vsv phan huy pah

I HC THI NGUYN TRNG I HC S PHM ---------------------------------

L TIN MNH

PHN LP, TUYN CHN V NGHIN CU KH NNG PHN HY SINH HC HYDROCACBON THM CA MT VI CHNG VI KHUN C PHN LP T NC NHIM DU TI QUNG NINH

LUN VN THC S SINH HC

Thi nguyn - 2008

I HC THI NGUYN TRNG I HC S PHM ---------------------------------

L TIN MNH

PHN LP, TUYN CHN V NGHIN CU KH NNG PHN HY SINH HC HYDROCACBON THM CA MT VI CHNG VI KHUN C PHN LP T NC NHIM DU TI QUNG NINHChuyn ngnh: Sinh hc thc nghim M s: 60.42.30 LUN VN THC S SINH HC

Ngi hng dn khoa hc: TS. NGHIM NGC MINH

Thi nguyn - 2008

Li cm n!Trc ht ti xin by t li cm n chn thnh, su sc ti TS. Nghim Ngc Minh tn tnh hng dn v du dt ti trong qu trnh nghin cu v hon thnh lun vn. Trong qu trnh nghin cu va qua, ti nhn c s gip v ch bo tn tnh ca PGS.TS. ng Th Cm H v cc anh ch Phng Cng ngh Sinh hc Mi trng, c bit l KS. m Thy Hng, Thc S Nguyn B Hu nhng ngi gip ti trong qu trnh thc hin lun vn ca mnh. Ti xin chn thnh cm n ban lnh o Vin Cng ngh Sinh hc, Vin Khoa hc v Cng ngh Vit Nam to mi iu kin thun li hon thnh bn lun vn ny. Ti cng xin by t lng bit n su sc ti Ban ch nhim khoa, cc thy c v cc bn ng nghip Khoa Sinh KTNN to iu kin, gip ti trong sut thi gian hc tp v nghin cu ti trng i hc S Phm - i Hc Thi Nguyn. Bn cnh , ti xin cm n nhng ngi thn trong gia nh v bn b to iu kin ng vin gip ti c v vt cht v tinh thn ti c th hon thnh bn lun vn ny. Vi lng bit n su sc, ti xin chn thnh cm n tt c s gip qu bu ! H ni, thng 9 nm 2008 Hc vin L Tin Mnh

S ha bi Trung tm Hc liu i hc Thi Nguyn

http://www.lrc-tnu.edu.vn

LI CAM OANTi xin cam oan y l cng trnh nghin cu ca ring ti, cc s liu, kt qu nghin cu trong lun vn ny l trung thc v cha c ai cng b trong mt cng trnh no khc.

Tc gi

L Tin Mnh



MC LC BNG CH VIT TT ....................................................................1 DANH MC CC BNG.................................................................2 DANH MC CC HNH ..................................................................3 M U .......................................................................................... CHNG I: TNG QUAN TI LIU .....................................................8 1.1 c im c bn ca hp cht hydrocarbon thm a nhn .............8 1.1.1. Tnh cht ha l ..................................................................8 1.1.2 Tnh c ca PAH v nh hng ca n ti mi trng sng .............................................................................................10 1.2. Ngun gc pht sinh PAH ..........................................................13 1.2.1. Hin trng nhim PAH trn th gii v Vit Nam ...........13 1.2.2. Ngun gc pht sinh ............................................................14 1.3. Cc bin php x l ty c PAH ................................................15 1.3.1 Phng php ha l ..............................................................16 1.3.2. Phng php phn hy sinh hc ...........................................16 1.4. Phn hy sinh hc cc PAH bi vi sinh vt ..................................19 1.4.1. Vi sinh vt phn hy PAH ...................................................19 1.4.2. C ch phn hy PAH bi VSV ...........................................21 1.5. Cc yu t nh hng ti qu trnh phn hy cc hp cht hydrocarbon thm a nhn .................................................................25 1.6. Cc phng php phn loi vi sinh vt ........................................29 1.6.1. Phng php phn loi truyn thng ...................................29 1.6.2. Phng php phn loi bng sinh hc phn t .....................30 CHNG II. VT LIU V PHNG PHP NGHIN CU ................33 2.1. Nguyn liu v ha cht .............................................................. 33



2.1.1. Nguyn liu.........................................................................33 2.1.2. Ha cht .............................................................................33 2.2. Mi trng nui cy....................................................................33 2.3. My mc v thit b nghin cu ...................................................34 2.4. Phng php nghin cu .............................................................34 2.4.1. Phn lp vi sinh vt trn mu nc nhim du ......................34 2.4.2. Nghin cu mt s c im sinh hc ca mt s chng vi khun .............................................................................35 2.4.3. nh gi kh nng s dng PAH ca vi khun......................36 2.4.4. Xc nh trnh t gen m ha 16S rRNA v catechol 2,3-dioxygenase............................................................................36 2.4.4.1. Tch chit DNA tng s ca vi khun theo phng php ca Sambrook, Russell ........................................36 2.4.4.2. Nhn on gen bng phng php PCR ........................37 2.4.4.3. Quy trnh bin np v chn dng ..................................38 2.4.4.4. Phng php xc nh trnh t gen bng my t ng ........................................................................................40 2.4.4.5. Phng php xy dng cy pht sinh chng loi ...........41 CHNG III: KT QU V THO LUN ............................................42 3.1. Phn lp v tuyn chn vi khun c kh nng pht trin trn mi trng cha PAH .................................................................42 3.2. c im hnh thi v t bo ca chng vi khun BQN31 .............44 3.3. Kh nng s dng cc loi PAH ca chng vi khun BQN31 .............................................................................................45 3.4. Xc nh trnh t on gen m ha 16S rRNA ca chng BQN31 .............................................................................................49



3.4.1. Tch chit DNA tng s v nhn on gen m ha 16S rRNA bng k thut PCR .......................................................49 3.4.2. Tch dng gen m ha 16S rRNA t chng BQN31 ............50 3.4.3 Tch DNA plasmid v kim tra cc dng khun lc thch hp ......................................................................................52 3.4.4. Trnh t gen 16S rRNA ca chng vi khun BQN31 .............54 3.5. Nhn on gen m ha catechol 2,3 dioxygenase t chng BQN31 .............................................................................................57 IV. KT LUN ........................................................................................62 TI LIU THAM KHO .........................................................................63



Lun vn tt nghip

1

Khoa Sinh - KTNN

BNG CH VIT TTbp USEPA LB PCR POP DNA RNA rRNA VSV X-gal l m PAH Base pair (cp baz) United State Environmental Protection Agency (Cc bo v mi trng Hoa K) Luria-Bertani Polymerase Chain Reaction (phn ng chui trng hp) Persistent Organic Pollutant Deoxyribonucleic acid Ribonucleic acid Ribosomal ribonucleic acid Vi sinh vt 5-bromo-4-chloro-3-indolyl--D-galactopyranoside Microlit Micromet Polycyclic aromatic hydrocacbon (hydrocarbon thm a nhn) ppm n v mt phn triu (mg/l)



Lun vn tt nghip

2

Khoa Sinh - KTNN

DANH MC CC BNGTrang Bng 1.1: Tnh cht vt l ca mt s loi PAH ......................................... 9 Bng 1.2: Mt s chng vi sinh vt c kh nng phn hy PAH ................. 20 Bng 1.3: Mt s phng php phn loi vi sinh vt ................................. 30 Bng 3.1: S lng vi khun phn lp c trn m i trng khong c b sung cc hp cht PAH .................................................................... 42 Bng 3.2. c im hnh thi ca cc chng vi khun ............................... 44 Bng 3.3: ph UV o kh nng phn hy cc PAH ca chng BQN31..................................................................................................... 46 Bng 3.4: Kh nng s dng cc PAH khc nhau ca chng BQN31 .......... 46 Bng 3.5: tng ng ca chng BQN31 so vi mt s i din c cng b trn ngn hng gen quc t .............................................. 56 Bng 3.6: tng ng ca on gen m ha enzyme catechol 2,3 dioxygenase ca chng BQN31 so vi mt s i din c cng b trn ngn hng gen quc t ................................................................... 60



Lun vn tt nghip

3

Khoa Sinh - KTNN

DANH MC CC HNHTrang Hnh 1.1: Cu trc ha hc ca mt s hydrocacbon thm a nhn (PAH) ...................................................................................................... 8 Hnh 1.2: Cc con ng phn hy PAH vi sinh vt ............................... 21 Hnh 1.3: Ba con ng phn hy hiu kh PAH chnh ca vi khun v nm .................................................................................................... 22 Hnh 3.1: Kh nng phn hy phenanthrene ca 4 chng BQN30, BQN31, BQN32, BQN33.......................................................................... 43 Hnh 3.2: Hnh thi khun lc chng vi khun BQN31............................... 45 Hnh 3.3: Hnh thi t bo vi khun BQN31 .............................................. 45 Hnh 3.4: DNA tng s ca chng BQN31 ............................................... 50 Hnh 3.5: Sn phm PCR nhn on gene m ha 16S rRNA ca chng BQN31........................................................................................... 50 Hnh 3.6: Kt qu bin np chng BQN31 ................................................ 51 Hnh 3.7: Sn phm in di kim tra DNA plasmid ca cc dng c la chn ........................................................................................... 52 Hnh 3.8: Sn phm ct DNA plasmid ca dng s 13 ............................... 53 Hnh 3.9: Sn phm lm sch DNA plasmid dng s 13 ca chng vi khun BQN31....................................................................................... 53 Hnh 3.10: Trnh t y on gen 16S rRNA ca chng BQN31 ........... 54 Hnh 3.11: Cy pht sinh loi da trn so snh trnh t cc on gen m ha 16S rRNA ca chng BQN31 v mt s chng vi khun i din. Thc o th hin hai nucleotide khc nhau trn 1.000 nucleotide so snh..................................................................................... 55



Lun vn tt nghip

4

Khoa Sinh - KTNN

Hnh 3.12: Sn phm PCR nhn on gen m ha enzyme catechol 2,3-dioxygenaza t DNA tng s chng BQN31 v cp mi C23OF v C23OR ................................................................................................ 58 Hnh 3.13: Trnh t nucleotide v trnh t axt amin suy din on gen m ha enzyme catechol 2,3-dioxygenaza ca chng Sphingomonas sp. BQN31 ........................................................................ 58 Hnh 3.14: Cy pht sinh loi da trn so snh trnh t cc on gen m ha enzyme catechol 2,3-dioxygenaza ca chng Sphingomonas sp. BQN31 v mt s chng vi khun i din. Thc o th hin mt nucleotide khc nhau trn 100 nucleotide so snh ................................ 59



Lun vn tt nghip

5

Khoa Sinh - KTNN

M UTrong thi i ngy nay, cng vi s pht trin nhanh chng ca x hi, nhu cu v nguyn liu v nhin liu ca con ngi ngy cng tng, ko theo s m rng cc ngnh cng nghip khai thc, ch bin nh : cng nghip du m, khai thc ch bin than, cng nghip sn xut sn, sn xut cc ha cht ty ra... Qung Ninh l mt tnh nm trong vng kinh t trng im Bc B, c tc tng trng kinh t cao so vi bnh qun chung ca c nc. Trong nhng nm gn y, cc ngnh cng nghip v dch v chim mt t trng ln, khong trn 80% GDP ca tnh. Cc ngnh cng nghip khai thc v ch bin than, xi mng, ng tu, nhit in, sn xut vt liu xy dng ang c tc pht trin nhanh. Qung Ninh l mt trong nhng im trung chuyn xng du ln nht c nc. Hot ng vn ti ng b, ng thy; cc hot ng du lch, vn ti khch du lch.din ra ht sc si ng. Cc hot ng trn to ra mt b mt kinh t rt a dng ca Qung Ninh. Tuy nhin, cc hot ng cng gy ra nhng hu qu khng nh v mi trng, c bit l cc hot ng vn chuyn v khai thc than, ch bin v s dng vt liu n, cc hot ng vn ti, trung chuyn xng du Qung Ninh. PAH l mt nhm cc hp cht hu c c hai hay nhiu vng thm. Chng c mt khp ni trong mi trng (t, khng kh, cc ngun nc v cc lp trm tch) v l mt trong cc thnh phn c trong cc sn phm ca du m [16]. Mt s PAH c kh nng gy ung th tim tng, gy t bin v l cht gy nhim mi trng nghim trng. T chc bo v mi trng M (USEPA) xp PAH vo nhm nhng cht nhim in hnh v tin hnh kim sot s c mt ca PAH trong cc h sinh thi di nc cng nh trn cn [16], [50].



Lun vn tt nghip

6

Khoa Sinh - KTNN

Trong nc thi ti nhiu c s sn xut cng nghip Qung Ninh, ngoi mt s hp cht hu c, v c (NH4 NO3) th s c mt ca du v mt s cc hp cht hydrocarbon thm a nhn (PAH) ang l vn ln t ra i hi cc cp chnh quyn a phng cn phi quan tm x l trit . Hin nay khc phc hu qu ny c nhiu phng php c th p dng nh s dng ha cht, hp ph, lng ng Tuy nhin cc phng php ny i hi chi ph ln v vn c th gy ra nhim th cp. Qua th nghim thc t, phng php x l bng cng ngh sinh hc v ang khng nh tnh u vit ca n. l gi thnh r, c th tin hnh thun li trong iu kin t nhin, an ton rt cao v thn thin vi mi trng. Do vy, trn th gii v Vit Nam c nhiu nh khoa hc tp trung nghin cu v iu tra v phn b, cu trc cc tp on vi sinh vt, kh nng phn hy PAH ca cc chng n cng nh cc tp on vi sinh vt. Cc gen tham gia qu trnh phn hy sinh hc PAH cng c quan trc trong qu trnh x l, ca cc tp on VSV bn a v trong cc nghin cu phn hy PAH iu kin phng th nghim. Catechol l mt trong cc sn phm trung gian ca qu trnh phn hy sinh hc cc hp cht vng thm, do vy cc gen m ha cc enzym chuyn ha catechol c nhiu nh nghin cu quan tm. Chnh v nhng yu cu thc tin trn, chng ti la chn nghin cu v thc hin ti Phn lp, tuyn chn v nghin cu kh nng phn hy sinh hc hydrocarbon thm ca mt vi chng vi khun c phn lp t nc nhim du ti Qung Ninh.



Lun vn tt nghip Mc tiu nghin cu:

7

Khoa Sinh - KTNN

Phn lp v tuyn chn ra c mt s chng vi khun c kh nng phn hu hp cht hydrocacbon thm. Ni dung nghin cu ca lun vn bao gm: 1. Phn lp v tuyn chn mt s loi vi khun c kh nng phn hy PAH. 2. Nghin cu, nh gi kh nng phn hy sinh hc PAH ca mt s chng vi khun c phn lp. 3. Nghin cu mt s c im hnh thi t bo ca mt i din vi khun c kh nng phn hy PAH. 4. Phn loi nh tn i din vi khun c kh nng phn hy PAH da trn vic xc nh trnh t on gen m ha 16S rRNA.



Lun vn tt nghip

8

Khoa Sinh - KTNN

CHNG I: TNG QUAN TI LIU1.1 c im c bn ca hp cht hydrocarbon thm a nhn 1.1.1 Tnh cht ha l Cc PAH c mt khp ni trong mi trng, nhng hp cht ny c 26 vng benzen kt hp vi nhau. Trng lng phn t vo khong 128 278 g/mol (Hnh 1.1).

Hnh 1.1: Cu trc ha hc ca mt s hydrocarbon thm a nhn (PAH) PAH l nhng cht k nc. Kh nng gy nhim mi trng ty thuc kh nng ha tan ca chng trong mi trng nc [16], [48]. c im v kh nng ha tan v p sut hi ca PAH l nhn t chnh nh hng n kh nng phn tn ca chng trong kh quyn, thy quyn v sinh quyn.



Lun vn tt nghip

9

Khoa Sinh - KTNN

S lng vng benzen trong cu trc ha hc ca cc PAH quyt nh kh nng ha tan ca cc PAH trong nc. PAH gim kh nng ha tan trong nc hay tng tnh k nc khi s lng vng benzen tng [54]. Kh nng ha tan ca cc PAH rt bin ng, t nhng cht kh ha tan nht l benzo(b)perylen c ch s ha tan l 0,003 mg/l cho n cht d ha tan nht l naphthalen c ch s ha tan ti 31 mg/l. Nu kh nng ha tan trong nc ca PAH thp, hay h s hp ph cao s dn n cc PAH c xu hng b hp ph trong cn bn, t v trm tch, do nh hng rt nhiu ti kh nng chng b phn hy sinh hc bi vi sinh vt [10]. Ngc li, kh nng ha tan trong nc ca PAH cao th kh nng b phn hy bi vi sinh vt cng cao. iu cho thy kh nng ha tan trong nc ca cc PAH c nh hng c bit quan trng trong qu trnh phn hy sinh hc PAH. Bng 1.1: Tnh cht vt l ca mt s loi PAH [31]Nhit S nng chy vng (o C) thm 3 3 4 4 4 4 5 5 6 101 216 111 158 149 255 179 262 222 Nhit si (o C) 340 340 250 400 360 488 496 524 - tan trong nc (mg/l) 1,29 0,07 0,26 0,24 0,14 0,02 0,0038 0,0005 0,0003 p sut hi 20o C (torr) 6,8x10-4 2,0x10-4 6,0x10-6 5,0x10-9 6,8x10-7 6,3x10-7 5,0x10-7 1,0x10-10 1,0x10-10

Loi PAH

LogKp 4,45 4,46 5,33 5,61 5,32 5,61 6,04 5,97 7,23

d

Phenanthrene Anthracene Fluoranthene Benzo[a]anthracene Pyrene Chrysene Benzo[a]pyrene (BaP) Dibenzo[a]anthracene Benzo[g,h,i]perylen

* Kp d= [octanol]/ [nc] Trong cc tnh cht vt l ca PAH, h s Kp d phn nh kh nng hp ph ln b mt vt liu rn. Nu h s Kp d cao, cc PAH c xu hng tng



Lun vn tt nghip

10

Khoa Sinh - KTNN

kh nng hp ph ln b mt cc vt liu rn, ng ngha vi s gim kh nng phn hy sinh hc. p sut hi v nhit si ca cc PAH cng c vai tr quan trng trong qu trnh x l loi b PAH khi cc vng b nhim do n nh hng n kh nng ha hi ca mi PAH. Khi p sut hi tng, kh nng bay hi ca cc PAH cng tng, m s bay hi cng l mt con ng loi b PAH khi ngun nhim. Kh nng bay hi ca cc PAH cng ph thuc vo kch thc v khi lng phn t. Naphthalene c kch thc nh nht nn c kh nng bay hi n 89%, trong khi benzo[a]pyrene (BaP) l hp cht c kch thc ln, ch c kh nng bay hi 1%. Phenanthrene l ng phn ca anthracene c bay hi thp hn do cu trc phn t cha cc vng thm khng thng hng nh trong cu trc ca anthracene (Hnh 1.1 v Bng 1.1). PAH cn b phn hy di nh sng t ngoi t bc x mt tri. Trong kh quyn, PAH c th phn ng vi nhng cht nhim nh nitrogen oxid, sulfur oxid to thnh cc dng dione, nitro v dinitro PAH v sulfonic acid. 1.1.2 Tnh c ca PAH v nh hng ca n ti mi trng sng Tnh c ca PAH c ngi ta bit n t nhng nm 30 ca th k XX, khi Hieger v Cook cng nhng cng s khc nghin cu v thy tinh th benzo(a)pyrene mu vng gy khi u ng vt th nghim [17]. Vi con ngi, PAH c th l tc nhn gy t bin v dn n ung th [16], [31], [11], [27]. quy m phng th nghim, mt s nghin cu ch ra rng, qu trnh gy ung th bi PAH l mt qu trnh phc tp, a giai on v ph thuc vo nhiu yu t : kch thc phn t PAH, phn cc trong phn t, ha hc lp th v cc phn ng xy ra trong qu trnh trao i cht, cc nguyn t mang in tch m c nh hng n s gn kt ca cc sn phm trao i cht vi cc i phn t l DNA, RNA. Mt s nghin cu



Lun vn tt nghip

11

Khoa Sinh - KTNN

khc a ra kt lun: cc PAH c khi lng phn t nh, cu to phn t ch c mt, hai, ba vng thm l rt c, trong khi cc PAH c khi lng phn t ln li c th gy c mc gen, hoc gy ra t bin, bi chng c kh nng gn vo cc phn t DNA, RNA, hoc protein, gy nn nhng bin i mc phn t [16], [42]. Naphthalene l mt cht nhim thuc nhm PAH gy nh hng ti mt lot cc c quan nh phi, thn v km hm qu trnh h hp. Nhim c naphthalene ngi dn ti bnh thiu mu v vim thn. Ngoi ra, s thay i v da v mt nhng ngi b phi nhim naphthalen cng c cng nhn. Phenanthrene c bit nh cht cm quang vi da ngi, cht gy d ng vi ng vt, t bin ti h thng vi khun trong cc iu kin c bit. Cht ny gy yu cc nhim sc th tng ng v km hm s ni lin cc k h gian bo. Ngoi ra, cc PAH khc nh acenaphthalene, fluoranthene, fluorene u gy c cho ng v thc vt. c tnh ca benzo(a)pyrene, benzo(a)anthracene, benzo(b)fluoranthrene, benzo(k)fluoranthrene, dibenzen (a,h)anthracene v indenol(1,2,3-c,d)pyrene c nghin cu chng minh gy ung th cho ngi. Trong t nhin him khi bt gp cc PAH n l m ch gp chng dng hn hp nhiu PAH, do c tnh ca chng cng c tng cng [53]. PAH thm nhp gin tip vo c th con ngi thng qua chui thc n, ng h hp hoc qua s tip xc trc tip vi ngun nhim, gy ung th, t bin gen. Rt nhiu PAH c cha vng bay v vng tng t n vng K. C hai vng ny u cho php trao i cht, to ra dng lin kt epoxit-vng bay v epoxit- vng K c kh nng hot ng cao v mt s chng l nguyn nhn gy ra ung th [53]. Khi xm nhp vo c th, PAH nhanh chng xm nhp vo cc m m v tip tc di chuyn n nhng c quan khc. Ty tng loi PAH vi liu



Lun vn tt nghip

12

Khoa Sinh - KTNN

lng v thi gian tc ng m mc nh hng n c th khc nhau. Chng hn, vi naphthalene, nu tip xc trong thi gian ngn, nng thp, n c th gy d ng, vim ty da, mt. Khi xm nhp vo h tiu ha, naphthalene s gy bnh thiu mu do chng ph v cc t bo hng cu. Nu tip xc vi naphthalene trong thi gian di vi nng ln hn 10 ppm s dn ti cc bnh kinh nin, gy ung th da phi v c th lm gim kh nng th thai ph n v c th lm nguy him ti s pht trin ca thai nhi [31]. Trong s cc PAH, ngi ta c bit ch n benzo[a]pyrene v tnh c hi ca n. Benzo[a]pyrene (BaP) l mt thnh phn c trong khi thuc l, v l mt trong nhng nguyn nhn dn n ung th phi [16], [31]. Nhiu nghin cu chng minh BaP c th chuyn ha thnh cc loi oxid vi s xc tc ca phc h cytochrome P450, m nhng oxid ny c th phn ng vi cc DNA gy t bin. BaP cng c xc nh l nguyn nhn gy ung th cho con ngi v ng vt [16], [31], [55]. Mt vi nghin cu trn i tng ng, thc vt cho thy, ng vt nu tip xc vi naphthalene nng cao, th ch trong thi gian ngn cng c th gy m mt, gy c mc va phi. Hiu ng mnh hn, naphthalene c th gy chm pht trin, thm ch gy cht vi ng thc vt. Nghin cu ngng c ca naphthalene i vi loi c vc, ngi ta xc nh LC50 l 240 g/l (LC50 l liu gy cht 50% mu sinh vt th nghim). Bng vic th nghim vi mt nhm chut cho s dng anthracene vi lng 1,8 g/l, ngi ta thy rng, sau 2 tun gy nhim, t l chut xut hin khi u l 40% [31]. S tn ti ca cc hp cht PAH trong mi trng vng b bin c th e da ti sc khe con ngi v mi trng bin thng qua cc tc ng trc tip hoc qua cc chui thc n trong chu trnh vt cht [61].



Lun vn tt nghip

13

Khoa Sinh - KTNN

Do tnh c hi nh vy, c quan bo v mi trng M (USEPA) xp PAH vo danh sch mt trong nhng cht nhim in hnh v tin hnh kim nh s c mt ca PAH trong h sinh thi di nc cng nh trn cn [16], [53]. 1.2. Ngun gc pht sinh PAH 1.2.1. Hin trng nhim PAH trn th gii v Vit Nam Cc hydrocarbon thm a nhn c tm thy nhiu mi trng sinh thi khc nhau, k c mi trng khng kh. Trong mi trng nc, PAH phn b rng ri. Ngi ta c tnh, hng nm c khong 2,3x105 tn cc hp cht ny xm nhp vo cc h sinh thi di nc. c bit, vi h sinh thi b nhim PAH vng ven b bin thng c ngun gc t ph thi v cng nghip ha du, cng nghip khai thc v vn chuyn du m, nc thi cng nghip v sinh hot, chy rng v chy ng c [61]. Ngi ta nh lng c nng BaP trong nc ung l 0,0002 n 0,024 g/l. Trong 90 mu nc kim tra M cho thy nng ca 6 PAH t 0,001 n 0,01 g/l, 1% mu nc kim tra cho thy nng trung bnh ln hn 0,1 g/l [31]. Nhiu cng trnh nghin cu cn cho thy, s c mt ca PAH trong cc mu trm tch vi nng ng k. Mt s ni c mc nhim PAH rt cao: nng cc hp cht PAH vnh Boston (M) c th ln ti 100.000 ng/g. Nguyn nhn ch yu l do trong mi trng bin hu ht cc hp cht PAH ha tan km trong nc dn n vic chng c tch ly trong cc lp trm tch [31]. Trong khng kh cng cha mt lng ng k PAH. c hn 500 PAH v cc hp cht lin quan c pht hin trong khng kh, c bit l BaP chim t l cao nht. Vo nhng nm 1970, M, nng BaP trong khng kh trung bnh l 1- 5 ng/m3. Trong 30 nm tr li y, nng BaP c



Lun vn tt nghip

14

Khoa Sinh - KTNN

xu hng gim, tuy nhin con s ny hin nay vn cn cao, khong 4 ng/m3 Copenhagen (an Mch). Trung Quc, vic t than trong cc h gia nh to ra mt lng BaP khong 14,7 g/m3, n l 4 g/m3 [31] Ngi ta cng tm thy hydrocarbon thm a nhn nhiu trong t b mt. Nng ca PAH trong t rng rt dao ng (t 5 - 100 g/kg), m ngun ch yu l t xc thc vt do qu trnh chy, hay hp ph PAH t khng kh. Vi t nng nghip, mc nhim PAH t 10 n 100 g/kg, ch yu do ma lm ngng t cc hp cht PAH t kh quyn v i vo t. Nu tnh cho c t rng v t nng nghip, lng nhim trung bnh l 1000 g/kg. i vi t b nhim du, nng nhim ln hn nhiu so vi vng t rng v t nng nghip, bi PAH l mt thnh phn chnh c trong du m [55]. Trong t thnh th, nng PAH khong 600 3000 g/kg, v c th cao hn na nhng vng c cc hot ng vn ti v sn xut cng nghip [55]. 1.2.2. Ngun gc pht sinh C nhiu nguyn nhn dn n s pht sinh ca PAH, nhng chng u c bn cht chung l do cc qu trnh t chy khng hon ton cc vt liu hu c trong t nhin hay cho hot ng sn xut ca con ngi. Trong t nhin, PAH c pht sinh l t cc qu trnh a cht t nhin, ha lng kh than, cc v chy ng c, chy rng thm ch trong cc trn ma [16], [28], [31], [38]. Con ngi cng c th gin tip hay trc tip lm pht sinh PAH vo mi trng t vic t nhin liu (than , du diesen, ...) trong sinh hot hay trong sn xut (t nhin liu cho cc phng tin my mc c ng c, qu trnh t rc ti cc nh l t). Cc v trn du do s c hay trong qu trnh vn chuyn cng lm pht sinh mt lng ng k PAH vo mi trng [31].



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15

Khoa Sinh - KTNN

Ti cc nc cng nghip pht trin, cc hot ng c lin quan n t chy to ra mt lng ln PAH. Cc PAH ny c tch t li trong t v lm tng nng PAH nhanh chng trong vng 100 -150 nm tr li y [31]. PAH l nhm hp cht c, nhng chng li kh quan trng trong cng nghip, c bit trong cc ngnh cng nghip dc, nhum, sn xut nha v sn xut thuc tr su [31]: naphthalene, anthracene, phenanthrene thng c s dng trong cng nghip sn xut thuc nhum. Naphthalene cng c dng nh thuc xng dit bm trong cc gia nh. c bit, phenanthrene cng c ng dng trong cng nghip dc dng sn xut thuc ng. New York (M), hn hp phenanthrene v anthracene c s dng chng r cho nhng thit b tr nc [31]. Benzo[a]pyrene c s dng hn ch mui v khi, trong cng ngh qut hc n, hay cn s dng lin kt cc phn tch in li vi nhau. Ngi ta cng c th tm thy trong hp cht Creosote, mt cht ha hc c s dng lm cht bo qun g (PAH chim n 85% khi lng ca creosote) [20], [31]. BaP cng c s dng l tc nhn gy t bin ln ng vt th nghim, kim tra c tnh gy ung th ca n trong thi gian ngn [31]. 1.3. Cc bin php x l ty c PAH Tnh cht c hi v cu trc kh b phn hy trong t nhin ca PAH tr thnh mt vn cp bch ang c ch . Ngi ta v ang nghin cu nhiu bin php ha hc, l hc, sinh hc v.vnhm phn hy PAH. Vic lm thay i cu trc ha hc ca cht c nhm to ra cc sn phm t c hoc khng c cho mi trng v con ngi ang l mt thch thc i vi cc nh khoa hc v cng ngh. gim c tnh ca cc cht ny, ngi ta hay ph v cu trc phn t ca chng bng cch s dng enzyme ct vng. n nay, trn th gii, ngi ta a ra mt s phng



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16

Khoa Sinh - KTNN

php tiu c nh: c lp, chn lp, x l ha hc, t nhit cao, l hc, sinh hc v.v. 1.3.1 Phng php ha l Trong cu to ha hc ca PAH, do c cu trc vng thm, PAH tng i kh phn hy trong t nhin. Phng php chn lp hay c p dng i vi nhiu cht thi, rc thi, k c cc cht thi nguy hi trong c cht c ha hc [22]. u im ca phng php ny l khng tn km nhng nhc im l cc cht c vn nm trong t ch khng c phn hy, cc cht c ha hc ny s l ngun tim tng gy nhim cho mi trng. Cng ngh thiu t cng c s dng trn th gii, phng php x l ny tng i trit song gi thnh li cao v c kh nng gy nhim th cp bi cc sn phm ph to trong qu trnh vn hnh. Cc phng php ha hc nh declo ha, oxy ha, phng php vt l nh quang ha, s dng tia bc x, tia cc tm, hay p sut cao cng mang li hiu qu nht nh. Theo Draper v cng s (1987), x l bng phng php quang ha, 80% cht c b phn hy di tc ng ca chm tia cc tm cng 20 W/cm3 nhit 20oC trong thi gian 3 ngy [25]. Tuy nhin, nhng phng php trn c nhc im l khng c tc dng vi lp t c su di vi milimet, do ch x l c lp t rt mng trn b mt [49]. Mc d lm sch PAH c th c tin hnh bng nhiu bin php l ha nu trn, nhng cc phng php ny c nhc im l gy nhim th cp nn khng an ton v khng x l trit . Mt khc, gi thnh ca vic s dng cc phng php l i cao nn xu hng s dng chng ngy cng gim. 1.3.2. Phng php phn hy sinh hc Hin nay, phng php sinh hc ang bt u c quan tm bi tnh an ton v hiu qu khng nhng v mt cng ngh m cn v kinh t. Cha



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17

Khoa Sinh - KTNN

kha ca cng ngh phn hy sinh hc l thc y tp on vi sinh vt bn a tham gia vo qu trnh phn hy mc cao nht. Cc nghin cu c bn u nhm mc ch thc y hiu qu qu trnh ty c thng qua vic kch thch tp on vi sinh vt trong cc iu kin phn hy khc nhau to ra kt qu cui cng l cc sn phm t c hoc hon ton khng c. Chnh v vy, cng ngh phn hy sinh hc tr thnh cng ngh thn thin vi mi trng. Phng php phn hy sinh hc c cc nh khoa hc trn th gii nghin cu v p dng trong nhng nm gn y v cng t c kh nhiu thnh tu. Cng ngh sinh hc m bo an ton cho mi trng hn tt c cc cng ngh khc. c bit, trong iu kin sinh thi a h, vic p dng cng ngh phn hy sinh hc PAH ni ring v cc ngun cht c ni chung s mang li hiu qu kinh t x hi cao nht [59], [61]. Qu trnh lm sch sinh hc c th thc hin quy m ln nh khc nhau v iu kin hiu kh hoc k kh. Vic ty c bng phn hy sinh hc c th c tin hnh ring r hoc kt hp vi cc phng php khc, sau vi thng hoc vi nm cc cht nhim c th c hon ton loi b. Phng php phn hy sinh hc khng i hi cc iu kin phc tp (nhit cao, p sut ln, qu trnh xc tc) khng gy ra nhim th cp, thn thin vi mi trng, chi ph thp, do rt ph hp vi iu kin nc ta. Tuy nhin, phng php sinh hc thng din ra vi tc chm, thi gian x l ko di. y chnh l mt nhc im c bn ca n, i hi c li gii p t pha cc nh khoa hc. X l cht nhim theo phng php sinh hc c th c tin hnh theo hai hng chnh: tng cng sinh hc v kch thch sinh hc. Tng cng sinh hc l phng php s dng tp on vi sinh vt bn a c lm giu hoc vi sinh vt s dng cc cht c t ni khc, thm ch vi sinh



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Khoa Sinh - KTNN

vt c ci bin v mt di truyn b sung vo cc mi trng b nhim. Tuy nhin, vn cn c nhng kh khn trong vic b sung vi sinh vt vo cc ni b nhim do chi ph ln; hiu qu phn hy nhiu khi khng cao do nhiu nguyn nhn (s cnh tranh ca vi sinh vt, c ca mi trng; s thiu ht ngun dinh dng, cc cht a lng v vi lng cn thit cho hot ng phn hy ca vi sinh vt) [26]. Kch thch sinh hc l qu trnh thc y s pht trin, hot ng trao i cht ca tp on vi sinh vt bn a c kh nng s dng cc cht c hi thng qua vic iu chnh cc yu t mi trng nh pH, m, nng O2, cht dinh dng, cc c cht, cc cht xc tc v.v. Kch thch sinh hc hin l khuynh hng c s dng rng ri trong x l nhim theo phng php phn hy sinh hc [26]. Trong hot ng sng, vi sinh vt cn nguyn t N, P, mt s cht dinh dng khc v cc iu kin sng thch hp. T ngun nhim, ngi ta c th phn lp nhng chng vi sinh vt c kh nng s dng PAH, nghin cu cc c tnh sinh l, sinh ha ca chng t tm ra iu kin sng ti u ca chng t ng dng cho vic kch thch hot ng sng ca tp on vi sinh vt bn a trong vic phn hy sinh hc PAH ti vng nhim. tng cng qu trnh phn hy sinh hc, vic b sung cc ngun dinh dng nh ngun cacbon, nit, photpho theo phi theo t l nht nh cp phn trn l C:N:P = 100:10:1. Ngoi ra, cc yu t mi trng cng cn iu chnh thch hp, m bo cho tc phn hy mc n nh v t hiu qu cao nht. i khi ngi ta cng kt hp c hai bin php c th tng cng s phn hy sinh hc. C ngha l va b sung cc chng vi sinh vt nui cy c kh nng phn hy cht nhim, ng thi cng to iu kin ti u cho tp on vi sinh vt bn a hot ng. Nh vy, hot ng ca tp on vi sinh



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Khoa Sinh - KTNN

vt bn a cng vi hot ng ca vi sinh vt ngoi lai s tng cng hiu qu ca qu trnh x l [26]. Qu trnh lm sch sinh hc c th thc hin vi cc quy m khc nhau v iu kin hiu kh hoc k kh. Vic ty c bng phn hy sinh hc c th tin hnh ring r hoc kt hp vi cc phng php khc. Sau khong thi gian nht nh (vi thng hoc vi nm), cc cht nhim c th c loi b hon ton [23], [32]. bo v s a dng vi sinh vt v an ton i vi mi trng, cn c s gim st cht ch khi a cc vi sinh vt t ni ny sang ni khc x l nhim, nht l vi cc vi sinh vt c chuyn gen. qu trnh b sung vi sinh vt t hiu qu, cn phi b sung thi im mi b nhim v vi lng ln vi sinh vt trng thi sinh trng tt trc khi cc vi sinh vt bn a kp thch nghi, tn cng v lm thay i cu trc c cht. Tuy nhin, vic ln men vi sinh vt vi khi lng ln l iu khng d dng v v cng tn km, mt khc cng khng m bo vi sinh vt gi nguyn c cc c tnh phn hy nhim [26], [27]. 1.4. Phn hy sinh hc cc PAH bi vi sinh vt 1.4.1. Vi sinh vt phn hy PAH Hin nay, c nhiu nghin cu v kh nng ca vi sinh vt s dng cc PAH c trng lng phn t thp nh naphthalene, phenanthrene v anthracene. Tuy nhin, cha c nhiu nghin cu v tim nng phn hy cc PAH c trng lng phn t cao nh chrysene v benzo[a]pyrene [16]. Vi sinh vt phn hy PAH phn b rng ri trong t nhin. S lng cc vi sinh vt c kh nng phn hy PAH ti cc vng nhim nhiu hn so vi cc vng khng nhim. Cc loi vi sinh vt trong vng nhim c xu hng thch nghi, bng cch thay i cu trc di truyn hng n vic phn hy PAH. Vi khun c vai tr quan trng trong tham gia phn hy sinh



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Khoa Sinh - KTNN

hc PAH trong nc v trm tch, trong khi , nm si v x khun ng vai tr quan trng phn hy PAH v cc cht nhim trong mi trng t [27]. Thng k bng 1.2 cho thy, vi sinh vt phn hy PAH thuc nhiu nhm vi sinh vt khc nhau [3], [6], [11], [27], [33], [42], [51], [60], [58], [61], [62]. Cc vi sinh vt c kh nng chuyn ha PAH phn ln thuc vi khun, vi khun lam v mt s vi to [16], [38], [58], [39], [60]. Bng 1.2: Mt s chng vi sinh vt c kh nng phn hy PAH Vi khun Acinetobacter sp. Aeromonas sp. Alcaligenes denitrificans Arthobacter sp. Bacillus cereus Beijerinckia sp. Cycloclasticus Corinebacterium renale Flavobacterium sp. Micrococcus sp. Mycobacterium sp. Staphylococcus auriculans Pseudomonas stuzeri Rhodococcus sp. Sphingomonas. sp Staphylococcus auriculans Vi khun lam Agmenellum quadruplicatum Anabena sp. CA Amphora sp. Aphanocapsa sp. Chlorella autotrophica Chlamydomonas angulosa Coccochloris elabens Cylindrotheca sp. Dunaliella tertiolecta Microcoleus chthonoplastes Navicula sp. Nostoc sp. Phorphyridium Scapricomutum Synedar sp. Ulva fasciata Vi nm Aspergillus sp. FVX5 Basidiobolus ranarum Bjerkandera adusta Candida maltosa Chrysosporium pannorum Claviceps paspali Gliocladium sp. Helicostylum catenoides Linderina pennispora Mucor hiemalis Penicillium chrysogenum Phycomyces blakesleeanus Ramaria sp. Rhizopus stononife Sordaria fimicola Trchoderma viride



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Khoa Sinh - KTNN

Trong mt thng bo tng quan gn y v cc phn hy sinh hc PAH ca Johnsen (2005) cho thy Sphingomonas, Burkholderia, Pseudomonas v Mycobacteria l cc vi khun chim u th [36]. 1.4.2. C ch phn hy PAH bi VSV Phn hy sinh hc cc PAH c th din ra theo hai c ch trao i cht v ng trao i cht. Qu trnh chuyn ha PAH bi vi sinh vt c th phn hy thnh cc dng khng c hoc chuyn ha hon ton thnh CO2. Cerniglia cho rng qu trnh phn hy PAH c th theo ba chiu hng: phn hy hon ton, ng phn hy v oxi ha khng c hiu [16]. Cerniglia (1993) tm lc phn hy PAH bi vi sinh vt theo 3 nhm chnh (hnh 1.2). Theo Cerniglia, catechol l cht trung gian ca phn hy sinh hc PAH bi vi khun v to, tip theo catechol li c ct vng v tr ortho v meta [16].Vi nm, to

Cyt P-450/Methan Monooxygenase

Nm trng PAH Ct vng

Lignin peroxidase, laccaseVi khun, to

Dioxygena se

C t

Dehydrogena se

C t

Hnh 1.2. Cc con ng phn hy PAH vi sinh vtS ha bi Trung tm Hc liu i hc Thi Nguyn http://www.lrc-tnu.edu.vn

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Khoa Sinh - KTNN

Suntherland (1995) cng a ra s tng t v cc con ng phn hy PAH bi vi khun v nm (Hnh 1.3)[51]. C th, cc PAH c phn hy bi vi sinh vt nh: - Chuyn ha PAH n cis-dihydrodiol, phenol v cc sn phm ct vng bi vi khun v vi khun lam - Chuyn ha PAH n phenol bi vi khun nhm methylotrophic - Chuyn ha PAH n trans-dihydrodiol bi vi nm, vi khun, v vi khun lam - Chuyn ha PAH n quinon bi nm mc trng (white-rot fungi)

Hnh 1.3. Ba con ng phn hy hiu kh PAH chnh ca vi khun v nm Chuyn ha PAH n trans-dihydrodiol bi vi nm, vi khun v vi khun lam. Hin nay ngi ta bit n cc enzyme cytochrome P450 monooxygenase c sinh ra bi mt loi vi nm, vi khun v vi khun lam.



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Khoa Sinh - KTNN

Cc enzyme ny tham gia chuyn ha PAH n dng aren oxit, sau dng hp cht trung gian ny c hydrat ha bi enzyme epoxyt hydrolase n dng trans-dihydrodiols hoc c ti sp xp khng c s tham gia ca enzyme to thnh dng phenol. Trong trng hp cc vi sinh vt ch c th thc hin theo cch chuyn ha ny, th chng s khng s dng PAH nh ngun cacbon m ch c th loi b tnh c ca PAH [16], [51]. Chuyn ha PAH n quinon bi nm trng (white- rot fungi): Mt s nm trng phn hy lignin v cellulo (c trong g) s chuyn ha PAH n quinon v cc cht khc m khng qua cis-dihydrodiol hoc transdihydrodiol, trong mt s trng hp qu trnh chuyn ha ny c s tham gia ca lignin peroxydase [16], [51]. Nhiu nghin cu cho thy, trong vi khun, cc gen m ha cc enzym tham gia vo qu trnh chuyn ha PAH c th nm trn plasmid hoc chromosome [29]. Trong hai thp k va qua, mt nhm cc gen bo th cao d ha PAH (cc gen ging nah) t cc loi Pseudomonas c iu tra y bao gm c quan h chc nng-cu trc v tin ha ca cc gen ny [29]. Tuy nhin, gn y cc gen d ha PAH m c s khc nhau v tin ha i vi gen ging nah c nghin cu cc vi khun Gram m khc v c vi khun Gram dng [29]. Theo Wikstrom (1996), bc u tin ca phn hy sinh hc hiu kh PAH ph thuc c s tham gia ca h enzyme a thnh phn xc tc s hydroxyl ha cc hp cht PAH v to ra dng cis-dihydrodiol [56]. Mt s tc gi khc cho rng, cc con ng phn hy hydrocarbon thm trong iu kin hiu kh u xy ra qua cc bc ct vng thm, loi vng theo con ng phn trn cng (upper pathway) v to thnh catechol [56], [46], [44]. Tip theo, qu trnh ct vng ca catechol bi enzyme dioxygenase c th xy ra ti cc v tr meta v ortho. Ct vng catechol v tr ortho c



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Khoa Sinh - KTNN

thc hin bi enzym catechol 1,2-dioxygenase to ra cis, cis-muconic acid, cht ny sau c phn hy theo con ng -ketoadipate [34]. Ct vng ti v tr meta ca catechol c thc hin bi enzyme catechol 2,3dioxygenase (C23O) to ra 2-hydroxymucomic semialdehyde [34], y c coi l qu trnh ph bin nht cc giai on tip sau con ng pha di (lower pathway) ca qu trnh phn hy cc PAH [56], [45], [44]. Enzyme catechol 2,3-dioxygenase l thnh vin ca lin h enzyme Extradiol dioxygenase. Cc enzyme extradiol dioxygenase c xem nh l cc enzyme cha kha trong rt nhiu con ng phn hy cc hp cht thm bi vi khun cng nh cc phn ng c xc tc bi cc enzyme ny [46]. Do v tr quan trng v trung tm ca catechol nn gen m ha cho enzyme catechol 2,3- dioxygenase c quan tm c bit trong cc nghin cu nh gi cng nh pht hin ra kh nng phn hy PAH ca cc chng vi sinh vt, tp on vi sinh vt ti cc im nhim hydrocarbon du m [45]. c rt nhiu nghin cu khai thc cc gi tr tim tng ng dng v l thuyt ca C23O trong bo v mi trng v trong cc lnh vc khc. Cc enzyme extradiol dioxygenase c hot tnh tt s nh hng trc tip n s gia tng cc hot ng ng ha PAH. Tnh n nm 2004 c trn 30 enzym C23O v nhiu trnh t nucleotide ca cc gen m ha C23O c xc nh [34]. Hin nay, cc nh nghin cu vn ang khai thc cc kha cnh ng dng v hc thuyt ca C23O v gen m ha C23O trong cc chng n, tp on v trong qu trnh phn hy cc cht vng thm t nhin v trong x l lm sch bng phn hy sinh hc. PAH l cc cht gy nhim quan trng trong nc ngm. Cc PAH c 2 v 3 vng c quan tm c bit do kh nng ha tan cao hn trong nc v d di chuyn vo nc ngm [43]. Hin nay, c rt t cng b v chuyn ha PAH bi vi khun k kh. Mc d naphthalene v acenaphthalene



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Khoa Sinh - KTNN

c loi b (rt t) bi hn hp vi khun kh nitrat, tuy nhin c ch trao i cht ca qu trnh cha c sng t. Gn y, Coates v cng s cng b v kh nng oxy ha PAH n CO 2 di iu kin kh sulphat ca cc mu trm tch vnh San Diego [18]. Tuy nhin qu trnh ny i hi nhiu thi gian v tc rt chm [16]. Phn hy k kh PAH c nghin cu vi NO3-, ion Fe hoc SO 42nh l cc cht nhn in t v di cc iu kin sinh methan. Cc con ng phn hy sinh ha c nghin cu vi cc tp on vi khun hoc chng sch phn hy napthalene v cho thy, 2-naphthoic acid l cht trao i cht trung gian. Naphthalene c hat ha bi b sung n v C1 to ra 2-naphthoic acid, trong khi methylnaphthalene c hot ha bi b sung fumarate i vi nhm methyl v c phn hy tip thnh 2-naphthoic acid. Phn hy 2-naphthoic acid c thc hin qua kh v ct vng to ra 5,6,7,8-tetrahydro-2-naphthoic acid. Sn phm trung gian ct vng nh l 2carboxyclohexylacetic acid cho thy s phn hy din ra qua cc dn x ut cyclohexan ch khng qua cc hp cht thm. Phn hy k kh PAH cng c chng minh trong mi trng nc nhim bng xc nh cc hp cht trao i cht c hiu v nghin cu ng v phng x. Cc sn phm trao i cht c hiu phn hy k kh PAH nh naphthyl-2-methylsuccinate c pht hin chng t s phn hy k kh ca 2-methylnaphthalene, trong khi 2-naphthoic acid l du hiu ca phn hy k kh naphthalene v 2methylnaphthalene [43]. 1.5. Cc yu t nh hng ti qu trnh phn hy cc hp cht hydrocarbon thm a nhn S phn hy sinh hc PAH ph thuc rt nhiu yu t. Ngoi cc yu t mi trng nh pH, nhit , dinh dng, m ca t, nng oxy, n cn



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Khoa Sinh - KTNN

ph thuc vo tnh cht vt l, ha hc ca cc PAH, s c mt ng thi hay ring r ca cc PAH trong mi trng. Ngoi ra n cn ph thuc vo bn thn cc vi sinh vt, phng thc m cc vi sinh vt chuyn c cht qua mng t bo. Thng nhng phn t tan c th c vn chuyn qua mng t bo v c kh nng phn hy sinh hc tt hn [16], [51], [31], [42], [61]. Cc yu t mi trng ti ni m vi sinh vt c phn lp nh hng rt ln n s pht trin ca chng. Do vy, trong qu trnh x l lm sch mi trng, vn ny ng vai tr quan trng v quyt nh hiu qu ca vic x l. Qu trnh phn hy cc hp cht hydrocarbon thm a nhn bi cc vi sinh vt thng xy ra vi tc chm, do vy vic to iu kin thch hp cho tp on vi sinh vt pht trin tt nht, c hiu qu phn hy sinh hc cao c th coi l cha kha ca cng ngh phn hy sinh hc. Theo Yuan v cng s (2000), iu kin ti u cho s phn hy cc hydrocarbon thm a nhn ca i tng m tc gi nghin cu l 30oC, pH 7. Hai chng vi khun Alcaligenes eutrophus JMP134 v Pseudomonas cepacia AC1100 pht trin tt nht 29oC [30]. Mt s nghin cu khc cho thy chng Streptomyces danangensis XKDN19 c kh nng pht trin tt nht 32oC, pH 6, nng NaCl 0 - 3% [9]; chng Streptomyces danangensis XKDN11 30oC, pH 7, nng NaCl 0,5% [1]; chng nm si FDN20 28oC, pH 6, nng NaCl 3% [3]. Cc yu t khc nh ngun nit, photpho, cacbon v cc khong khc cng nh hng n s pht trin v phn hy cht c ca cc vi sinh vt. Nghin cu ca Nguyn B Hu v cng s (2002) v nh hng ca cc yu t mi trng n s phn hy cc hp cht hydrocarbon thm a nhn ca mt s chng vi khun thuc chi Pseudomonas, Sphingomonas phn lp t t nhim du cho thy cc chng ny c kh nng pht trin tt nht nhit 30oC, pH 7 - 7,8 v nng mui NaCl t 0% n 3% [4]. Mt s



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Khoa Sinh - KTNN

tc gi khc cng pht hin iu kin ti u cho phn hy sinh hc PAH 30oC v pH 7 [31], [51], [61]. Trn y l nhng kt qu quan trng trong vic kch thch tp on vi sinh vt bn a trong c vi khun tin ti x l vng t nhim c ha hc. Trong t nhin, thng khng tn ti mt loi hydrocarbon thm a nhn m thng tn ti di dng hn hp. Do vic nghin cu kh nng phn hy hn hp cc PAH l iu cn thit xem nh hng qua li ca chng trong hn hp cng nh nng ca chng. S tn ti ca cc PAH khc nhau c th thc y hoc c ch qu trnh phn hy sinh hc, nng ca PAH cao s lm gim qu trnh phn hy sinh hc v gy c cho vi sinh vt. Cc PAH c trng lng phn t thp, cu trc n gin d dng phn hy hn so vi cc PAH c trng lng phn t cao v c cu trc phc tp. Theo Yuan, iu kin ti u cho s phn hy cc hydrocarbon thm a nhn i vi i tng tc gi nghin cu l ti nhit 30 oC, pH 7 [52]. Tc gi Zaidi cng cc cng s nghin cu nh hng ca pH n s phn hy phenanthrene v nhn thy ti pH t 6 n 7 th khng nh hng mnh n kh nng phn hy, nhng ti pH 10 th gy c ch kh nng phn hy phenanthrene ca vi sinh vt [14],[61]. Bn cnh cc yu t quan trng k trn phi k n cc yu t dinh dng. l ngun nit, pht pho, cc ngun cacbon c th b sung nh cao men, glucoza, axetat, pyruvat.v.v. y cng s l nhng nghin cu rt cn thit phc v cho qu trnh to cc yu t cng ngh thch hp trong x l ty c [37]. S phn hy sinh hc cc hydrocarbon thm a nhn ring r trong mu th nghim khng ngng c nghin cu v ngy cng thu c nhiu kt lun c gi tr [16], [42], [58]. Cc PAH c cu trc phn t n gin, trng lng phn t thp d dng phn hy sinh hc hn so vi cc



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Khoa Sinh - KTNN

PAH c trng lng phn t cao v cu trc phc tp [11], [16], [58], [59]. Tuy nhin, chng ta cn t hiu bit v kh nng phn hy sinh hc cc PAH khi chng c mt ng thi trong cc vng nhim. Thng trong t nhin khng ch tn ti mt loi PAH m tn ti hn hp cc PAH. Do , vic nghin cu kh nng phn hy hn hp PAH l iu cn thit c th nh gi c nh hng qua li v tng quan nng ca chng n s phn hy hn hp. S tn ti ca cc PAH khc nhau c th thc y hoc c ch qu trnh phn hy sinh hc [58]. Yuan cng cng s (2000) tin hnh nghin cu kh nng phn hy ng thi cc PAH trong bn sng c nhim PAH. Kt qu cho thy, tc phn hy Phenanthrene gim khi c mt thm bt k mt PAH no trong mu. Khi 6 PAH c mt ng thi, tc phn hy phenanthrene, acenaphthalene gim nhng tc phn hy Anthracene, Fluorene v Pyrene li tng [58]. Ngoi ra, tc phn hy PAH cng cn b nh hng mnh bi cc cht hot ng b mt khng phn cc nh Bij 30, Bij 35, triton X100, triton N110. Cc cht ny gy c v c ch hot ng ca vi sinh vt v chng c kh nng tng tc vi mng t bo hay chnh xc hn l cc phn t cht hot ng b mt tng tc vi protein mng t bo [40]. Cc cht ny cng c th trc tip gy c ch hot ng ca cc enzyme lin quan ti con ng chuyn ha PAH. C th chng cn lin kt vi c cc enzyme hay vi cc c cht . Mt l do lm gim kh nng phn hy sinh hc cc hp cht ny l chng hn ch s tip xc ca t bo vi sinh vt vi cc cht hu c ha tan trn b mt [61]. Tm li, vic nghin cu cc yu t nh hng n s phn hy sinh hc cc hp cht hydrocarbon thm a nhn l rt quan trng. C th coi y l c s cng ngh cho qu trnh x l nhim PAH bng phng php phn



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Khoa Sinh - KTNN

hy sinh hc (bioremediation), m bo cc iu kin thun li lm cho tc phn hy cc hydrocarbon a nhn c th t ti mc ti a. 1.6. Cc phng php phn loi vi sinh vt Vi sinh vt rt a dng trong t nhin. Tuy nhin, ch mt phn nh trong s chng l c th nui cy c trong iu kin phng th nghim. Do , vic phn loi chng gp nhiu kh khn. Hin nay c nhiu phng php phn loi vi sinh vt. Cc phng php c in ch da vo cc c im sinh l, sinh ha v hnh thi cu to bn ngoi phn loi vi sinh vt, cn phng php hin i li ch trng nhiu n vic nghin cu cc phn t axit nucleic nh DNA, RNA p dng trong phn loi. T c th thnh lp cy pht sinh chng loi, phn nh mi quan h v mt tin ha gia cc loi vi nhau. 1.6.1. Phng php phn loi truyn thng Phng php phn loi c in s dng hnh thi kt hp nhng c im hnh dng bn ngoi v cc c im sinh l phn loi. Cc phng php c in thng dng cc ch tiu phn loi l: mu sc, kch thc ca khun lc pht trin trn mi trng; cc c im ging nhau v hnh dng, kch thc, c hay khng c tin mao ca t bo vi khun; s khc nhau v c im sinh ha: phn ng nhum Gram, s khng cht khng sinh, s trao i cht v dinh dng, i tng vt ch, phn tch phn t axit teichoic. Ngoi ra, cn rt nhiu phng php ph bin khc na nh: kim tra cc phn ng sinh ha ca vi sinh vt vi cc cht phn ng khc nhau, cc phn ng min dch ca cc khng nguyn l thnh phn cu to ca t bo nh khng nguyn O ca lipopolysaccharid hay ca bao nhy thng c s dng phn bit cc chng ca mt loi [15].



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Khoa Sinh - KTNN

1.6.2. Phng php phn loi bng sinh hc phn t Hin nay, cc phng php phn loi hin i thng da trn vic nh gi mc phn t axit nucleic. Trn c s trnh t DNA, ngi ta c th c nhiu cc phng php khc nhau tin hnh phn loi. T cui th k 20, c bit l nhng nm 1980 tr li y, vi s pht trin mnh m ca sinh hc phn t, ngi ta s dng mt phng php nghin cu mi cho phn loi vi sinh vt l phn loi hc phn t. Phng php mi ny c th pht hin, m t v gii thch tnh a dng sinh hc mc phn t gia cc loi v trong phm vi loi trong thi gian ngn v c chnh xc cao. Jesus v Silvia (1999) tm tt cc phng php phn tch v kh mc s dng trong phn loi vi sinh vt (Bng 1.3) [35] Bng 1.3: Mt s phng php phn loi vi sinh vt Thnh phn t bo Phng php phn tchThnh phn baz (%G+C) Bin tnh DNA :DNA Cc phn DNA c ct bng enzyme gii hn a hnh chiu di cc on gii hn ca RNA riboxom Trnh t nucleotide Lai DNA : rRNA Trnh t axit amin So snh bng phn ng huyt thanh Cc kiu in di in di enzyme a v tr Cu trc peptidoglycan Polysacharid Axit teichoic

Phm vi phn loiChi Loi Loi v di loi

DNA nhim sc th

RNA riboxom

Loi, chi v trn chi Chi v trn chi Loi v chi

Protein

Cc dng trong loi Loi v chi

Thnh t bo



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Mng

Axt bo Lipid phn cc Axit mycolic Isoprenoid quinonones

Loi v chi

Trc y, vic phn loi vi sinh vt i khi gp kh khn v thiu chnh xc. Vi s pht trin ca k thut sinh hc phn t, vic phn loi ngy nay da ch yu vo nghin cu trn cc phn t axit nucleic (DNA, RNA). Cc phng php ny phn nh chnh xc hn mi quan h v mt tin ha gia cc nhm sinh vt. Tuy nhin, cng khng th ph nhn vai tr ca cc phng php phn loi da trn cc c im bn ngoi. Do vy, cn kt hp c hai phng php kt qu phn loi c chnh xc. Ngy nay, vic nghin cu phn t rRNA c coi l phng php hu hiu nht xc nh mi quan h trn cy tin ha ca cc vi sinh vt, v rRNA c mt tt c cc loi vi sinh vt, c chc nng xc nh v l trnh t c tnh bo th cao, chng ch khc nhau rt t gia cc nhm vi sinh vt. Da vo s khc nhau ny, ngi ta c th nh gi c mi quan h pht sinh chng loi v phn loi cc chng vi sinh vt. Trong 3 loi gen rRNA ca vi khun (5S, 16S, 23S) th gen 16S rRNA l ph hp nht cho vic nghin cu phn loi. Gen m ha cho 5S rRNA c kch thc khong 120 nucleotide, d c v so snh trnh t, nhng li khng phn bit mt cch chi tit gia cc chng. Ngc li, gen m ha 23S rRNA li c kch thc ln (3000) nucleotide do gy kh khn cho vic tch dng, c v so snh trnh t. Ch c gen 16S rRNA vi kch thc khong 1500 nucleotide va phn loi chi tit gia cc chng vi sinh vt v cng khng gy kh khn trong nghin cu. Do , n c u tin chn la trong vic phn loi vi khun. Gen m ha cho cu trc 16S rRNA c cc nh khoa hc nghin cu k lng v h thit lp c rt nhiu cc cp mi nhn on



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chng bng k thut PCR. y l mt thun li ln cho cc nghin cu phn loi da trn gen m ha 16S rRNA [35].



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Khoa Sinh - KTNN

CHNG II. VT LIU V PHNG PHP NGHIN CU2.1. Nguyn liu v ha cht 2.1.1. Nguyn liu Nguyn liu s dng trong ti nghin cu ny l mu nc nhim du thu thp ti b thu gom ca x nghip khai thc m Qung Ninh. 2.1.2. Ha cht Cc ha cht c s dng trong nghin cu ny c tinh khit cao ca cc hng trn th gii nh New England Biolabs, Sigma, Merk v.v. Cp mi 27F v 1492R c tng hp ti hng InvitrogenTM , vector PCR2.1 (Invitrogen). Mi xui (27F ): 5 - AGA GTT TGA TCM TGG CTC AG - 3 Mi ngc (1492R) : 5 - GGY TAC CTT GTT ACG ACT T - 3 Cp mi catechol 2,3-dioxygenase c tng hp ti hng Alpha DNA (Canada). Mi xui (C23OF) : 5 - ATG GAT DTD ATG GGD TTC AAG GT - 3 Mi ngc (C23OR) : 3 - ACD GTC ADG AAD CGD TCG TTG AG - 5 2.2. Mi trng nui cy + Mi trng mui khong dch (g/l) NH4NO3 KH2PO4 MgSO4 NaH2PO4 4 0,3 0,4 0,7 NaCl pH 1,2 6,5 Nc my va 1 lt

+ Mi trng mui khong thch (g/l): Thnh phn cc loi ha cht ging nh mi trng khong dch nhng c b sung thm 20g agar.



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Khoa Sinh - KTNN

+ Mi trng mui khong c b sung thm hn hp vitamin, hn hp vi lng v cao men. Mi trng trc khi c s dng c kh trng 121oC, 1 atm trong 25 pht. + Mi trng LB dch (g/l) NaCl Trytone Cao men 10 10 5,0 Nc ct va 1 lt pH 7

+ Mi trng LB thch (g/l): Thnh phn cc ha cht tng t mi trng LB dch nhng c b sung thm 20g agar. + Nc mui sinh l (0,85%) NaCl Nc ct 2.3. My mc v thit b nghin cu Trong qu trnh nghin cu, cc thit b, my mc c s dng c chnh xc cao ti phng th nghim Cng ngh sinh hc mi trng v phng th nghim trng im Quc gia v cng ngh Gen - Vin Cng ngh sinh hc bao gm: knh hin vi, cn k thut, my o pH Hanna, t cy v trng Laminar ca Php, t sy, ni kh trng, my nui lc cc nhit khc nhau, t nui n nhit, my ly tm Eppendorf, my PCR, my soi DNA, my in di Bio-Rad, my chp nh Gel-Doc, t lnh cc loi 4oC, -20oC, 80oC, my xc nh trnh t t ng ABI PRISM 3100 Avant Genetic Analyzer, l vi sng, pipet man ca hng Eppendorf, bnh nn, u cn, ng ly tm v.v. 2.4. Phng php nghin cu 2.4.1. Phn lp vi sinh vt trn mu nc nhim du Lm giu vi sinh vt ln mt bng cch ht 5 ml mu nc nhim du chuyn vo trong bnh tam gic cha 45 ml mi trng khong dch c b 8,5g 1 lt



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Khoa Sinh - KTNN

sung vitamin, vi lng, cao men v PAH vi nng 50 ppm. Nui lc bnh 200 vng/pht, 30oC trong 5 ngy. Lm giu vi sinh vt ln 2 v ln 3 tng t nh mu lm giu ln 1. Tin hnh pha long mu lm giu ln 3, ri gt mu pha long trn mi trng mui khong c b sung vitamin, vi lng, cao men v 100 ppm PAH. Mu c em nui tnh 30oC. Chn chng vi khun c kh nng pht trin trn mi trng c PAH ri tin hnh nui lc v lm sch. 2.4.2. Nghin cu mt s c im sinh hc ca mt s chng vi khun * Phng php nhum Gram Lam knh c ra sch bng cc dung dch ra, sau lm kh trn ngn la n cn. Ly mt git dch sau 16 gi nui nh ln lam knh ri dng que cy v trng dn u git dch v c nh t bo trn ngn la n cn. Nhum tiu bn bng dung dch tm kt tinh (Cystal violet) trong thi gian 1 pht. Ra bng nc ct ri nhum tiu bn vi lugon trong thi gian 2 pht. Tip tc ra tiu bn bng cn 70 % khong 20 giy, sau ra bng nc sch ri kh. Nhum tip vi safarin trong 1 pht, ra li bng nc ct 2 ln v kh t nhin. Mu c quan st bng knh hin vi quang hc vi phng i 100 ln di vt knh du. Nu vi khun bt mu xanh tm thuc loi Gram (+) cn bt mu hng l Gram (-). Vi khun s dng lm i chng cho nhm Gram (-) l E. coli v Gram (+) l Bacillus. * Quan st hnh thi t bo bng knh hin vi in t qut Vi khun c nui cy 5-7 ngy trn mi trng mui khong cha PAH. Dch nui cy c lc qua giy lc v trng loi b cn, ri ly tm 5000 vng/pht trong 5 pht thu sinh khi t bo. Ra li sinh khi vi khun bng nc ct v trng 2 ln loi cc cht cn bn trong mi trng nui cy. T bo vi khun c ha tan trong glutaraldehyt 2,5 % trong m



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photphat natri 100 mM (pH 7,2) trong 30 pht. Ly mt git t bo x l a ln li ng khong 1 pht cho mu bm c vo li. Ra nh bng nc sch sau lm kh mu qua cn 25, 50, 75, 100 % T-butyl. Sau lm kh mu bng my ng kh v ph mu bng vng. Mu c quan st di knh hin vi in t qut JEOL 5410 LV. Hnh thi t bo vi khun c chp nh v o kch thc. C nh mu v soi knh c thc hin ti Vin 69, B t lnh Lng H Ch Minh. 2.4.3. nh gi kh nng s dng PAH ca vi khun PAH c xc nh bng phng php o quang phn t, da trn s dch chuyn in t trn cc orbital ca cc ni i lin kt trong vng thm ca PAH. Kh nng chuyn ha PAH ca vi khun trong dch nui cy c xc nh trn my quang ph t ngoi kh kin UV-VIS: GBC - CINTRA 4. 20 ml dch nui cy vi sinh vt s dng PAH c chit bng 180 ml acetone, sau lc ton b, thu dch lc a i phn tch. Nng ca mi PAH trc v sau x l c tnh ton da vo chiu cao peak hp th ti cc bc sng, cc PAH hp th ti cc bc sng nh: pyrene 275 nm; fluorene: 257,7 nm, fluoranthrene: 286,7 nm, phenanthrene: 292,6 nm, anthracene: 252 nm, naphthalene: 296 nm. 2.4.4. Xc nh trnh t gen m ha 16S rRNA v catechol 2,3-dioxygenase 2.4.4.1. Tch chit DNA tng s ca vi khun theo phng php ca Sambrook, Russell [47]. Bc 1: Thu sinh khi t bo bng cch ly dch nui cy chuyn vo ng li tm ri li tm vi vn tc 6.000 vng/pht 4oC trong 10 pht. Bc 2: Ha tan mu trong 400 l m lysis ri lc k cho tan ta. Bc 3: B sung 50 l lysozym v 37oC trong 30 pht. Bc 4: B sung 20 l protease K ri 56oC trong 2 gi.



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Bc 5: B sung phenol (t l 1:1 v/v), lc u v ly tm 12.000 vng/pht trong 15 pht 4oC. Bc 6: Ht pha trn chuyn sang eppendorf mi, b sung CI (24:1) vi t l 1:1 v/v, lc nh sau ly tm 12.000 vng/pht trong 15 pht 4oC. Bc 7: Ht pha trn chuyn sang eppendorf mi v ta DNA bng cn tuyt i, gi -20oC trong khong 2-3 gi. Bc 8: Ly tm 12.000 vng/pht trong 15 pht 4oC, thu ta DNA. Bc 9: Ra ta bng cn 70 %, ly tm 12.000 vng/pht trong 10 pht 4oC Bc 10: Lm kh ta v ha tan ta trong nc kh ion v trng 2.4.4.2. Nhn on gen bng phng php PCR Phng php PCR do Kary Mullis v cng s pht minh nm 1985. Phng php ny to ra bc nhy vt trong k thut sinh hc phn t v c s dng ph bin n nay. Phng php ny cho php nhn ln s lng ln on gen cn thit trong thi gian ngn. thc hin c phn ng PCR cn c cc nguyn liu chnh l on DNA khun, cp mi c hiu, enzyme DNA polymerase, cc nucleotide t do, my PCR v.v. * Thnh phn phn ng: Buffer Taq 10X MgCl2 25 mM dNTPs 2,5 mM Mi xui 20 M Mi ngc 20 M Taq DNA polymerase (5 U/l) H2O DNA khun Tng th tch 2,5 3,0 2.5 1 1 0,2 1,5 25 l l l l l l l l

13,3 l



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i vi nhn on gen m ha 16S rRNA s dng cp mi 27F v 1492R th chu trnh nhit cho phn ng PCR nh sau: Bc 1 Bc 2 Bc 3 Bc 4 Bc 5 Bc 6 Bc 7 95oC trong 5 pht 94oC trong 1 pht 55oC trong 1 pht 72oC trong 2 pht Lp li 35 chu k t bc 2 n bc 4 72oC trong 7 pht 4oC bo qun mu

Trong trng hp nhn on gen m ha catechol 2,3-dioxygenase s dng cp mi C23OF v C23OR vi chu trnh nhit di y: Bc 1 Bc 2 Bc 3 Bc 4 Bc 5 Bc 6 Bc 7 94oC trong 7 pht 94oC trong 1 pht 55oC trong 1 pht 72oC trong 1 pht Lp li 35 chu k t bc 2 n bc 4 72oC trong 10 pht 4oC bo qun mu

2.4.4.3. Quy trnh bin np v chn dng Bin np: b Kit TA Cloning(R) ca hng InvitrogenTM (M) c s dng cho qu trnh bin np. Nguyn tc: on DNA cn thit nhn ln trong phn ng PCR c s dng gn vo vector PCR 2.1 to thnh vector mang on gen 16S rRNA.



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* Thnh phn phn ng: Dung dch m Vector pCR2.1 Sn phm PCR (on DNA cn thit) Enzyme T4 ligase Nc Tng th tch 1,0 l 1,5 l 1,2 l 1,0 l 5,3 l 10 l

Phn ng gn on DNA cn thit vo vector c thc hin trong iu kin nhit 14oC, thi gian 18 gi. Sau sn phm ny c bin np vo t bo kh bin (vi khun E. coli INV F). * Quy trnh bin np: Bc 1: Ly 4 l vector ti t hp trn cho vo ng ng t bo kh bin E. coli INV F ri trn 30 pht. Bc 2: Sc nhit 42oC trong thi gian 45 giy. Bc 3: t ng t bo ln trong 2 pht. Bc 4: Thm 250 l SOC vo ng ng t bo ri em nui t lc 37oC trong khong 1 gi. Bc 5: Ht dch nui gt trn a mi trng LB c b sung 50 mg/l ampicilin, v 50 mg/ml X-Gal. Sau nui 37oC trong khong 16-18 gi. Chn cc dng khun lc mu trng v mt dng khun lc mu xanh nui trn mi trng LB dch. Trn mi trng LB c X-gal nhng khun lc mu trng l nhng c th c th mang DNA plasmid gn on DNA ngoi lai, nhng khun lc mu xanh l nhng c th khng nh on DNA plasmid v DNA ngoi lai. * Quy trnh tch chit DNA plasmid: Bc 1: Thu sinh khi t bo bng cch ly tm dch nui 6.000 vng/pht trong 10 pht 4oC, dch v thu ta.



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Bc 2: B sung 100 l Sol I ri vontex k cho tan ht ta. Bc 3: Thm 200 l Sol II v o nh, khong 5 pht. Bc 4: B sung 150 l Sol III, o u ri 10 pht. Bc 5: B sung C:I theo t l 1:1 v/v, lc u ri ly tm 12.000 vng/pht trong 10 pht. Bc 6: Ht pha trn chuyn sang ng mi v thm 500 l isopropanol 100 % ta DNA, khong 2-3 gi. Bc 7: Ly tm 12.000 vng/pht trong 15 pht. thu ta. Bc 8: Ra ta bng cn 70 %, ly tm 12.000 vng/pht trong 10 pht. Bc 10: Lm kh ta bng cch ly tm kh, sau ta c ha tan trong nc kh ion. * Phn ng ct vector ti t hp bng enzym e EcoRI: *Thnh phn phn ng: m EcoRI 10X Enzyme ct EcoRI (5 U/l) RNase (1 mg/ml) H2O DNA plasmid Tng th tch 2,0 0,2 0,5 2,0 20 l l l l l

15,3 l

Trn u cc thnh phn phn ng vi nhau ri t ng phn ng vo b n nhit 37oC trong 16 gi. 2.4.4.4. Phng php xc nh trnh t gen bng my t ng Xc nh trnh t hai on gen m ha 16S rRNA v catechol 2,3dioxygenase ca vi khun BQN31 trn my c trnh t t ng ABI PRISM 3.100 Avant Genetic Analyzer t ng theo phng php ca Sanger. Trnh



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Khoa Sinh - KTNN

t hai on gen 16S rRNA v on gen m ha catechol 2,3-dioxygenase ca vi khun BQN31 c ng k trn GenBank. 2.4.4.5. Phng php xy dng cy pht sinh chng loi S dng chng trnh Blast so snh mc tng ng gia trnh t cc on gen m ha 16S rRNA, gen m ha catechol 2,3-dioxygenase ca vi khun BQN31 vi cc trnh t gen lin quan trn ngn hng gen. Tip theo, s dng phn mm Clustal X, NJ xy dng cy pht sinh chng loi on gen m ha 16S rRNA, gen m ha catechol 2,3-dioxygenase ca vi khun BQN31 v cc on gen i din.



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CHNG III: KT QU V THO LUN3.1. Phn lp v tuyn chn vi khun c kh nng pht trin trn mi trng cha PAH T mu ban u, vi sinh vt s dng PAH c phn lp theo phng php lm giu trn mi trng mui khong cha cc ngun PAH nh ngun cacbon v nng lng duy nht. VSV s dng PAH trong mu nc nhim du khng a dng, trn mi loi PAH ch c t 1 n 4 loi khun lc, trong loi vi khun c mu trng, trn, li bng v nh chim u th (Bng 3.1). Kh nng pht trin ca vi khun c nh gi s b bng s thay i mu mi trng. Bng 3.1: S lng vi khun phn lp c trn mi trng khong c b sung cc hp cht PAH Mi trng cha PAH v hn hp PAH Phenanthrene Anthracene Fluorene + Fluoranthene Pyrene + Chrysene Naphthalene S lng vi khun phn lp 5 4 1 3 2

Cc s liu trong bng 3.1 cho thy, t mu lm giu vi sinh vt trn hai ngun phenanthrene v anthracene c s lng vi khun phn lp c nhiu nht. Tng s 15 chng vi khun c kh nng pht trin tt trn mi trng cha cc ngun PAH khc nhau c phn lp v c k hiu t BQN20-BQN34. Trong s 15 chng vi khun ny, 4 chng vi khun BQN30, BQN31, BQN32, BQN33 c kh nng pht trin mnh hn c trn ngun phenanthrene (Hnh 3.1).



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BQN30

K

K

BQN31

BQN32

K

BQN33

K

Hnh 3.1: Kh nng phn hy phenanthrene ca 4 chng BQN30, BQN31, BQN32, BQN33 (Ghi ch: K: mu i chng) Mi trng trong cc bnh nui cy vi khun v cc PAH c mu vng nu. Trong s 15 chng vi khun, chng BQN31 phn hy phenanthrene mnh nht, mi trng i mu ch sau 32 gi nui cy. Cc chng vi khun cn li chuyn ha mc yu hn, sinh khi vi sinh vt t hn, mi trng khng i mu hoc i mu sau nhiu ngy. Hnh thi khun lc ca 4 chng vi khun s dng phenanthrene mnh c trnh by tm tt Bng 3.2.



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Khoa Sinh - KTNN

Bng 3.2. c im hnh thi ca cc chng vi khun Chng vi khun BQN30 BQN31 BQN32 BQN33 c im khun lc trn mi trng mui khong cha phenanthrene Trn, trn, trng trong Trn, trn, bng, li, trng c, nh xanh Trn, trn, nh vng Trn, trn, bng, nh xanh

Trong s cc chng vi khun k trn, chng vi khun BQN31 c khun lc to, mc dy, dch nui cy chuyn mu nhanh hn nhng chng khc. Do , chng BQN31 c chn nghin cu v xc nh kh nng phn hy PAH. S tn ti ca cc vi khun bn a s dng cc loi PAH khc nhau cho thy tim nng ln trong vic p dng thnh cng cng ngh phn hy sinh hc x l nhim du v PAH. 3.2. c im hnh thi v t bo ca chng vi khun BQN31 Chng vi khun BQN31 c nui cy trn mi trng mui khong b sung phenanthrene, nui nhit 30oC. Khun lc chng BQN31 sau 3-5 ngy nui cy c hnh trn, trng, trn, li, bng, c nh xanh, kch thc khong 1-2 mm (Hnh 3.2). Chng BQN31 thuc nhm vi khun Gram m, t bo hnh que, b mt tng i nhn v kch thc khong 0,29 - 0,5 m x 0,95 - 1,1 m (Hnh 3.3).



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1 m

Hnh 3.2: Hnh thi khun lc chng vi khun BQN31

Hnh 3.3: Hnh thi t bo vi khun BQN31 (Quan st trn knh hin vi in t qut JEOL 5410 LV vi phng i 15.000 ln)

3.3. Kh nng s dng cc loi PAH ca chng vi khun BQN31 Trong mi trng t nhin, cc PAH thng tn ti dng hn hp v rt c. Do , vic nh gi kh nng phn hy PAH bi cc chng vi sinh vt bn a c ngha rt quan trng trong qu trnh lm sch nhim du ni chung v PAH ni ring. Sau 5 ngy nui lc trn mi trng khong b sung cc PAH khc nhau, mu ca mi trng cha cc vi sinh vt thay i r rt, mi trng chuyn mu nu vng. c bit, trong cc bnh nui cy cha phenanthrene v naphthalene mi trng c hn v c nhiu sinh khi hn (Bng 3.3).



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Bng 3.3: Ph UV o kh nng phn hy cc PAH ca chng BQN31

Ngun PAH

Ph UV C

Hnh nh minh ha

BQN31 Phenanthrene

C

Naphthalene

BQN31

Hiu qu s dng mt s loi PAH ca chng BQN31 c trnh by trong bng 3.4.



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Bng 3.4: Kh nng s dng cc PAH khc nhau ca chng BQN31 Cc PAH Phenanthrene Fluorene Fluoranthene Pyrene Naphthalen Anthracene Hiu sut phn hy (%) 60,24 18,52 18,75 69,38 25,9 Mu mi trng nui cy vng nu vng nht vng chanh nu nht, nh vng mu vng mu nu Ghi ch

khng phn tch

Kt qu bng 3.4 cho thy, chng BQN31 s dng tt c cc PAH th nghim. i vi mi loi PAH, mu mi trng nui cy c s thay i khc nhau. Da trn kt qu phn tch, sau 5 ngy nui lc nhit 30oC v 200 vng/pht, chng BQN31 c kh nng s dng 69,38% naphthalene, 60,24% phenanthrene, 18,52% fluorene, 25,9% anthracene v 18,75% pyrene vi nng ban u l 100 ppm mi loi PAH. Kh nng phn hy ca chng BQN31 vi naphthalen l cao nht v fluorene l thp nht. Kt qu ny chng t vi cc PAH t vng th kh nng b phn hy sinh hc cng mnh. Trn th gii, c nhiu cng trnh nghin cu v kh nng phn hy cc loi PAH ca cc chng vi sinh vt. Weissenfels v cng s (1991) cng b chng Alcaligenes denitrficans WW1 phn hy fluoranthene vi tc 300 mg/l mi ngy, ng thi chng ny s dng cc loi PAH khc nh pyrene, benzo(a)anthracene theo c ch ng trao i cht [53]. Chng Mycobacterium sp. PYR-1 phn hy 74% hn hp PAH gm phenanthrene, anthracene, fluoranthene, pyrene, chrysene v benzo(a)pyrene sau 7 ngy nui cy. Trong 95% fluoranthren b loi b sau 24h nui cy, tuy nhin nng ban u ch c 17 mg/l [21]. Hai chng Cycloclasticus N3 v W ca Gelsenbrecht v cng s khng s dng c PAH cao phn t nh fluoranthene, pyrene v chrysene nh l ngun carbon v nng lng duy



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Khoa Sinh - KTNN

nht, tuy nhin theo c ch ng trao i cht chng Cycloclasticus N3 c th phn hu khong 50% cc PAH cao phn t trn vi nng ban u 1ppm khi b sung 10ppm phenanthrene sau 7 ngy [12]. Hin nay, c nhiu cng b kt qu nghin cu v kh nng phn hy fluoranthene v pyrene nh l ngun cacbon v nng lng duy nht ca cc chng x khun thuc cc chi Gordonia, Mycrobacterium [36] . Theo nghin cu ca Lors v cng s v x l sinh hc mu t nhim PAH t mt khu vc cng nghip min Bc nc Php ti phng th nghim vi 3 loi hn hp c cht PAH gm cc PAH c 3 hoc 4 vng thm cng vi 16 loi PAH (theo quy nh ca UEPA - Hoa K) cho kt qu loi b ln lt l 96%, 80% v 80%, sau 245 ngy [19]. Chng Rhodococcus sp. c phn lp t mu trm tch c kh nng phn hy 53% anthracene sau 24h nng 3g/ml [21]. Xue-Jing Zheng v cng s nghin cu kh nng chuyn ho sinh hc ca tp on vi sinh vt hiu kh c trong bn cng, b sung cht hot ng b mt tween 80 cho hiu qu loi b PAH trn 95% i vi PAH cha 3 vng thm v tng tc loi b PAH cha 4 vng thm sau 21 ngy [57]. Cng b sung tween 80 (0,5%), Dhenain v cng s cho thy kh nng loi b t 35 50% benzo(a)anthracene, benzo(a)pyrene, benzo(b)fluoranthene, benzo(j)fluoranthene, benzo(k)fluoranthene,

fluoranthene trong bn en ca cng nghip nhm [13]. Vit Nam, chng vi khun Sphingomonas yanoikuyae MXL-9 phn lp t cn du th m Bch H c kh nng phn hy 64,5% phenanthrene v 61,4% anthracene sau 7 ngy nui cy vi nng ban u l 12mg/l [8]. Hong Th M Hnh v cng s (2003) phn lp v nghin cu kh nng s dng PAH ca chng nm Aspergillus sp. FVX5 phn lp t th nghim x l lm sch cn du th ca tu Ch Linh. Chng nm ny c kh nng phn



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Khoa Sinh - KTNN

hy 266,69 mg/l phenanthrene, 33,34 mg/l anthracene, 19,16 mg/l fluoranthene sau 7 ngy nui cy iu kin 37oC [2]. Sau 7 ngy nui lc trn mi trng khong c b sung glucose, hai chng x khun XKDN12 v XKDN13 phn lp t t nhim cht c ho hc c kh nng s dng PAH, 32,72% v 45,05% vi phenanthrene, 39,01% v 52,22% vi anthracene, 23,32% v 41,41% vi fluoranthen [7]. Chng vi khun Chromobacterium sp. BPQ1 phn lp t nc thi nhim du cng ty xng du B12, Qung Ninh c th s dng phenanthrene v fluorene [5]. Trong mt nghin cu khc, chng vi khun KCP8 phn lp ti Khe Ch c kh nng phn hy hn hp PAH (phenanthrene, anthracene v fluoranthene). Sau 7 ngy nui lc chng KCP8 phn hy 76,12% hn hp PAH, trong kh nng phn hy phenanthrene trong hn hp l 79,96%, anthracene l 71,09% v fluoranthene l 41,01% [4]. So snh vi cc kt qu cng b trn th gii v Vit Nam, cho thy rng, chng vi khun BQN31 c kh nng phn hy trung bnh cc PAH cng b. Tuy nhin, kh nng s dng nhiu loi PAH khc nhau chng t vai tr nht nh ca chng vi khun ny trong nc thi nhim du khu vc Qung Ninh. Chng ny c th c s dng xy dng tp on ging vi sinh vt s dng trong qu trnh lm sch nhim PAH cc iu kin c kim sot. 3.4. Xc nh trnh t on gen m ha 16S rRNA ca chng BQN31 3.4.1. Tch chit DNA tng s v nhn on gen m ha 16S rRNA bng k thut PCR Tch chit v lm sch DNA tng s vi khun l khu quan trng v cht lng ca DNA s nh hng n hiu qu ca cc th nghim tip theo. DNA tng s ca vi khun BQN31 c tinh sch cao, hm lng ln, bng gn khng t gy (Hnh 3.4).



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Khoa Sinh - KTNN

DNA tng s

Hnh 3.4: DNA tng s ca chng BQN31 S dng DNA tng s ca vi khun BQN31 lm khun, cp mi c hiu (27F v 1492R) v chu trnh nhit nh trnh by phn 2.4, sn phm PCR thu c l mt bng DNA duy nht, sc nt v c kch thc khong 1.500 bp ng nh l thuyt (Hnh 3.5). kb M BQN31 Hnh 3.5. Sn phm PCR nhn on gene m ha 16S rRNA ca chng BQN31. M: thang DNA chun 1 kb1,5 1

3.4.2. Tch dng gen m ha 16S rRNA t chng BQN31 Hin nay, xc nh trnh t gen c th s dng hai phng php nh xc nh trnh t nucleotide trc tip t sn phm PCR hoc gn sn phm PCR vo mt vector sau bin np vo t bo kh bin nh E. coli, nui cy t bo E. coli ti t hp, tch v lm sch DNA plasmid v xc nh trnh t on DNA c chn vo vector s dng cc mi ca vector.



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Khoa Sinh - KTNN

u im ca phng php xc nh trnh t trc tip l tit kim thi gian. Phng php xc nh trnh t DNA thng qua bc bin np cho kt qu r rng hn. Ngoi ra, tch dng gen gip cc nh nghin cu ch ng hn v gen hoc on gen nhn s dng cho cc mc ch nghin cu khc. Trong nghin cu ny, sn phm PCR nhn on gen 16S rRNA t chng BQN31 c xc nh trnh t theo phng php gn sn phm PCR vo mt vector sau bin np vo t bo kh bin E. coli. *Gn sn phm PCR vo vector pCR 2.1 v bin np vo E. coli DH5 Sn phm PCR nhn on gen 16S rRNA c gn vo vector pCR 2.1 nh enzyme T4 DNA ligase v bin np vo t bo kh bin E. coli DH5. Sau khi nui 37 oC trong 18 gi trn mi trng LB c cha ampicillin v X-Gal xut hin nhng khun lc trng xen k nhng khun lc xanh (Hnh 3.6). S lng khun lc mu trng nhiu hn so vi khun lc mu xanh, chng t hiu sut bin np l tt. Cc khun lc trng E. coli DH5 c th mang plasmid c chn sn phm PCR nhn on gen 16S rRNA. Cc khun lc trng E. coli DH5 c chn la, tch DNA plasmid. Sn phm DNA plasmid thu c t cc dng t bo chn la c in di kim tra trn gel agaros 1%. Hnh 3.6: Kt qu bin np chng BQN31 (Khun lc trng: l nhng khun lc c th mang DNA plasmid gn on DNA ngoi lai. Khun lc xanh: l nhng khun lc khng nh on DNA plasmid v DNA ngoi lai)



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Khoa Sinh - KTNN

Cc khun lc mu trng c th mang vector cha on DNA cn thit c la chn tch DNA plasmid cng vi mt vi khun lc xanh c s dng lm mu i chng. 3.4.3. Tch DNA plasmid v kim tra cc dng khun lc thch hp Cc khun lc la chn c nui trn mi trng LB dch tch DNA plasmid. Sn phm DNA plasmid c in di kim tra trn gel agarose la chn dng t bo mang vector ti t hp (Hnh 3.7 ). C 1

Hnh 3.7. Sn phm in di kim tra DNA plasmid ca cc dng c la chn C: DNA plasmid dng khun lc mu xanh (i chng) 1: DNA plasmid ca dng khun lc s 13 mu trng Kt qu hnh 3.7 cho thy, DNA plasmid ca dng s 13 nm v tr cao hn mu i chng. Dng vi khun s 13 ny c th mang vector ti t hp cha on gen 16S rRNA nhn ln t DNA tng s ca vi khun BQN31. Enzyme gii hn EcoRI c s dng kim tra liu thc s DNA plasmid ca dng s 13 c mang sn phm PCR hay khng. Sn phm ct ca dng 13 cho bng c kch thc ph hp khong 1.500 bp (Hnh 3.8).

M 1

S ha bi Trung tm Hc liu i hc Thi Nguyn 1,5 Kb


1 Kb

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Khoa Sinh - KTNN

Nh vy, sn phm PCR nhn on gen 16S rRNA c chn vo vector v c bin np vo E. coli DH5. DNA plasmid dng s 13 c lm sch phc v cho vic xc nh trnh t gen m ha 16S rRNA ca vi khun BQN31 (Hnh 3.9). C 1

Hnh 3.9. Sn phm lm sch DNA plasmid dng s 13 ca chng vi khun BQN31 Ging C: Mu i chng (DNA plasmid dng mu xanh) Ging 1: DNA plasmid dng s 13 Kt qu hnh 3.9 cho thy DNA plasmid dng s 13 sau khi loi RNA c sch cao, iu kin s dng xc nh trnh t gen m ha 16S rRNA ca chng vi khun BQN31. 3.4.4. Trnh t gen 16S rRNA ca chng vi khun BQN31



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Khoa Sinh - KTNN

Trnh t on gen m ha 16S rRNA chng BQN31 c xc nh hai chiu vi s dng cp mi M13R v M13F. Sau khi phn tch v x l s liu, trnh t y ca on gen m ha 16S rRNA ca vi khun BQN31 c xc nh (Hnh 3.10)1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 AGAGTTTGAT GAGATCTTCG GGAATAACAT GCCCAGGGAT CGATCCTTAG TCCTACGGGA CCGCGTGAGT GTACCGGGAG CTAGCGTTGT TGAAAGCCCG AGGTGAGTGG GCGAAGGCGG GGATTAGATA GCACTTCGGT TAAAACTCAA AGCAACGCGC CCTTCAGTTC GATGTTGGGT GGGTACTCTA TCATGGCCCT CCTCGCGAGG GAGAGCATGA CCAGGCCTTG GCTAACCGCA AGGTAACCGT CATGGCTCAG GATCTAGTGG CGGGAAACTG GAGCCCGCGT CTGGTCTGAG GGCAGCAGTA GATGAAGGCC AATAAGCTCC TCGGAATTAC GGGCTCAACC AATTCCGAGT CTCACTGGAC CCCTGGTAGT GGCGCAGCTA AGGAATTGAC AGAACCTTAC GGCTGGCTGG TAAGTCCCGC AAGGAACCGC TACGCGTTGG GGAAGCTAAT AGGCGGAATC TACACACCGC AGGAGGCAGG AAA AACGAACGCT CGCACGGGTG ATGCTAATAC AGGATTAGCT AGGATGATCA GGGAATATTG TTAGGGTTGT GGCTAACTCC TGGGCGTAAA CCGGAATAGC GTAGAGGTGA CGGCATTGAC CCACGCCGTA ACGCATTAAG GGGGGCCTGC CAACGTTTGA GTGACAGGTG AACGAGCGCA CGGTGATAAG GCTACACACG CTCCAAAAGT GCTAGTAATC CCGTCACACC CGACCACAGT GGCGGCATGC CGTAACGCGT CGGATGATGA AGTTGGTGGG GCCACACTGG GACAATGGGG AAAGCTCTTT GTGCCAGCAG GCGCACGTAG CCTTGAGACT AATTCGTAGA GCTGAGGTGC AACGATGATA TTATCCGCCT ACAAGCGGTG CATCCCCAGT CTGCATGGCT ACCCTCGCCT CCGGAGGAAG TGCTACAATG CGTCTCAGTT GCGGATCAGC ATSGGAGTTG GGGTTTAGCG CTAATACATG GGGAATCTGC CGTAAGTCCA GTAAAGGCTC GACTGAGACA GCAACCCTGA TACCCGGGAT CCGCGGTAAT GCGGCGATCC GGATTGCTTG TATTCGGAAG GAAAGCGTGG ACTAGCTGCT GGGGAGTACG GAGCATGTGG ATGGTTTCCA GTCGTCAGCT TTAGTTGCCA GTGGGGATGA GCGACTACAG CGGATTGTTC ATGCCGCGGT GATTCACTCG ACTGGGGTGA CAAGTCGAAC CCTTGGGTTC AAGATTTATC ACCAAGGCGA CGGCCCAGAC TCCAGCAATG GATAATGACA ACGGAGGGAG AAGTCAGRGG AATCCGGGAG AACACCAGTG GGAGCAAACA GGGGTGCATG GTCGCAAGAT TTTAATTCGA GAGATGGATT CGTGTCGTGA TCATTCAGTT CGTCAAGTCC TGGGTAGCGA TCTGCAACTC GAATACGTTC AAGGCGTTGA AGTCGTAACA

Hnh 3.10: Trnh t on gen 16S rRNA ca chng BQN31

Trnh t on gen m ha 16S rRNA ca chng BQN31 c xc nh v ng k trn GenBank vi m s EU814953. Kt qu so snh trnh t on gen 16S rRNA ca chng BQN31 vi trnh t ca cc VSV prokaryote c cng b trn GenBank cho thy, chng BQN31 quan h gn gi vi cc chng vi khun thuc chi Sphingomonas (Hnh 3.11). Chng BQN31 c tng ng cao vi cc chng Sphingomonas sp. JQL4-5 (98%), Sphingomonas sp. A4 (98%), Sphingomonas sp. AMS7 (98%), Sphingomonas sp. VX-MXL9 (97%),



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Sphingomonas paucimobilis EPA 505 (96%). Bn chng Sphingomonas sp. A4 , Sphingomonas sp. AMS7, Sphingomonas sp. VX-MXL9, Sphingomonas paucimobilis EPA 505 u l cc chng vi khun s dng PAH c phn lp t cc ngun nhim khc nhau. Cc vi khun phn hy PAH chim u th trong t thng thuc cc chi nh Sphingomonas, Burkholderia, Pseudomonas v Mycobacterium [36]. Trong s cc chng vi khun phn lp t mi trng, s lng vi khun thuc chi Sphingomonas chim t l cao trong s cc chng c kh nng phn hy nhiu loi cht c hi trong c c PAH, cc hp cht dioxin, cc hp cht phenol cha clo. Cc vi khun Sphingomonas cng phn b rng ri trong cc mi trng khng nhim nh cc h thng phn phi nc v mi trng bin [36].Sphingomonas sp. CFO6 S. yanoikuyae B1 Sphingomonas sp. P2 Sphingomonas sp. A4

Hnh 3.11. Cy pht sinh loi da trn so snh trnh t cc on gien m ha 16S rRNA ca chng BQN31 v mt s chng vi khun i din. (Thc o th hin hai nucleotide khc nhau trn 1.000 nucleotide so snh)

Sphingomonas sp. BQN31Sphingomonas sp. JQL4-5 Sphingomonas sp. AMS7 S. paucimobilis EPA 505 Sphingomonas sp. 3Y

0.002Sphingobium sp. 2F5-2



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Bng 3.5: tng ng cc on gen 16S rRNA ca chng BQN31 v mt s chng vi khun i din S nucleotide tng ng 1353* /1374# 1433* /1456# 1392* /1420# 398*/408# 1385* /1430# Mc tng ng (%) 98,47 98,42 98,02 97,54 96,85

STT 1 2 3 4 5

Chng vi khun Sphingomonas sp. AMS7 Sphingomonas sp. JQL4-5 Sphingomonas sp. A4 Sphingomonas sp. VXMXL9 S. paucimobilis EPA 505

Ch thch: *- s nucleotide ca on gen 16S rRNA chng BQN31 #- s nucleotide ca on gen 16S rRNA cc chng i din Theo mt s cng trnh nghin cu c cng b trong v ngoi nc, cc chng vi khun Sphingomonas sp. A4, Sphingomonas sp. VXMXL9, S. paucimobilis EPA 505 u c kh nng phn hy cc hp cht PAHs kh tt. Chng vi khun S. yanoikuyae MXL-9 phn lp t cn du th m Bch H phn hy phenanthrence v anthracene [8]. Chng Sphingomonas sp. A4 c kh nng s dng acenaphthene v acenaphthylene nh ngun cacbon v nng lng duy nht. [41]. Chng vi khun S. paucimobilis EPA 505 c kh nng s dng fluoranthene nh ngun cacbon v nng lng duy nht, ngoi ra chng cn c th chuyn ha c nhiu loi PAH khc. Dingyi Ye v cng s ch ra rng, sau 16h nui cy vi nng 10 ppm mi PAH, chng vi khun S. paucimobilis EPA 505 phn hy 80.0, 72.9, 31.5, 33.3, 12.5, v 7.8% ca pyrene, benz[a]anthracene



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(B[a]A), chrysene, benzo[a]pyrene (B[a]P), benzo[b]fluoranthene (B[b]F), and dibenz[a,h]anthracene (DB[a,h]A) [24]. Nh vy, da trn mt s c im hnh thi v so snh trnh t on gen 16S rRNA, chng vi khun BQN31 c xp vo chi Sphingomonas v c t tn l Sphingomonas sp. BQN31. Chng vi khun Sphingomonas sp. BQN31 cng thuc trong s cc vi khun s dng PAH thng gp trn th gii v Vit Nam. 3.5. Nhn on gen m ha catechol 2,3 dioxygenase t chng BQN31 Phn hy sinh hc ca hydrocarbon thm iu kin hiu kh thng xy ra qua cc bc ct vng thm v to thnh catechol, y l mt sn phm trung gian ph bin nht ca qu trnh phn hy cc hp cht hydrocarbon thm. Qu trnh ct vng ca catechol bi enzyme dioxygenase c th xy ra hai v tr meta v ortho. Tuy nhin, ct vng ti v tr meta c thc hin bi enzyme C23O c coi l qu trnh ph bin nht cc giai on tip theo ca phn hy sinh hc PAH. Xut pht t v tr quan trng ca gen m ha enzyme C23O trong vic phn hy sinh hc PAH, gen m ha C23O c nhiu tc gi nghin cu quan trc. Vic nghin cu s tn ti ca gen ny trong t nhin cng nh trong cc chng vi khun s dng PAH l rt cn thit. Trong nghin cu ny, trnh t on gen m ha enzyme catechol 2,3-dioxygenase ca chng BQN31 c xc nh nhm mc ch tm hiu s a dng gen chc nng cng nh bn cht qu trnh lm sch du v PAH nhim mc phn t. Cc kt qu thu c s gip chng ta hiu r hn v qu trnh lm sch nhim du cng nh s a dng cc gen chc nng trong nc nhim du ti khu vc Qung Ninh. Sn phm DNA c tch sch nh phn 3.3.1 c dng lm khun tin hnh phn ng PCR vi cp mi C23OF v C23OR. Sn phm



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PCR thu c c kch thc khong 750 bp ph hp vi tnh ton l thuyt (Hnh 3.12).kb M 1

Hnh 3.12. Sn phm PCR nhn on gen m ha enzyme catechol 2,3-dioxygenase t DNA tng s chng BQN31 v cp mi C23OF v C23OR. M-

0,75 0,50

thang DNA chun 1kb.

atggatgttatgggtttcaaggtcgccaaggacgcggacttggaccattttaccgagcgc M D V M G F K V A K D A D L D H F T E R ttgctcgatatcggtgtccatgtcgacgtgatcccggcgggggaagatcccggtgtaggc L L D I G V H V D V I P A G E D P G V G cgcaagattcggtttaacacgccgacacagcacgtcttcgaactttacgccgagatggcg R K I R F N T P T Q H V F E L Y A E M A ctgtcggccaccggtccggccgtcaagaaccccgatgtctgggtcgtggagccacgtggc L S A T G P A V K N P D V W V V E P R G atgcgtgccacccgctttgatcactgtgcgctcaacggcgtggatatagccagttcggcc M R A T R F D H C A L N G V D I A S S A aagatttttgtcgatgcgcttgatttctcagtcgccgaggaactggtcgatgaaacca gc K I F V D A L D F S V A E E L V D E T S ggcgcccggctcggcatctttcttagctgcagcaacaaagcacacgatgtcgccttctta G A R L G I F L S C S N K A H D V A F L ggctatcccgaagacggtaagatccaccatacctcgttcaacctggaatcctggcacgat G Y P E D G K I H H T S F N L E S W H D gttggccatgccgccgacatcatcagccgctacgatatttcgctggatatcgggccgacc V G H A A D I I S R Y D I S L D I G P T cgtcatgggatcacccgcgggcagacgatctacttcttcgatccctcgggcaaccgcaac Hnh 19 l cy pht sinh chng vi khun i din. R H G I T R G Q T I Y F F D P S G N R N gaaaccttcagcggcggttacatttattatccggacaatccgcagcgcctgtggcaggca E T F S G G Y I Y Y P D N P Q R L W Q A gagaacgccggcaaggccatcttctactacgaaaaggcgctcaacgaccgcttcctgaca E N A G K A I F Y Y E K A L N D R F L T gt

Hnh 3.13. Trnh t nucleotide v trnh t axt amin suy din on gen m ha enzyme catechol 2,3-dioxygenase ca chng Sphingomonas sp. BQN31S ha bi Trung tm Hc liu i hc Thi Nguyn http://www.lrc-tnu.edu.vn

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Sn phm PCR c gn vo vector pCR 2.1 nh enzyme T4 ligase 14oC, bin np vo t bo E. coli INV F, tch chit v kim tra DNA plasmid theo Sambook v cng s. Xc nh trnh t on gen m ha C23O trn my xc nh trnh t nucleotide t ng ABI PRISM 3100 Avant Genetic Analyzer, c x l trn phn mm Bioedit 6.0.7 (5/17/04), v suy din ra trnh t axt amin, so snh mc tng ng bng chng trnh Blast (Hnh 3.13). Trnh t nucleotide v axit amin suy din on gen m ha C23O ca chng BQN31 c ng k trnh t cc on gen trn GenBank vi m s ln lt l EU814954 v ACF24468.1C23O-Sphi. A8AN3 (U73127) C23O-Sphi. B2-7 (EU596533) C23O-Rhiz- ZJF08 (DQ534019) xylE-Sphi- KMG425 (AF494100) xylE-Sphi- B1 (U23375)

cmpE-Sphi- HV3 (Z84817)catE-Sphi. agrestis - HV3 (L10655) phnE-Pseu- DJ77 (U83882) C23O-Pseu- ZP2 (EU082778)

C23O-Sphi. BQN31C23O-Sphi- C230 (DQ873681)

Hnh 3.14. Cy pht sinh chng loi cc on gen m ha enzyme catechol 2,3-dioxygenase ca chng Sphingomonas sp. BQN31 v mt s chng vi khun i din (Thc o th hin mt nucleotide khc nhau trn 100 nucleotide so snh)



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So snh mc tng ng gia trnh t on gen m ha enzyme catechol-2,3 dioxygenase ca chng BQN31 v ca cc chng khc trn GenBank v EMBL cho thy, on gen ny chng BQN31 c tng ng rt cao i vi cc on gen m ha enzyme catechol-2,3 dioxygenase tng t cc chng vi khun thuc chi Sphingomonas, Pseudomonas (Hnh 3.14, Bng 3.6). on gen m ha catechol 2,3-dioxygenase ca chng BQN31 c mc tng ng 99 % vi cc on gen phnE chng vi khun Pseudomonas sp. DJ77, cmpE chng Sphingomonas sp. HV3, catE chng Sphingomonas agrestis HV3 v C23O chng Pseudomonas sp. ZP2 (Bng 3.6). Bng 3.6: tng ng ca on gen m ha enzyme catechol 2,3 dioxygenase ca chng BQN31 so vi mt s i din c cng b trn ngn hng gen quc t

STT

Chng vi khun Sphingomonas sp. strain HV3 Pseudomonas sp. DJ77 Pseudomonas sp. ZP2

S nucleotide tng ng 716*/719# 716*/719# 714*/719#

Mc tng ng (%) 99,58 99,58 99,30

1 2 3

Ch thch: *- s nucleotide ca on gen 16S rRNA chng BQN31 #- s nucleotide ca on gen 16S rRNA cc chng i din Cc trnh t nucleotide cng b v gen m ha C23O ca cc i din thuc chi Pseudomonas (P. aeruginosa JI104, P. putida CF600, P. putida P 35X, P. putida pDK1, P. putida H, P. putida pW0, P. putida NAH, v Pseudomonas sp. IC) cho thy mc tng ng t 75% n trn 95% [45]. Trong khi , mc tng ng v trnh t nucleotide ca gen ny cc vi



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khun thuc nhm Sphingomonas (Sphingomonas sp. HV3 v S. yanoikuyae B1) l 76,5%. S ging nhau v trnh t nucleotide gen m ha C23O cc chng vi khun ca hai chi ny vo khong 50% [45]. Trnh t C23O ca cc chi khc nh Ralstonia (R. pickettii U20258, R. eutropha JMP 52415, R. rhodochous X69504, R. rhodochous NCIB 13064 v Bacillus (B. stearothermophilus FDTP-3) khng ng nht. Cc trnh t ny cng khng hon ton ging nhau gia cc thnh vin, vi nhm Sphingomonas v nhm Pseudomonas. Kt qu thu c trong nghin cu ca chng ti mt ln na khng nh s phn b rng ri ca vi khun s dng PAH thuc nhm Sphingomonas. Chng vi khun BQN31 c th l vi sinh vt hu ch trong qu trnh t lm sch hoc x l nc thi nhim du bng phng php phn hy sinh hc hoc c s dng xy dng tp on ging phc v cho cc mc ch x l nhim PAH trong cc im c th kim sot c.



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KT LUN 1. Trong s 15 chng vi khun c kh nng s dng cc ngun PAH c phn lp, chng BQN31 c kh nng phn hy mnh nht. 2. Chng BQN31 thuc nhm vi khun Gram m, khun lc hnh trn, trn, li nh, trng c c nh xanh, kch thc khong 1-2 mm. T bo vi khun BQN31 c hnh que, b mt tng i nhn, kch thc khong 0,29 0,5 m x 0,95 - 1,1 m. 3. Sau 5 ngy nui lc nhit 30oC v 200 vng/pht, trong mi trng c nng ban u 100ppm cho mi loi PAH, chng BQN31 c kh nng s dng 69,38% naphthalene, 60,24% phenanthrene, 18,52% fluorene, 25,9% anthracene v 18,75% pyrene. 4. Da trn mt s c im hnh thi v so snh trnh t on gen 16S rRNA, chng BQN31 c xp vo chi Sphingomonas v c t tn l Sphingomonas sp. BQN31. Trnh t on gen m ha 16S rRNA ca chng BQN31 c ng k trn GenBank vi m s EU814953. 5. on gen m ha enzyme catechol 2,3-dioxygenase chng BQN31 c tng ng rt cao so vi cc on gen tng t cc chng vi khun thuc chi Sphingomonas, Pseudomonas. N c mc tng ng 99 % vi cc on gen phnE chng vi khun Pseudomonas sp. DJ77, cmpE chng Sphingomonas sp. HV3, catE chng S. agrestis HV3 v C23O chng Pseudomonas sp. ZP2. Trnh t nucleotide v trnh t axt amin suy din on gen m ha enzyme catechol 2,3-dioxygenase ca chng Sphingomonas sp. BQN31 c ng k trn GenBank vi cc m s ln lt l EU814954 v ACF24468.1



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TI LIU THAM KHOTing Vit. 1. ng Th Cm H, Nguyn ng Nh, Nghim Ngc Minh, Nguyn Thnh c (2003) Phn loi x khun XKDN11 s dng dibenzofuran, hyrocarbon thm a nhn phn lp t t nhim cht c ha hc, Tp ch Cng ngh sinh hc, 3(1), tr. 377-386. 2. Hong Th M Hnh, Nguyn ng Nh, ng Th Cm H (2003), Nm si phn hy hydrocarbon thm a nhn phn lp t cn du th ca ging khai thc du, Vng Tu, Tp ch Cng ngh Sinh hc, 2(1), tr. 255-264. 3. Hong Th M Hnh, Nguyn Thanh Thy, Ng Xun Qu, Nghim Xun Trng, Nghim Ngc Minh, ng Th Cm H (2004), Kh nng phn hy 2,4-D v dibenzofuran ca chng nm si FDN20, Tp ch Cng ngh Sinh hc, 2(4), tr. 517-528. 4. Nguyn B Hu (2002), Nghin cu cc nhm vi sinh vt v kh nng phn hy hydrocacbon thm a nhn ca mt s chng vi khun trong qu trnh x l nhim du ti Khe Ch, Qung Ninh, Lun vn Thc s Sinh hc, Vin sinh thi v ti nguyn sinh vt, H Ni. 5. Nguyn B Hu, Trn Th Tng Vi, Nghim Ngc Minh, ng Th Cm H (2007), Phn hu sinh hc du diesel v hydrocarbon thm a nhn ca mt s chng vi khun phn lp t nc thI nhim du kho cng B12, Qung Ninh, Tp ch Khoa hc v Cng ngh - i Hc Thi Nguyn. S 2 (42). 6. Nghim Ngc Minh, Nguyn Thnh c, (2004), Phn loi chng vi khun HDG1 phn lp t mu nc thi nh my giy Hi Dng, Tp ch Cng ngh Sinh hc, 2(2), tr. 245-252.



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7. Nguyn ng Nh (2004). Nghin cu c im sinh hc v kh nng phn hu dibenzofuran, hydrocarbonthm a nhn ca hai chng x khun phn lp t t nhim cht c ho hc. Lun vn thc s sinh hc, Vin sinh thi v ti nguyn sinh vt, Vin Khoa hc v cng ngh Vit Nam nm. 8. La Th Thanh Phng, Nguyn B Hu, ng Th Cm H (2003), Phn hy sinh hc hydrocarbon thm a nhn (PAHs) bi chng vi khun MXL-9 phn lp t cn du th ca m Bch H Vng Tu, Tp ch Cng ngh Sinh hc, 1(1), tr. 109-117. 9. Mai Anh Tun, Nghim Ngc Minh, ng Th Cm H (2004), Nghin cu phn loi v kh nng s dng hydrocarbon thm a nhn bi chng x khun XKDN19 phn lp t t nhim cht c ha hc, Tp ch Cng ngh Sinh hc, 2(3), tr. 389-396. Ting Anh. 10. Ahn Y, Sanseverino J, Sayler GS (1999), Analyses of polycyclic aromatic hydrocarbon-degrading bacteria isolated from contaminated soil, Biodegradation, 10, pp. 149-157. 11. Albert L., Juhasz, Ravendra Naidu (2000), Bioremediation of High molecular weight polycyclic aromatic hydrocarbons: a review of the microbial degradation of Benzo[a]Pyren, International

Biodeterioration

Biodegradation 45, pp. 57-88.

12. Allison D. Gelsenbrecht, Brian P.Hedlund, Mary A.Tichi v J. T. Staley (1998), Isolation of Marine Polycyclyc Aromatic Hydrocarbon Degrading Cycloclasticus Strain from Gulf of Mexico and Comparison of Their PAH Degradation Ability with That of Puget Sound Cycloclasticus Strains, Applied and Environmental

Mycrobiology, pp.4703-4710.



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13. A Dhenain , G Mercier , J F Blais , M Bergeron (2006), PAH removal from black sludge from aluminium industry by flotation using nonionic surfactants, Environ Technol. Sep ;27 (9), pp. 1019-30 17067128. Bioinfobank Library. 14. BAQAR R. ZAIDI* and SYED H. IMAM (1999), Factors Aecting Microbial Degradation of Polycyclic Aromatic Hydrocarbon Phenanthrene in the Caribbean Coastal Water, Marine Pollution Bulletin,

vsv phan huy pah

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