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

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
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
Chuyn ngnh
M s
: Sinh hc thc nghim

: 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
MC LC
M U
PHN 1 TNG QUAN
1. S nhim ca 2,4,5-T v 2,4-D
2. c im v tnh cht ca 2,4,5-T v 2,4-D
2.1 Cht dit c 2,4,5-T
2.2 Cht dit c 2,4-D
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
3.2 nh hng ca 2,4,5-T, 2,4-D n con ngi
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
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
Phm Ngc Long
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
Phm Ngc Long
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
Phm Ngc Long
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.
Phm Ngc Long
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
Phm Ngc Long
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].
S dng
trong
trong nm
S lng
c tnh
ri (lt)
961-1081 g/l
acid tng
ng
1961-1965
503.121;
413,852
Cht n-Butylester ca
xanh 2,4,5-T
l cy
Ging nh cht
hng
Cha r,
ri cng
thi gian
vi cht
hng
31.026
Cht
tm
1033 g/l acid

tng ng
1962-1965
1.892.773
Tn
cht
Thnh phn ho
cht
Cht
hng
60% - 40% n-Butyl:

isobutylester ca
2,4,5-T
50%n-Butylester
2,4,D
m c
tng ng
30% Butylester
Phm Ngc Long
2,4,5-T
20% isobutyl ester
2,4,5-T
Cht
da
cam
(1)
50% n-Butyl ester

2,4-D
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
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% triisopropanolamine
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
1962-1964
25.650
Cht
xanh
(dng
dch)
21% Natri
cacodylat+ acid
cacodylic t nht
chim 26% tng
lng acid tng
ng
1964-1971
4.715.731
50% n-Butyl ester

2,4,5-T
50% n-Butyl ester

2,4,5-T
Mui: 70%
tng ng
Khi lng
acid: 360,3 g/l
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
Phm Ngc Long
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 30 oC 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
Phm Ngc Long
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.
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 1,2,4,5tetrachlorobenzene, 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].
Phm Ngc Long
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
10
Phm Ngc Long
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 .
11
Phm Ngc Long
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 2,4,5T 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
12
Phm Ngc Long
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
13
Phm Ngc Long
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].
14
Phm Ngc Long
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 EnviroGrard TM
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
15
Phm Ngc Long
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
16
Phm Ngc Long
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].
17
Phm Ngc Long
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
18
Phm Ngc Long
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]
19
Phm Ngc Long
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
20
Phm Ngc Long
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]
21
Phm Ngc Long
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 2,4D [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
22
Phm Ngc Long
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 3,4,5,6tetrachlorocatechol 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
23
Phm Ngc Long
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 lsinh 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
24
Phm Ngc Long
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
Phng php
Phm vi
t bo
phn tch
phn loi
DNA
Thnh phn baz(%G+C)
Chi
nhim sc th
Bin tnh DNA:DNA
Loi
Cc phn DNA c ct
Loi v di
bng enzyme gii hn
loi
a hnh chiu di cc on gii

hn ca RNA riboxome
25
Phm Ngc Long
Trnh t nucleotid
Loi, chi v
Lai DNA: rRNA
trn chi
RNA riboxom
Trnh t amino acid
Loi, chi v trn

chi
So snh bng phn ng huyt

Protein
thanh
Loi v chi
Cc kiu in di
in di enzyme a v tr
Cc dng trong
loi
Cu trc peptidoglycan
Thnh t bo
Polysaccharid
Loi v chi
Acid teichoic
Acid bo
Mng
Lipid phn cc
Loi v chi
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.
26
Phm Ngc Long
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].
27
Phm Ngc Long
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
28
Phm Ngc Long
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 BioRad, t lnh cc loi 4 oC, -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
- CaSO4
- K2HPO4
0,5
- Lactat Natri
1 ml
- MgSO4
- Axt Butyric
10 l
- NaCl
- Axt Propionic
10 l
- NH4Cl
- Axt Isobutyric
1 l
- NH4NO3
- Axt Succinic
3,5 l
- CH3COONa
- 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
Na2HPO4
0,7
MgSO4
0,4
NaCl
0,4
KH2PO4
0.3
pH
7-7,2
29
Phm Ngc Long
2.1.4 Mi trng LB dch (g/l)

NaCl
10
Nc ct va 1 lt
Tryptone
10
pH
Cao men
5,0
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 57 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 30 oC, nhng
30
Phm Ngc Long
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.
31
Phm Ngc Long
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 N 2
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.
32
Phm Ngc Long
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), 37 oC 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
DNA
1 l
dNTPs ( 10mM )
2,5 l
MgCl2 ( 10 mM )
3 l
Mi xui 27 F
1 l
H2O
13,8 l
Mi ngc 1492 R
1 l
Tng th tch
25 l
Taq polymeraza
0,2 l
Chu trnh nhit

Bc 1
95oC trong 5 pht
Bc 5 Lp li 30 ln t bc
Bc 2
94oC trong 1 pht
2 n bc 4
Bc 3
55oC trong 1 pht
Bc 6
72oC trong 8 pht
Bc 4
72oC trong 1 pht 30
Bc 7
4oC qua m
giy
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
33
Phm Ngc Long
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 14 oC 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 42 oC trong 30 giy.
Bc 3: B sung 250 l mi trng SOC, nui lc 200 vng/pht 37 oC
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.
34
Phm Ngc Long
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)
Nc ct kh ion, kh trng
14.75
Buffer PCR (NH+4)
2.5
Mgcl2
dNTPs
2.5
Mi xui 27F
Mi ngc 1492R
Taq polymerase
0,25
Template
Khun lc
Tng
25
35
Phm Ngc Long
Chu k nhit cho phn ng PCR

Bc
Phn ng
Nhit (OC)
Thi gian
Chu K
Bin tnh
95
5 pht
Bin tnh
94
1 pht
Gn mi
52
1 pht
Ko di chui
72
1 pht 30 giy
Hon tt ko di
72
8 pht
Kt thc phn ng
32
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.
36
Phm Ngc Long
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.
37
Phm Ngc Long
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.
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)
38
Phm Ngc Long
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.
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.
39
Phm Ngc Long
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).
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.
40
Phm Ngc Long
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.
Khun lc hnh trn b, li, mu
HR4.1
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.
41
Phm Ngc Long
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
42
Phm Ngc Long
DNA
tng s
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
43
Phm Ngc Long
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).
44
Phm Ngc Long
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
45
Phm Ngc Long
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
46
Phm Ngc Long
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).
47
Phm Ngc Long
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).
48
Phm Ngc Long
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.
49
Phm Ngc Long
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 30 oC lc 200vng/pht,
chng HR5.1 pht trin mnh trn c 3 c cht b sung vo (bng 3.3).
50
Phm Ngc Long
Bng 3.3 Kh nng pht trin ca chng HR5.1 trn PAH

Kh nng pht trin
+++
+++
+++
Ch thch - : Khng pht trin

++ : Pht trin bnh thng
C : i chng
51
+
: Pht trin yu
+++ : Pht trin tt
Phm Ngc Long
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.
52
Phm Ngc Long
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.
53
Phm Ngc Long
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 bnh thng
C : i chng
54
+
: Pht trin yu
+++ : Pht trin tt
Phm Ngc Long
55
Phm Ngc Long
56
Phm Ngc Long
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.
57
Phm Ngc Long
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.
58
Phm Ngc Long
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
59
Phm Ngc Long
TI LIU THAM KHO

Ti liu ting Vit
1. ng Th Cm H, Nguyn B Hu, Mai Anh Tun, Nguyn ng
Nh, Nguyn Quc Vit, Nguyn Nguyn Quang (2008), Kho st vi sinh vt
trong vng nhim cht dit c cha dioxin khu vc sn bay Nng v kh
c t nhim iu kin phng th nghim, Tp ch Cng ngh Sinh hc,
6(4A), tr. 837-846.
2. ng Th Cm H, Phm Hu L, Nguyn B Hu, Nguyn Th ,
Nghim Ngc Minh, Nguyn ng Nh, Mai Anh Tun, La Thanh Phng,
Nguyn Th Snh, Nguyn Thu Thy, Bch Thanh, Ngc Tuyn,
Nguyn Vn Minh, Nguyn Vn Hng (2005), Nghin cu pht trin cng
ngh phn hy sinh hc v k thut nh chm lm sch cht c ha hc
trong t, Bo co nghim thu ti nh nc thuc chng trnh 33, H
Ni.
3. ng Th Cm H (2008). 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. Bo co
nghim thu ti cp Vin Khoa hc v Cng ngh Vit Nam, H Ni.
4. Hong Anh Cung (1993). nh hng ca 2,4,5-T n cy la v vi sinh
vt trong t. Cht dit c, tc hi lu di i vi con ngi v thin nhin.
Hi tho quc t ln II: 139-141.
5. Hong Th M Hnh, Nguyn Thanh Thy, Ng Xun Qu, Nghim
Xun Trng, Nghim Ngc Minh, ng Th Cm H (2004), Kh nng phn
hy 2,4-D v dibenzofuran ca chng nm si FDN20, Tp ch Cng ngh
Sinh hc, 2(4), tr. 517-528.
60
Phm Ngc Long
6. La Th Thanh Phng, Nghim Ngc Minh, ng Thi Cm H (2005).

Mt s c im sinh hc v kh nng phn s dng 2,4,5-T ca chng vi
khun BDN15 phn lp t vung t nhim cht c ha hc. Tp Ch Cng
ngh Sinh hc 3(3): 389-396, 2005.
7. L Trn Bnh, Phan Vn Chi, Nng Vn Hi, Trng Nam Hi, L
Quang Hun (2003). p dng cc k thut phn t trong nghin cu ti
nguyn sinh vt Vit Nam. Nh xut bn KH&KT H Ni: 325-329.
8. Nguyn B Hu, ng Th Cm H (2007), Nghin cu mt s c im
sinh hc phn t ca ba chng vi khun s dng 2,4-D phn lp t t nhim cht
dit c cha dioxin ti Nng, Tp ch Sinh hc, 29(4), tr. 80-85.
9. Nguyn B Hu. 2002. Nghin cu cc nhm vi sinh vt v kh nng
phn hy hydrocacbon thm a nhn ca mt s chng vi khun trong qu
trnh x l nhim du ti Khe Ch, Qung Ninh. Lun n thc s sinh hc.
10. Nguyn ng Nh, Nghim Ngc Minh, Nguyn Ngc Bo, ng
Th Cm H (2005), Kh nng phn hy hydrocarbon thm a nhn v
dibenzofuran ca chng x khun XKDN12, Tp ch cng ngh sinh hc,
3(1), tr. 123-132.
11. Nguyn Thanh Thy, Hong Th M Hnh, Nghim Ngc Minh, ng
Th Cm H (2006), Nghin cu phn loi v kh nng phn hy cht c ca
chng nm si FDN22 phn lp t t x l nhim cht c ha hc, Tp
ch cng ngh sinh hc, 4(1), tr. 125-132.
12. Nguyn Thanh Thy, Hong Th M Hnh, V Xun t, Nghim
Ngc Minh, ng Th Cm H (2007). Phn loi v kh nng phn hy cht
dit c 2,4,5-trichlorophenoxyacetic acid ca chng nm si FDN41 phn lp
t t nhim cht dit c/dioxin. Tp ch cng ngh sinh hc, 6(1), tr. 119-126
13. Nguyn Th Snh, Nghim Ngc Minh, ng Th Cm H (2005).
Phn loi v nghin cu mt s yu t nh hng ln s pht trin ca chng
61
Phm Ngc Long
vi khun k kh khng bt buc SETDN1 t t nhim c ha hc ti

Nng. Tp ch Cng ngh Sinh hc. 3(2): 257-264.
14. Nguyn Vn Minh (2003). Nghin cu ty c Vit Nam. Hi tho
Vit Nam-Hoa K v cc phng php xc nh, x l v nh gi vng
nhim dioxin: 80-85.
15. Trn Xun Thu (2003). Bc u nh gi mc nhim dioxin
trong mi trng Vit Nam. Hi tho Vit Nam-Hoa K v cc phng php
xc nh, x l v nh gi vng nhim dioxin: 38-47.
16. Trnh Ngc Bo, Phan Th Hoan, o Ngc Phan, Nguyn Th Vnh
(1993). Nghin cu nhim sc th th h F2 ca nhng ngi tip xc vi
cht c ha hc trong cuc chin tranh Vit Nam. Cht dit c, tc hi lu
di i vi con ngi v t nhin. Hi tho quc t ln II: 399-402.
17. V Qu, ng Huy Hunh, Mai nh Yn, Phng Tu Bi, Phm Bnh
Quyn (2002). Th nh gi li hu qu ca cht mu da cam/dioxin ln mt
trng ti vng a li sau gn 30 nm kt thc chin tranh. Cht dit c, tc hi
lu di i vi con ngi v thin nhin. Hi tho quc t ln II: 205-213.
Ti liu ting nc ngoi

18. Barry Dellinger (2003). Treatment and prevention of formation of
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19. Bunge, M., Adrian, L., Klaus, A., Opel, M., Lorenz, W.G., Andresen,
J.R., Gorisch, H., Lechner, U (2003). Reductive dehalogenation of chlorinated
dioxins by an anaerobic bacterium. Nature. 421: 357-360.
20. Dang T. C. H., Mai A. T., Nguyen Q. V., Nguyen T. S., Trinh K. S.,
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66

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I HC THI NGUYN

Phm Ngc Long

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

LUN VN THC S SINH HC

Thi Nguyn - 2009

S ha bi Trung tm Hc liu i hc Thi Nguyn

Phm Ngc Long

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

: Sinh hc thc nghim

LUN VN THC S SINH HC

Thi Nguyn - 2009

S ha bi Trung tm Hc liu i hc Thi Nguyn

Phm Ngc Long

S ha bi Trung tm Hc liu i hc Thi Nguyn

2,4,- dichlorophenoxyacetic acid

Polycyclic aromatic hydrocarbon

Polymerase Chain Reaction

Ribosomal ribonucleic acid

Lun vn thc s sinh hc

Phm Ngc Long

PHN 1 TNG QUAN

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

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

2.1 Cht dit c 2,4,5-T

2.2 Cht dit c 2,4-D

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

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

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

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

4.2 Phng php phn hy sinh hc

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

6. Phn loi vi sinh vt

6.1. Phn loi theo phng php c in

6.2 Phng php phn loi bng sinh hc phn t

PHN 2 VT LIU V PHNG PHP

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

2. Phng php nghin cu

2.1 Mi trng nui cy

S ha bi Trung tm Hc liu i hc Thi Nguyn

Lun vn thc s sinh hc

Phm Ngc Long

2.1.1 Mi trng SH1 dch (g/l)

2.1.2 Mi trng SH1 thch

2.1.3 Mi trng mui khong

2.1.4 Mi trng LB dch

2.1.5 Mi trng LB thch

2.1.6 Nc mui sinh l 27

2.2.1 Nui cy lm giu vi sinh vt

2.2.2 Phng php phn lp vi khun

2.3 Nghin cu hnh thi t bo ca chng vi khun

2.3.1 Nhum Gram

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

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

2.5.1 Phng php tch DNA tng s t vi sinh vt

2.5.2 Nhn on gen 16S rRNA bng phng php PCR

2.5.3 in di kim tra trn gel agarose

2.5.4 Tch dng on gen m ha 16S rRNA

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

2.5.6 PCR trc tip t khun lc (colonyPCR)

2.5.7 Tch DNA plasmid theo Kit ca hng Fermentas

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

2.5.9 Xy dng cy pht sinh chng loi

S ha bi Trung tm Hc liu i hc Thi Nguyn