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Nghiên cứu khả năng phân hủy sinh học Hydrocarbon thơm đa vòng của một số chủng vi khuẩn phân lập từ Bioreactor xử lý đất nhiễm chất độc hóa học

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Đất nhiễm hỗn hợp 2,3,7,8-TCDD (dioxin), các chất diệt cỏ 2,4,5-T,2,4-D, sản phẩm phân hủy của chất diệt cỏ TCP, DCP, PAH được xử lý bằng bioreator kị khí không bắt buộc. Từ đất đang xử lý, 4 chủng vi khuẩn sử dụng chrysene và 2 chủng vi khuẩn sử dụng pyrene đã được phân lập. Chủng vi khuẩn BDNR1 có thể phát triển trên môi trường tương tự với nguồn carbon duy nhất là dịch chiết đất chứa dioxin và BDNR4 có thể phát triển trên môi trường tương tự với nguồn carbon là 2,4-D. BDNR1 thuộc nhóm vi khuẩn Gram âm, khuẩn lạc tròn, lồi, trơn, bề mặt ướt, màu trắng đục, kích thước từ 2-5mm.

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Nội dung Text: Nghiên cứu khả năng phân hủy sinh học Hydrocarbon thơm đa vòng của một số chủng vi khuẩn phân lập từ Bioreactor xử lý đất nhiễm chất độc hóa học

Tgp chi Cdng nghi Sinh hgcl{l):l 17-124, 2008<br /> <br /> <br /> <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 /> <br /> <br /> 118<br /> Tgp chi Cdng nghi Sinh hgc 7(1); 117-124, 2008<br /> <br /> 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 /> <br /> <br /> Chat 6 nhiem Moi truang muoi khoang Mdi trud'ng CNSHl<br /> <br /> BDNRl BDNR4 BDNRl BDNR4<br /> <br /> 2,4 D<br /> <br /> 2,4,5 T<br /> <br /> 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 /> <br /> <br /> <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 /> <br /> <br /> <br /> 120<br /> Tgp chi Cdng nghi Sinh hgc 7(1); 117-124, 2008<br /> <br /> <br /> PAH PhoUV<br /> <br /> <br /> <br /> <br /> Chrysene<br /> <br /> <br /> <br /> <br /> Pyrene<br /> <br /> <br /> <br /> <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 /> <br /> 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 /> <br /> 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 /> Yamamoto H (2004) Root nodule Bradyrhizobium spp.<br /> 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 /> <br /> Field JA, Sierra-Alvarez R (2008) Microbial degradation Nguyen LH, Itoh K, Suyama K (2007) Diversity of 2,4-<br /> of chlorinated dioxins. Chemosphere 71: 1005-1018. dichlorophenoxyacetio acid (2,4-D) and 2,4,5-<br /> trichlorophenoxyacetic acid (2,4,5-T)-degrading bacteria<br /> Gray KA, Hilarides RJ (1995) Radiolytic tteattnent of in Vietaamese soil. Microb Environ 22(3): 243-256.<br /> dioxin contaminated soils. Radiat Phys Chem 46(4-6):<br /> 1081-1084. Nguyen Ba Hiiu, Dang Thi Cam Ha (2003) Kha nang phan<br /> huy hydrocarbon thom da nhan cua mpt so chimg vi khuan<br /> Hofrnann KH, Polnisch E, Kreisei H, Mach H, Schubert M<br /> phan lap ta thit nghiem xit ly 6 nhiem dau tai bai trieu Khe<br /> (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 /> Symposium on Chlorinated Dioxin and Related<br /> Compounds, Tampere, Finland. Nguygn Ba Hiiu, Dang Thi Clm Ha (2007) Xac dinh doan<br /> gen ma hoa dioxygenaza ta chung vi khuan phan buy<br /> Hong HB, Chang YS, Choi SD, Park YH. (2000)<br /> dibenezofuran Terrabacter sp. DMA phan lap ta dat nhiem<br /> Degradation of dibenzofuran by Pseudomonas putida PH-<br /> chat diet co chiia doxin tai Da Nang. Tgp chi Sinh hgc<br /> OI. Wat Res (34)8: 2404-2407.<br /> 29(3): 83-89.<br /> Armenegaud J, Timmis KN (1997) Biodegradation of<br /> Nguyen Duong Nha, Nghiem Ngpc Minh, Nguyen Ngpc<br /> dibenzo-p-dioxin and dibenzofiiran by bacteria. J<br /> Bao, Dang Thi Cam Ha (2005) Kha nang phan buy<br /> Microbiol 35(4): 241-252.<br /> hydrocarbon thom da nhan va dibenzofiiran cua chung xa<br /> La Thi Thanh Phuang, Nguyen Ba Hiru, Dang Thi Cam khuin XKDN12. Tgp chi Cong nghe Sinh hoc 3(1): 123-<br /> Ha (2003) Phan buy sinh hpc hydrocarbon thom da nhan 132.<br /> (PAHs) bai chung vi khuan MXL-9 phan lap ta can dau<br /> tho cua mo Bach Ho Viing Tau. Tgp chi Cong nghe Sinh Nguyen Ngpc Bao, Dam Thuy Hang, Vii Diic Lpi, Dang<br /> hoc 1(1): 109-117. Thi Thu, Dang Thi Cim Ha (2008) Phan buy sinh hpc<br /> hydrocarbon thom da vong cua mpt so chimg vi khuan<br /> Lee K, Park JW, Ahn IS (2003) Effect of additional carbon phan lap ta nudc thai nhiem dau. Tgp chi Khoa hgc vd<br /> source on naphthalene biodegradation by Pseudomonas Cong nghe 46(6): 67-75.<br /> putida G7. J Hazard Mater (105)1-3:157-67.<br /> Paul D, Pandey G, Panday J, Jain KR (2005) Assessing<br /> Leach LH, Lewis TA (2006) Identification and microbial diversity for bioremediation and environmental<br /> characterization of Psedomonas membrane ttansporters restoration. Trends Biotechnol 23: 135-142.<br /> necessary for utilization of the siderophore pyridine-2,6-<br /> bis(thiocarboxylic acid) (PDTC). Microbiology 152: 3157- Rodrigues AC, Wuertz S, Brito AG, Melo LF (2005)<br /> 3166. Fluorene and phenanthrene uptake by Pseudomonas putida<br /> ATCC 17514: kinetics and physiological aspects.<br /> Mai Anh Tuan, Nghiem Ngpc Minh, Dang Thi Cam Ha Biotechnol Bioeng 90(3): 281-289.<br /> (2004) Nghien cira phan loai va kha nang sit dung<br /> hydrocarbon tham da nhan bai chimg xa khuan XKDN19 Sambrook J, Russell DW (2001) Molecular Cloning. A<br /> phan lap tir dat nhiem chat doc hoa hoc. Tgp chi Cong Laboratory Manual, 3''' ed. Cold Spring Harbor<br /> nghe Sinh hgc 2(3): 3%9-396. Laboratory Press. Cold Spring Harbor, NY.<br /> <br /> 123<br /> Nguyin Ngpc Bio et al.<br /> <br /> Scbecter A, Gasiewicz A (2003) Dioxin and Health, 2"^ Vallaeys T, Persello-Cartieaux F, Rouard N, Lors C,<br /> ed. A John Wiley & Sons, Inc, New York. Laguerre G, Soulas G (1997) PCR-RFLP analysis of 16S<br /> xi.icxri A..iv.f* J T T.r j D c i T ^ rRNA, tfdA and tfdB genes reveals a diversity of 2,4-D<br /> Takada S, Nakamura M, Matsueda T, Kondo R, Sakai K A^^.,A • -r . rr'it.rc t,r;^..„h;^i F^-^I 'yd-<br /> /inn£\ T>, J *• .£• 1 t.1 • . J JU J- • degraders m soil aggregates. FEMS Microbiol Ecol 24.<br /> (1996) Degradation ot polychlormated dibenzo-p-dioxms 269 278<br /> and polychlorinated dibenzofiirans by the white rot fungus<br /> Phanerochaete sordida YK-624. Appl Eneviron Microbiol White PA (2002) The genotoxicity of priority polycychc<br /> 62: 4323-4328. aromatic hydrocarbons in complex mixtare. Mut Res 515:<br /> 85-98.<br /> Takizawa N, lida T, Sawada T, Yamauchi K, Wang YW,<br /> Fukuda M, Kiyohara H (1999) Nucleotide sequences and Yang Y, Chen RF, Shiaris MP (1994) Metabolism of<br /> characterization of genes encoding naphthalene upper naphthalene, fluorene and phenanthrene: preliminary<br /> pathway of Pseudomonas aeruginosa PaKl and characterization of a cloned gene cluster from<br /> Pseudomonas putida 0VSS2. J Biosci Bioeng 87(6): 721- Pseudomonas putida NCIB 9816. J Bacterial 176(8):<br /> 31. 2158-2164.<br /> 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 />
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