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Báo cáo "Phân lập đánh giá khả năng phân hủy Hexachlorocyclohexane của chủng nấm sợi FNA33 từ đất xử lý khử độc thuốc trừ sâu bằng Bioreactor hiếu khí "

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Phân lập đánh giá khả năng phân hủy Hexachlorocyclohexane của chủng nấm sợi FNA33 từ đất xử lý khử độc thuốc trừ sâu bằng Bioreactor hiếu khí

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Nội dung Text: Báo cáo "Phân lập đánh giá khả năng phân hủy Hexachlorocyclohexane của chủng nấm sợi FNA33 từ đất xử lý khử độc thuốc trừ sâu bằng Bioreactor hiếu khí "

  1. Tgp chi Cdng nghi Sinh hpc 1(2): 257-264, 2009 P H A N L A P V A D A N H GIA K H A N A N G P H A N H U Y H E X A C H L O R O C Y C L O H E X A N E C U A C H U N G N A M SOI FNA33 TlT D A T X\S L Y K H i y D O C T H U O C TRlT S A U B A N G BIOREACTOR HIEU KHI Dang Thj Cam Ha, Pham Quoc Hiep, Nguyin Nguyen Quang, TrSn Thi Nhu Hoa, Nghiem Ngoc Minh Viin Cdng nghi sinh hpc TOMTAT Tir mau xu ly khir ddc dat nhiem hdn hpp cac thudc trvt sau gdm Dichloro-diphenyl-trichloroethane (DDT), Hexachlorocyclohexane (HCH), Dieldrin, Aldrin... frong bioreactor hi6u khi da phan lap duac ba chiing nam sai FNA31, FNA32 va FNA33. Cac chung nam sai deu phat trien duac frong mdi trudng chi cd mudi khoang chiia 300 ppm HCH. Chung FNA33 cd kha nang phat trien manh nhat so vdi hai chung cdn lai, do do chiing FNA3 3 da duac su dung frong cac nghien ciiu tiep theo. Dua vao cac dac diem hinh thai khuan lac, cudng sinh bao tit va bao tii, trinh tu doan gen ma hda 18S rRNA chimg nam sai FNA33 duac xep vao chi Aspergillus va duac dat ten la Aspergillus sp. FNA33. Doan gen ma hda 18S rRNA cua chiing Aspergillus sp. FNA33 da diiac dang ky fren GenBank vdi ma sd EU684231. Sau 14 ngay nudi lac 200 vdng/phiit d 30°C frong mdi trudng mudi khoang cd bd sung 0,5% glucose, chung nam sai FNA33 da phan huy duac 88% HCH vdi ndng dp ban dau la 243 ppm. Budc dau nghien ciiu kha nang sinh enzyme ngoai bao cho thay, sau 7 ngay nudi cay fren mdi trudng chiia HCH chung nam sai FNA3 3 sinh tdng hap hai loai enzyme la mangan peroxidase (434,5 U/l) va laccase (4,3 U/l), hoat tinh enzyme mangan peroxidase van tdn tai frong mdi trudng sau 20 ngay nudi cay. Tit khod: Ndm sai, phdn huy sinh hoc, thuoc trie sdu, 18S rRNA M6DAU qudc gia tren the gidi. Cho den nay, nhieu phuang phap hda ly va sinh hpc da duoc ap dung de xir ly cac chdt ddc thudc POP tiong cd HCH. Phuang Viec sir dyng rdng rai cac thudc bao ve thye vat phap phan huy sinh hpc dupc nhin nhan la phuang chlorine hiiu ca d nhieu nudc dang phat trien nhu phap him hieu, khdng gay d nhiem thii cap cho mdi Viet Nam da va dang dan den tinh frang mdi trudng trudng va phu hop cho viec khir ddc vdi dieu kien bi d nhiem do dac tinh khd phan huy cua chiing kinh t6 cua cac nudc dang phat ttien (Dang Thi tiong ty nhien. Hexachlorocyclohexane (HCH) la Cam Ha et al, 2007). mdt ttong 12 hap chat hiiu ca khd phan hiiy nhdm POP dupc UNEP quy dinh phai quan ly an toan va Cac loai vi sinh vat ddng vai trd quan ttpng giam thieu theo cdng udc Stockholm 2001 ma Viet nhit trong qua trinh phan huy sinh hpc. Cac nha Nam da ky (Dang Thi Cam Ha et- al, 2007). HCH khoa hpc da chung minh mpt sd loai vi khuan nhu thudng tdn tai d hai dang chinh la HCH ky thuat va Xanthomonas sp. IHC12, Microbacterium sp. HCH thuang mai hay cdn gpi la Lindane (d Viet ITRCl; Sphingomonas paucimobilis UT26 va Nam gpi tat la thudc 666). Mac du HCH da bi cim chung Sphingomonas paucimobilis B90A da san xuat va sir dung tir nhiing nam 70 - 80 d hau het khoang hda hoan toan cac ddng phan HCH thanh cac nudc ciing nhu d Viet Nam nhimg van cdn rat CO2 va H2O (Nagata et al, 1999; Kumar et al, nhieu d dang qua han gay d nhiem mdi trudng dat, 2002; Manickam et al, 2007; Middeldorp et al, nudc, khdng khi (Dang Thi Cam Ha et til, 2007; 2005). Dya tren nhiing nghien ciiu ve cac gen Nguyin Thiiy Binh et al, 2003). Cac ddng phan chiic nang va kha nang phan huy ciia chung B90A, cua HCH xam nhap va tich tu ttong ca the con thi chung nay cd the dupc su dung de loai bd HCH ngudi thdng qua chudi thiic an, dudng hd hap hay thdng qua phuang phap kich thich sinh hpc do tidp xiic tryc ti6p vdi ngudn d nhiem gay nen cac (RupLal et al, 2008). Mdt nghien ciiu gan day cua can benh nguy hi8m. De giai puyet thuc trang d Benimeli va ddng tac gia (2006) da thdng bao ve nhiem nay, viec nghien ciiu tim ra giai phap xir ly, chung xa khuan Streptomyces sp. M7 cd kha nang giam thieu va tay ddc chat d nhiem la mdt nhiem vu phan huy Lindane khi nudi cay trong mdi trudng hdt site can thiet khdng chi d nudc ta ma cdn nhieu chiia glucose 0,6 g/l va 100 mg/l Lindane 257
  2. Dang Thi Cim Ha et al (Benimeli et al, 2006). Ben canh dd, kha nang NGUYEN LIEU VA PHUONG PHAP NGHIEN phan huy HCH ciing da dupc nghien ciiu d dieu CUU kien ki khi dac biet la trong he thdng UASB bdi cac loai vi sinh vat ki khi nhu la Dehalobacter sp., Nguyen lieu Sedimentibacter sp. ... (Doesburg et al, 2005; Praveena et al, 2008). Tuy nhien, cac nghien ciiu Dit dang dupc xir ly khu ddc hon hap HCH, deu chiing minh rang d dieu kien ki khi khdng cd DDT,... ttong bioreactor hieu khi bang phuang phap sinh vat nao cd the khoang hda hoan toan cac ddng phan huy sinh hpc (kich thich sinh hpc) da dupc sit phan HCH (Nagata et al, 1999; Middeldorp et al, dung de phan lap va tuyen chpn vi sinh vat cd kha 2005; Praveena et al, 2008). nang phan huy HCH. Sii dung cac mdi trudng mudi khoang va mdi trudng Czapek (Atlas, 1995) duoc cai Mdt sd chiing nam dam nhu Phanerochaete, tien cd chiia HCH ky thuat, dudng glucose de nghien Trametes, Bjerkandera va Pleurotus ciing cd kha ciiu phan loai va phan huy sinh hpc. nang chuyen hda va dan tdi khoang hda hoan toan HCH nhd he enzyme ngoai bao peroxidase Phuong phap nghien ciru (Mougin et al, 1997; Singh, Kuhad, 2000; Phan lap nam bang phuang phap cay ttai tten dia Rajashekara, Manonmani, 2007). Singh va Kuhad thach vdi mdi trudng mudi khoang co ban chiia 100 (2000) da chitng minh loai Phanerochaete ppm HCH. Sau do, tiep tuc lam giau 3 lan de chpn chrysosporium cd kha nang phan hiiy Lindane dupc chung nam cd kha nang su dung HCH. thap hon so vdi loai Trametes hirsutus sau 4 ngay nudi cay. Varima va ddng tac gia (2008) cdng bd Hinh thai cudng sinh bao tii va bao tii cua chung chiing nam spi Conidiobolus 03-1-56 cd kha nang FNA33 dupc quan sat dudi kinh hien vi quang hpc phan huy y-HCH nhd he enzyme ngoai bao cao vdi dp phdng dai 40, 100 lan. Phuang phap phan loai hon chiing nam dam Pleurotus ostreaus 1200, dya vao trinh ty doan gen ma hda 18S rRNA dupc Trametes versicolor 1086 (Varima et al, 2008). thye hien nhu sau: tach chiet DNA tdng sd, tach Tuy nhien, sd lupng cac loai nam spi, dac biet la chiet DNA plasmid theo md ta cua Sambrook va cac chiing cd kha nang sinh enzyme ngoai bao Russell (2001), nhan doan gen 18S rRNA bing ky duoc phat hien cdn rat khiem tdn, chua phan anh thuat PCR vdi viec sit dung cap mdi EF4f va Fung5r thye sy vai trd cua nam spi trong chuyen hda, (Elsas et al, 2000), phuang phap tach ddng gen phan huy, khoang hda hoan toan cac ddng phan dupc thye hien theo hudng dan cua nha san suat TA cua HCH. He ezyme ngoai bao ciia cac chung nam Cloning® JCit (InttovigenTw). Viec xac dinh trinh ty ndi chung ngoai kha nang tham gia xiic tac cac dupc thye hien theo phuang phap cua Sanger, sir phan ling phan huy cac hpp chat lignin chung cdn dung may xac dinh trinh ty gen ty ddng ABI Prism cd kha nang phan huy va khoang hda rat nhieu hpp 3100. Cac budc tien hanh cy the da dupc md ta tiong chat thupc POP hay cac chat hiiu co mach vdng cd cac cdng trinh nghien cuu trudc day (Hoang Thi My cau triic gidng thanh phan cau tao nen phan tir Hanh et al, 2004; Nguyen Thanh Thuy et al, 2006). lignin. Enzyme lignin peroxidase, mangan Nghien cim, xac dinh hoat tinh enzyme ngoai peroxidase va laccase ddng vai trd quan trpng nhat bao thudc nhdm peroxidase va laccase bang phuang trong hai nhdm enzyme ngoai bao peroxidase va phap hap thy quang phd d cac budc sdng thich hop oxidoreductase. Day la chia khda cho viec nghien vdi cac C chat phan iing 2,4-DCP, phenol red, O ciiu sir dung cac loai vi nam, dac biet la nam spi syringaldazine tuong iing chd mdi loai enzyme trong cdng nghe phan huy sinh hpc cac chat hiiu (Deny et al, 1996; Niladevi, Prena, 2005). Cac C gay d nhiem (Reineke, 2001; Dang Thi Cam Ha O chung nam sau 7 ngay va 20 ngay nudi lac dupc thu et al, 2007; Mougin et al, 1997; Rajeshekara, dich de xac dinh kha nang sinh enzyme ngoai bao. Manonmani, 2007). Phan tich lupng tdn lim HCH tit dich nudi cay vi Trong bai bao nay, chung tdi trinh bay cac kdt sinh vat dupc tien hanh nhu sau: thu nhan dich nudi qua phan lap va xac dinh kha nang sinh tdng hop cay cd chimg nam spi FNA33 va mau nudi cay enzyme ngoai bao MnP va laccase va kha nang khdng cd vi sinh vat (ddi chiing) cd chiia HCH, tien phan huy HCH cua chiing nam soi phan lap tir hanh ehiit hai lin bing dung mdi CH2CI2, tach liy bioreactor hieu khi xii ly dat nhiem hdn hop thudc phan dung mdi cd hda tan HCH. Loai nudc trong trir sau HCH, DDT, Aldrin, Dieldrin... d quy md dung mdi bing Na2S04 khan sau dd cat quay chan 100 kg dit. khdng va chiSt lai bing dung mdi n-hexane, tiSp tuc 258
  3. Tgp cki Cong nghi Sinh hpc 1(1): 257-264, 2009 cat quay chan khdng dd loai bd hoan toan dung mdi dau mau vang nhat sau mau vang ram (Hinh IA). n-hexane, lupng HCH tdn luu dupc phan tich tten Dau sinh bao tii fran dang tia, cudng sinh bao tii fran may sac ky khi khdi phd (GC/MS). cd cudng dai, cd cac the bmh dan le tten the binh ldn hinh cau. Hai tang the binh, cudng the binh 6 - 9 pm X 3 - 3,5 pm; the bmh 7,5 - 9 pm x 3 - 3,5 pm. Bao KET QUA VA THAO LUAN tir hinh cau, nhan 4 pm x 3,5 - 4,5 pm, khi gia gai rap (Hinh IB). Xet cac dac diem hinh thai khuan lac, Phan lap va tuyen chon vi sinh vat phat trien tren cudng sinh bao tii va bao tir thi chung nam spi moi triromg chira HCH FNA33 cd the thudc chi Aspergillus. Tir mau dat cua bioreactor hieu khi xii ly dat nhiem hdn hop cac thuoc trii sau ttong dd cd HCH, 3 chimg nam, 2 chung vi khuan da dupc phan lap va tuyen chpn. Cac chung vi sinh vat cd hinh thai khac nhau tten mdi trudng mudi khoang chiia HCH khdng bd sung glucose va tten mdi trudng tuong ty cd bd sung cd bd sung 0,1%. Cac chimg nam sgi dupc dat ten FNA31, FNA32, FNA33. Sau 7 ngay nudi lac 200 vdng/phiit d nhiet dp 30°C, kha nang phat trien cua 3 chiing n i m FNA31, FNA32 va FNA33 tten mdi trudng mudi khoang Hinh 1. A. Hinh thai khuan lac tren Czapek; B. cuong sinh dich chiia 300 ppm HCH cd bd simg 0,1% glucose bao tu va bao tu cua chung nam sai FNA33. va khdng cd glucose dupc trinh bay d bang 1. Bang 1. Kha nang ph^t trien cua 3 chung nam tren mdi Phan loai FNA33 dua tren trinh tu doan gen ma trudng chiia HCH. hoa 18S rRNA Sau khi DNA tdng sd da duoc lam sach, gen ma Chung Mdi tru'd'ng l(iioang + HCH hda 18S rRNA cua chung nam soi FNA3 3 da dupc Khong co glucose Co glucose tach ddng va xac dinh, so sanh trinh ty vdi cac trinh FNA31 + ty tuong ung cua cac chung vi nam da dupc cdng bd ++ fren GenBank. Chung FNA33 cd miic tuong ddng FNA32 + ++ toi 99% so vdi cac dai dien thudc chi Aspergillus, cd FNA33 ++ +++ mdi qua he gan giii vdi chi Trichodermaceae (98%), Penicillium (97%) (Hinh 2). Dya vao dac diem hinh Gili chu: (+): phat trien; (++): phat trien trung binh; (+++): thai va ket qua so sanh trinh ty doan gen ma hda 18S phat trien tdt. rRNA, chung FNA33 dupc dat ten la Aspergillus sp. FNA33. Trinh ty doan gen nay dupc dang ky fren GenBank vdi ma sd EU684231. Ket qua thu dupc cho thay, fren hai loai mdi trudng, ca 3 chung deu phat trien. Khi mdi trudng cd Chimg Aspergillus sp. FNA3 3 cd quan he gan bd sung glucose chiing cd kha nang phat trien nhatih giii vdi cac chiing Aspergillus oryzae 112822, hon. Chung FNA33 cd kha nang phat trien tdt hon so Aspergillus sp. FS132, Aspergillus alliaceus SIIA vdi hai chung cdn lai nen da dupc chpn de khao sat 1572, Aspergillus flavus UCSP1768 hay cac chiing cac dac diem sinh hpc khac. Trichocomaceae sp. LM253, Penicillium tardum KCTC 16051 (Hinh 2). Tuy nhien, cho den nay chua Phan loai chung ndm soi FNA33 dua tren cac dac cd cac thdng tin ve kha nang su dung hay phan huy diem hinh thai HCH ciia cac chimg dai dien fren. Chi cd mdt vai cdng bd nghien ciiu ve chung nam spi FDN20, Sau 3 ngay nudi cay fren mdi trudng Czapek, Aspergillus terreus FDN22 cd kha nangg su' dyng khuin lac chimg FNA33 cd mau xanh reu dam, cd chat diet cd 2,4-Dichlorophenobcylacetic va vidn soi fring xung quanh. Sau 7 ngay dudng kmh dibenzofuran phan lap tir dat nhiem chat ddc hda hpc khuin lac 2,5 cm, bd mat phdng dai, nhan tiheo cd (Nguyen Thanh Thiiy et al, 2006; Hoang Thi My gipt tiit, bao tii chuyen sang mau den, mat frai luc Hanh et al, 2004). 259
  4. Dang Thi Cim Ha et al Penicillium tardum KCTC16051 Aspergillus oryzae 112822 Aspergillus sp. FS132 Aspergillus alliaceus SIIA 1572 Aspergillus flavus UPSC 1768 Aspergillus sp. FNA33 Tricfiocomaceae sp. LM253 Aspergillus terreus FDN22 0.002 Aspergillus terreus ATCC1012 I 1 Aspergillus cen/inus ATCC16915 Hinh 2. Cay phat sinh chung loai cua chung FNA33 (Thudc do the hien su sai khac cua 2 nucleotide tren 1000 nucleotide so sanh). Kha nang sinh enzyme ngoai bao cua chung ddi chat ciia ca 3 chung nam, ddng thdi cac enzyme FNA33 dupc sinh ra sau 7 ngay va van tdn tai sau 20 ngay. De xac dinh kha nang sinh tdng hop nhdm Bang 2. Hoat tinh enzyme ngoai bao LiP, MnP, Lac cua cac chung nim sai. enzyme ngoai bao peroxidase va laccase cua ba chung nam spi, dac biet la chimg FNA33, dich nudi cay cua cac chung dupc thu nhan va tien hanh xac Ten Sau 7 ngay Sau 20 ngay ditih hoat tinh enzyme tien may quang phd hap thy. chung LiP MnP Lac LiP MnP Lac Ket qua xac dinh kha nang sinh tdng hpp enzyme (U/l) (U/l) lignin peroxidase, mangan peroxidase va laccase cua FNA31 - - 4.0 - 168 0,9 ba chimg nim spi FNA31, FNA32 va FNA33 dupc trinh bay d bang 2. FNA32 - - 12,3 110,8 2,3 Enzyme laccase da dupc ca 3 chung sinh tdng FNA33 - 434,5 4,3 - 269 hpp sau 7 ngay va deu giam hoac mat hoat tinh sau Ghi chu: (U/l): dan vi do enzyme sinh ra trong 1 I djch mdi ngay thii 20. Sau 7 ngay nudi cay, chung FNA33 da trudng nudi cay; (-): khdng sinh enzyme. sinh tdng hpp enzyme mangan peroxidase vdi hoat tinh kha cao 434,5 U/l cdn enzyme laccase cd hoat tinh thip hon (4,30 U/l). Sau 20 ngay thi hoat tinh Ba loai enzyme ngoai bao da dupc tim thay d ba cua enzyme MnP va laccase khdng cdn phat hien chung nam spi ttong nghien ciiu nay md ra mdt duoc. Chiing FNA32 sinh tdng hop enzyme lignin hudng mdi cho viec xii ly hdn hpp POP d ttong dat peroxidase sau 20 ngay nudi cay, ciing tiong thdi vdi cac ndng dp khac nhau bang nam spi ttong gian nay, enzyme mangan peroxidase cua chiing bioreactor hidu khi, khi cac yeu td mdi trudng cd the FNA31 dupc phat hien vdi hoat tinh 168 U/l nhung didu khidn dupc. Day la budc khdi diu dl cd thl van thap han chung FNA33 la 269 U/l. Nghien ciiu thuc hien cac nghien ciiu sau hon, hudng tdi xay budc dau cho thay sy da dang trong kha nang frao dung cac quy trinh xir ly dat, ttim tich, nudc d 260
  5. Tgp chi Cdng nghi Sinh hpc 1(2): 257-264, 2009 nhiem khdng chi cho HCH ma cdn cho cac loai POP ngoai bao dac biet dupc quan tam va cd iing dung khac nhu dioxin, PAH, PCB... Cac enzyme nay nhieu nhat cho xir ly cac hpp chat xenobiotic la khdng dac hieu cho timg loai POP va la he enzyme laccase va mangan peroxidase (Timothy, Jeffer, ngoai bao nen md ra triln vpng dl tang cudng hieu 2006). qua xii ly hdn hop POP ttong dd cd thudc tini sau cd chira gdc chlorine. Kha nang phan huy HCH cua chung nam soi 0 dieu kien hieu khi, tiong len men pha rin thi FNA33 nhdm enzyme ngoai bao thudng dupc sinh tdng hop Kha nang phan huy HCH cua chimg nam spi da nhieu han la enzyme ndi bao, dieu nay ciing giai dupc nghien cim de phan nao ly giai mdi quan he thich kha nang khoang hda HCH hay cac hpp chit giiia he enzyme ngoai bao dupc sinh tdng hop frong phenolic nhd he enzyme nay va chi cd the xay ra d mdi trudng va phan huy HCH. Ket qua phan tich dieu kien hieu khi (Singh, Kuhad, 2000; Reineke, lupng HCH cdn lai ttong mau nudi cay chiing nam 2002; Timothy, Jeffer, 2006). Khi phat tiiln tiong spi FNA33 tten mdi trudng mudi khoang cd bd sung cac loai mdi trudng ran khac nhau, cac chung nim 0,5% glucose so sanh vdi mau tuong ty khdng cd spi thuan loi hon vi khuan vi chung cd he soi va cdn FNA3 3 dupc trinh bay d bang 3. cd kha nang sinh tdng hpp cac enzyme khdng dac hieu khac nhu monooxygenase, dehydrogenase, Bang 3. Kha nang phan huy HCH cua chiing nam sai oxidoreductase tang cudng xiic tac phan ung sinh FNA33. hpc (Reuieke, 2001; Timothy, Jeffer, 2006). Cac he enzyme ngoai bao ma nam spi sinh ra khdng chi d HCH Miu piian tich Lvang HCH bj dieu kien in vitro ma ngay nhiing dieu kien in vivo (ppm) loai bo (%) ciing dupc kich thich nhd cac yeu td dinh dudng hay khdng cd Cd FNA33 FNA33 nhu cau cho va nhan dien tir. Cac minh chiing tien gdp phan giai thich sy xuat hien cac chung nam spi a-HCH 0,8 0,3 65,0 cd kha nang phat trien manh va hoan toan chiem uu Y-HCH 241,7 28,8 88,1 the khi xit ly bang phuang phap phan hiiy sinh hpc frong bioreactor hieu khi sau 7 den 15 ngay (Dang 5-HCH 0,5 0,06 88,5 Thi Cam Ha et al, 2007). Chae chan tap doan nam Tong so 243 29,1 88,0 spi ddng mdt vai frd nhat dinh frong giai doan dau khi -sdi ly nham chuyen hda, phan huy va khoang hda hdn hop cac chat ddc thudc nhdm POP frong dd cd Sau 14 ngay nudi cay, chung nam soi FNA33 da HCH. Cd the enzyme ngoai bao d nam spi tiong dieu phan huy, loai bd 88,0%) lupng HCH tiong mdi kien hieu khi ddng vai frd chu dao, day la mdt van de trudng nudi ciy. Tdc dp phan buy HCH bdi chiing can cd them cac nghien ciiu khang dinh (Dang Thi nam sai Aspergillus sp. FNA3 3 kha cao. Cam Ha era/., 2007). Trong cac cdng tritih da cdng bd ve kha nang Trong cac nghien ciiu ve enzyme ngoai bao thuy phan huy HCH bdi nam soi cho thay mdt vai loai cd phan hpp chat lignin d nam spi phan lap tir dat, kha nang chiu dyng tiong mdi trudng cd ndng dp chimg nam spi Aspergillus terreus LD-1 gan giii vdi HCll thap nhu Aspergillus niger, Trichoderma sp., chimg Aspergillus sp. FNA33 da dupc cdng bd, dac Penicillium camemberti, Aspergillus awamori, biet chiing nay sinh tdng hop 2 loai enzyme tuoftg ty Thermoascus aurantiacus nhimg chua cd nhieu minh nhu chimg Aspergillus sp. FNA33. Hai enzyine thu chiing thuylt phuc ve kha nang phan buy HCH duoc cd kha nang hoat ddng d dilu kien rat kilm pH (Mougm et al, 1997; Smgh, Kuhad, 2000; Reineke, tir 11 din 12,5, hoat tinh enzyme trung binh 0,384 2001). U/mg doi vdi timg loai co chat dung de xac dinh (Kanayama et al, 2002). Ngoai ra, 3 loai nam Theo nghien ciiu cua Nguyen Thiiy Binh va Aspergillus nidulan, A. oryzae, A. niger ciing dupc ddng tac gia (2003), chiing nam spi Aspergillus niger sir dung lam vat chii de bieu hien cac gen ma hda cho IFO 31125 va chung No2-500 cd kha nang chiu cac enzyme ngoai bao peroxidase va laccase tir cac dyng ddc tinh ciia y-HCH lan lupt la 1, 2, 3 ppm. chimg nim dam Phanerochaete chrysosporium, Sau'lO ngay, hai chimg nay chuyen hda dupc khoang Pleurotus ostreatus, Cyathus bulleri, Trametes sp. ... 80%) vdi ndng dp ban dau la 1,0 pmm (Nguyen Thuy (Singh, Kuhad, 2000; Reineke, 2001). Cac enzyme Binh et al, 2003). Cdn chimg Aspergillus sp. FNA33 261
  6. Dang Thi Cam Ha et al ttong nghien ciiu nay cd kha nang phan huy manh KETLUAN han hai chiing tten khoang 200 lin. Gupta va ddng tac gia (2000) khi nghien ciiu kha Nam chung vi sinh vat cd kha nang sir dung nang phan huy HCH d 2 chung Bacillus circulans va HCH da dupc phan lap, ttong dd cd 3 chimg nam sgi Bacillus brevis, sau 2 nam nudi cay tich liiy, chiing FNA31, FNA32 va FNA33. Chung niin sgi FNA33 ciing da phan huy dupc cac ddng phan HCH vdi cd kha nang sinh trudng va phat triln tdt hon so vdi ndng dp ban dau rat thap 1,0 ppm (Gupta et al, hai chimg nam sgi cdn lai. 2000). Mdt nghien ciiu khac d chiing Pseudomonas Tren mdi trudng mudi khoang dich cd chiia aeroginosa ITRC5, Kumar da xac dinh dupc tten HCH d ngay iiudi ciy thii 7, chung Aspergillus sp. 90% ddng phan a, y-HCH, 45% |3-HCH, 55% 5- FNA33 sinh tdng hop 2 loai enzyme ngoai bao la HCH dugc loai bd khi sir dung chiing nay de xir ly mangan peroxidase va laccase vdi hoat tinh lin lupt HCH ttong dit bi d nhilm (Kumar et al, 2006). Mdt la 434,5 U/l, 4,3 U/l. Enzyme laccase ciing dupc sinh cdng bd khac ve kha nang phan huy y-HCH cua tdng hop bdi hai chimg nam spi FNA31 (4,0 U/l) va chung Xanthomonas sp. ICH12, tac gia Manickam FNA32(12,3 U/l) Chung nim sgi FNA32 sinh tdng cho biet chimg ICH 12 sir dung y-HCH nhu la ngudn hop enzyme lignin peroxidase (118,8 U/l) d ngay carbon va nang lupng duy nhat ttong sudt qua trinh nudi cay thir 20, d cung thdi diem nay enzyme phat trien. Sau 8 ngay, chimg nay phan huy dupc 100 mangan peroxidase ciing dugc sinh ra tit chimg ppm y-HCH va lupng sinh khdi cao nhat thu dupc FNA31 cd hoat tinh 168 U/l nhung vin thip hon ttong ngay nudi cay thii 6 (Manickam et al, 2007). chung FNA33 la 269 U/l. Singh va Kuhad (2000) so sanh kha nang phan huy HCH cua hai chimg nam dam Phanerochaete Sau 14 ngay nudi cay chimg Aspergillus sp. sordida va Cyathus bulleri da ket luan rang khi nudi FNA33 loai bd dugc 88%o HCH vdi ndng dp ban diu cay tiong mdi trudng cd ngudn carbon thii hai la 243 ppm. Chiing nam sgi FNA33 dugc xlp vao chi glucose thi cac ddng phan HCH mdi bi phan huy. Aspergillus, dugc dat ten la Aspergillus sp. FNA3 3, Sau 4 ngay nuqi cay, hai chung nay bat dau phan huy tritih ty doan gen ma hda 18S rRNA dugc dang ky y-HCH cd ttong mdi trudng vdi ndng dp ban dau la tien GenBank vdi ma sd EU684231. 79,4 ppm (Singh, Kuhad, 2000). Ltri cam on: Cong trinh ndy duoc thuc hiin voi tdi So sanh vdi nhung ket qua ve kha nang phan tra kinh phi cua di tdi ddc lap cdp Viin Khoa hpc vd huy HCH cua vi sinh vat ma chiing tdi dupc bilt thi Cdng nghi Viet Nam "Nghiin cieu xie ly tdy doc mpt chung nam spi Aspergillus sp. FNA33 cd toe dp sd hap chdt him co chua Clo bdng phuong phdp hda phan hiiy tuong ddi cao, sau 2 tuin 213,9 ppm HCH hpc, sinh hpc tiin tiin ". da bi loai bd. Ngoai ra, mdt sd chung nam spi khac cd kha nang phan buy DDT ciing da dupc phan lap tii bioreactor hieu khi xii ly dat nhilm hdn hop nhilu TAI LIEU THAM KHAO loai thudc trii sau. Mdt tiong sd -dd la chung Aspergillus sp. FNA4 da loai bd dupc 94,5% hdn Atlas RM (1995) Handbook of Media for Enviromental Microbiology, CRC press, Boca Raton, Florida. hop DDT, DDD, DDE vdi ndng dp ban diu la 52.6 ppm (Nguyen Nguyen Quang, Dang Thi Cim Ha, Benimeli CS, Casfro GR , Chaile AP, Amoroso MJ 2009). Chung FNA4 cung da dupc khao sat kha nang (2006) Lindane removal induction by Streptomyces sp. sinh tdng hop enzyme ngoai bao va chi cd enzyme M7. J Basic Microbiol 46 (5): 348-57. laccase dupc tim thiy, boat tinh enzyme la 15,4 U/l Dang Thi Cam Ha, Nguyen Ba Huu, Nghiem Ngoc Minh, cao hon so vdi chung FNA33 (4,3 U/l). Dilu nay Nguyen Nguyen Quang, Nguyen Thanh Thuy, Dam Thuy cQng cd thl giai thich duoc rang vi sinh vat khdng Hang, Tran Nhu Hoa, Nguyen Ngoc Bao (2007) chi phan huy HCH ma chiing cdn cd kha nang phan Application of bioremediation technology for huy DDT va cac thudc trir sau khac. Tir dac tinh detoxification of herbicide/dioxin contaminated soil. In phan huy cac ddng phan HCH frong mdi trudng nudi proceccdings: "P'* International HCH and Pesticides Forum for CEECCA countries" Sept. 20 - 22, 2007, ISBN cay da chiing minh dugc chung nim spi FNA33 978-9975-70-707-7: 270-274. ddng mdt vai frd nhit dinh ttong qua trinh phan hiiy hdn hgp cac thudc trir sau ttong dit d nhilm xit ly Deny KM, Iqbal M, Miller PGG, McCathy A (1996) bang bioreactor hilu khi. Screening Actinomycetes for extracellular Peroxidase Activity. J Appl Environ Microbiol 62: 2186-2190. 262
  7. Tgp chi Cdng nghe Sinh hpc 1(2): 257-264, 2009 Doesburg WV, Miriam HA van Eekert, Middeldorp PJM, degradation in Sphingomonas paucimobilis UT26. India J Balk M, Gosse S, Alfons JM (2005) Reductive Microbiol Biotechnol 23: 380-390. dechlorination of P-hexachlorocyclohexane (P-HCH) by a Nguyin Nguyen Quang, Dang Thi Cam Ha (2009) Phan Dehalobacter species in coculture with a Sedimentibacter lap, phan loai va kha nang phan buy DDT, DDD, DDE cua sp. Appl Microbiol Biotechnol 69(5): 80-588. chung nam soi Aspergillus sp. FNA4. Tgp chi Cong nghe Elsas JD, Duarte GF, Keizer-Welters A, Smit E (2000) Sinh hpc 7(1): 125-132. Analysis of dynamics of fungal communities in soil via Nguyen Thuy Binh, Dang Diic Nhan, Dd Thi Loan, Dinh fimgal specific PCR of soil DNA followed by denaturing Thu Hang, Nguyen Ngpc Diing, Van Nhu Ngpc, Tran Van gradient gel elecfrophoresis. J Microbiol Method 43: 133- Nhi (2003) Nghien cim kha nang chiu dung va chuyen hda 151. Lindane (y-HCH) cua mpt sd vi sinh vat. Hpi nghi Cong Gupta A, Kaushik CP, Kaushik A (2000) Degradation of nghe sinh hpc Todn quoc: 172-174. hexachlorocyclohexane (HCH; a, P, y, 8) by Bacillus Niladevi KN, Prena P (2005) Mangrove Actinomycetes as circulans and Bacillus brevis isolated from soil the source of ligninolytic enzymes. Actinomycetologica 19: contaminated with HCH. Soil Biol Biochem 32: 1803- 40-47. ---_:••, 1805. Praveena B, Suresh Kumar M, Sandeep M, Tapan C Hoang Thi My Hanh, Nguyen Thatih Thuy, Ngd Xuan (2008) Enhanced biodegradation of Quy, Nghiem Xuan Trudng, Nghiem Ngoc Minh, Dang hexachlorocyclohexane in upflow anaerobic sludge blanket Thi c i m Ha (2004) Kha nang phan huy 2,4-D va reactor using methanol as an elecfron donor. Bioresour dibenzofiiran cua chimg nam sai FDN20. Tgp chi Cong Technol 99(7): 2594-2602. nghe Sinh hpc 2(4): 517-528. Rajashekara MHM, Manonmani HK (2007) Aerobic Kanayama N, Suzuki T, Kawai K (2002) Purification and degradation of technical hexachlorocyclohexane by a characterization of an Alkaline manganese peroxidase defined microbial consortium. J Hazardous Material 149: from Aspergillus terreus LD-1. J Biosci Bioeng 93(4): 18-25. 405-410. Reineke W (2001) The Handbook of Environmental Kumar M, Gupta SK, Garg SK and Kumar A (2006) Chemistry Vol. 2 Part K. Biodegradation and Persistence. Biodegradation of hexachlorocyclohexane-isomers in Springer-Verlag Berlin Heidelberg. contaminated soils. Soil Biol Biochem 38: 2318-2327. Sambrook J, Russell DW (2001) Molecular Cloning: A . Manickam N, Misra R, Mayihaj S (2007) A novel pathway Laboratory Manual 3"* ed. Cold Spring Harbor for the biodegradation of y-HCH by Xanthomonas sp. Laboratory. Cold Spring Harbor, NY. Sfrain ICH12. J App Microbiol 102: 1468-1478. Singh BK, Kuhad RC (2000) Degradation of insecticide Middeldorp PJM, Doesburg WV, Gosse S, Alfons IM lindane (y-HCH) by white-rot fungi Cyathus bulleri and (2005) Reductive dechlorination of Phanerochaete sordida. Pest Manage Sci 56: 142-146. hexachlorocyclohexane (HCH) isomers in soil under anaerobic condition. Biodegradation 16: 283-290. Timothy PR, Jeffer WT (2006) Enhancing bioremediation with enzymatic processes: A review. Pratiee periodical of Mougin C, Peri Caud C, Dubroca J, Asther M (1997) hazardous, toxic and radioactive waste Management 10: Enhanced mineralization of lindane in soils supplemented 73-85. with the white rot basidiomycete Phanerochaete chrysosporium. Soil Biol Biochem 29(9-10): 1321-1324. Varima N, Scrinivasan MC, Paknikar KM (2008) Biodegradation of y-hexachlorocyclohexane (lindane) by Nagata Y, Miyuchi R, Tagaki M (1999) Complete analysis non-white rot fungus Conidiobolus 03-1-56 isolated from of gens and enzyme for y-hexachlorocyclohexane litter. India J Microbiol 48: 134-141. 263
  8. Dang Thi Cam Ha et al ISOLATION AND CHARACTERIZATION OF HEXACHLOROCYCLOHEXANE DEGRADATION OF FILAMENTOUS FUNGAL STRAIN FNA33 FROM PESTICIDE DETOXIFICATION BY AEROBIC BIOREACTOR Dang Thi Cam Ha*, Pham Quoc Hiep, Nguyen Nguyen Quang, Tran Thi Nhu Hoa, Nghiem Ngoc Minh Institue ofBiotechnology SUMMARY Three filamentous fungal sfrains were isolated of soil bioremediation freatment by aerobic bioreactor in order to detoxify DDT, HCH and other pesticides. These fimgal strains FNA31, FNA32 and FNA33 cultivated in mineral salt medium containing 300 ppm HCH and FNA33 showed the best grovrth. Based on colony morphology, sporephore observed through microscope and sequence of a partial 18S rRNA gene, the sfrain FNA33 could be classified Aspergillus sp. FNA33. The 18S rRNA gene fragment sequence oi Aspergillus sp. FNA33 was deposited in the GenBank database with assession number EU684231. After 14 days incubation in mineral salt medium containing 0.5% glucose, Aspergillus sp. FNA33 degraded 88% HCH with initial concenfration 243 ppm. Furthermore, after 7 days in the same condition this sfrain also produced exfracellular enzyme mangan peroxidase (434.5 U/l) and laccase (4.3 U/l). After 20 days of cultivation the activity of manganese peroxidase detected still at high level (269 U/l). Keywords: Aspergillus, bioremediation, hexachlorocyclohexane, filamentous fungi, ISSrRNA * Author for correspondence: Tel: 84-4-38360892; Fax: 84-4-38363144; E-mail: danscamha&.ibt ac. vn 264
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