Tgp chi Cdng nghi Sinh hgcl{l):l 17-124, 2008<br />
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<br />
N G H I E N ClTU K H A N A N G P H A N H U Y SINH H O C H Y D R O C A R B O N T H O M D A V O N G<br />
CUA M O T SO C H U N G VI K H U A N P H A N L A P TlT B I O R E A C T O R X\f L Y D A T N H I E M<br />
CHAT DOC HOA HOC<br />
<br />
Nguyin Ngoc Bao, Phan Thi Hoang Hao, Nguyin Ba Hihi, Nguyin Thanh Thiiy, Vu Dire Loi, Dang Thi<br />
d m Ha<br />
Viin Cdng nghi sinh hgc, Viin Khoa hgc vd Cdng nghi Viet Nam<br />
<br />
TOM TAT<br />
<br />
Dat nhiem hon hpp 2,3,7,8-TCDD (dioxin), cac chat diet cd 2,4,5-T, 2,4-D, san pham phan buy cua chat<br />
diet cd TCP, DCP, PAH dupc xu ly bang bioreactor ki khi khong bit bupc. Tir dat dang xix ly, 4 chimg vi<br />
khuan su dung chrysene va 2 chimg vi khuan sit dung pjrene da dupc phan lap. Chimg vi khuan BDNRl co the<br />
phat ttien tten moi trudng muoi khoang vdi nguon carbon duy nhat la dich chiet dat chiia dioxin va BDNR4 co<br />
the phat trien fren moi trudng taong ty vdi nguon carbon la 2,4-D. BDNRl thupc nhdm vi khuan Gram am,<br />
khuan lac ttdn, loi, tton, be mat udt, mau ttang due, kich thudc ta 2 - 5 mm. Te bao BDNRl hinh hat dau, kich<br />
thudc dao dpng ta chieu ngang ta 0,2 - 0,4 pm va chieu dai ta 0,6 - 0,9 pm. Khuan lac cua chiing vi khuan<br />
BDNR4 CO kich thudc ta 3 - 6 mm, te bao thupc nhom Gram am, kich thudc dao dpng 0,4 - 0,6 pm. Sau 7 ngay<br />
nuoi cay, chimg BDNRl phan buy 86,2% pjnrene, 50% anthracene, naphthalene va phenanthrene, 44,4%<br />
fluoranthrene va 20% fluorene; chimg BDNR4 phan buy 61,5% pyrene, 50% anthracene va naphthalene,<br />
65,3%fluoranthrene,47% fluorene va 23,1% naphthalene. Dya fren mpt so dac diem hinh thai va trinh ty doan<br />
gen 16S rRNA, chiing vi khuan BDNRl dupc xep vao chi Pseudomonas va c6 ten la Pseudomonas sp.<br />
BDNRl. Trinh tu doan gen 16S rRNA cua chung BDNRl dupc dang ky fren GenBank vdi ma so EU726633.<br />
<br />
j!i Tif khoa: Bioreactor ki khi khong bdt buoc, dioxin, PAH, phdn huy sinh hoc, Pseudomonas<br />
<br />
<br />
MCJDAU Na/NHs d 25°C (Paul et al, 2005). Phdng Cdng nghe<br />
sinh hpc mdi tradmg, Vien Cdng nghe sinh hpc dang<br />
Sau chien franh, frong dat v i fram tich d mpt sd nghien cim xir ly dat nhiem chat doc hda hpc bang<br />
dia diem tai khu vyc d san bay Da Nang van bi d phan buy sinh hpc frong cic bioreactor hieu khi v i<br />
nhiem cic chat diet cd chiia dioxin (2,3,7,8- ki khi khdng bat bupc. Cic tap doan va chiing vi sinh<br />
tefrachloredibenze-p-diexin), 2,4,5-trichlore- vat cd khi nang phan buy 2,3,7,8-TCDD; 2,4,5-T;<br />
phenoxyacetic acid (2,4,5-T); 2,4-dichlorophenoxy- 2,4-D da dugc cdng bd frong nhieu cdng trinh lien<br />
acetic acid (2,4-D); ttichlorephenol (TCP), quan den leai dat d nhiem nay. Cac vi khuan sit dung<br />
dicblorophenel (DCP). Diexin dugc biet den nhu la 2,4-D dugc biet den thugc cic chi Achromobacter,<br />
mpt chat hum co ben vflmg, gay ung thu v i dot bien, Arthrobacter, Burkholderia, Corynebacterium,<br />
dupc xep vao loai chat doc sinh thai. Do do, nhu cau Delftia, Flavobacterium, Halomonas, Pseudomonas,<br />
kbit doc dat nhiem chat diet cd chita dioxin (DCD) Ralstonia, Rhodoferax, Variovorax, Bradyrhizobium<br />
vi cic chat dec khic ttong dd cd cic hydrocarbon va Sphingomonas (Vallaeys et al, 1997; Iteh et al,<br />
tham da vdng (PAH) tai khu vyc bai nhiem l i bet 2004; Nguyen et al, 2007), tuy nhien nhiing nghien<br />
siic can thiet. ciiu ve cic chung vi khuan su dung PAH tir dat<br />
nhiem chat diet cd chiia diexin chua nhieu. Nham<br />
Abe v i ding tic gia (2001) da sit dung phuomg tim hieu khi nang leai bd PAH cua cic chimg vi<br />
phip vat ly de giim thieu diexin d nhiem frong dat. khuan tir cic mau dang xir ly khu doc, cic nghien<br />
Gray va ddng tic gii (1995) da sir dyng y-radiolysis<br />
ciiu pban lap, phan loai v i cic dac tinh sinh hpc cua<br />
de bien 2,3,7,8-TCDD thanh san pham da doc hom.<br />
chiing da dugc tien hinh.<br />
Phuomg phip cho diexin bay hoi cung vdi hei nudc<br />
cung da dupc nghien cuu bdi Mino va dong tic gii Trong bai bio nay, chiing tdi trinh biy ket qui<br />
(2001). Dit nhiem dioxin d thanh phd Tokorozawa, nghien cim met sd die diem sinb hpc, kha nang<br />
Nhat Ban da dupc lam sach bang phuang phip nay. chuyen hda PAHs cua mpt sd chung vi khuan phan<br />
Cic nha khoa hpc ciing da khir loai Cl cua PCB, lap tit bioreactor ki khi khdng bat budc xir ly dat<br />
PAH, chit diet cd frong dat bang each sit dung nhiem DCD. Nghien ciru nay se la ca sa khoa hpc<br />
117<br />
Nguyin Ngpc Bio et al<br />
<br />
gdp phan quan frgng frong cic qui trinh cdng nghe phdng dai 10.000 lln.<br />
xii ly kbit dec va tien tdi lam sach cic chat 6 nhiem<br />
bang pban huy sinh hpc d ty nhien cflng nhu trcng he Xdc dinh khd ndng chuyen hda PAHs<br />
thdng xir ly kin nhu bioreactor.<br />
Phuomg phip xie dinh khi nang chuyin hda<br />
PAH li phuang phip do quang phan tii thee phuomg<br />
NGUYEN LIEU VA PHUONG PHAP<br />
phip da mo ta trade day (Nguyin Ngpc Bie et al,<br />
2008).<br />
Nguyen lieu<br />
<br />
Mau dat sau met thing xir ly ttong bioreactor Phdn loai<br />
ban ki khi nham kbit doc DCD va cac chat d nhiem<br />
Nhan dean gen 16S rRNA, xie dinh frinh tu<br />
ttong dd cd PAH da dugc sit dung de phan lap vi<br />
dean gen 16S rRNA theo phuang phap cua<br />
khuan.<br />
Sambrook va Russell (2001) vi da dugc md ta trong<br />
cdng trinh trade day cua Nguyen Bi Hiiu va Dang<br />
Hoa chat Thi elm Hi (2007).<br />
Cic hda chat tii hang Merck va Sigma; 2,4,5-T;<br />
dibenezofuran (DBF); 2,4-D; PAH nhu naphthalene, BCET QUA NGHIEN CUtJ<br />
phenanthrene, anthracene, fluorene, fluoranthene,<br />
pyrene, chrysene va cac hda chat tinh khiet khic da Vi sinh vat co Idia nang phat trien tren moi<br />
dugc sit dyng trong cic nghien ciiu cua cdng trinh truong chiia pyrene va chrysene<br />
nay. Cap moi 341F va 907R dugc tong hgp tai hang<br />
Invifrogen^'^, vector pCR®2.1 (Invifrogen)... Moi Bang phuomg phip lam giau fren mdi ttudng<br />
tradmg LB su dyng frong nudi cay E. coli trcng tich mudi khoing chiia pyrene li nguon carbon va nang<br />
ddng nghien ciiu dean gen ma hda 16S rRNA lugng duy nhat vdi mau dat dang xit ly DCD ttong<br />
(Sambrook, Russell, 2001). Dich chilt dit (DC) chiia biereacter ki khi khdng bat bupc, da phan lap dupc<br />
2,3,7,8-TCDD tucmg duomg vdi tdng dp doc tir hai chung vi khuan ky hieu BDNRl va BDNR2, cdn<br />
30.000 - 40.000 pg TEQ/ml. fren cimg moi tradmg bd sung chrysene, phan lap<br />
dugc bin chimg vi khuin BDNR3, BDNR4, BDNR5<br />
Phuong phap nghien ciru va BDNR6. Sd di 2 loai PAH fren dugc chpn la do<br />
chiing rat khd bi vi sinh vat tan cdng va cung cd mat<br />
Cic hydrocarbon tham da vdng nhu frong dat nhiem chat diet cd chiia diexin dang xir ly<br />
naphthalene, phenanthrene, anthracene, fluorene, khir doc.<br />
fluoranthene, pyrene, chrysene d ndng dp ban dau Trong siu chung vi khuan niy, hai chiing<br />
100 ppm da dugc sii dung nhu li ngudn carbon va BDNRl va BDNR4 cd khuan lac te, mpc day, dich<br />
nang lupmg duy nhat de dinh gii kha nang phan buy nuoi cay chuyen mau nhanh vi dye hem nhiing chiing<br />
sinh hpc ciia cic vi khuan phan lap dugc trong moi khic. Hai chimg vi khuan nay dugc lya chpn che cic<br />
tradmg mudi kheang (Redrigues et al, 2005). Mdi nghien cim tiep thee nham tim hieu kha nang sir<br />
trudng CNSHl la mdi tradng cho nghien ciiu khi dung PAH va cic chat d nhiem khic.<br />
nang phan buy theo co che ddng trao ddi chat, cd<br />
them cic nguon carbon khic ngoai chat d nhiem<br />
Kha nang phat triln tren DC; 2,4,5-T; 2,4-D;<br />
(Dang Thi Cim Hi et al, 2005). Dinh gii dinh tinh<br />
DBF va PAH cua BDNRl va BDNR4<br />
kha nang phan buy diexin, 2,4,5-T; 2,4-D, DBF va<br />
PAH (vdi nlng dp ban diu mdi chit la 100 ppm),<br />
Kha nang phdt trien tren DC, 2,4,5-T, 2,4-D vd<br />
dya tten sy thay dli miu cua mdi tradmg nudi cay.<br />
DBF<br />
Phan lap, phan leai vi danh gii khi nang sii Hai chiing BDNRl va BDNR4 dugc nudi ddng<br />
dung PAH, cua cic chung vi khuin theo cic phuomg thdi tten hai mdi tradmg mudi khoing vi CNSHl<br />
phip da mo ti trade day (Redrigues et al, 2005). chira cic loai chat d nhiem khic nhau de tim hilu<br />
Kich thudc tl bao vi khuin dugc quan sat dudi kinh kha nang pban huy vi kha nang phit triln ciia chiing<br />
hiln vi dien tii quet SEM JEOL 5410 LV vdi do fren hai mdi tradng khac nhau (Bing 1).<br />
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118<br />
Tgp chi Cdng nghi Sinh hgc 7(1); 117-124, 2008<br />
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Bang 1. Kha nang phat trien cua chung BDNRl va BDNR4 tren moi truang muoi khoang va CNSHl co bo sung cac chat 6<br />
nhiem khac nhau.<br />
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Chat 6 nhiem Moi truang muoi khoang Mdi trud'ng CNSHl<br />
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BDNRl BDNR4 BDNRl BDNR4<br />
<br />
2,4 D<br />
<br />
2,4,5 T<br />
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DBF ++ ++<br />
<br />
DC ++ ++<br />
<br />
Ghi chu: +: Phat trien yeu, moi truang doi mau nhe, co it sinh khoi; ++: Phat trien t6t, moi truang doi mau ro, dam, co nhieu<br />
sinh khoi; -; Khong phat trien, moi tru'ang khong doi mau, khong co sinh khoi.<br />
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<br />
Tren moi truong mudi khoang chua DBF; 2,4,5T; D dudi dang ding trae ddi chat va cic tap doan vi<br />
2,4-D va DC, chung BDNRl chi sit dung DC (Bang khuin phan buy 2,4,5-T cflng da dugc phan lap.<br />
1) v i mau cua mdi tradmg chuyen thanh nau, con<br />
Da sd cic cdng trinh nghien cuu cdng bd cho<br />
chimg BDNR4 chi su dung 2,4-D va mdi tradmg<br />
thiy che thiy, phan buy sinh hpc cua vi khuan hieu<br />
chuyen miu xim. Day cd the la mau cua cac san<br />
khi cic hgp chit dioxin chiia Cl xiy ra thdng qua qui<br />
pham phan buy sinh hpc. Thudmg cic sin pham cat<br />
trinh ding trao dli chit (Nguyen B i Hiiu, Dang Thi<br />
vdng cd mau ving nhat hay cic mau nau khic nhau<br />
d m Ha, 2007).<br />
phy thupc vac cic chit khd phan buy (Field, Sierra-<br />
Alvarez, 2008). So vdi mdi tradmg mudi khoing, sinh<br />
Kha ndng su dung PAH ciia BDNRl vd BDNR4<br />
khii hai chimg BDNRl, BDNR4 deu nhieu hem, mau<br />
sac cua dich nudi cay dam hom khi nuoi cay hai chimg<br />
Tai dat nhiem chat diet cd chiia dioxin d khu vuc<br />
vi khuin nay tten mdi tradmg CNSHl (Bing 1).<br />
san bay Da Nang va trong tu nhien, tai cic diem d<br />
DBF khdng bi vi sinh vat chuyen hda tten mdi nhiim dau md, PAH tdn tai d dang hdn hgp ciia<br />
tradmg mudi khoing cd the vi DBF khdng cd mat nhilu PAH khic nhau. De vay, cic vi sinh vat c6 khi<br />
tteng dit d bioreactor ki khi khdng bat bupc cung nang phan buy nhieu PAH khic nhau se ddng vai trd<br />
nhu frong dit nhiem d D i Nang, tuy nhien chat niy quan trpng frong qua trinh lim sach chat 6 nhiem<br />
lai cd kha nang bi hai chiing fren chuyen hda fren blng phan buy smh hpc d ngay tai khu vuc 6 nhiem<br />
mdi tradmg CNSHl, dung dich nudi cay ddi mau nau cung nhu frong he thong xd ly kin nhu biereacter.<br />
nhat, cd nhilu sinh khdi... Hai chiing nghien ciiu<br />
Hai chung BDNRl va BDNR4 diu cd k h i nang<br />
khdng su dung DBF nhu nguon carbon va nang<br />
loai bd c i 7 loai PAH tit 2 vdng tham frd len a cac<br />
lugng duy nhat. Nhiing chung nay can ngudn carbon<br />
mirc dp khic nhau sau 7 ngay nuoi cay fren moi<br />
khic dl phit trien va sau do phan buy DBF. Day la<br />
tradmg khoing cd chiia PAH vdi nong dp ban dau<br />
qui ttinh ding ttao ddi chat cua vi sinh vat va luon<br />
100 ppm (Bang 2, Hinh 1).<br />
xay ra trong ty nhien. Die tinh qui nay cua hai chiing<br />
vi khuan phan lap dugc la mgt ca sd khoa hpc ddng C i hai chung deu cd kha nang phan buy tdt<br />
gdp them de chiing minh sy thinh cdng cua cdng pyrene v i fluoranthrene. Chrysene la chat rat khd bi<br />
nghe phan buy sinh hpc bang tang cudmg sinh hpc vi sinh vat tin cdng ciing bi BDNRl v i BDNR4<br />
hay kich thich sinh hpc khir doc dit nhiim chat diet phan buy d miic dp thap. Dac biet, hai chiing niy sir<br />
cd chiia dioxin d quy md hien tradmg hoac frong dung cic PAH da vdng tham nhu ngudn carbon va<br />
bioreactor. nang lupmg duy nhat.<br />
Tir dit nhilm chit diet cd chua dioxin ciing nhu Cic nghien cim ve phan lap v i khi nang phan<br />
dit frong cac cong thiic kbit ddc loai dat nhiim nay d buy da dugc nghien cuu rat nhieu. Hai chimg<br />
khu vuc san bay Da Nlng, trong s l chin chimg vi Cycloclasticus N3 va W (Allison et al, 1998) khdng<br />
khuin sit dung DC tbi 5 chung vi khuan cd kha nang sir dung dugc PAH nhu fluoranthene, pyrene va<br />
phit triln tten mdi tradmg muoi khoing cd chiia 2,4- chrysene nhu l i ngudn carbon va nang lugng duy<br />
Nguyin Ngpc Bae et al.<br />
<br />
nhat, tuy nhien theo ca che ddng frao doi chat chung dien PAH khd phan buy nhit la chrysene, pyrene ya<br />
Cycloclasticus N3 cd the phan buy khoang 50% cac cic d nhiim khic da dugc nghien ciiu a cimg dieu<br />
PAH cao phan tit fren vdi nong dp ban dau 1 ppm va kien. Trong cong ttinh nay chi cd khi nang phan buy<br />
bd sung tdi 10 ppm phenanthrene sau 7 ngiy. chrysene va pyrene dugc trinh biy d hinh 1. Mau<br />
mdi trudng da thay dli rit ro sau 7 ngay nuoi cay<br />
Bang 2. Kha nang phan buy PAH cua BDNRl va BDNR4. BDNRl va phi sic ky cho thiy kha nang phan buy<br />
sinh hgc 2 PAH tten d miic dp khac nhau, 34,9% dli<br />
vdi chrysene, va 86,2%) pyrene.<br />
PAH Kha nang phan huy (%)<br />
Trong met cdng bl trade day (Nguyen Ngpc<br />
BDNRl BDNR4 Bie et al, 2008), chung BHNl ciia tic gii phan lap<br />
Pyrene 86,2 61,5 tir nuac thii nhiim diu tai khe chiia xang dau Ha<br />
Nam cd kha nang sir dyng 7 loai PAH d ndng dp ban<br />
Chrysene 34,9 14,3 diu 100 ppm moi PAH sau 2 ngay nudi cly tten moi<br />
Phenanthrene 50 50 tradmg muli khcing, ttong dd naphthalene bi phan<br />
buy 38%, phenanthrene 41%, fluorene va<br />
Fluoranthrene 44,4 65,3 fluoranthene 40%i va pyrene li 52%).<br />
Fluorene 20 47 Hai chiing BDRRl va BDNR4 cd kha nang sit<br />
Anthracene 50 50 dung nhilu loai PAH khac nhau d mirc trang binh.<br />
Tuy nhien, chung vi khuan BDNRl su dyng pyrene,<br />
Naphthalene 50 23,1 la PAH chiia 4 vdng tham, tdt horn se vdi chimg<br />
BDNR4.<br />
<br />
Lers va ddng tic gii (2005) da nghien ciiu xir ly Phan loai hai chung vi khuan BDNRl va BDNR4<br />
sinh hpc mau dat nhiem PAH tit mpt khu vyc cdng<br />
nghiep d mien Bic nudc Phap a dieu kien phdng thi Dac diim khudn lac vd te bdo hai chung vi khuan<br />
nghiem vdi 3 leai hdn hgp PAH. Sau 245 ngay xir ly,<br />
96% hdn hgp PAH 3 vdng tham, 80% bin hgp PAH Sau 4 ngay nudi cay tten mdi tradmg khoing bo<br />
4 vdng tham va 80% hdn hgp 16 loai PAH khic sung pyrene, khuan lac chimg vi khuan BDNRl cd<br />
nhau da lin lugt bi leai bd dang frdn, loi,from,be mat hoi udt, mau frang due,<br />
cd vdng frdn ddng tam, dudng kinh khuan lac tit 2 -<br />
Tai Viet Nam, chimg vi khuan Sphingomonas<br />
5 mm. Dudi kinh hien vi dien tu quet, te bie chiing<br />
yanoikuyae MXL-9 phan lap tii can dau tho md<br />
BDNRl thupc nhdm Gram am, dang hat dau, kich<br />
Bach Hd phan buy 64,5% phenanthrene va 61,4%<br />
thudc tl bio tii 0,6 - 0,9 pm x 0,2 - 0,4 pm (Hinh<br />
anthracene sau 7 ngay nudi cay d ndng dp thap<br />
2A).<br />
(La Thi Thanh Phuang et al, 2003). Chimg KCP8<br />
phan lap tai Khe Che (Quing Ninh) cd khi nang Tren mdi tradmg khoing chira chrysene, khuan<br />
phan buy 76,12% hdn hgp PAH (phenanthrene, lac ciia BDNR4 cd dang frdn, ldi, be mat udt, mau<br />
anthracene va fluoranthrene) 79,96%) trang due, kich thudc 3 - 6 mm. Te bae BDNR4<br />
phenanthrene, 71,09% anthracene va 41,01%) thupc nhdm Gram am, hai frdn, dp dai khcing tit 0,4<br />
fluoranthrene trong hdn hpp PAH sau 7 ngay nudi pm tdi 0,6 pm (Hinh 2B).<br />
lac d mdi tradng mudi khoing (Nguyen Bi Hu:u,<br />
Chiing BDNRl khdng nhiing cd kha nang phan<br />
Dang Thi Cam Ha, 2003). Streptomyces sp.<br />
buy PAH dac biet la pyrene ma cdn cd khi nang phat<br />
XKDN12 phan lap cflng tit dat nhiem DCD phan<br />
triln tit fren mdi tradmg chiia dich chiet. Vi vay,<br />
buy dugc 32,72% phenanthrene, 49,84%<br />
frong ngbien ciiu niy, BDNRl dugc chpn phin loai<br />
anthracene, 17,85% fluoranthrene trong hdn hgp 3<br />
theo phuomg phip xac dinh trinh ty gen ma hda 16S<br />
PAH va 32,1% DBF sau 7 ngay nudi cay tren mdi rRNA vdi kich thudc fren 500 bp. Sin phim PCR<br />
tradmg mudi khoing vdi nguon carbon thii hai la nhan doan gen 16S rRNA da dugc gan vao vector<br />
glucose (Lee et al, 2003). pCR®2.1 nhd T4 DNA ligase va biln nap vao tl bae<br />
Khi nang phit trien cua BDNRl tren hai dai khi biln E. coli DH5a.<br />
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120<br />
Tgp chi Cdng nghi Sinh hgc 7(1); 117-124, 2008<br />
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<br />
PAH PhoUV<br />
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Chrysene<br />
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<br />
Pyrene<br />
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<br />
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<br />
2DD.D 220.0 2*40.0 260.D 2SO .D<br />
C O S T P v r e n e B D N R 1 2 1/3AJS<br />
<br />
Hinh 1. Kha nang phan huy PAH cua chiing BDNRl.<br />
<br />
<br />
<br />
<br />
Hinh 2. A. Hinh thai khuan lac va te bao chCing BDNR1. Kinh hien vi phdng dai 10.000 lan. B. Hinh thai khuan lac va te bao<br />
chung BDNR4. Kinh hien vi phong dai 10.000 lan.<br />
<br />
<br />
<br />
Xdc dinh vi tri phan loai chiing BDNRl bang so tdng sd chiing BDNRl dugc dang ky tren GenBank<br />
sdnh mgt ph&n trlnh tugen md hda 16S rRNA vm ma sd EU726633.<br />
Doan gen 16S rRNA d chung BDNRl cd mirc<br />
Vi tri tren cay phit sinh chiing loai ciia BDNRl tuomg ddng 100% vdi dean gen nay a nhilu chiing vi<br />
da dugc xac dinh bang trinb ttr nucleotide doan gen khuin thupc loai Pseudomonas putida. Ngoai ra,<br />
16S rRNA (560 nucleotide) nhan len dupc tti DNA doan gen 16S rRNA d chimg BDNRl co mirc ttrang<br />
Nguyin Ngpc Bao et al.<br />
<br />
ddng tit 95 den 97%) vdi nhieu vi khuan thupc cic gilng DD. Cic vi khuin su dyng PAH thudc chi<br />
loai khac nhau cua chi Pseudomonas. Pseudomonas pbii k l din nhu; Pseudomonas<br />
cepacia, P. fluorescenes, P. paucimobilis, P. putida,<br />
Tic gii Leach va Lewis (2006) cho bilt, cic<br />
P. testeroni, P. vesicularis, Pseudomonas sp. Mpt sd<br />
chiing P. putida DSM 3601 va DSM 3602 va P.<br />
vi khuan thudc loii P. putida cd khi nang phan buy<br />
stutzeri KC cd the tiet ra PDTC (pyridine-2,6-<br />
PAH nhu P. putida G7 (Lee et al, 2003), P. putida<br />
bis(thiocarbexylic acid) giiip chimg cd the leai bd<br />
ATCC 17514 (Rhodrigues et al, 2005), P. putida<br />
hoan toan carbon tetrachloride (CT) khdi vimg nudc<br />
KT2442 (Newton et al, 2005), P. putida NCIB<br />
nhiem chat nay. Trong khi do, thee Chang va ddng 9816, P. putida OUS82 (Takizawa et al, 1999).<br />
tic gii (2007) chung P. putida OS-19 la vi khuan<br />
bin dia cd khi nang oxy hda, leai bd arsene d ndng Nhieu thanh vien cua chi Pseudomonas nay cd<br />
dp cao trong tat c i cic mau ttam tich va ric lay tai the sinh tradmg dugc a c i dieu kien cd va thieu oxy.<br />
viing Myoung-bong (Han Qudc). Hai chimg vi khuan Hom nira, chung BDNRl dugc phan lap tir bioreactor<br />
P. putida ATCC 17514 va P. putida NCIB 9816 diu ki khi khong bat budc xir ly dat nhiem chat diet cd<br />
cd khi nang phan buy cic PAH. Pseudomonas chira dioxin va cd the sit dung cic chat 6 nhiem d ca<br />
putida ATCC 17514 phan buy tdc dp tdi da dieu kien hieu khi. Trong bioreactor kieu nay oxy<br />
phenanthrene 1,4 ± 0,1 mg/l/h cao hom se vdi phan cflng ludn ludn cd d ndng do thap. Diy la dac diem<br />
buy fluorene 0,8 ± 0,07 mg/l/h (Rodrigues et al, rat quan trpng m i cac dai dien cua chi Pseudomonas<br />
2005). Yang v i ddng tic gii (1994) cung da tich cd dugc, vi vay chimg la vi khuan "an tap" nhat va<br />
ddng cym cic gen tham gia chuyen hda naphthalene, cd vai trd vd cimg quan trpng ddi vdi chu ttinh<br />
fluorene va phenanthrene tii P. putida NCIB 9816 va chuyen hda vat chit, die biet li cic chat d nhiem da<br />
bieu hien trong Escherichia coli HBIOI. vdng tham. Vai cic dac tinh ke tren chiing BDNRl<br />
trinh bay trong bii b i e niy ddng nhiing vai frd quan<br />
trpng trcng qui frinh chuyen hda cac chat d nhiim d<br />
-Acinetobacter haemolyticus LH168 dieu kien ty nhien cung nhu trong cic be xir ly kin.<br />
Chung nay cd the dugc sir dyng nhu met img cir vien<br />
P. putida ATCC 17514 de bd sung che xir ly dit nhiem DCD hay PAH d cic<br />
dieu kien cd the dieu khien dugc nhu frong<br />
P. putida DSM 3602 bioreactor.<br />
<br />
BDNRl<br />
KET LUAN<br />
Pseudomonas sp. K2<br />
Da phan lap dugc 4 chimg vi khuan sit dung<br />
P. putida OS-\9 chrysene v i 2 chiing vi khuan sir dung pyrene tii dat<br />
trong bioreactor ki khi khdng bat buoc xir ly dat<br />
P. aeruginosa LMG 1242^<br />
nhiem chat diet cd chua dioxin.<br />
<br />
^P. stutzeri DSM 5190^ Chiing BDNRl cd the phat trien fren mdi tradng<br />
chira dioxin va BDNR4 fren mdi tradmg chira 2,4-D.<br />
P. chlororaphis LMG 5004T<br />
Sau 7 ngay nuoi cay, chimg BDNRl phan hiiy<br />
86,2% pyrene, 34,9%. chrysene, 50% anthracene,<br />
— P . fluorescens DSM 50090"^<br />
naphthalene v i phenanthrene, 44,4% fluoranthrene<br />
Hinh 3. Cay phat sinh loai ciia chiing BDNRl. v i 20% fluorene; chiing BDNR4 phan buy 61,5%<br />
pyrene, 14,3% chrysene, 50%) anthracene va<br />
phenanthrene, 65,3% fluoranthrene, 47%o fluorene va<br />
Cic hop chat dioxin chiia it Cl hem cd thl bi<br />
23,1% naphthalene.<br />
phan buy bdi cic vi khuan hilu khi, chiing thupc cic<br />
chi cua Sphingomonas, Pseudomonas vd Dya tren mdt sd dac diem hinh thii va trinh ty<br />
Burkholderia. Tuy nhien, cd rat it cdng b l chi tilt vl dean gen 16S rRNA, chung vi khuin BDNRl dugc<br />
vi khuan thupc leai P. putida cd khi nang sir dyng xlp vao chi Pseudomonas v i cd ten li Pseudomonas<br />
DD va DBF. Hong va ding tic gii (2000) thdng bao sp. BDNRl vdi ma s l dang ky tren GenBank la<br />
chiing P. putida PH-01 cd khi nang phan buy DBF EU726633.<br />
<br />
122<br />
Tgp chi Cdng nghi Sinh hgc 7(1); 117-124, 2008<br />
<br />
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Ltri c a m o n : Cdng trinh da dugc hd trg kinh phi tit Meyer S, Moser R, Neef A., Stahl U, Kampfer P (1999)<br />
di tdi dgc lap cdp Viin Khoa hgc vd Cdng nghi Viet Differential detection of key enzyme of polyacromatic<br />
Nam "Nghiin ciru xir ly tdy ddc mdt sd hgp chdt hiru hydrocarbon-degradation bacteria using PCR and gene<br />
ca chira clo bang phuang phdp hda hgc, sinh hgc probes. Microbiology 145: 1731-1741.<br />
tien tien " vd dugc thirc hiin mgt phdn tgi Phdng thi Mino Y, Moriyama Y (2001) Possible remediation of<br />
nghiem trgng diim Cdng nghi gen. dioxin-polluted soils by steam distillation. Chem Pharm<br />
Bull 49(8): 1050-1051<br />
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TAI LIEU THAM KHAO Gomes NC, Kosheleva IA, Abraham WR, Smalla K (2005)<br />
Effects of the inoculant sfrain Pseudomonas putida<br />
Abe T , Mizuno H, Noguchi K (2001) A new remediation KT2442 (pNF142) and of naphthalene contamination on<br />
technique for soils contaminated with dioxins employing the soil bacterial community. FEMS Microbiol Ecol 54:<br />
vaccuum pyrorisis. In: 21*' Intemational Symposum on 21-33,<br />
Halogenated Environmental Organic Pollutant & POP 54:<br />
Itoh K, Tashiro Y, Uobe K, Kamagata Y, Suyama K,<br />
157-159.<br />
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Chang JS, Ren X, Kim KW (2008) Arsenic-oxidizmg Harbor tfdAa and cadA, homologous with genes encoding<br />
bacteria for indicating the Arsenic-contaminated soil. J 2,4-dichlorophenoxyacetic acid-degrading proteins. Appl<br />
Environ Sci 20(11): 1348-1355. Environ Microbiol 70: 2110-2118.<br />
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huy hydrocarbon thom da nhan cua mpt so chimg vi khuan<br />
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(1992) Degradation of dibenzofuran and dibenzodioxins<br />
Che, Cita Luc, Quang Ninh. Tgp chi Khoa hoc vd Cong<br />
by fiingi. Presented at: Dioxin'92,12th intemational<br />
nghe 42(3): 37-44.<br />
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Compounds, Tampere, Finland. Nguygn Ba Hiiu, Dang Thi Clm Ha (2007) Xac dinh doan<br />
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Degradation of dibenzofuran by Pseudomonas putida PH-<br />
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bis(thiocarboxylic acid) (PDTC). Microbiology 152: 3157- Rodrigues AC, Wuertz S, Brito AG, Melo LF (2005)<br />
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P O L Y C Y C L I C A R O M A T I C H Y D R O C A R B O N B I O D E G R A D A T I O N O F BACTERIAL<br />
STRAINS I S O L A T E D F R O M H E R B I C I D E / D I O X I N D E T O X I F I C A T I O N B I O R E A C T O R<br />
<br />
Nguyen Ngoc Bao, Phan Thi Hoang Hao, Nguyen Ba Huu, Nguyen ThanhThuy, Vu Due Loi, Dang Thi<br />
Cam Ha<br />
Institute of Biotechnology, Vietnam Academy ofSience and Technology<br />
<br />
SUMMARY<br />
<br />
From detoxification tteatment of herbicide/dioxin contaminated soil in semianaerobic bioreactor, four<br />
chrysene degraders and two pyrene degraders were isolated. Among these bacterial strains, only two sfrains,<br />
BDNRl and BDNR4, were able to utilize soil exfraction containing 2,3,7,8-TCDD and 2,4-D, respectively.<br />
Both bacterial sttains belong to Gram negative. BDNRl and BDNR4 degraded seven different PAHs such as<br />
phenanthrene, anthracene, fluoranthene, naphthalene, fluorene, pyrene and chrysene. After 7 days of<br />
incubation, BDNRl degraded 86.2% pyrene, 50% anthracene, 50% naphthalene, 50% phenanthrene, 44.4%<br />
fluoranthrene and 20% fluorene; BDNR4 bacterial strain also degraded 61.5 % pyrene, 50% anthracene, 50%<br />
naphthalene, 65.3% fluoranthrene, 47% fluorene and 23.1% naphthalene. Based on several morphological<br />
properties and partial sequence of 16S rRNA gene, BDNRl sfrain should belong to the genus Pseudomonas<br />
and named as Pseudomonas sp. BDNRl. The partial sequence of 16S rRNA gene ofthe sfrain BDNRl was<br />
deposited in Genbank with an assession number EU726633.<br />
<br />
Keywords: Biodegradation, herbicide/dioxin, PAHs, Pseudomonas, semianaerobic bioreactor<br />
<br />
<br />
<br />
<br />
'Authorfor correspondenece: Tel; 84-4-38360892; Fax; 84-4-38363144; E-mail; danscamha(a)iht nr -.^<br />
124<br />