33(4): 86-91 Tap chi SINH HOC 12-2011
NGHI^.N c u t KIIA NANG PlIAN HUY DAU DIESEL CUA CHUNG VI KHUAN BTLDS PHAN LAPTU NU6C THAl KHU
C 6 N G N G H I $ PCUNC; THI NGOC MAI, NGUYf.N THUY LINH, NGUYEN VAN HAC, VU THI THANH,
NGIII^.M NGOC MINI!
Sau than da, ddu mu la nguyen liCu hda thach thfi hai dugc con ngudi bill din vd dua vao khai thae, sfi dung. Kl tfi khi phat hien ra din na\. ddu md vdn dugc coi nhu Id mdt ngudn nang lugng khdng thi thiOu cung nhu chua thi thay the'. Chinh vl cd mdt vi tri quan trgng dd'i vdi loai ngudi ma nganh edng nghiep ddu md ngay mdt phat triln manh me va da trd thdnh thi manh kinh t l dd'i vdi nhfing nude ed tilm ndng dau md.
Tu\ nhien, ben canh ngudn Igi vl kinh t i d o nganh cong nghiep ndy dem lai la hilm hga d nhilm mdi trudng cd nguyen nhdn tfi nhfing su cd khai thae, van chuyen ddu md tren biin... gdy ra. Ngoai su cd tran ddu, phai kl din mdt so lugng khdng nhd can thai xdng ddu tdn dgng trong cac kho ehfia, eung nhu ham lugng ddu dugc su dung cho cde ddng co, cde loai ddy chuyen san xud't edng nghiep eung lam lugng dau d nhilm ed trong nudc thai cdng nghiep va sinh boat ngay cang gia tang. Vdn dl d nhilm ddu ngay nay dang trd thanh ndi bfie xuc toan edu. Dfing trude nhfing hilm hga d nhilm ddu md va cac san phdm cua nd, d l cd thi giai quylt mdt each triSt dl, ddi hdi phai ed sU kit hgp nghien cfiu ciia nhilu nha khoa hge, edng nghe va cac nha quan li mdi trudng eung nhu su hgp tac gifia cac don vj vdn ehuyin, kinh doanh vd sfi dung ddu md.
Hien nay, edng nghe phdn buy sinh hge (Bioremediation) da dugc dp dung rdng rai dd'i vdi xfi ly d nhilm ddu va eae ehd't ddc hda hge, eung nhu cac ehd't d nhilm khae do cd hiiu qua cao, chi phi thd'p va an toan vdi mdi trudng. Ban ehd't eiia edng nghe phdn buy sinh hge la kieh thieh su phdn buy, su phat triln eua vi sinh vdt ban dia cd kha ndng phdn buy ddu bode cac ehd't gdy d nhilm khae ed sdn trong tu nhien, bdng
Vien Cong nghe sinh hoc
each thay ddi cde ylu td mdi trudng nhu do thdng khi, cde ehdt dinh dudng nhu ngudn nito vd photpho, eae ehd't vi lugng, cac ehd't boat ddng bl mat sinh bgc... cd nghla la tao ra diiu kien td'i uu d l vi sinh vdt sfi dung cde thanh phdn ddu md phat triln va boat ddng phdn buy.
Vl vdy, d l gdp phdn xfi ly d nhilm ddu diesel (ddu DO) trong nude thai cdng nghiep, chung vi khudn BTLD5 da duge phdn ldp tfi nudc thai khu edng nghiep (KCN) Tfi Liem vd danh gia kha nang sfi dung dau DO eua nd.
I. PHUONG PHAP NGHIEN ClTU 1. Nguyen lieu
Chung vi khudn BTLD5 dugc phdn lap tfi nudc thai tai bl ehfia tap trung eua KCN Tfi Licm Ha Ndi d edc vj tri khac nhau.
2. Phuong phdp a. Phdn lap
Phuang phap lam gidu 3 Idn duge dung dl phdn ldp chung vi khudn BTLDS, Hut 5 ml mdu nude thai sau khi trdn diu vao binh tam giac 250 ml ehfia 45 ml mdi trudng khoang ed bl sung 5% ddu DO, nudi Idc 5 7 ngdy d 3 0 T vdi td'c dd 200 vong/phut, Sau 5 7 ngdy, chuyin 10% gid'ng d Idn lam gidu thfi nhd't sang binh lam gidu Idn hai vd Idn ba. Sau Idn lam gidu thfi ba, hut 0,5 ml djch nudi ed'y, pha loang vd ed'y gat tren mdi trudng hiiu khi tdng sd, mdu dugc nudi tTnh d 30"C, Sau 7 ngay, cac khuan lac mge rieng re se dugc tdch rieng ra nudi tren 20 ml mdi trudng khoang djch thi,
b. Quan sdt hinh thdi khudn Ige vd hinh thdi te'bdo
Hinh thai khu^n lac cua chung vi khuiti
BTLD5 duge quan sat tren mdi trudng bilu khi tdng sd thach.
Hinh thai t l bao eua chung vi khudn dugc quan sat dudi kinh hiin vi dien tfi quit JSMLV5410 vdi su phdi hgp cua Vien 69, Bd Tu Lenh Ldng.
c. Nghien cuu khd ndng phdn huy ddu DO cua chung BTLDS
Chung vi khudn BTLD5 duge nudi Idc 200 vong/phut tren mdi trudng mudi khodng cd bd sung 5% ddu DO d 30"C. Kha nang phan buy dau ciia chung BTLD5 dugc thue hien theo phuong phap phan tich khdi lugng cua TCVN 4582-88. Qua trinh phan tich dau dugc thuc hien v6i su phdi hgp cua Vien Hda hge cdng nghiep.
d. Phdn loai vi khudn dUa tren viee so sdnh trinh tu gen 16S iRNA
DNA tdng sd cua chung BTLD5 duge tach chie't theo hudng ddn eua bd kit (hang Bioneer), sau do DNA tdng sd dugc nhan len vdi cap mdi 27f va 1492r vdi ehu trinh phan fing Id': 94°C trong 5 phut; lap lai 32 ehu ky: 9 4 ^ trong 1 phut, 55"C trong 1 phut 30 gidy, 72"C trong 1 phut 30 gidy; 72°C trong 7 phut; 4°C d l gifi.
Tilp dd san phdm PCR (kich thudc khoang 1500 bp) dugc gdn vao vector tach ddng, bien nap vao t l bao kha biin E. coli DH5a. Ddng piasmit ehfia doan gen mong mud'n dugc tach ehilt, tinh sach vd xae djnh trinh tu tren may xac dinh trinh tu gen tu ddng (ABI PRISM 3100 Avant'Genetic Analyzer). Viee so sanh trinh tu nucletit va xay dung cay phat sinh chung loai ciia chung BTLD5 vdi cac chung vi khudn dai dien khac sfi dung phdn mim Blast, Bioedit, Clustal X va Treeview.
II. KfeT QUA VA THAO LUAN
1. Phan Idp ehiing BTLDS tfi nude thai KCN Tfi Li^m, Ha Ndi
Sau 3 Idn lam gidu, chung tdi nhdn thd'y mau sde vd dd due eua mdi trudng thay ddi rd ret so vdi mdu nudc thai ban ddu. So sanh kit qua cac Idn lam gidu thfi nhdt, Idn lam gidu thfi 2 va thfi 3 chung tdi quan sat tha'y sau 24 gid, mau moi trudng thay ddi rd ret va sinh khdi ngay cang tang len, diiu dd cho thdy su phat trien nhanh ehdng cua cac vi sinh vat trong mdu nudc thai (hinh 1).
L d n l Lan 2 Lan 3
Hinh 1. Mau lam siiau lan 1. lan 2, liin 3 tren moi trudng khoang co bd sung 59c dau DO
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Hinh 2. Hinh thai khudn lac (A) va hinh thai te bao chung BTLDS vdi do phdng dai 7500 lan (B)
Sau khi pha loang Idn lam gian thfi 3 va ed'y gat trcMi inOi tiirdng bilu khi king sd thach, rdi cay chuyin sang moi trudng khoang djch ehfia 57, ilaii DO, chiing tOi nhdn tliA'y chiing BTLD5 la cluing vi khudn phat triln nhanh nhd't, sau 24h lugng sinh khdi ham tren thdnh binh va lugng ckiu nOi tien hi mat dieh cung biCn mdt nhanh nhat. Vi vay, chiing loi sfi dung chiing vi khudn nay dl dung clu) cac nghiCn cini tilp theo.
2. Hinh thai khuun lac vu hinh thai tc bdo chiing BTLDS
Tren mdi tiudng bilu khi tOng sd' thach, khudn lac cluing vi khudn BTLD5 cd mdu trdng due. nhdt, U)i, dudng kinh khoang 2 mm vd tilt ra mfti trudng sjic td xanh lue (hinh 2A). Dudi kinh hien vi diCn tfi quit vdi dd phdng dai 7500 Idn, t l bao chiing Bn^D5 ed dang hinh que ngdn. kich thudc tfi 0,73 0,91 pm x 1 1,8 fi.m
(hinh 2B).
3. Nghiln curu kha ndng phdn huy dau DO cua chiing BTLDS
Chung BTLD5 duge nudi Idc tren mdi trudng khoang djch ed bd sung 5% ddu DO. Sau 7 ngay nudi lac d 30"C, so vdi mdu dd'i chfing khdng eo vi sinh vdt (mdu K), mdi trudng nudi cdy chung BTLD5 da cd su ddi mdu rd ret vd lugng sinh khd'i bam vao thanh binh cung Idn (hinh 3). Vi vdy, ed thi phdn nao khang dinh duge kha ndng phdn huy ddu eua chung vi khudn ndy. Tuy nhien, d l danh gid chinh xac kha ndng phdn buy ddu DO, chung tdi da gfii phdn tich b Vien Hda edng nghiep. Bdng phuong phap phdn tieh khdi lugng, sau "7 ngdy nudi Ide ham lugng ddu cdn lai la 3 g/1 giam 25 g/1 so vdi mdu ddi chfing (28 g/l).
Tfi dd, ehiing tdi tinh toan duOc luong ddu do BTLD5sfidungla89,3%.
K
Hinh 3. Kha naiiti sinh IriioiiL: eiia ehuiiii BThD5 lien moi irudii!: elura S', dau DO
1500bp
Hinh 4, San phdm nhdn doan gene ma hda 16S rRNA cua chung BTLDS
M. Thang DNA chudn kich thudc 1 kb;
1. San phdm PCR cua chung BTLDS.
Hinh 5. San phdm dien di DNA plasmid cua ddng sd 2 tren gel agarose 1 %
C. DNA plasmid ddng khudn lac mau xanh (ddi chimg); 1. DNA plasmid ddng khudn lac sd 2.
4. Phdn loai vi khudn dua tren trinh tu doan tach chie't, nhdn doan gen 16S rRNA vdi cap gen 16S rRNA mdi 27f vd 1492r vdi chu tnnh nhiet phan fing DNA tdng sd ciia BTLDS dugc chung tdi "^" ^ P ^ ^ phuong phap. Dien di dd san ph£m
thudc khoang 1500 bp, hoan todn phfi hgp vdi tinh toan ly thuylt (hinh 4), San phdm PCR duge tinh sach, gdn vdo vector tach ddng pBT vd biin nap vao t l bdo kha biin E. coli DH5a, Mdt ddng khudn lac trdng dugc tach ehilt DNA piasmit (hinh 5). DNA plasmid eua ddng khudn lac ndy dugc kiim tra bang each PCR Idn 2 vdi edp mdi 27f vd 1492r vdi DNA plasmid cua ddng "khudn lac sd 2 lam khudn (thdnh phdn phan fing vd chu
trinh nhiet nhu Idn PCR thfi nhd't), kit qua cho thd'y ddng khudn lac trdng dd ehfia dung doan gen edn thilt (1500 bp). Do dd, DNA plasmid eua ddng khudn lac dd dugc tdch ehilt, tinh sach vd xde djnh trinh tu gene,
Trinh tu doan gen 16S rRNA eiia chung BTLDS da dugc xde dinh tren may doc trinh tu tg ddng ABI PRISM 3100, Sau khi phdn tieh vd xfi ly sd lieu, trinh tu doan gen 16S rRNA eua chung vi khudn nay duge trinh bdy tren hinh 6.
1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321
tcctggattc aacgtccgga ggacctcacg ccaaggcgac ggtccagact ccagccatgc aagggcagta cttcgtgcca taaagcgcgc ctgcatccaa gtgaaatgcg tgacactgag ccgtaaacga ataagtcgac gcccgcacaa ccttgacatg tgctgcatgg caacccttgt aaaccggagg cacgtgctac aaaccgatcg taatcgtgaa ccaccatggg Hinh
agcggcggac gggtgagtaa tgcctaggaa tctgcctggt agtgggggat aacgggcgct aataccgcat acgtcctgag ggagaaagtg ggggatcttc ctatcagatg agcctaggtc ggattagcta gttggtgggg taaaggccta gatccgtaac tggtctgaga ggatgatcag tcacactgga actgagacac cctacgggag gcagcagtgg ggaatattgg acaatgggcg aaagcctgat cgcgtgtgtg aagaaggtct tcggattgta aagcacttta agttgggagg agttaatacc ttgctgtttt gacgttacca acagaataag caccggctaa gcagccgcgg taatacgaag ggtgcaagcg ttaatcggaa ttactgggcg gtaggtggtt cagcaagttg gatgtgaaat ccccgggctc aacctgggaa aactactgag ctagagtacg gtagagggtg gtggaatttc ctgtgtagcg tagatatagg aaggaacacc agtggcgaag gcgaccacct ggactgatac gtgcgaaagc gtgggggagc aaacaggatt agataccctg gtagtccacg tgtcgactag ccgttgggat ccttgagatc ttagtggcgc agctaacgcg cgcctgggga gtacggccgc aaggttaaaa ctcagatgaa ttgactgggg gcggtggagc atgtggttta attcgaagca acgcgaagaa ccttacctgg ctgagaactt tccagagatg gattggtgcc ttcgggaact cagacacagg ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gtaacgagcg ccttagttac cagcacctcg ggtgggcact ctaaggagac tgccggtgac aaggtgggga tgacgtcaag tcatcatggc ccttacggcc agggctacta aatggtcggt acaaagggtt gccaagccgc gaggtggagc taatcccata tagtccggat cgcagtctgc aactcgactg cgtgaagtcg gaatcgctag tcagaatgtc acggtgaata cgttcccggg ccttgtacac accgcccgtc atcgttgctc cagaagtatc tag
6. Trinh tu doan gen 16S rRNA eua chung vi khudn BTLDS
Dua vao viee so sanh trinh tu doan gen 16S Standing in Nomenclature), chfing tdi da thdng rRNA ciia chung vi khudn BTLDS vdi trinh tu ke mfic dd tuong ddng eua edc chung so sanh cac chiing vi sinh vdt prokaryote chudn khac (bang 1) va xdy dung cdy phat sinh chung loai tren LPSN (List of Prokaryotic names with eua chfing vi khudn nay (hinh 7),
Bdng 1
STT 1 2 3 4 5 6 7 8 9
Mure do tuong ddng i Ten chung vi khuan Pseudomonas aeruginosa Pseudomonas agarici Pseudomonas acidovorans Pseudomonas abietaniphila Pseudomonas alealophila Aminobacter aminovorans Pseudomonas amygdale Pseudomonas andropogonis Pseudomonas anguilliseptica
ciia BTLDS so vdi mot sd chiing vi khuan Ma sd tren
NCBI X06684 Z76652 AB02147 AJOl1504 AB030S83 AJOl1759 Z766S4 X67037 X99S40
Sd nucleotit so sdnh 1329/1355 1294/1371 1147/1354 1297/1369 1311/1363 806/956 1276/1359 1160/1372 1300/1349
Miifcdg tuong ddng
98%
94%
85%
95%
96%
84%
94%
85%
96%
~ Pseudomonas amygdale (Z766S4)
— Pseudomonas alealophila (AB030S83)
— Pseudomonas anguilliseptica (X99540)
— BTLDS (JF750922)
— Pseudomonas aeruginosa (X06684)
— Aminobacter aminovorans (AJO11759)
— Pseudomonas acidovorans (AB02147)
— Pseudomonas andropogonis (X67037)
— Pseudomonas amygdale (Z766S4)
— Pseudomonas abietaniphila (AJO 11504)
Hinh 7. Cdy phat sinh chimg loai eua chung BTLDS so vdi cac loai cd hg hang gdn giii Quan sat tren bang 1 vd hinh 7 chung tdi
nhdn thd'y, ehiing BTLDS ed quan he gdn giii vdi cac chiing thuOc ehi Pseudomonas, dua tren viee so sanh nucleotit thi chung vi khudn nay ed dd tuong ddng eao vdi chung Pseudomonas aeruginosa (98%). Do dd, chung tdi tam ddt ten chiing vi khudn nay la Pseudomonas sp.
BTLDS. Chung vi khudn nay da dugc ddng ky tren ngdn hang Genbank (NCBI) vdi ma sd' la JF750922.
Mdt sd tac gia tren thi gidi da edng bd' vl kha ndng phan buy ddu DO cua ehi Pseudomonas. Nghien efiu mdi nhd't ciia nhdm tac gia Kaezorek vd nnk. (2011) khi nghien eiiru vl kha ndng phan buy sinh hge eae hgp ehd't hydrocarbon da phdn ldp duge chung vi khudn Pseudomonas alcaligenes S22 ed kha ndng phdn hiiy 92% ddu DO sau 21 ngdy nudi ed'y ed bd sung them Triton X-100 [3]. Ndm 2009, tde gia Adeline va nnk., da phdn ldp duge chung Pseudomonas lundensis UTAR FPE2 tfi Id nhien lieu cua nhd mdy ed kha ndng phdn buy 69% ddu diesel trong 3 ngay ddu tien [1], Ndm 2006, nhdm tac gia Ueno A vd nnk., da phdn ldp duge ehiing Pseudomonas aeruginosa WatG trong dd't d nhilm ddu ed kha ndng phdn buy 51% tong lugng hydrocarbon mach thdng cd trong ddu DO [5]. Ndm 2004, nhdm tac gia Hong va nnk, da phdn ldp duge chung vi khudn
Pseudomonas aeruginosa lUS ed kha ndng phdn buy 60% ddu DO (8500 mg/kg) sau 13 ngay nudi ed'y [2].
O Viet Nam da cd cdng bd cua mdt sd nhdm tac gia vl cde chung vi khudn cd kha ndng sfi dung ddu DO. Ndm 2010, nhdm tac gia tai Phdng Vi sinh vdt Ddu md, Vien Cdng nghe sinh hge da nghien ciiru kha ndng tao chat hoat ddng bl mat tfi chung vi khuan Pseudomonas pseudomalei H24 gifip tang cudng kha nang phan buy ddu DO cua vi sinh vdt tfi mdu cat biin ed kha nang phdn buy 67% va 37% vdi ndng dd ddu ban ddu la 39,2 g/1 [4].
Nhu vdy, so sanh vdi cac chung vi khudn co kha ndng phdn buy ddu DO tren the' gidi vd Viet Nam, chung BTLDS cua chung tdi cd kha nang phan buy ddu kha Idn. Chung vi khuan nay eung da chfing minh uu the' xfi ly ddu trong nudc thai cdng nghiep. Do dd, cd thi bd sung chung nay vdo tap dodn gid'ng tao bun hoat tinh dl ndng eao hieu qua xu ly nudc thai cdng nghilp,
in. K^T LUAN
Chung vi khudn BTLDS cd khudn lac tron, mau trdng due, nhdt, Idi, dudng kinh khoang 2 mm vd tie't ra mdi trudng sac td xanh lue. Dudi kinh hiin vi dien tfi quit, te' bdo chung BTLDS cd dang hinh que ngdn, kich thudc tfi 0,73 - 0,91
^im X 1 - 1,8 |Lim.
Cnung vi Knuan C I L O J co Kna nang pnan hiiy 89,3% ddu DO vdi ham lugng ban ddu Id 28 g/1 sau 7 ngdy nudi ed'y.
Kit qua phdn loai thdng qua viee xac djnh trinh tu nucleotide eua doan gen ma hda 16S rRNA cho thd'y, chung BTLDS cd md'i quan he gdn giii vdi eae loai thude chi Pseudomonas.
Trinh tu nucleotide eua doan gen ma hda 16S iRNA d chung nay ed dd tuong ddng 98% so v6i loai Pseudomonas aeruginosa (X06684). Vi vay, chiing BTLDS dugc ddt ten la Pseudomonas sp. BTLDS. Trinh tu nucleotit eua doan gen ma hda 16S rRNA cua chung ndy da dugc dang ky tren Ngan hang gen qud'c t l NCBI v6i ma sd la JF750922.
Ldi cam an: Bai bdo duge hoan thanh nhd su h6 trg kinh phi tfi dl tai: "Ung dung edng nghe vi sinh dl \u ly nude thai cd ehfia eae hgp chat hull CO hydroeacbon tham da vdng'', ma sd OlC-09/02-2009-2, do Sd Khoa hge vd Cdng nghe Ha Ndi tdi trg.
TAI LIEU THAM KHAO
1. Adeline S. Y. Ting, Carol H. C. Tan and Aw C. S., 2009: Hydrocarbon-degradation by isolate Pseudomonas lundensis UTAR FPE2. Malaysian Journal of Microbiology, 5(2): 104-108.
Hong J. H., Kim J., Choi O. K., Cho K. S.
and Ryu H. W., 2005: Characterization of a diesel-degrading bacterium, Pseudomonas aeruginosa lUS, isolated from oil- contaminated soil in Korea. Worid Journal of Microbiology and Biotechnology, 21:
381-384.
Kaezorek E., Moszynska S., Olszanowski A., 2011: Modification of cell surface properties of Pseudomonas alcaligenes S22 during hydrocarbon biodegradation.
Biodegradation, 22(2): 359-366.
Lai Thuy Hien, Nguyin Thi Thu Huyln, Dd Thu Phuong, Pham Thi Hdng, Vuong Thi Nga, Le Thi Nhi Cdng, Nguyin Thi Yen, Nguyin Bd Tu, Hoang Vdn Thdngi 2010: Nghien cim tao ehd't boat hda bl mat sinh hge tfi vi sinh vat nham ung dung trong eae ngdnh edng nghiep vd xfi ly mdi trudng:
199-209. Hdi nghj Khoa hge ky niem 35 ndm Vien Khoa hge vd Cdng nghe Viet Nam.
Ueno A., Yukiya I., Isao Y., Hidetoshi O., 2007: Isolation and characterization of bacteria from soil contaminated with diesel oil and the possible use of these in autochthonous biogaugmentation. World Journal of Microbiology & Technology, 23(12): 1739-1745.
STUDYING DIESEL OIL BIODEGRADATION OF THE BACTERIAL STRAIN BTLDS ISOLATED FROM INDUSTRIAL WASTEWATER
CUNG THI NGOC MAI, NGUYEN THUY LINH, NGUYEN VAN BAC, VU THI THANH, NGHIEM NGOC MINH
SUMMARY
The bacterial strain BTLDS was isolated from wastewater of Tu Liem industrial zone. This strain had round and smooth colony with 2 mm diameter. The cell morphology of the strain BTLDS was observed under the Scaning Electron Microcopy showed that it was a short rod with 0.73 - 0.91 pm in wide and 1 1.8 pm in length.
The strain BTLDS is able to utilize diesel oil as sole energy and carbon sources. After 7 days in shake cultivation, 89.3% adding oil was removed. The nucleotide sequence of the 16S rRNA gene had been used for taxonomy of the strain BTLDS. The result of the amplification of the nucleotide sequence with the primer 27f/1492r indicated that the strain BTLDS had high homology with the species of the genus Pseudomonas and was close to the Pseudomonas aeruginosa strain (X06684). Based on the morphology and the 16S rRNA gene nucleotide sequence, this strain belong to genus Pseudomonas and named Pseudomonas sp. BTLDS. The nucleotide sequence of the 16S rRNA gene was deposited in the NCBI genbank database whh assession number JF750922.
Ngdy nhgn bdi: 26-4-2011
