pht trin; di pH

v pH ti thch cho sinh trng, cc sn phm trao i th cp v.v.

Kha phn loi vi khun ca Bergey l v d in hnh ca phng

php phn loi vi sinh vt theo phng php truyn thng, kha phn loi ny

hin vn ang c cp nht v c nhiu nh vi sinh vt s dng.

6.2 Phng php phn loi bng sinh hc phn t

Phn loi bng sinh hc phn t l phng php mi nhng c chun

xc cao. Phng php ny c th pht hin m t v gii thch tnh a dng

sinh hc mc phn t v quan h gia cc loi v trong phm vi loi [7].

Bng 1.2. Phn tch cc thnh phn ha hc ca t bo theo h thng ha hc

Thnh phn

t bo

Phng php

phn tch

Phm vi

phn loi

DNA

nhim sc th

Thnh phn baz(%G+C) Chi

Bin tnh DNA:DNA Loi

Cc phn DNA c ct

bng enzyme gii hn Loi v di

loi

a hnh chiu di cc on gii

hn ca RNA riboxome



RNA riboxom Trnh t nucleotid Loi, chi v

trn chi Lai DNA: rRNA

Protein

Trnh t amino acid Loi, chi v trn

chi

So snh bng phn ng huyt

thanh Loi v chi

Cc kiu in di

in di enzyme a v tr Cc dng trong

loi

Thnh t bo

Cu trc peptidoglycan

Loi v chi

Polysaccharid

Acid teichoic

Mng

Acid bo

Loi v chi

Lipid phn cc

Acid mycolic

Isoprenoid quinones

Ngy nay phng php phn loi vi khun bng phng php xc nh v

so snh trnh t gen m ha 16S rRNA ang c p dng ph bin. Vic

nghin cu phn t rRNA l phng php hu hiu nht xc nh mi

quan h, tin ha ca cc vi sinh vt, v rRNA c mt tt c cc loi vi sinh

vt, c kh nng xc nh, c tnh bo th cao, chng ch khc nhau rt t gia

cc nhm vi sinh vt. Tuy nhin, da vo s khc nhau ny, ngi ta c th

nh gi c mi quan h pht sinh chng loi v phn loi vi sinh vt.

Trong ba gen m ha rRNA ca vi khun 5S rRNA, 16S rRNA v 23S rRNA

th gen 16S rRNA l ph hp nht cho vic nghin cu phn loi hin nay.



Gen m ha 5S rRNA c kch thc khong 120 nucleotid do c kch thc

nh nn thng tin cha ng t do kh phn bit c chnh xc s khc

nhau gia chi, gia loi v trong loi ca vi sinh vt cng nh v tr phn loi

ca chng. Gen m ha 23S rRNA c kch thc khong 2900 bp qu ln cho

nn khng thun li cho tch dng, xc nh trnh t v phn loi vi khun.

Gen m ha 16S rRNA c kch thc khong 1500 nucleotid va phn

loi chi tit gia cc chng vi sinh vt khng gy kh khn trong cc bc

xc nh trnh t gen v c u tin chn la trong phn loi vi khun. Cu trc

ca gen m ha 16S c cc nh khoa hc nghin cu k v thit k rt

nhiu cc cp mi chung dng cho nhiu nhm vi khun cng nh cc cp mi

c hiu ring cho cc chi v loi. y l mt thun li ln cho cc nghin cu

phn loi vi khun v x khun da trn gen m ha 16S rRNA [29].



PHN 2 VT LIU V PHNG PHP

1. Vt liu, ha cht, cc thit b s dng trong nghin cu

1.1 Vt liu

Mu t t cc bioreactor x l t nhim cht c ha hc sn bay

Nng thuc ti Nghin cu x l ty c mt s hp cht hu c cha clo

bng cc phng php ha hc v sinh hc tin tin do PGS.TS ng Th

Cm H l ch tr c s dng nghin cu phn lp chng vi sch vt

c kh nng s dng 2,4,5-T v 2,4-D.

1.2 Ha cht

Cc ha cht s dng trong th nghim c tinh khit cao ca cc hng

Roche, Sigma, Merk v.v. Cc dung dch tch DNA plasmid (Sol I (glucose:

50 mM; tris - HCl (pH 8): 25 mM; EDTA (pH 8): 10 mM); Sol II (NaOH:

0,2N; SDS: 1%); Sol III (CH3COOK 5M: 60 ml; CH3COOH: 11,5 ml; H2O:

28,5ml). Vect pCR2.1 (Promega), m TAE 1X in di (Tris- acetate:

40 mM; EDTA: 1 mM). Loading dye 6x (bromophenol blue: 0,25%; xylen

cyanol FF: 0,25%; glyxerol: 30%) v.v. Dch chit t cha hn 99% l

2,3,7,8-TCDD, ngoi ra cn c cc cht khc nh 2,4,5-T, 2,4-D v.v. c

chit t t nhim ca sn bay Nng.

Trnh t cp mi

Mi xui 27F: 5 AGA GTT TGA TTC MTG GCT CAG 3

Mi nguc 1492R: 5 GGY TAC CTT GTT ACG ACTT 3

1.3 Thit b, my mc

Cc thit b, my mc s dng ti phng th nghim Cng ngh sinh hc

mi trng v phng th nghim trng im quc gia v cng ngh gen



Vin Cng ngh sinh hc bao gm Box cy v trng, knh hin vi, cn in

t, ni kh trng, t sy, my nui lc nhit 30oC, t nui n nhit 30oC,

my ly tm, my PCR, my xc nh trnh t gen ABI PRISM 3100 Avant

Genetic Analyzer, my soi DNA, my chp nh Gel-Doc, my in di Bio-

Rad, t lnh cc loi 4oC, -20oC, -80oC, cc dng c th nghim nh bnh tam

gic, pipet, u typ, ng ly tm v.v.

2. Phng php nghin cu

2.1 Mi trng nui cy

2.1.1 Mi trng SH1 dch (g/l)

- KH2PO4 0,5

- K2HPO4 0,5

- MgSO4 2

- NaCl 1

- NH4Cl 1

- NH4NO3 1

- CH3COONa 1

- CaSO4 1

- Lactat Natri 1 ml

- Axt Butyric 10 l

- Axt Propionic 10 l

- Axt Isobutyric 1 l

- Axt Succinic 3,5 l

- V cc cht b sung khc

2.1.2 Mi trng SH1 thch

Thnh phn cc loi ha cht ging nh mi trng khong dch nhng c

b sung 18g agar/l.

2.1.3 Mi trng mui khong (g/l)

KNO3 3

MgSO4 0,4

KH2PO4 0.3

Na2HPO4 0,7

NaCl 0,4

pH 7-7,2



2.1.4 Mi trng LB dch (g/l)

NaCl 10

Tryptone 10

Cao men 5,0

Nc ct va 1 lt

pH 7

2.1.5 Mi trng LB thch (g/l)

Cng thc cc ha cht tng t mi trng LB dch nhng c b sung

thm 18g agar/l.

2.1.6 Nc mui sinh l (0,85%)

NaCl 8,5g

Nc my va 1 lt

2.2 Phng php nui cy v phn lp vi khun t mu t

nhim cht dit c/dioxin trong bioreactor hiu kh

2.2.1 Nui cy lm giu vi sinh vt

Vi khun c phn lp theo phng php lm giu trn mi trng

SH1/5 c b sung 2,4,5-T. Cn 5g t t bioreactor cho vo bnh tam gic

250ml cha 50ml mi trng SH1/5 c b sung 200 ppm 2,4,5-T, nui lc 5-

7 ngy 30oC. Chuyn 10% ging ln lm giu th nht sang bnh lm giu

ln hai cha mi trng SH1/5 b sung 200 ppm 2,4,5-T. Qu trnh lm giu

ny c lp li 3.

2.2.2 Phng php phn lp vi khun

T mu lm giu ln th 3, pha long t 101 n 107, sau bm 100l

dung dch pha long c cha vi khun ln cc a mi trng SH 1/5 thch c

b sung 200 ppm 2,4,5-T. Sau 5 n 7 ngy nui iu kin ti 30oC, nhng



khun lc mc ring r trn a thch c tch v chuyn sang nui cy

trong bnh tam gic cha 20 ml mi trng SH 1/5 dch c b sung 200 ppm

2,4,5-T sau nui lc 200vng/pht 7 n 10 ngy 30oC.

2.3 Nghin cu hnh thi t bo ca chng vi khun

2.3.1 Nhum Gram

Nh mt git mi trng nui cy c cha vi sinh vt vo gia lam knh,

gin u dch, c nh mu trn ngn la n cn. Tip theo, nh dung dch

tm kt tinh (crystal violet) ln mu, 1 pht. Mu c ra bng nc ct 2

ln, thm kh, sau nh dung dch lugon ln mu, yn mu trong 1 pht.

C nh mu bng cn 900 trong 30 giy sau bay hi t nhin n kh

b mt. Tip theo, nh dung dch safarin ln mu, yn trong 1-2 pht v

ra li mu bng nc ct 2 ln, kh t nhin. Mu c quan st bng

knh hin vi quang hc vi vt knh du vi phng i 100 ln. Vi khun

gram m bt mu hng, vi khun gram dng bt mu xanh.

2.3.2 Quan st hnh thi t bo di knh hin vi in t qut

Vi khun c nui cy 7 ngy trn mi trng CNSH1 dch c cha dch

chit t. Dch nui cy c lc qua giy lc th loi b cn bn ri ly

tm 5000 vng/pht trong 5 pht thu sinh khi t bo. Sau ra li sinh

khi bng nc ct 2 ln loi cc cht cn bn trong mi trng nui cy.

T bo vi khun c ha trong glutaraldehyt 2,5 % trong m phosphat natri

100 mM (pH 7,2) trong 30 pht. Tip theo, ly mt git t bo x l

(khong 106- 108 t bo/ ml) a ln li ng v 1 pht mu bm vo

li. Ra nh nhng vi vi git nc v mu c lm kh qua cn 25, 50,

75 v 100%, T-butyl. Sau , cc mu c lm kh bng my ng kh v

ph vng v quan st di knh hin vi in t qut JEOL 5410 LV.



2.4 Phng php phn tch kh nng phn hy 2,4,5-T

Bc 1 Ly 22 ml mu b sung thm 2 gam NaCl, 30 ml n-hexan v lc

trong 30 pht.

Bc 2 Chit ly tng hu c v cht chuyn ha, cho isopropanol v

mt hai git H2SO4 c, ra bng nc ct n pH = 7, thi kh mu bng N2

sch n 1 ml. Bm dch vo my sc k kh (GC) vi detechter ECD.

2.5 Phn loi vi khun da trn so snh trnh t gen m ha 16S rRNA

2.5.1 Phng php tch DNA tng s t vi sinh vt

Mng t bo c ph bng enzyme lyzozym. Enzyme protease K c

dng loi b protein. Vic b sung thm cc ha cht nh phenol,

chloroform, isoamylalcohol nhm loi protein v cc tp cht ra khi dung

dch cha DNA. Thu hi DNA bng cch ta trong cn hay isopropanol v ly

tm. Cc bc c tin hnh theo th t sau:

Bc 1: Thu sinh khi t bo vo eppendorf 1,5 ml bng cch ly tm 6000

vng trong 10 pht.

Bc 2: Ha tan mu trong 400l dch m lysis. Thnh phn m:

20 mM Tris-Cl (pH 8)

50 mM NaCl

10 mM EDTA

Bc 3: B sung lyzozyme, 37oC trong 15-30 pht.

Bc 4: B sung protease K, 56oC trong 1 gi.

Bc 5: B sung phenol (t l 1:1 v/v), ly tm 12000 vng/pht trong 15 pht.

Bc 6: B sung chloroform : isoamylalcohol (t l 1: 1 v/v), ly tm 12000

vng/pht trong 15 pht.

Bc 7: Ht dch pha trn chuyn sang eppendorf mi. Ta DNA bng

ethanol 100% gi -20oC trong 2-3 gi.



Bc 8: Ly tm 12000 vng/pht trong 15 pht thu kt ta DNA.

Bc 9: Ra DNA bng cn 70%.

Bc 10: Lm kh, ho tan trong nc kh ion v trng.

Bc 11: Loi RNA bng RNase (10 mg/ml), 37oC trong 2 gi.

2.5.2 Nhn on gen 16S rRNA bng phng php PCR

K thut PCR c Kary Mullis pht minh vo nm 1985. Cho n nay k

thut PCR c coi l mt trong nhng phng php nn quan trng nht ca

cng ngh sinh hc hin i.

Thnh phn phn ng

Buffer MgCl2 2,5 l

dNTPs ( 10mM ) 2,5 l

Mi xui 27 F 1 l

Mi ngc 1492 R 1 l

Taq polymeraza 0,2 l

DNA 1 l

MgCl2 ( 10 mM ) 3 l

H2O 13,8 l

Tng th tch 25 l

Chu trnh nhit

Bc 1 95oC trong 5 pht



Bc 4 72oC trong 1 pht 30

giy

Bc 5 Lp li 30 ln t bc

2 n bc 4


Bc 7 4oC qua m

Trnh t cp mi

Mi xui 27F: 5 AGA GTT TGA TTC MTG GCT CAG 3

Mi nguc 1492R: 5 GGY TAC CTT GTT ACG ACTT 3



2.5.3 in di kim tra trn gel agarose

Phng php ny da vo c tnh cu trc ca axit nucleic. Axit nucleic

l nhng i phn t lun tch in m, di tc dng ca dng in mt

chiu chng s di chuyn t cc m v cc dng. Trong cc th nghim ny

chng ti s dng gel agarose 1%, nhum bng Ethidium Bromide v tin

hnh soi DNA di nh sng t ngoi.

2.5.4 Tch dng on gen m ha 16S rRNA

Sn phm PCR c hiu c gn vo vector pCR2.1 nh T4 DNA ligase.

Phn ng thc hin 14oC trong 18 gi. Thnh phn phn ng nh sau:

Nc ion v trng 4,0 l

m T4 DNA ligase 1,0 l

Sn phm PCR 2,5 l

Vector pCR 2.1 (25mg/l) 1,5 l

T4 DNA ligase 1,0 l

o trn ri cho vo t lai 14oC trong 18 gi.

2.5.5 Bin np DNA ti t hp vo t bo E.coli

Bc 1: Cho 1,5 l dch vector pCR2.1 gn sn phm PCR vo ng

cha 50 l t bo kh bin Escherichia coli INVF, trong 30 pht.

Bc 2: Sc nhit 42oC trong 30 giy.

Bc 3: B sung 250 l mi trng SOC, nui lc 200 vng/pht 37oC

trong 1 gi.

Bc 4: Cy gt dch sn phm trn ln a cha mi trng chn lc (mi

trng LB c b sung ampicillin 100 mg/ml v X-Gal 50 mg/ml).

Bc 5: Nui 37oC trong 18 gi.

Bc 6: Bo qun 4oC thc hin cc th nghim tip theo.



Trn mi trng LB c b sung X-gal, nhng khun lc c mu trng l

nhng dng c th c nh on DNA ngoi lai, cn nhng khun lc mu

xanh c th l nhng c th khng c nh on DNA ngoi lai.

2.5.6 PCR trc tip t khun lc (colonyPCR)

Sau khi nui khun lc bin np trn mi trng chn lc, khun lc

trn mu trng c chn ra chy phn ng colony-PCR vi cp mi

27F-1492R nhm xc nh khun lc c plasmid mang on gen quan tm

hay khng. in di kim tra sn phm trn gel agarose 1% chn ra nhng

khun lc mang gen c kch thc nh mong mun.

Thnh phn v chu k nhit cng ging nh phn ng PCR, nhng ch

khc l mu DNA c thay bng khun lc.

Thnh phn phn ng PCR

STT Thnh phn Th tch (l)

1 Nc ct kh ion, kh trng 14.75

2 Buffer PCR (NH+

4) 2.5

3 Mgcl2 3

4 dNTPs 2.5

5 Mi xui 27F 1

6 Mi ngc 1492R 1

7 Taq polymerase 0,25

8 Template Khun lc

Tng 25



Chu k nhit cho phn ng PCR

Bc Phn ng Nhit (O

C) Thi gian Chu K

1 Bin tnh 95 5 pht

2 Bin tnh 94 1 pht

3 Gn mi 52 1 pht 32

4 Ko di chui 72 1 pht 30 giy

5 Hon tt ko di 72 8 pht

6 Kt thc phn ng 4

2.5.7 Tch DNA plasmid theo Kit ca hng Fermentas

Bc 1: Cho 1,5ml dch nui cy ca dng vi khun c chn vo

eppendorf, ly tm 12000 vng/pht trong 2 pht.

Bc 2: dch, p eppendorf trn giy thm. B sung 250l m P1

vortex cho n khi sinh khi tan ht.

Bc 3: B sung 250l m P2 v o nh 15 ln cho ti khi dch tr nn trong.

Bc 4: B sung 350l m N3 v o nh 15 ln.

Bc 5: Ly tm 13000 vng/pht trong 10 pht.

Bc 6: Chuyn dch sang ct QIAprep, ct t trn eppendorf 2ml.

Bc 7: Ly tm 13000 vng/pht trong 1 pht. Chuyn ct sang eppendorf mi.

Bc 8: B sung 500l m PB v ly tm 12000 vng/pht trong 1 pht,

chuyn ct sang eppendorf mi.

Bc 9: B sung 750l m PE, trong 3 pht.

Bc 10: Ly tm 12000 vng/pht trong 1 pht. Chuyn ct sang

eppendorf mi.

Bc 11: Ly tm li mt ln na 12000 vng/pht trong 1 pht.

Bc 12: Chuyn ct sang eppendorf mi, b sung 60ul nc deion.

trong 1 pht.



Bc 13: Ly tm 13000 vng/pht trong 1 pht. B ct v gi li

eppendorf cha DNA plasmid.

2.5.8 Xc nh trnh t on gen m ha 16S rRNA

c trnh t on gen 16S rRNA theo hng php ca Sanger s dng

my c trnh t t ng ABI PRISM 3100 Avant Data Analyzer. Trnh t

nucleotide c x l bng phn mm ABI PRISM 3100 Avant Data

Collection v1.0 v DNA Sequencing Analysis.

2.5.9 Xy dng cy pht sinh chng loi

So snh trnh t on gen 16S rRNA thu c vi on gen c kch thc

v v tr tng t cc vi sinh vt khc thuc nhm prokaryote c cng

b trn ngn hng d liu gen th gii EMBL (European Molecular Biology

Laboratory) v s dng phn mm tin sinh hc Clustal xy dng cy pht

sinh chng loi.



PHN 3 KT QU V THO LUN

1. Nui cy, phn lp chng vi sinh vt t mu t nhim cht

dit c/dioxin trong bioreactor hiu kh

1.1 Nui cy, lm giu tp on vi sinh vt

Mu t ca ba bioreactor hiu kh HKR3, HKR4 , HKR5, c tin hnh

lm giu trong mi trng SH1/5 c b sung hai ngun c cht l 2,4,5-T,

2,4-D. Cc mu t c tin hnh lm giu 3 ln trn mi trng SH1/5 vi

nng cc c cht l 100ppm. Kt qu lm giu tp on vi sinh vt nui

cy lm giu ln mt trn c cht 2,4,5-T, 2,4-D c trnh by hnh 3.1.

A

B

Hnh 3.1 Tp on vi sinh vt ca bioreactor HKR3, HKR4, HKR5 trn

mi trng cha 2,4,5-T (A) v 2,4-D (B)



Trn hnh 1.A v 1.B tp on vi sinh vt trong cc mu t cc

bioreactor HRK3, HRK4, HRK5 pht trin rt tt trong ln lm giu u tin,

cc bnh nui cy u thy c vin sinh khi trn thnh bnh. Qu trnh lm

giu c tin hnh hai ln na v kt qu ca ln lm giu cui cng c

th hin trn hnh 3.2.

A B

C

Hnh 3.2 Tp on vi sinh vt ca bioreactor HKR3(A), HKR4 (B), HKR5

(C) trong mi trng cha 2,4,5-T v 2,4-D.

Quan st hnh 3.2 nhn thy cc bnh nui cy tp on vi sinh vt pht

trin mnh, vin sinh khi r rng v mi trng c mu trng c. iu ny

cho thy tp on vi sinh vt c nghin cu ca ba bioreactor HKR 3,

HKR4, HKR5 c kh nng pht trin trn mi trng c b sung hai ngun

carbon l 2,4,5-T v 2,4-D. nghin cu, tm hiu kh nng s dng 2,4,5-T

hoc 2,4-D ca tng chng n, cc nghin cu sng lc tm kim v phn

lp cc chng vi khun t cc bnh nui cy lm giu ln 3 c tin hnh.



1.2 Phn lp chng vi khun

T cc mu lm giu ln 3 tp on vi sinh vt ca bioreactor HKR3,

HKR4, HKR5, c nui trn mi trng thch. T cc a thch ny mt

s khun lc c chn chuyn sang nui cy trn mi trng dch th.

Sau qu trnh phn lp v chn lc cc chng vi khun sch HR3.1 , HR4.1,

HR5.1 c phn lp (hnh 3.3, bng 3.1).

A B

C

Hnh 3.3 Hnh thi khun lc cc chng HR3.1 (A), HR4.1 (B), HR5.1 (C)

trn mi trng thch c 2,4,5-T.



Bng 3.1 c im hnh thi khun lc ca chng HR3.1, HR4.1, HR5.1 trn

mi trng cha 2,4,5-T

Chng vi khun c im khun lc

HR3.1 Khun lc hnh trn, li, mu trng

c, ng knh khong 2 mm.

HR4.1 Khun lc hnh trn b, li, mu

trng, ng knh khong 0,5 mm.

HR5.1 Khun lc hnh trn, mu trng c,

ng knh khong 1,9-2,1 mm

T kt qu nhn c hnh 3.3 v bng 3.1 cho thy, trong 3 chng n

phn lp c, chng HR5.1 c kh nng pht trin tt hn hai chng

HR3.1 v HR4.1. V vy chng HR5.1 c chn tip tc nhin cu

phc v cho mc ch cng nh ni dung ca lun vn.

2. c im phn loi ca chng HR5.1

2.1 Hnh thi t bo

Chng HR5.1 l vi khun Gram m, quan st v chp nh di knh hin

vi in t qut c phng i 10.000 ln cho thy chng HR5.1 t bo ca

chng HR5.1 c dng hnh que ngn kch thc khong 1,6-1,9 x 0,5-0,6 m.



Hnh 3.4 Hnh thi t bo ca chng vi khun HR5.1

xc nh v tr phn loi ca chng vi khun HR5.1 Cc nghin cu

tch dng gen m ha 16S rRNA, gii trnh t v so snh vi cc d liu trn

ngn hng Gen c tin hnh.

2.2 Phn loi da trn trnh t gen m ha 16S rRNA

2.2.1 Tch chit DNA tng s

tin hnh cc nghin cu phn loi vi khun, vic thu c DNA tng

s khng b t gy, c tinh sch cao l rt quan trong. Hin nay c nhiu

phng php tch chit DNA tng s ca vi sinh vt c s dng, ph thuc

vo loi mu nghin cu. Mu vi khun HR5.1 c tin hnh tch chit

DNA tng s theo m t ca Sambrook v Russell [40]. Qu trnh tch chit

c thc hin theo cc buc m t phn phng php, sn phm c in

di kim tra trn gel agarose 1%. Kt qu trnh by hnh 3.5



Hnh 3.5 Ph in di DNA tng s chng HR 5.1

Kt qu hnh 3.5 chng t DNA tng s ca chng HR5.1 khng b t

gy, sch c th s dng cho cc nghin cu tip theo.

2.2.2 Nhn on gen 16S rRNA ca chng HR5.1 bng k thut PCR

S dng DNA tng s tch t chng HR5.1 lm khun, cp mi c

hiu (27F v 1492R) v chu trnh nhit nh trnh by phn phng php

nhn gen 16S rRNA ca chng HR5.1 c nhn ln nh PCR. Sau

phn ng, sn phm PCR c in di kim tra trn gel agarose 1% kt

qu c th hin trn hnh 3.6

DNA

tng s



Hnh 3.6 Ph in di sn phm PCR ca chng HR5.1

Ging M Thang DNA chun 1Kb

Ging HR5.1 Sn phm PCR ca chng HR5.1

Trn in di sn phm PCR l mt bng duy nht v c kch thc

khong 1500bp, ng vi kch thc theo tnh ton l thuyt. iu ny chng

t phn ng PCR nhn kh c hiu on gen 16S rRNA c kch thc

mong mun.

2.2.3 Tch dng gen 16S rRNA trong vector pBT

Sn phm PCR c tin hnh gn sn phm vo vector pBT nh T4

DNA ligase v bin np vo t bo kh bin E. coli. Sau khi nui qua m

37oC, trn a bin np xut hin cc khun lc xanh v trng xen k nhau

(hnh 3.7).



Hnh 3.7 a bin np sn phm ligation ca chng HR 5.1.

kim tra nhanh t bo vi khun c mang gen mong mun hay khng,

phng php colony-PCR c s dng. Mt s khun lc trng c s

dng lm khun cho phn ng colony-PCR vi chu trnh nhit v thnh phn

phn ng nh phn phng php. Kt qu phn ng colony-PCR c th

trnh hnh 3.8.

Hnh 3.8 Ph in di sn phm colony-PCR

Ging M Thang DNA chun 1Kb

Ging 1,2,3 Th t cc dng c chn tin hnh colony-PCR



Quan st trn hnh 3.8 ta thy sn phm PCR ca ba khun lc c chn

PCR kim tra l mt on DNA c kch khong 1500bp ph hp vi kch

thc ca sn phm PCR on gen 16S rRNA. iu ny chng t c ba

khun lc c chn u c DNA plasmid mang on gen 16S rRNA. Mt

dng DNA plasmid c chn tch DNA plasmid bng Kit Plasmid

DNA purification. Sau khi tch DNA plasmid, enzyme BamHI c s dng

ct dng DNA plasmid ny. Kt qu in di sn phm ct cho thy c mt

on DNA c kch thc khong 1500bp c ct (hnh 3.9). Kch thc

on DNA ny ph hp vi kch thc on gen cn nghin cu.

Hnh 3.9 in di sn phm ct DNA plasmid bng enzyme

BamHI

Ging M: Thang DNA chun 1kb Ging pBT-HR5.1: dng Plasmid c chn

Ging pBT: dng plasmid i chng

2.2.4 Xc nh trnh t gen 16S rRNA ca chng HR5.1

Dng vi khun mang DNA plasmid c gen mong mun c tch DNA

plasmid v lm sch sn phm. Sau , DNA plasmid c xc nh trnh t



nucleotide theo phng php ca Sanger. Sau khi x l s liu trnh t chng

HR5.1 nh sau:

Kt qu c trnh t cho thy, on gen c kch thc 1503kb l ng vi

tnh ton l thuyt. So snh trnh t on gen 16S rRNA ca chng HR5.1 vi

cc trnh t gen ca cc vi sinh vt nhn s c cng b trn ngn hng

gen th gii v phn tch bng phn mm Clustal, ng thi s dng chng

trnh Blast, tng ng gia chng HR5.1 vi mt s chng vi sinh vt

khc c xc nh (bng 3.2).



Bng 3.2. Mc tng ng on gen m ha 16S rRNA ca chng HR5.1

vi mt s chng vi khun khc

Tn vi sinh vt Mc tng ng

Pseudomonas putida strain ZB-16A 98%

Pseudomonas putida strain ppnb1 98%

Pseudomonas putida isolate PD39 98%

Pseudomonas putida strain YJF3-34 98%

Da trn trnh t on gen 16S rRNA ca chng HR5.1 v trnh t

nucleotide tng ng ca cc chng VSV thuc nhm prokaryate, cy pht

sinh chng loi c xy dng (hnh 3.10).



Hnh 3.10 Cy pht sinh chng loi ca vi khun Pseudomonas sp. HR5.1

T v tr ca chng HR5.1 trn cy pht sinh chng loi cho thy chng

ny gn gi vi cc chng vi khun thuc chi Pseudomonas. Trnh t gen 16S

RNA ca chng HR5.1 c mc tng ng 98% vi cc vi khun

Pseudomonas putida sp.ZB-16A, Pseudomonas putida isolate PD39,

Pseudomonas putida strain ppnb1, Pseudomonas putida strain YJF3-34,

Pseudomonas sp. PHD-8. Kt hp vi mt s c im hnh thi v trnh t

on gen 16S rRNA ca chng vi khun HR5.1, chng vi khun ny c th

c xp vo chi Pseudomonas , v c t tn l Pseudomonas sp. HR5.1.



Mt s nghin cu gn y cng cho thy mt s loi vi khun thuc chi

Pseudomonas c kh nng phn hy 2,4,5-T v cc hp cht thm cha clo.

La Th Thanh Phng v cng s phn lp c chng Pseudomonas sp.

BDN15, t ngun t nhim cht dit c/dioxin. Chng BDN15 c kh

nng pht trin trn mi trng c b sung 2,4,5-T [6]. Chng Pseudomonas

pseudoalcaligenes strain NRRL B-18087 c Dipak Roy phn lp phn

hy 2,4,5-T, 2,4D v 2,4,5-T 2,4D l ngun cung cp carbon cho chng ny

[23]. Chng vi khun HR5.1 cng thuc chi Pseudomonas vy liu chng ny

c kh nng phn hy 2,4,5-T hay khng cc nghin cu tip theo c

tin hnh.

3 Nghin cu mt s c im ca chng HR5.1

3.1 Kh nng pht trin ca chng HR5.1 trn PAH

Trong t nhim cht dit c sn bay Nng ngoi thnh phn nhim

l cht dit c 2,4,5-T v 2,4-D cn c mt s cc thnh phn nhim khc

nh dichlorphenol, trichlorophenol, PAH .v.v. kim tra kh nng pht

trin ca vi khun HR5.1 trn ngun PAH, chng HR5.1 c nui trn mi

trng SH1/5 c b sung 100ppm cc PAH nh Pyren, Napthalen,

Phananthren. Kt qu cho thy sau mt tun nui 30oC lc 200vng/pht,

chng HR5.1 pht trin mnh trn c 3 c cht b sung vo (bng 3.3).



Bng 3.3 Kh nng pht trin ca chng HR5.1 trn PAH

Kh nng pht trin

+++

+++

+++

Ch thch - : Khng pht trin + : Pht trin yu ++ : Pht trin bnh thng +++ : Pht trin tt C : i chng



Trn th gii, ngi ta pht hin ra kh nhiu chng Pseudomonas c

kh nng phn hy PAH nh Pseudomonas putida NCIB 9816-4 c kh nng

phn hy napthelen [38], Pseudomonas rhodesiae KK1c kh nng pht trin

trn ngun c cht trn ba loi PAH anthracene, naphthalene v phenanthrene

[31]. Nh vy chng HR5.1 c kh nng pht trin tt trn ngun PAH. Tuy

nhin khng nh chc chn kh nng phn hy PAH ca chng ny cn

phi c cc nghin cu xc nh bng HPLC.

3.2 Kh nng phn hy 2,4,5-T ca chng HR5.1

3.2.1 nh hng ca mi trng nui cy c cha 2,4,5-T ln s pht

trin ca chng HR5.1

Qu trnh sinh trng v pht trin ca vi sinh vt ph thuc vo rt nhiu

iu kin nh nhit , m, pH, thnh phn mi trng .v.v. c bit l

thnh phn mi trng nui cy c nh hng ln n kh nng pht trin ca

vi sinh vt. xc nh nh hng ca mi trng nui cy ln s pht trin

ca vi khun HR5.1, chng ny c nui trn ba mi trng khc nhau l

mi trng SH1 (mi trng giu dng cht), mi trng mui khong

ngho cht dinh dng v mi trng SH1/5 (mi trng trn gia mi

trng SH1 v mi trng mui khong). Kt qu c ch ra hnh 3.12.



Hnh 3.12 nh hng ca mi trng ln s pht trin

ca Chng HR5.1

Chng HR5.1 pht trin tt nht trn mi trng SH1, chng ny lm i

mu mi trng sang mu xanh c nh hunh quang (hnh 3.12). Trn mi

trng mui khong chng HR5.1 tuy c mc nhng yu hn rt nhiu so vi

khi nui trn mi trng SH1 hay SH1/5.

3.2.2 nh hng ca nng 2,4,5-T ln s pht trin ca chng

HR5.1

Mi chng vi sinh vt u c mt ngng nht nh i vi mt cht c

m ti n c th pht trin c. Ty thuc vo tng chng cng nh loi

cht c m ngng ny c th cao hay thp. tm hiu s b ngng ca

2,4,5-T i vi s pht trin ca chng HR5.1, chng ny c nui trn mi

trng SH1/5 vi cc nng 2,4,5-T khc nhau. Kt qu bng 5 cho thy,

sau 7 ngy nui cy trong mi trng c nng 300 ppm chng vn pht

trin nhng yu. Chng ny pht trin tt t nng 50 ppm n 200 ppm.



Bng 3.4 nh hng ca nng 2,4,5-T ln s pht trin ca chng HR5.1

Kh nng pht trin

+++

+++

+++

+

Ch thch - : Khng pht trin + : Pht trin yu ++ : Pht trin bnh thng +++ : Pht trin tt C : i chng



3.2.3 Kh nng phn hy 2,4,5-T ca chng vi khun HR5.1

nh gi kh nng s dng 2,4,5-T ca chng vi khun HR5.1 chng

ti tin hnh phn tch lng 2,4,5-T cn li trong dch nui cy c v

khng c vi sinh vt (mu i chng) bng phng php sc k. Mu em i

phn tch c nui lc 30oC v b sung 200ppm 2,4,5-T. Kt qu c

trnh by bng 3.5 v hnh 3.13.

Bng 3.5 Kh nng phn hy 2,4,5-T bi HR 5.1

Hm lng thu hi 2,4,5-T b loi b (%)

Khng c vi sinh vt (ppm) C vi sinh vt (ppm)

183,35 117,9 35,7%

T kt qu bng 5 v hnh 3.13 ta thy chng HR5.1 sau mt tun nui cy

s dng c 35,69% lng 2,4,5-T a vo mi trng nui cy.

Lin quan ti kh nng s dng 2,4,5-T tc gi Nguyn Thanh Thy v

cng s cng b chng nm FDN41 c kh nng loi b 43,46% lng

2,4,5-T a vo sau 20 ngy nui lc. Chng ny phn hy 2,4,5-T trong mi

trng ch cha 2,4,5-T l ngun cacbon v nng lng duy nht [12]. La

Thanh Phng v cng s phn lp c chng Pseudomonas sp BDN15

cng t ngun t nhim ti sn bay Nng, chng ny c kh nng phn

hy 39,37% 2,4,5-T trong 90 ngy iu kin tnh v nng 2,4,5-T ban

u l 1000 ppm y cng l ngun nng lng v carbon duy nht trong mi

tng nui cy [6]. Khi so snh trnh t nucleotid ca chng BDN15 v

chng HR5.1 cho thy trnh t ca chng ging nhau 94%. iu ny cho thy

tuy hai chng BDN15 v HR5.1 cng thuc chi Pseudomonas nhng chng

c th l hai chng khc nhau, y l s a dng ca sinh vt c mt trong

vng b nhim.



Trn th gii c kh nhiu nghin cu cng b v sinh vt c kh nng

phn hy 2,4,5-T. Theo Haugland v cng s cng b kh nng phn hy

mnh 2,4-D v 2,4,5-T ca mt s chng vi khun. Vi c tnh sinh trng

nhanh ca vi khun, ch sau 24h nui, chng Pseudomonas cepacia AC1100

phn hy c 960 g/ml 2,4,5-T, 280 g/ml 2,4-D. Chng ny thm ch

cn c kh nng phn hy hn hp 2,4,5-T v 2,4-D tuy nhin kh nng phn

hy hai cht ny gim hn xung cn 26 g/ml 2,4,5-T v 24 g/ml 2,4-D

[26]. Ngi ta to ra mt chng ti t hp c ngun gc t chng

Pseudomonas cepacia AC1100 c nhn thm plasmid pJP4 quy nh kh

nng phn hy 2,4-D t chng Alcalugenes eutrophus JMP134, chng ny

c t tn l RHJ1. Chng ti t hp RHJ1 c kh nng phn hy mnh

cht c mi trng c b sung ring r 2,4,5-T, 2,4-D cng nh mi

trng c b sung hai loi c cht ny [26].

Nh vy chng HR5.1 c kh nng pht trin tt trn cc ngun c cht

khc ngoi 2,4,5-T nh PAH, 2,4-D, iu ny rt ph hp vi cng ngh x

l bng bioreactor. Vi khun HR5.1 c kh nng phn hy kh tt 2,4,5-T

iu ny ng gp thm hiu bit v vi sinh vt c mt trong bioreactor nhm

hon thin cng ngh ny.



KT LUN V KIN NGH

Kt Lun

1. T 3 mu t trong bioreactor HRK3, HRK4, HRK5, 3 chng vi

khun HR3.1, HR4.1, HR5.1 c phn lp. Chng HR3.1 c kh nng

pht trin trn mi trng SH1/5 c b sung 2,4-D, Chng HR4.1 v HR5.1

c kh nng pht trin trn mi trng SH1/5 c b sung 2,4,5-T.

2. Chng vi khun HR5.1 l vi khun Gram m, khun lc hnh trn, mu

trng c, ng knh t 1,9-2,1 mm. Chng HR5.1 c dng que ngn kch

thc khong 1,6-1,9 x 0,5-0.6 m. Chng HR5.1 c mi quan h gn gi vi

cc loi thuc chi Pseudomonas c bit trnh t gen 16S rRNA ca chng

HR5.1 c tng ng cao ti 98% vi mt s loi thuc chi Pseudomonas.

Chng HR5.1 c t tn l chng Pseudomonas sp. HR5.1.

3. Chng HR5.1 pht trin tt nht trn mi trng SH1 v pht trin yu

trn mi trng khong c cha 2,4,5-T. Vi khun HR5.1 c kh nng pht

trin tt trn mi trng SH1/5 c cha PAH.

4. Chng HR5.1 pht trin tt nng 2,4,5-T b sung t 50 ppm n

200 ppm, gim dn nng 2,4,5-T 300 ppm.

5. Trn mi trng SH1/5 cha 200 ppm 2,4,5-T sau 1 tun, chng HR5.1

phn hy 35,7%.

Kin ngh

1. Tip tc nghin cu kh nng phn hy cc ngun carbon khc nh

PAH, 2,4-D, dioxin .v.v. ca chng vi khun HR5.1 nhm nh gi kh nng

phn hy nhiu loi cht c ca chng ny.

2. Nghin cu su hn v gen chc nng tham gia qu trnh phn hy

2,4,5-T v 2,4-D ca chng HR5.1



TI LIU THAM KHO

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