HOI NGHI KHOA HOC CONG NGHE SINH HOC T O A N Q U 6 C . 2 0 1 3
I KHA NANG PHANTHDY PHENOL CDAIMNG SINH HOCTSTHON
kN TAO T H A N Ha Nghiem Ngoc Minh
) hgc, Vi0n Hin lim Khoa hgc va Cdng nghg Viet Nam
c hien bao g6m v i ^ xac dinh kha a3ng phan Imy phenol cua m^t so chiing vi khuin tao mang sinh hpc
;n hilnh danK gid kha nang pMn hiiy phenol ciia mang do hon hgp cac chiing nhy t^-th&nli bang phuong phap iiig cao Sp (HPLC). Qua nghiSn CUM, chung toi ^ xSc dinh dirge 04 chung vi khuan tao biofilm co kha nang ien tot trSn nguon co chat la phenol, c6 chilag chju nSng dg len toi 200 ppm. B6 sung 150 ppm phenol vao ic chiing (B2, B8, B15 v^ B16) tao thinh trong mo blnh 5 lit a quy mo phong da nghiSm, sau 07 ngay thi phap do quang da xac ajnh duoc ham lirpng phenol dugc phSn hiiy dat trSn 99%. DS khing dinh ket qui nay, ighiem khae nhau, djch nu8i ciy ciia mang nay da dugc tach chilt va phan tich danh gia bing phuong phap fy. sau 7 ngiy thi nghifm trSn sac ki d6 xuSt hi?n cac san phim trung gian mgt each ro rang va kh6ng c6n phat let qua nay cho thay linh uu viet ciia vi?c svr dyng biofilm do hon hgp cac chimg vi khuSn Ipo thinh trong vieo a xuat klidc ciia phenol gSy f nhiem moi tmong. • JC, phan hiiy sinh hgc, phenol, s5c k^ long cao ap, vi khuin
nhij-ng d?ng chit hOu co cd mgt pho bien nh^t trong tg nhl&n va thudng co trong nguon chit thai 1 nghlfp nhU' hoa hgc, h6a dau, du'g'c phim, d§I, gang th^p,... (Basha et al., 2010). Phenol va eke I du'g'c xep vao logl chit gSy 6 nhilm. BSy la nhdm tucng d6i bin, c6 khS nang tich luy trong co-thl mS<)cc^p tinh, man tinh cho con ngiroi. Khixam nh|p vaoco'thl, cac hgp cfilt nay thu'&n^ g'ayra 10 eke ca quan v^ hk th6ng kh^c nhau nhung chu yeu la tac dgng len h i thin kinh, hg thdng tim ly, vl|c logi bo phenol da va dang dug^ nhilu nh'a khoa hgc trong va ngoai nude quan tam nghlSn phkp da dug'c dua ra, chlng han nhu hip phy (Rengaraj ef al.. 2002), UV/Fe*^(Zhou ef al.. 2001), ig long (Lin etaf., 1999), phSnhOyslnh hQc(yamagaeta/.,201D). Trong s l cdc bl3n phap nay, phan
;em Ik bi^n phdp cd thi lo^i bo ho^n toan phenol khdi nucrc thai l-Ii#n nay, mkng slnh hgc dug'c xem phuo'ng phap phkn huy sinh hgc an toan, hieu qua va than thlgn vol moi trudng. Mkng slnh hgc - ig'p bao g6m nhilu t l bko vi sinh vM ci!ia mdt hoac mgt so loai dugc tao th^nh tr^n m$t be mgt hogc idl trudng nude vk khflng khi (vl du nhu mat nude ao, hi, blln) (Davey. OToole, 2000). Cac nghien rong cac diSu ki^n mfll trudng khic nghiSt, cac vi sinh vat se hlnh thinh biofilm. Nhd do, cac ca the
chju dug'c eke knh hudng hgl, I;? khic nghi|t ciia mdi trudng-. Th&m vao do, nhd qua trinh hinh nh vkt nky co thi hip thf^ du^rc c^cchlt dinh dudng tit hon, tao dugc mil liSn hk glija ckc tk bao i dug'c su cgnh tranh cCia ckc vi sinh v^t khae trong ciing dieu kien mdi trudng song (Kumar, Anand, ig logi bd cac hgp chat hi>u cc n6i chung. phenol noi rieng da duo'c cac nha i^hoa hgc tren t h i gidi u tir lau (Yamaga etal., 2010). Tuy nhien, chuacd nhieu cdng blvlphSn hiiy phenol tir mang slnh bift, cho tdi nay d nude ta eung chua co nhilu bai bao ve phan huy va ctiuyin hda phenol cua
^ c chiing vi khuan tao .thSnh Do do trong bai nay, chung toi trlnh bay kit qua nghidn cdu kha nang hda phenol cua mdng sinh hoe do hdn hg'p 4 chiing vi khuln tgo ra
PHI/ONG PHAP
(VK) sii" dyng trong thi nghifm n^y dugc lay tip bO suu t$p cac chiing vi slnh vat ciia phdng Cdng Tjdng, Vl$n Cflng nghf sinh hgc, Vign Han Lam KH&CN Vi6t Nam. - "
t6ng s6 vk mdi hirdng mull khocing eo ban co tra sung cac nong dd phenol khae nhau (LeThi Nhi
ldp ddnh gid si.r hlnh thdnh mdng sinh hpc cOa Morikawa efa/. (2006)'chiing nufli cly sau 12-16 gid tnJdng dgc hifu. Sau 2 ngay, mang sinh hgc tgo thanh se duge rira bang nude c^t va nhugm vdi
hod tan bing dimetyl sulfoxide (DMSO) hogc add acetic vd do trgn may do quang pho d budc sdng cd mit dd quang hgc cao se dug'c lya chgn:
I sinh trudng vd phat triln cOa cac chOng vi khuan tgo mang sinh hoc trong moi tru-d-ng co ng phap do m|it d9 quang hoc (OD)
1 dugc nufll cly trong mdi trudng muoi khodng Gost (50ml j ed bo sung cdc'ning dd phenol khde 100,150, 200 vd 250 ppm phenol. Sau 1, 3,5 vd 7 ngdy nudi cly, 1 ml-dloh dugc hO! ra de tien hdnh
600 nm de danh gia kha nang sinh tnrdng va phdt triln trdn ngufln eg chat phenol cua chiing.
HQ\ NGHI KHOA HOC C G N G N G H E SINH HQC T O A N Q U 6 C 2013
Xiy dimg md hlnh mang hon hgp da chung (quy m6 5 lit)
Phmmg phdp xdy d^mg mfl hinh da dirge t i l n hdnh theo mfl ta eCia Le Thi Nhi Cong etal. (2012).
Do quang xic djnh ham lugng phenol cdn lai trong thi nghi$m
Mdng sinh hgc da chung sau khi tgo thdnh, dugc n>a b i n g nude e l t vfl t r i n g tia l l n v^ ^ u g c b 6 s u muirkhodng c c bdn ciing 150 ppm phenok SaU 7 ngdy. hdm lugng phenol cfln lai trong tht nghiem d phuong phdp do quang theo tifiu c h u i n ngdnh TCN 102-97 (http://vww.vinachem.com.ynmEU 97,hbnl. Qud trlnh phdn tlch kha nang si> dyng phenol dugc thyc h l | n vdi sg phfli hgp cua Vi§n ^ Ldm KH&CN V1$t Nam.
s i c k9 ling cao ip (HPLC)
D\d\ nu6l c l y cua mdng vdi phenol da dugc phdn tich HPLC trSn mdy Hewlett-PatAard (Bad Hombu hO t h i n g UChroCart 125-4 RP-1B end-capped (5-pm) column (Merek, DamistadL Gennany) cdc san f chuyin hod phenol se d u g c phdn tdch. Nflng dO dung mdi chgy m l u ban d i u gom 30% methanol-70 (0,1%), sau 14 phiit thi dat 100% methanol, tOcdOchay 1 ml/phiit.
K£T QUA VA THAO LUAN
Ddnh gid khd ndng tgo mdng cua cdc chiing v l k h u l n
Cdc chiing vi khuln {k^ hi#u B2, B5. B6, B8, B13. B15, 816) dugc «en hdnh nufli c l y hoat hda Idm t cdc Chung ndy dugc n>a sach vd do OD d l c6 s g d i n g n h l t v l m$t d^ nhu mfi ta d p h l n phuong | khd ndng t?o mdng cua vi k h u l n , cdc chiing ndy dirge nufli trong mdi trudng LB vd ci> sau 24 g i d dugc ri>a vd nhuOm vdi tlm tinh t h i Mdng tgo thdnh ciia cdc chiing ndy sau do dugc bo sung axit ace do OD d budc sdng 570 nm d l xdc djnh df h i p thy ciia mdng. Ket qud sau 24 gid,, khd ndng tgo mi.
dugc t h i hl$n trong Bdng 1.
Bdng 1. Ddnh gtd k h i nSng I90 biofilm cua m$t s6 chung vi khuin Iga chgn bang phuong phap do OD sau ChOng vl khuln Ldn 1 L^n 2 Lin 3 Kh
0.283 0.213 0188 0 369 0.732 0.377 0.162 0.223
0.222 0.645 0.268 0.472 0,183 0 224 0 280 0.281
0.288 0,114 0.260 0.241 0.149 0.233 0,153 0 278 o i l chi>ng
Sau 3 ngdy nghidn,ci>u khd ndng tgo mdng eua cdc chiing cd khd nang phdn hOy PAHs, phenol hogr tai dd Iga chgn dirge 4 chiing vl k h u l n B2, B8,815 vd B16 Id nh&ng chung tao mdng cao hgn hai e h i trdn 15. So sdnh vdi chijng d i i chi>ng duong Id Adnetobader calcoaceticus P23 thi ddy d u g c xem mdng t i t (Yamaga et al.. 2010). Do v$y. ede chung ndy sd dugc lya eh<?n cho cdc nghidn c i i u tiep thet .Ddnh gid khd ndng slnh t n e d n g v d phdt t r i l n ciia cdc chiing v l khuan tgo mang slnh h g c t r o n
phenol bang phucrng phdp do m^ d$ quang hgc (OD)
D l ddnh gid khd ndng sinh trudng vd phdt triln o i a cdc ehiing cfl khd ndng tgo biofilm trdn n g u l n chiing tdi da tidn hdnh nufli I l c cdo chung ndy trong mdi trudng mu6i khodng Gost (50ml) c6 b l sung vd 250 ppm phenol. Sau 1, 3. 5 vd 7 ngdy nudi c l y , 1ml djch dugc hiit ra d l tien hdnh do OD 6 b u d c 7 ngdy thi nghi$m, vdi nflng dO 50 ppm, cdc chung d i u c6 khd ndng phdt triln lot; cfln vdi ndng dd I1 c d c c h u n 9 B 2 , B 8 v d B 1 6 d l u e d k h d n a n g s i n h t a r d n g c a o ( H l n h 1 a & 1 b )
i
t h o i gian (ngay)
• B 3 0615 " B i e
HOI NGHI KHOA HOC C O N G NGHg SINH HQC T O A N Q U 6 C 2013
Dl kilm tra khi ndng phdn hiiy phenol cua MSH da chung vi khu^n. chOng tfli da t?o mdng h;ong binh 50ml c6 b l sung T M p p ^ 7 h e ^ o i . ^ ^ t d c h ^ K c h nufli d y vd sd dyng phuong phdp HPLC d l danh gia sy cfl mjit CL« ^^^^ ^ cdc ttSi * l m th nghifim khde nhau. Kit qua cho thiy, sau 7 ngdy trdn sac ki dfl cua blnh thi nghi?m xuat hi§n cdc sdn
^ l ^ n g l n X S T r d n g vd khfl^ cfln phdt hign co chit phenol (Hinh 5). Kit h ' = ' P . ; - P ^ ^ f P ^^ ^ ° chiing Ifl. thiy ring hdm lugng phenol trong binh thi nghiem da gidm 99%; efln trong binh dli chung. hdm lugng phenol thay dll khflng ddng kl.
Dl xdc djnh sdn phim trung gian Id chit gi. chiing ta cin tiln hdnh phdn tfch GCMS djch nufljcly, tir * i c6 t h | so sdnh vdi thu vi$n chit chuin d l dy dbdn. Sau dd ddt mua hda chit dy dodn vd phan tich GCMS cQng nhu HPLC dg so sdnh Igi thdi gian luu, phi. tn?ng lugng phan ti> oia sdn phim tmng gian vd san phim dy dodn. Cdc nghifin ci>u ndy dang dugc thyc hi§n vd kit qud sd cflng b l trong m$t bdi bdo khae. Con dudng diuyln hda phenol da dudng nhom tdc gid Basha (2010) trlnh bdy chl tilt, cy thi: budc diu.tidn, phenol se dugc hydroxy hda d l tgo thdnh catechol, ti> ddy qud trinh chuyin hfla cfl thi diln ra theo hai ccr chl khde nhau, tiiy thu^c vdo vj tri md vdng Id meta hay ortho. Nlu hgp chdt catechol dugc d t d vj trf meta, en^me catechol 2,3-dioxygenase sd tham gia.dl tgo thdnh sdn pham Id 2- hydroxymuconic semlaldehyde. Sdn phim ndy sau dd sd duge khCr lln lugt tO-ng nhflm hydroxyl (-OH) vd carboxyl (- COOH) d l tgo thdnh 2-oxopent-4-enoate. Sin phim culi ciing cua con dudng ndy Id acelaldehyde vd pynivic acid. Cfln nlu catediol dugc elt d vj tri ortho thi wzyme catechol 1,2-dloxygenase sd chuyin hda catechol thdnh cis.cis-muconic add. Sau dd, cdc enzyme md vflng lactone. Isomarase vd cdc nhflm enzyme transferase se tham gla vdo con duflng chuyin hda ndy. Sdn phim cull cCing eiia con dudng ndy Id acetyl CoA vd suceinyl CoA.
Kit qud thu dugc cho thiy sau 7 ngdy nufli-ITnh, Irong thi nghi$m mdng da chung cfl b l sung 150 ppm phenol, hdm lugng phenol gin nhir dd dugc phdn hiiy vd chuyin hfla hodn todn.
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won OT cnai pnenol ban aSu hen mil hoan to4n, cac sSn phim tnjng
HQ\ NGH! KHOA HOC C O N G NGHE SINH HQC T O A N Q U 6 C 2013
-gian-iculthi§nTdrdncrtrenxdrsac"k:?-dlrCdc-klrqua-nay-cho-pMpxhOngi61i!inh'mrdfTgT'i0xrs^^
d quy md Ign hgn. dong thdi cho thay t l l m nang ciia mdng ndy trong x i i ly cdc hgp chat hydrocarbon thgm khde K^T LUAN
T i r nhOng k i L q u d nhgn d u g e , chiing tfli CO mOt sfi k i t ludn n h u sau. , . .^ . - - = — Da xdc djnh d u g c 4 Chung vi khuan vfli k)? hieu chung B2, 8 8 . B 1 5 v d B16 vira tgo mdng smh hoe t i t vira cd khd ndng phdn hiiy vd c h u y i n hda phenol cao.
Mang slnh hgc do h i n h g p 4 chiing vl khuan cd khd nang phdn hiiy vd chuyen hfla phenol tao thdnh dd cho t h i y cfl hlSu qua xi> Ij phwiol rdt cao trong md hinh 5 lit sau 7 ngdy thi nghi#m. Bang phucmg phdp sac k^ Idng cao dp da xdc djnh hdm l u g n g phenol d u g c phdn hiiy d ^ t l r d n 99% v d i nflng dO ban dau Id 150 ppm
Cdc k i t qua trdn ddy da cho t h i y ede chiing vi k h u l n tao mdng sinh hgc d u g e phdn Igp d Vi§t Nam cfl t i l m ndng irng dung Irong x u \i phenol vd cdc d i n x u l t khae cOa phenol gdy 6 n h i l m mdi t r u d n g .
hid ciim on
Cong oinh dupe thirc hiin vdi sv ho trp lank phi tir de tdi do gwp Phdt triin Khoa hgc vd Cong nghi quic gia cap, ma so 106.03- 2011.53 (04-Khoa hoc stf sdng) vd su dung trang thiet bj Phong Thi nghi§m trong diem Cong nghf gen. Vi^n Cong nghi sinh hpc;
Vifn Hod hgc, Vipt Hdn ldm Khoa hpc vd Cdng ngii? Vi$t Nam
TAI LI$U THAM KHAO
Basha KM. Rajenran A, Thangavelu V (2010) Recent advances in the biodegradaSon of phenol: A rewew. Asian J Exp Biol Sd 1(2):219-234.
Cheng KC, Demircl A and Catchmark JM (2010) Advances in biofilm reactore for production of value - added products. AppI Microbiol Sibtec/inol 87:445-456
Davey ME and OToola GA (2000). Microbial biofilms" from ecology to moletular genetics Microbiol Md BidRev&A-MlSI.
JeriyBJ, Makin SA, KadurugamuwaJL and Zusheng L (1997). Interactions between biofilms and the envlnanmant FEMS Microbiol Rev 20:291-303
Kumar CG and Anand SK (1998) Significance of microbial biofilms in food industry: a review, inter J Food MkmUdA2:S-27.
Lin Shi, Pan CL and Leu HG (1999) Liquid membrane extraction of Z-chlorophenot from aqueous solutions, J Hazard Mater 65, 289- 304.
LeTh^ Nhl Cong, Ho Thanh Huyen, NghiemNgocMinh(2012)Phenotdegradationof a biofilm formed bya mixture of marine bacteria, VNU Journal of Science. Natural Sciences and Techndogy, 28(2SJ'75-81.
Monlcawa M. Kaglhiro S. Haruki M. Takano K, Branda S. Kolter R and Kanaya S (2006) Biofilm fdnnation by a Bacillus subtilis strain thatpipducesgamma-polygiutamate. M'c/Oib/o/152:2801-7.
Rengaraj S, Moon S,-!. Sivablan R. Arabind B and Mumgesan V (2002) Removal of phenol from aqueous soiuHon and resiii' manui^cturing industry wastewater using an agnculturai waste rubber seed coat. J Hazard Mater 89.185-196
Vamaga F. Washio K and Morikawa M (2010). Sustainable biodegradatlon of phenol by Aeinetobacter calcoaceticus P23 isolated from the ihizosphere of duckweed Lemne aoukikusa. Environment Scl Technol, 44;6470-e474
Zhou M, Wu Z end Dahui W (2001) Promoted UV/Fe'^ process for p-nitraphenol degradahon by electrocalalyas. In: Proceedings of The international Water Association Conference an Water and Wastewater Management for Developing Countiies. PWTC, Kuala Lumpur, Malaysia, 29-31 October 2001,1:222-237
http://www.vinachemcom.V(VTIEU_CHUAN/TCN/102-97.htm - . . .
PHENOL DEGRADATION OF BIOFILM FORMED BY MIXTURE OF BACTERIA
Le Thi Nhl C o n g ' a n d Nghiem Ngoc Mlnh
institute of Biotechnology. Vietnam Academy of Sdence and Techndogy
S U M M A R Y
Phenol and other Bromatic compounds are ubiquitously detennined in nature as pollutants originating from oil exploitatian, gas oil transforation and other industrial activities. T o overcome this problem, scientists have used bacteria which have subsequently developed metabolic pathways for purposes of phenol degradation and transformatioB. Using biofikn-forming bactena is considered as new method to degrade and transform these components. In this study, the capacity of phenol degradation b y several biofilm- forming bacterial strains was investigated. After that, the phenol degradation of Inofibn formed b y mixture of these bacteria was also estimated b y UV-absorbance and high performance liquid chromatography (HPLC) mediods As a result, 04 biofilm-forming bacterial strains which could well grow on phenol were selected, 2 strains could grow up to 200 ppm. 150 ppm of phenol was supplemented to biofilm formed by mixture of bacterial suains including B2, B8, B15 a n d B 1 6 in a module 5 lilte in-situ. After 07 days, 99% of phenol was degraded by using UV-absorbance method. To confirm this result, at deferent times of experiments, the cultivation w a s extracted and analyzed b y HPLC. Intermediates were e}qx)sed on the chromatographies during 07 days of expffliments and the substrate - phenol was totally degraded. These results enhance our understanding of using biofilm formed b y mixture of bacteria to degrade and transfomi phenol and Other aromatic compounds; and illustrate the potential a p p l i c ^ o n of this biofilm in the bioremediMJon of these common environmental pollutants.
Keywords: bacteria, biodegradation, biofilm, phenol, high performance liquid chromatography.
'Author for correspondence. Tel: •84-932360809, Fax +84-4-38363J44, E-mail: [email protected]
