phaiv lap va tuyeiv chom cac chijivg vi khuaixi

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KHOA HOC CONG NGHE

P H A I V L A P V A T U Y E I V C H O M CAC CHIJIVG V I KHUAIXI BAIXI D ! A CO K H A I V A I X I G P H A I X I H U Y IVIAIXIH CAC H O P C H A T HLTU CO T I J IVLTOC T H A I LAIVG IVGHE I V A U R U O U D A I L A M IXIHAIVI IJIXIG DLJIMG TROIXIG XLT L Y IXIUOC T H A I

D^o Thi H6ng Van\ D6 Phuong Khanh\ Nguyfe Van Hi^u^

T6MTAT

Lang nghS n^u rugu tniyen thong 6 Viet Nam thirong ket hop voi chan nuoi lpn, n^n nuoc thai a day la t6ng hop tir cac nguon san xuat ruou, chan nuoi va smh hoat cua hp gia dinh. TTianh phan nunc thai phong phu, trong do chiia luong lon hop ch^t huu co kh6ng duoc xu ly ma thai ra moi truong. Nghien ciiu sir dung vi smh vSt ban dia de xu ly nuoc thai co nhieu im the va dang dugc quan tam. Tir 7 mau nuoc thai thu thap tai lang nghe Dai LSm (tinh BSc Ninh) da phan lap duoc 35 chung vi khuan hieu khi. Da tuy^n chon duoc 3 chung vi khuan ki hi^u BHl, BH2, BH3 co kha nang phan huy tdt hop chat him co (tmh hot, protein, cellulose). Nghien cuu hinh thai khuan lac va te bao, kha nang sir dung nguon duong va nito, k^t qua phan tich trinh tu gen 16S rDNA va so sanh cho thay muc do tuong dong cao (> 99%) voi mgt sd loai tr&n GenBank. 3 chung da dupe dinh danh va dang ky tren GenBankvoima sd: Bacillus sp. BHl {MN519478), Baallussp. BH2 (MN519479) va Pseudomonassp. BH3 (MN519480).

Tirkh6a: Bacillus, Pseudomonas, xu ly nuac thai lang nghS.

I.DATVAniDE

6 Viet Nam, su phat trien ciia cac nganh che bien thuc pham thoi hOi nhap da keo theo nhi^u van de v^ 6 nhi6m m6i trtrong. Nuoc thai ttr cac co so che bi^n Itrong thuc, nau nrgu, lam my, banh da, lam bun CO luong dinh dtrong kha 16n (protein, tmh bpt, cellulose) day la nhimg hop chat hang ngay v§n diroc thai true tiep ra moi trirong xung quanh hoac cac nuoc thai chi duoc xir ly so bp. Qua phan tich moi trudng niroc mat cua Trung tam Quan trSc va Phan tich moi trudng tinh Bac Ninh cho thay nuoc thai b lang nghe Dai Lam (B^c Nmh) co ham lupng COD vuot tir 12-15 lan QCVN. K^t qua da gay nen hien tupng phii dirPng trong nuoc, lam giam n6ng dp oxy hoa tan, gay min thdi,...[2]. Loai bo tinh bpt, cellulose va protein c6 trong nude thai bing phuong phap sinh hoc da va dang duoc chii y trong thdi gian gan day, do cd tinh hieu qua cao, an toan vdi mdi truong. Hien nay, da cd nhieu cdng bo v6 vai trd khac nhau cua nhieu nhdm vi sinh vat khac nhau tham gia vao qua trinh chuyen hda tinh bdt, cellulose, protem ttr dang cao phan tii cd trong nuoc thai thanh dang d§ tan d^ VI sinh vat su dung tao sinh khoi, sinh khi sinh hpc (Choi etal., 2002; Lalloo etal., 2007). Trong cac nhom vi sinh vat, chi Bacillus dugc ung dimg pho bi^n trong nhieu nganh tir nudi trdng thuy san, chan

' Khoa Cong nghe sinh hoc, Trucmg Dai hgc Md Ha Noi

^ Vien Cong nghe Sinh hoc. Vien Han iam KH&CN Viet Nam

nudi, xu ly nude thai, fhuc pham chiic nang, dac bi$t la trong nganh nudi trdng thiiy san va xu ly ntrdc thai ngay cang ung dung nhieu (Choi etal., 2002), nhat la d cac nude cd cac he thdng nu6i ca tham canh. Cac loai thupc chi Bacillus la thanh phan cua cac san pham cd kha nang tieu didt cac nhdm vi khuan gay benh, day la hdn hop cac hoat chat duoc cac loai sinh ra va cac chung khi bo sung vao hd thong nudi trong dem lai hieu ring cd lpi cho he thdng (Lalloo et al., 2007). Cac loai thudc chi Bacillus cd kha nang smh trudng nhanh, kha nang sinh bao tu, giiip chiing chiu dung dugc nhidu dieu kien bat loi cua mdi trudng (Lalloo etal., 2007; Lalloo etal, 2009). Nhieu bao tu cua cac loai thupc chi Bacillus nhu B. subtih's; B.

hcheniformis; B. clausu... duoc ban trdn toan the gidi dugc sii dung nhu la mdt san pham thuc p h ^ chuc nang cho ngudi va dong vat (Sanders et al, 2003).

IVIdt sd loai nhu B. subtih's cd the phat tri^n trong dieu kien hidu khi, vi hieu khi va ky khi, day la cac dieu kien trong chuyen hda nito nhu nitrat hda va khu nib-at (Choi etal, 2002; Sakai etal, 1996; Lm et al., 2006). Wang va cdng su (2008), su dung mpt sd chung vi khuan thudc chi Pseudomonas, Aeromonas va Bacillus de loai bd tmh bdt, cellulose, nita tu nguon cdng nude thai, ho tap trung, hieu qua loai bd tdng cacbon hiru co, tdng nita va tong phospho lan lugt la 80,2%, 81,6% va 86,8%. Trong nghien ciiu nay, trinh bay mpt sd ket qua nghien ciiu phan lap, tuyen chpn va phan loai cac chung vi khuan cd kha nang

NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 4/2020

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p h a n h u y t i n h b d t , c e l l u l o s e v a p r o t e m d u p e t h u n h a n n g u o n n u o c t h a i ciia c a c c d s d n a u n r p n thu c d n g tai l a n g D a i L a m (tinh B a c N i n h ) .

2. PHUDNG PHAP IWGEN CUU 2.1. V4t hfyi vk mdi trudng

- Vat lieu : 7 mau nude thai dupe thu nhan tai co sd nau rupu tai lang nghd nau rupu Dai Lam (xa Tam Da, huyen Yen Phong, tinh Bac Ninh).

- Mdi trudng: MPA (g/1): cao thit 5,0; peptone 10,0; NaCl 5,0; glucose 2,0; agar 20,0; pH 7,2+0,2;

IQioang ca ban. (^H^^SO^ 0,5; Y^R-^O^ 2,5;

MgS04.7H202 0,2; vi luang 1 ml (^^SO.'.mzO 0.1;

MnCl2.4HaO 0,1; ZnSO^.yH^O 0,1; nude cat 100 ml).

pH 7,0-7,2. A/o/ trudng kidm tia boat tinh protease, amylase, cellulase (g/1): (casein, tinh bpt, CMC) 2,0;

FeS04.7H20 0,1; KCl 0,5; K^HPO, 1,5; MgS04.7H202 0,5; Glucose 10; agar 20,0 pH 6,8-7,2.

2.2. Phtrcmg phip nghidn cuu

2.2.1. Xac dinh kha nang phan giai cac hop chat hiru ca

Cac chung vi khuan dupe nudi d nhiet dd 37°C trdn cd bd sung cac ca chat khac nhau: casein, tinh bdt tan, carboxymethyl cellulose (CMC). Sau 48 gid, xac dinh hoat tinh enzym ngoai bao bang phuong phap khuech tan tren thach su dung cac dung dich thudc thir tuong ling: Lugol 1% vdi mdi trudng chiia tmh hot, Congo dd cho mdi tnrdng chira CMC, Trichloro acetic acid 5% vdi mdi trudng chira casein (EgorovNX, 1983).

2.2.2. Xac dinh hoat luc enzym

Cac chiing dugc nudi tren mdi trudng MP sau 48 gid, ly tam 10000 vdng/phiit d 4°C trong thdi gian 10 phiit, loai smh khdi va thu dich. Dich enzym ngoai bao se duoc xac dinh hoat luc enzym: protease theo Anson, 1938; amylase theo Hitoshi va cdng sir 1988;

cellulase theo Ryu va Mandels 1980; trong dd xac dmh dudng tao thanh theo phuong phap Miller, 1959.

2.2.3. Nghien cuu mdt sddac diem smh hoc Theo khda phan loai Berge/s Mannual of Determinative Bacteriology (1989).

2.2.4. Phuong phap dinh danh bing sinh hoc phan tir

Tach chidt DNA tdng sd theo Sambrook va Russell (2001); Chung tijyen chon ti-en mdi tiirdng MP b "iTC trong 24 gid. Lay ImL dich len men vao dng eppendorf 1,5 mL. Ly tam 5000 vong/phiit trong 10 phiit, thu smh khdi td bao de tach DNA Hda sinh

khdi t^ bao h-ong 0,5 mL ddm TE va SDS (TE buffer:

15 mM Tris-HCl * 1 mM EDTA (pH=7.5). U o nhiet dp phdng 10 phiit Bo sung lysozym 50 ul/ml va proteinase K. Trdn nhe nhang h-ong 3 phtil, u 65"C h-ong 1 gid. Sau do them 0,15 ml CH3COOK va ly tam 10000 vdng/phiit trong 15 phiit b 4''C. Dich ndi dugc dua sang dng eppendorf mdi va bo sung mpt lupng tuong duong isopropanol. Sau dd, ly tam d 13000 vdng/phiit b-ong 10 phut d 4°C thu tiia. Rua tiia bang EtOH 70% rdi lam khd va hda tan lai DNA vdi 30 (iL trong nude.

• Phan ungPCRnhan doan gen IGSrDNA -I- Thanh phin cua phan ling PCR chuan vdi th6 tich phan ling 20 pi la: 1,0 pi DNA template; 0,2 Taq polymerase; 1,0 pi GFl (10 pmol/pl); 1,0 ^il GRl (10 pmol/pl) 2,0 ul Taq Buffer (lOX); 0,6 ^1 MgCI^ (50 mM); 0,4 pi dNTP (10 mM); 13,8 pi H^O.

-H Trinh tu mdi xudi va mdi ngugc nhu sau:

GFl (5'-TAACACATGCAAGTCGAACG-3') GRl (5'-GG(C/T)TACCTTGTTACGA(rrr-3') + Chu trinh nhiet cua phan ung PCR. Bien tinh b chu ky ddu d 94''C trong 5 phiit. Sau dd chay 30 chu ky (94X ti-ong 60 giay, GO^C ti-ong 60 giay; 72''C trong 90 giay) va chay d 72"C trong 10 phut., giii mau d4"C.

- Dien di gel agarose: San pham cua phan ling PCR duoc ki^m tra bang dien di trdn gel agarose 1%, che do chay 80V va 40 mA trong 40 phiit va nhupm EtBr, tinh sach bang kit PureUnkTM DNA Purification (Invitrogen, My).

- Giai tiinh for Trinh tir nucleotide doan gen IBS rDNA dugc phan tich tren may dpc tu ddng ABI PRISM 3100 Avant Genetic Analyzer, yyx ly bang phan mdm CLC Main Workbench 7.1. Muc dp tuong ddng cua gen 168 rDNA a i a cac chung vi khuan dugc so sanh vdi cac trinh hi gen 16S rDNA tren co sd da heu GenBank. Mtic do tuong ddng di tiny^n cua cac chung dugc xay dung dua tren phan m^m CLC Mam Workbench 7.1.

3 . KET QUA VA THAO LUAN

3.1. Phan lap vk tuy&i chpn cAc chung vi khu5n CO kha nang phfin hiiy hgp ch^t hint co

Tir 07 miu nude thai thu thap tir cac ca sd niu ruou da phan lap duoc 35 vi khuan. Trong sd nay, cac chung BHl, BH2, BH3 co kha nang suih ddng thdi 3 enzyme ngoai bao hoat tinh cao. Hoat dd enzyme phan giai casern, tinh bdt, cellulose ciia 3 chung duoc the hien dbang 1 va hinh 1.

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KHOA HQC COWO NGHE

Bang 1. Kha nSng sinh tdng h(ip enzyme ngoai bdo ikhufa

Chung BHl BH2 BH3

Hoat luc enzvme CU/ml) Protease

25,9 10,8 15,4

Cellulas 0,8 1,6 1,2

Amylase 1,4 2,9 3,2

trgng, phii hpp vdi muc dich nghien cuu, tim ki^m nhimg chitng cd kha nang ling dung cao trong xu li nude thai trong nau rugu truyen thdng va chan nudi, vdn ton tai kha nhieu cac hpp chat nhu: protein, chat xa, nita va tinh hot vdi ham lugng kha cao. Vdi kha nang sinh enzyme manh ciia cac BHl BH2 va BH3 se gdp phan thiic day nhanh qua trinh phan huy cac hgp chat gay d nhidm ntrdc thai tai cac ca sd nau rugu va chan nudi.

Cac chung vi khuan phan lap chinh tir ngudn thai budc dau cho thay tiem nang ling dung chting vao qua trinh xtr ly hpp chat hiiu ca trong nude thai.

3.2. Hinh thai khuSn lac cua ba chiing vi khuan tuyfo chgn

Cac chiing vi khuan BHl, BH2 va BH3 dugc cay na trdn dia petri cd mdi trudng MPA nudi 2448 gid d Hinh 1. Khd nang phan giai casern {A), tinh bOt (B) nhiet dp 37°C. Ket qua quan sat dac diem hinh thai vk cellulose (C) cua chung BNl khuan lac ctia 3 chiing vi khuan dugc th^ hidn d bang Kha nang phan giai cac hap chat hiru ca thdng 2.

qua he enzyme la mdt trong nhiing dac diem quan

Bang 2. D$c dilm hinh thai k h u ^ lac cua ba chung vi khuan sau thdi gian 48 gid a 37''C trSn mdi trudng MPA

H

(A) CB) (C)

Dac diem Kha nang phat tnen Hinh thai khuan lac Mep khuan lac Mau sac khuan lac Bd mat khuan lac Mat dudi khuan lac Tiet sac td

Kha nang phat trien ciia cac chung BHl

Tdt Khdng trdn Rang cua Vang hai nau Phang nhan Vang Khdng

BH2 Tdt Trdn Rang cua Trang hoi nau Phang nhan Vang nhat Khdng

BH3 Tdt

Trdn deu Trdn Tring siia

Loi, khdng nhan dang nhao Tring due

Khdng Ket qua the hien tren hinh 2 va bang 2 cho thay,

khuan l^c chung BHl va BH2 cd mau vang hoi nau, bd mat phing, mep rang cua va khdng tidt sac td tren moi trudng thach. Chung BH3 cd mau trang due, Idi, mep ti-dn va khdng tiet sdc td tren mdi tnrdng thach.

3.3. Hinh thai td bao cua ba chung vi khuin tuy^n chgn

Nhudm Gram la mdt phuong phap cho phep phan biet cac loai vi khuan thanh 2 nhdm Gram dtrong (Gram +) va Gram am (Gram -) dua tren cac dac tinh hoa ly cua thanh te bao bat mau vdi thudc nhudm, ddng thai khi quan sat ta cd the biet hinh thai te bao ciia cac chiing vi khuan (Egorov NX,

1983). Tidn hanh nudi cay 3 chimg BHl, BH2 va BH3 tren mdi trudng KB long, sau 16 gid dem nhudm Gram, soi tren kinh hi^n vi quang hpc. Ket qua nghien ciiu dac diem hinh thai cua 3 chung vi khuan tuyen chgn duoc the hien trong bang 3.

Bang 3. D§c di^m hinh thai t^ bao ciia ba chung vi khuan.

Chung BHl BH2 BH3

Hinh thai te bao Que dai Que dai Que ngan

Gram + +

Ghi chu: "-" vi khuan Gram am ; "-/- Gram duong

vi khuan

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KHOA HOC CdNG NGHt

Bang 4. Khi nang su dijng die nguin cacbon cua 3

Hinh 2. Hinh thai khuin lac 3 chung id khuan sau 48 gidr nuoi trtn moi tniemg MPA a 37°C

/ ' : M

'

Hinh 3. Hinh thai t^ bao cua 3 chiing vi khuan (xlOOO)

Tir ket qua hinh 3 cho thay te bao cac chung vi khuan BHl va BH2 cd hinh que dai, bat mau tim;

chung BH3 cd hinh que ngSn bat mau hdng. Theo dd, chung BHl va BH2 la chung vi khuan Gram (+), cdn chting BH3 la chung vi khuan Gram (-).

3.4. Kha ndng sii dung ngudn cacbon ciia ba chiing vi k h u ^ tuy^n chgn

Cacbon la ydu td cau tao nen nhieu hgp chat can thiet khac nhau cho su sdng va la thanh phan diiih dudng rat quan trong ddi vdi vi sinh vat. Vi sinh vat sir dung cac ngudn cacbon de sinh trudng va phat trien. Do vay, viec nghien ciiu kha nang sir dung ngudn cacbon ciia cac chung vi khuan la het siic can thiet. Tren mdi trudng khoang bd sung cac ngudn dudng khac nhau vdi ti le 1% (w/v). Khii trimg cac dng nghiem chiia mdi trudng thach theo phuong phap Pasteur. Tiep do, cay cac chung vi khuan va nudi ti-ong thai gian 48-72 gid d nhiet dp 37"C. Ket qua kha nang phat trien ctia 3 chiing vi khuan dugc th^ hidn b bang 4.

Ngudn cacbon Ddi chung (-) Glucose (+) Maltose Arabinose Trehalose D-Fructose D-Sorbitol Lactose Rhamnose D-Mannose Raffinose Xylose

Chiing vi klu..

BHl

+ + -H- + -1- -1- + +-H

+ -1-

BHli

+ + + +

+

111-13

-H-

• H -

+ -1- ++

+-I- + -H- +-f -H

• H -

Hinh 4. Kha nSng su dung cdc ngudn cacbon ciia chiing BHl sau 48 gid d 3?'C

Ghi chu. (-): Khdng phat tii^n; (+): Phat tilln;

(++): Phat ti-ien tdt

Viec nghidn cuu kha nang sOr dung cac nguon dudng la chi tieu quan trgng de phan loai vi khuan.

Tir ket qua d bang 4 va hinh 4 cho thay, 3 chung vi khuan BHl, BH3 va BH2 cd kha nang phat tn^n tdt tren cac ngudn cacbon: Glucose, Maltose, Arabinose, Trehalose-Dihydrate, D-Fructose, D-SorbitoJ, Lactose, Rhamnose, D-Mannose, Raffinose va Xylose.

Kha nang sii dung nhieu ngudn cacbon se giiip cho cac chung vi khuan cd kha nang thich nghi vdi dieu kien mdi trudng da dang, ddng thdi tao ra dinh hudng tdt cho viec tim ngudn cacbon thay the cho vide san xuat d qui md cdng nghiep.

3.5. Kha nang sii dung ngudn nito cua ba chiing tuy^n chgn

Trong thuc te, vi sinh vat can nita trong cac qua trinh sdng de xay dung te bao nhung chi cd mpt sd it cd kha nang cd dinh nita. Trong tat ca cac mdi tnrdng nudi cay can tiiiet phai cd cac loai hop chat nita ma vi sinh vat cd kha nang sir dung duoc. Cac ngudn nito him co dimg trong nudi cay vi sinh vat d qui md cdng nghiep thudng la cao ngd, bdt dau tuong, hot lac, cac loai khd dau. Khi dd vi sinh vat phai tidt ra he protease de thiiy phan protein thanh cac axit amin va sii dung. Kei qua danh gia su phat ti-ien ciia 3 chung tuyen chpn tren cac ngudn nito khac nhau sau 72 gid nudi b nhiet dp 3? C. dugc the hien d bang 5 va hinh 5.

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JJ.IiWlttH

imim

thai dac biet la nude thai giau dmh dudng nhu protein, tinh hot, cellulose, lipid...

3.6. Phan loai vi khudn bang smh hpc phan tii

* Tach chiet DNA tdng sd cua vi khuan dupe tach theo phuong phap duoc md ta bdi Sambrook va Russell (1989), ket qua dugc the hien d hinh 6.

Hinh 5. Kha nang phat tri^n cua 3 chiing tr^n cac ngudn nita khac nhau sau 72 gid nuoi cay b 37''C Bang 5. Kha nang phat triln cua 3 chung tuyfo chpn trfin cac ngudn nita khac nhau sau 72 gid nudi

cayd37''C Ngudn Nita Ddi chiing (-) Histidin L - tryptophan (NH,)2S0, (NH4)2Fe(S04).6H20 NH4NO3 NH4CI KNO3 (NH4),HP04

Chung vi khuan BHl

-t-

-H- + ++

+ +

BHl

+ +

+

++

-H

BHl + + ++

+ ++

+ -H + Ghi chu:{-) : Khdng phat trien; (+): Phat tnen;

(++): Phat ti-ien tdt

Ket qua the hien trdn bang 5 va hinh 5 cho thay, 3 chung BHl, BH3 va BH2 cd kha nang phat tiien tdt tren mdi tnrdng cd ngudn nito la KNO3 va (NH4)2Fe(S04).6H20 va histidin. Ddi vdi chiing BHl va BH2 cd kha nang manh tren mdi trudng cd ngudn nita NH4NO3 va KNO3, chung BH3 cd kha nang manh tren mdi tnrdng cd ngudn nita KNO3.

Tir cac ket qua nghien ciiu thu duac d vd hinh thai, dac diem sinh ly- smh hda va cac dieu kien vd mdi trudng, so sanh vdi khda phan loai cua Bergey's 1989 budc dau cd the ket luan rang:

- Chiing VI khuan BHl, BH2 thudc chi Bacillus - Chimg vi khuan BH3 thudc chi Pseudomonas.

Tir ket qua nghidn ciiu thu nhan duac tien hanh nghidn ciiu kha nang img dung cua 3 chung vi khuan trong xir ly nude thai. Theo cac nghien ciiu trude day mdt so loai thudc nhdm vi khuan Bacillus nhu: B.

niegatenuni; B. subtih's va B. hcheniformis va chi Pseudomonas da dugc img dung ti-ong xu ly nude

1 2 3 I M

kt : M l^A. 2_-=i. 3 - - ^

Hinh 6. Didn di dd DNA tong sd va san pham PCR khudeh dgi gen IBS rDNA cua cac chung BHl; BH2,

BH3

[fiiinsllKirSfrP'WfllSlI titrlitaliixiaHlitmimil

ttmimaiiil smiaiiimm.Min'imiaiici

_jWusm

— SidftltM^KSIfSmiWKTSU

— BioiiomuiJM-rSWHJM!)

Hinh 7a. Mdi quan he tuong ddng di truydn giira chung BHl vdi cac chung trdn (^nBank

* Khuech dai gen WSrDNA

Gen 16S rDNA cua cac chung vi khuan dugc khuech dai bang phan ling PCR sir dung cap mdi GRlva GFl (nhu phan phuong phap). San pham cua phan ling PCR duoc kiem tra, tinh sach, giai trinh tir.

DNA tdng sd cua cac chiing vi khuan lua chpn dugc kidm ti-a bang dien di tren gel agarose 1% (Hinh 6) va sir dung lam khudn cho phan img PCR khuech dai gen 163 rDNA San pham cua mdi phan img PCR cho 1 bang duy nhat cd kich thudc 1,4 kb ti-en gel agarose 1%, tuong ling vdi kich thudc mong dpi. Vi vay, san pham PCR sau khi tinh sach duac su dung dd giai trinh tij hai chidu cho doan gen 16S rDNA (Hinh 6).

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KHOA HOC CONO NGHl

H i n h 7 b . M d i q u a n h ^ t u o n g d d n g di t r u y d n gifta Tir k e t q u a p h a n tich t r i n h tiJ g e n 16S r D N A va s o s a n h vdi c a c t r i n h t u g e n t u o n g u n g t r d n n g a n h a n g dir lidu c o s d G e n B a n k b a n g c o n g cu B L A S T ( N C B I ) c h o t h a y : g e n 16S r D N A c h i i n g B H l va B H 2 cd d p t u o n g d d n g c a o (99%) s o vdi g e n t u o n g ling ciia c h i Bacillus, t r o n g d d g e n 16S r D N A c u a c h u n g B H l c d d o t u o n g d d n g c a o (100%) so vdi g e n t u o n g l i n g c u a c h i i n g Bacillus s p . B K T (KX257799) va g e n 16S r D N A c u a c h i i n g B H 2 cd d o tirang d d n g c a o (99%) s o v d i g e n tirong u n g c u a c h u n g B. amylohquefaciens B l l ( M F 1 2 5 2 8 0 ) . C h u n g B H 3 cd d o t u o n g d d n g c a o (99%) s o vdi g e n t u o n g i m g c u a c h i Pseudomonas n h u Pseudomonas fulva ZIO (MN515149) va Pseudomonas parafulva L ^ 1 4 3 (MK414838).

Tir c a c k e t q u a nudi cay, m d t sd d a c d i ^ m s i n h h d a v a giai t r i n h t u g e n 16S r D N A , cd t h e d i n h ten c a c c h i i n g vi k h u a n p h a n giai h p p c h a t h i i u c a t u y e n c h g n d u a c n h u s a u :

- C h u n g B H l t h u d c chi Bacillusva d u g c d i n h ten la Bacilluss^. B H l (MN519478)

- C h u n g B H 2 t h u d c chi Bacillus va d u g c d i n h tdn la Bacillus sp. B H 2 (MN519479)

- C h u n g B H 3 t h u d c c h i Pseudomonas va d u o c d i n h tdn la Peudomonas sp. B H 3 (MN519480)

4 . KETLUAN

- Tir 7 m a u n u d e thai d a p h a n lap d u o c 35 c h i i n g vi k h u a n hidu k h i .

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617.

ISOLATION AND SELECTION OF INDIGENOUS BACTERIA CAPABLE IN HIGH RATE ORGANIC MATTER DECOMPOSION WITH THE PURPOSE OF TREATING WASTEWATER

FROM DAI LAM UQUOR PRODUCTION VILLAGE

Dao Thi Hong VanS Do Phuong KhanhS Nguyen Van Hieu^

'Department of Biotechnology, Hanoi Open University

^Institute of Biotechnology, Vietnam Academy of Science and Technology Summary

In Vietnam, the traditional liquor production villages are often associated with pig farming, so the wastewater here is a combinaOon of alcohol producpon, animal husbandry and household acUvities. The composiPon of wastewater is abundant, which contains large amounts of untreated organic compounds that discharge mto the environment Researchs on using indigenous microorganisms to treat wastewater has many advantages and is being paid attention. From 7 samples of wastewater collected m Dai Lam liquor production village (Bac Ninh province), 35 aerobic bactena strains were isolated. Three strains of these bacteria, namely BHl, BH2 and BH3, were selected to be capable of decomposing organic compounds (starch, protein, cellulose). Results of study on colony and ceU morphology, ability to use sugars and nitrogen sources, analysis of 16S rDNA gene sequence and related comparisons showed high similanty levels (> 99%) with several species on GenBank.

The three strains were identified and registered on GenBank with the codes as feciffl/ssp. BHl (MN519478);

Bacillussf. BH2 (MN519479) and Pseudomonai sp. BH3 (MN519480).

Keywords: Bacillus, Pseudomonas, wastewater treatment for craft villages.

Ngir6i phan bita: PGS.TS. Khuat Hiiu Thanh Ngiy nhan bai: 13/12/2019

Ng4y thang qua phan hi$n; 14/01/2020 Ng4y duyet dang: 21/01/2020