Tgp chi Cong nghi Sinh hgc 12(1): 373-380, 2014
L A M G I A U V A P H A N L ^ P VI S I N H V A T N H O M M E T H A N O G E N TU' T R A M T I C H B I E N V I E T N A M
Nguyen Thu Hoai', Nguyen Thi Tuy^n\ Nguyin LSn Dung^, Dinh Thuy HSng^
' Trung ldm nhtit dai Viit - Nga, Bd Quoc phdng
^Viin Vi sinh vdt vd Cdng nghi sinh hgc. Dgi hgc Quoc gia Hd Ngi Ngay nhan bai: 08. 4 2014
Ngay nhan dang: 03.6,2014 TOM T A T
Vi sinh v^t thupc nhom Methanogen dong vai fro quan trgng trong chu trinh chuyen hoa chat huu co d dieu kien khong co oxy, tiio san pham cuoi ciing la methane. O Viet Nam hien nay, cac cong nghe xu ly ky khi nhu be khi sinh hoc (biogas), UASB (upflow anaerobic sludge blanket) hay AF (anaerobic filter) dugc ap diing tuong doi hieu qua trong dieu kien nuoc nggt, tuy nhien lai khong thanh cong trong dieu kien nuac lg va nudc mSn do miic hoat dgng kem ciia vi sinh vat trong do c6 methanogen. D^ ho trg cac cong ngh^ xii ly ky khi 6 dieu kien nudc nhiem man, trong nghien ciru nay chiing toi t?io nguon methanogen co hoeit tinh cao thong qua phuong phap lam gi^u va phan lap chiing thuan khiet tir cac mau tram tich bien 6 Viet Nam Tir hai mau tram tich bien Nha Trang va Cat Ba, 4 quSn xa methanogen co hoat tinh sinh khi cao va on djnh da dugc thiet lap thong qua phucmg phap lam giau su dung hai nguon co chat acetate va methanol ket hop voi moi tnrdng nudc la va nudc mSn. Phan tich 16S rDNA bang phuong phap PCR-DGGE cho thay cac mau lam giau vdi acetate CO Methanosarcina chiSm uu thS, trong khi do cac mau lam giau vdi methanol c6 Methanolobus chiem uu the Tong so 10 chiing methanogen thuan khiet da duoc phan lap tir cac mau lam giau nay. Dua Iren vi tri ciia doan gen 16S rDNA tren gel dien di bien tinh, cac chiing nay dugc x^p vao 3 nhom, d;ii diSn ciia mSi nhom co dg tuong dong cao ve Irinh tir ciia doan 16S rDNA (1000 bp) ian luct vdi Methanosarcina semetsiae (97%), Methanolobus profundi (96%) va Methanosarcina vacuolala (96%) Cay la cac chiing methanogen dau tien dugc phan lap tir moi truong bien d Viet Nam.
Tir khoa: Tram tich bien, ldm gidu, methanogen. Methanosarcina. Melhanolobtis
MO DAU
Su dung c6ng nghe biogas de khai thac nguon nang lugng sach td chat thai hdu ca la mgt trong nhung bien phap gop phan quan trgng trong viec ho trg an ninh nang lugng va bao ve moi trudng. Tam diem CLia c6ng nghe la qua trinh phan huy chat hiiu eg trong dieu kien ky khi de tao biogas (gom CH4 va CO2), la mpt qua trinh smh hgc do nhieu nhom vi sinh vat dam nhiem, trong do vi sinh vat sinh methane (methanogen) dugc coi la nhom "chia khoa", NhOng vi smh vat nay thuoc vi^c (domain) co khuan (Archaea), rdt mSn cam vdi oxy va thong thudng sinh methane tir hydro, formate, acetate va m6t sd hgp ch5t carbon cd nhdm methyl (Whitman el ai, 1992), Chung co mat d nhieu dang moi trudng khac nhau, td moi trudng nirdc nggt (day thuy vuc, rugng ngap nudc, trong he tieu hoa ciia cac dgng vat nhai Igi), den mdi trudng nudc lg (rimg ngap man) va ca moi.trudng bien (hydrothermal vent, trfim tich bi£n) (Whitman e/a/., 1992).
Trong nudc bien vdi nong do 28 - 30 mM, sulfate la chat nhan dien tu cd ham lugng ldn dung thu hai sau oxy, do vay vi khuan khir sulfate dugc coi la nhdm ky khi chiem uu the trong moi trudng bien (Widdel, Bak, 1992). Mac dii bj canh tranh cao bdi vi khuan khu sulfate, methanogen van ton tai va dugc phat hien ngay mgt da dang d moi trudng bien, Trong 2 thap ky gan day nhieu loai methanogen mdi da dugc phan lap tu cac mau tram tich bien kJiac nhau nhu Methanococcoides alaskense, Melhanacoccoides burlgmi. Methanosarcina baltica, Methanoculleus submarinus, Methanoculleus marisnigri (Mikuki et ai, 2003; Singh el ai. 2005). Mgt s6 nghien cdu dua tren phan tich gen 16S rDNA bang dien di bien tinh cGng cho thay sir co mat pho bien ciia methanogen trong moi trudng bien (Ganzert et al., 2007; Lazar el ai, 2011). Ben canh do, sd dung cac thiet bi do microsensor va dong vi phong xa cacbon cac nha khoa hgc con xac dinh dugc toe do hinh thanh methane frong moi trudng bien kha cao, chirng to nhom vi sinh vat nay dong vai tro quan trpng trong chu trinh phan
373
Nguyen Thu Hoai et ai giai chit hiru ca d day (Kuivila el ai, 1990; Cowell et
ai, 2008; Yoshioka et ai. 2009).
CJ Viet Nam hien nay, vdi sir giiip dd ciia chinh phil Ha Lan thong qua "Chuang trinh khi sinh hgc cho nganh chan nuoi Viet Nam", cong nghe biogas da dugc tri^n khai thanh cong tai nhieu tinh thanh trong ca nudc (http://210.245.92.22/english/Home.aspx) Tuy nhien cong nghe nay gap kho khan khi trien khai d cac viing nudc lg va nudc man do (i) toe do phan hiiy cham, (ii) bj canh tranh bdi vi khuan khu sulfate va dac biet la (iii) thi^u iigu6n methanogen co tinh canh tranh cao trong dieu kien nhi^m man.
Nhiing y^u t6 neu iren khdng nhirng chi ton tai trong hg th6ng ht biogas ma con thudng gap trong cac he th6ng xu ly chit thai tai cac viing ven bien va hai dao, dac bigt la he th6ng bfi tir hoai hay cac he th6ng xu ly nudc thai sinh boat theo cong nghe yem khi.
Vdi muc tieu tao ra nguon methanogen co hoat tinh cao trong moi trudng nhiem man de ho trg cho c4c h6 th6ng xu ly chit thai neu tren, trong nghien cdu nay chiing toi tifin hanh khao sat va phan lap vi iinh vat sinh methane tu mpt so mau tram tich d bien Viet Nam. Methanogen la nhom vi sinh vat sinh trudng trong dieu kien ky khi ngat ngheo raa phan Idn cac phong thi nghigm d Viet Nam chua dap ung dugc, do vay day la nghien cim co tinh tien phong trong linh vyc nay d nudc ta.
VAT Lifu VA PHU'ONG PHAP Miu tr^m tich bien
Mau tram tich dugc thu thap tai viing bien Nha Trang va Cat Ba d dp sau 20 - 30 cm, dua vao cac dung cy chua ky khi, bao quan trong nudc da cho den khi ve phong thi nghiem de lam giau va phan lap vi sinh vat.
Lam giau va phan lap methanogen
Mdi trudng khoang co ban (Widdel, Bak, 1992) de lam giau va phan lap methanogen gom 26,4 g NaCI (doi voi moi trudng nudc lg: 13,7 g NaCl );
11,4 g MgCL.dHzO; 1,47 g CaCL 2H2O; 0,66 g KCl;
0,09 g KBr; 0,25 g NH4CI, 0,2 g KH3PO4; trong I lit nudc cat.
M6i trudng sau khi khii triing 20 phiit d I21°C dupc dg ngupi xu6ng 80°C, sue khi N2/CO2 (90,10, v/v) trong 5 phut, r6i lam ngugi den nhiet dp phong bang nudc may. Cac chat bo sung sau dd cho 1 lit moi trudng g6m 30 ml 1 M NaHCO}; 1 ml hon hgp vitamin, 1 ml hon hgp vi lugng (Widdel, Bak, 1992);
374
1 ml I M NajS; 10 ml I M ca chit (Na-acetate ho$c methanol). Trong khi b6 sung cac chat, don^ khi N2/CO2 van dupc duy tri sue qua moi trudng de logi oxy, pH cua moi trudng dugc chinh den 7,2-7,4 bSng cac dung djch 1 M Na3C03 ho|ic I M HC1._ M6i trudng sau do dupc san vao cac binh serum the tich 100 ml, ddng kin bSng nip cao su va su dyng de nuoi methanogen.
Trong thi nghiem lam gi^u, mau trdm tich bien dugc dua vao cac binh serum (theo ty le 10% the tich m6i trudng, w/v) chira mdi trudng khoang ky khi va mpt CO chat Na-acetate hoac methanol, nu6i d 30°C.
Sinh trudng ciia methanogen trong cac binh nuoi cay dugc danh gia thdng qua the tich khi tao thanh sau m6i 7 ngay (do thS tich cot nuoc) va quan sat mat dg tk bao dudi kinh hien vi huynh quang (nhd tinh ty phat sang cua coenzyme F420 d methanogen) (Dolfing, Mulder, 1985). Sau 4 tuSn, dich nudi dugc cay truyen sang moi trudng tuai. Qua trinh lam giau dugc thuc hien qua 3 lan cay truyen, mau d lan cu6i ciing dugc sd dung dl phan 13p chung methanogen thuan khifit.
Methanogen dugc phan lap trong day dng thgch ban long chua m6i trudng khoang ca ban nhu tren bo sung 10 g/l thgch va ca chat tuong ung vdi mau lam giau (Widdel, Bak, 1992). Khuin lac methanogen dupc tach bang dau pipet Pasteur keo dai va chuyen sang moi trudng ky khi djch the. Qua trinh tinh sach tren moi trudng thgch ban long dugc lap lai vdi m6i chiing phan lap tu 2-3 lan.
Tich DNA tong so va nhan doan gen 16S rDNA DNA tong so tir cac miu lam giau methanogen duac tach chiet theo phuang phap do Zhou et ci/.,(l996) m6 ta. De phan tich cau tnic quan the methanogen trong cac mau lam giau, doan gen 16S rDNA (350 bp) duac khuech dgi PCR su dyng cap m6i dgc hieu 0348Af- TCCAGGCCCTACGGG va 0691R-GGATTACARGATTTCAC (Wanatabe etai, 2004). De 6n dinh muc di chuyen ciia cac san pham PCR tren gel dien di bien tinh, kep GC (Muezer et ai, 1993) duac gan vao dau 5' ciia doan moi 0348aF, PCR dupc thyc hien vdi che d6 nhiet: 94°C/3', 40 chu ky ciia 94°C/r, 49°C/1' va 72°C/2'; 72''C/8'. San pham PCR dupc dien di tren gel 1% agarose, dem TAE, n o v , 15 phiit.
Di^n di biln tinh
Dien di bien tinh (DGGE - Denaturing Gradient Gel Electrophoresis) dugc thuc hien tren gel 8%
polyacrylamide trong dem IxTAE vdi do bi^n tinh
ciia gel (urea/formamide) tu 25 dan 60%, chfi dp dien
di la 200 V d eCC trong 3,5 h. Gel dien diduoc
Tap chi Cong nghi Sinh hgc 12( 1): 373-380, 2014 nhugm vdi edithidium bromide (5 mg/ml) trong 15 phut, rda bing nudc cit trong 5 phiit va quan sat dudi den UV tren may soi gel GelDoc (Biorad, USA).
Phan tich trinh ty 16S rDNA cua cac chung methanogen thuan khilt
Cac chung methanogen thuin khilt dupc nudi tren m6i tnrdng dich thi, sau do ly tam thu sinh khdi de tdch DNA tong so theo phuong phap cua Marmur (1961), Doan gen 16S rDNA ciia cac chiing nay dugc khuylch dai vdi cgp moi dac hieu A109f- ACKGCTCAGTAACACGT va A934b- GTGCTCCCCCGCCAATTCCT (Groskopf et ai,
1998). San phim PCR dugc tinh sgch bing Kit (Bioneer, Han Qu6c), va su dung lam khuon trong phan irng dgc trinh ty vdi ABI Prism BigDye Terminator cycle sequencing kit va dgc trinh ty tren may ty dgng 3110 Avant Appied Biosystems. Trinh
tu gen dugc phan tich, so sanh vdi trinh tu 16S rDNA cua cac Ioai co lien quan tren GenBank su dyng Blast Search. Cay phan logi dugc dimg theo phuang phap neighbour-joining (Saitou, Nei, 1987), trong do dinh dang cay dugc tien hanh dua tren 1000 phep so sanh da chieu (Felsenstein, 1985), KET QUA VA THAO LUAN
Lam giau methanogen tir trSm tfch biln Cat Ba
va Nha Trang
Cat Ba va Nha Trang la hai viing bien tigu bieu ciia mien Bac va mien Trung Vift Nam dugc lya chgn de thu mau tram tich lam nguon vi sinh vat ban dau cho thi nghiem lam giau methanogen. Doi vdi m6i miu tram tich, ca hat m6i trudng nudc lg va nudc man dugc sir dyng, ciing vdi nguon co chat la methanol hoac acetate.
E r ao.o
^ Jilli
D Lam giau 1 a Ldm g i a m
• LSmgiSulU
1
T h 6 i gian ( t u i n ) Thi^i gian ( l u i r
60 0
1
o 20,0
10 0
1 1
1
1 1
1 luin 2 luin 3 tuin 4 tuin ThM atan ( l u i n )
O i a m g i a u l
Hinh 1 . Lam giSu methanogen tir cac m i u tr^m tich bien Nha Trang ( A B) va Cat Ba (C, D) trong moi t r u d n g n u o c man v a i methanol (A, C) va acetate (B. D)
Nguyin Thu Hoai el al Ct moi trudng nudc man, cac mau Iam giau vdi
ca hai ioai ca chit diu sinh khi tot, lugng khi smh ra tang diu theo thdi gian (Hinh I). Tuy nhien, hien tugng tang din ctia khi sinh ra khong quan sat dugc khi so sanh gitra cac lan cay truyen vai nhau.
D6i vdi miu tram tich tu Nha Trang thi ting lugng khi sinh ra trong cac lin cay truyin sau giam so vdi cac lin ciy truyin trudc (Hinh 1; A, B), trong khi do d6i vdi miu trim tich tir Cat Ba thi ting lupng khi tang sau lin ciy truyen dau tien, sau do
tuong d6i 6n dinh qua cac lan lam giau 2 va 3 (Hinh 1; C, D), Nguyen nhan co thi do anh hudng ciia nong do mudi trong moi trudng nudc man den so tugng va hoat tinh ctia methanogen co mat trong mau tram tich bien Nha Trang.
Kit qua tuong tu ciing thu dugc trong thi nghiem lam giau d dieu kien mdi trudng nudc lo, T6ng lugng khi sinh ra trong cac mau lam giau tang diu theo thdi gian, kh6ng phan biet Ioai ca chat sit dyng (Hinh 2)
f SO'"
i " 50,0 • r 40,0 ,a 30,0
^ 20,0 HO 10,0
n rrl ll i
1 tuan 2 Ij^n 3 tuan 4 Uan Thd'i gian (tuan)
DLSmgisjII
• Lam giau III
Hinh 2. Lam giau methanogen td cac miu tram tich bien Nha Trang (A, B) va Cat Ba (C,D) trong m6i trudng nudc lg vo-i methanol (A, 0) va acetate (B, D).
Khi so sanh gida cac lan lam giau vdi nhau cho Qua hai thi nghiem lam giau co thi nhan thiy
thay thi nghiem lam giau vdi raau Nha Trang lai co methanogen cd mat trong mau trim tich biln Nha Trang
tinh on dinh han, lugng khi sinh ra tang deu d cac sinli trudng tot ban trong moi ttudng nudc la trong khi
lan cay truyen sau (Hinh 2; A, B) Trong khi do d thi do methanogen tu tram tich bien Cat Ba lai thich hop
nghiem lam giau vdi mau Cat Ba, tong lugng khi ban vdi moi trudng nudc man Dieu nay phan anh muc
sinh ra d !an lam giau 3 giam dang kl so vdi trudc do dg anh hudng ciia nguon nudc tu dat Hen tdi khu vuc ven
(Hinh 2; C, D). biln Nha Trang ldn hem so vdi tai dao Cat Ba
376
0 chi Cdng nghe Sinh hoc 12( 1): 373-380, 2014 QuSn xa methanogen du'O'c lam giau tir cac mau t r a m tich bifin C a t Bii va Nha T r a n g
Cac mau Iam giau methanogen vdi ca hai loai ca chat va m6i trudng nudc Ig hoac nudc man sau 3 lan cay truyen deu the hien kha nang sinh khi cao va mat dp te bao dn dinh. De danh gia dugc hieu qua cua thi nghiem lam giau, dong thdi xac dinh cac nhom methanogen thich ung cao nhat vdi dieu kien lam giau, quan xa methanogen trong cac mau Iam giau lan thu 1 va 3 da dugc phan tich thong qua phuang phap DGGE doi vdi doan gen 16S rDNA khuech dai tu DNA tong so ciia cac m i u lam giau vdi cap moi
dac hieu cho nhdm co khuan la 0348aF-GC va 0 6 9 I R ( H i n h 3 ) .
Nhu vgy qua 3 budc lam giau, cac quan xa methanogen da dupc thiet lap va co miic da dang tuong doi cao Co the thay qua trinh lam giau da tgo dieu kien cho mot so nhdm methanogen co tinh thich nghi cao nhat dugc duy tri va tich liiy trd thanh nhom chiem da s6 trong quan x§ (cac bang dien di bl va b2). Khong co sir khac biet r6 rang giiia cac miu lam giau tu tram tich bien Nha Trang (2, 3, 4) va Cat Ba (5, 6, 8) hay gifta dieu kien nudc lg (2, 5, 6) va nudc mgn (3, 4, 8).
2.1 3.1 4.1 5,1 6.1 8.1
r - y - - ^ "f |P^
. ' t
• I M M
A B Hinh 3. PhSn tich qu^n xa methanogen trong cac m i u lam giau vdi trim tich tip biln Nha Trang va Cat Ba bing phuong
ph^p PCR-DGGE gen 16S rDNA A - Cac mau lam giau lan 1 (.1), B - Cac miu lam giSu l i n 3 (.3), Tgn miu, 2 - Nha Trang/nu'oc Ig/methanol; 3 - Nha Trang/nLFoc man/acetate, 4 - Nha Trang/nudc man/methanol; 5 - Cat Ba/nuo'c lo/acetate, 6 - Cat Ba/nudc Ig/methanol, 8 - Cat Ba/nudc man/methanol.
Moi lien quan kha ro co the thay dugc giira dieu kien lam giau va quan xa methanogen thu dupc la moi lien he giiia nguon ca chat lam giau va nhom chiem uu the, Cy the, vdi co chat methanol (mau 2, 6, 8) nhom dai dien bang bl chiem uu the, trong khi do vdi CO chat acetate (mau 3, 5) thi nhom dai dien bang b2 chiem uu the. Cac nhom nay da co mat trong m i u lam giau lan 1 nhung vdi so lugng thap (bang dien di md) va dugc tang them ve so lupng sau 3 budc lam gi^u (bang dien di dam net) Trudng hgp ngoai le la mau 4, tgi thdi diem ban dau c6 bang bl (dudng gel so 4 . 1 , hinh 3A) nhung sau qua trinh lam giau vdi nguon ca chat la methanol bang nay bien mat, thay v^o do la bang b2 xuat hien. Hai bang b l va b2 dupc c5t, thoi gel va giai trinh tu. Ket qua so sanh vdi GenBank cho thay bang bl va b2 tuang dng dgi dien cho cac loai Methanolobus sp. va Methanosarcina sp,
P h a n lap methanogen ttr c^c mau lam giau T6ng so 10 chung methanogen thuin khilt da dupc phan lap tir cac mau lam giau su dung ong
thach ban long vdi co chat acetate hoac methanol.
De danh gia muc dp linh sach ciia cac chiing phan lap va danh gia sa bg moi lien quan ve vi tri phan loai cua chiing, mgt ian nda doan gen 16S rDNA dugc khuech dai tir DNA genome ciia cac chung nay su dung cap m l i 0348aF-GC va 0691R, va dupc phan tich bing DGGE (Hinh 4)
Dua tren vi tri phan tach ciia cac bang tren gel dien dl, 10 chiing methanogen da phan lap co the dugc xep vao 3 nhom, trong dd nhom 1 gom cac chting so 7, 20a, 2 1 , nhom 2 gom cac chiing 23a, 23b, 24 va nhom 3 gom cac chiing 18a, 25a, 25b, Trong thi nghiem nay chung so 10 khong the hien duac thanh bang tren gel dien di bien tinh, Ba chiing dgi dien cho moi nhom ia 2 1 , 23b va 25a duac giai trinh tu gen I6S rDNA va xac djnh vj tri phan logi. Dogn gen 16S rDNA dai 1000 bp d cac chiing nay dugc khuech dai nhd sd dyng cap moi A109f va A934b dac hieu doi vdi co khuan. So sanh vdi cac trinh tu d GenBank, chiing 21 (KC571195) co dp tuong dong cao nhat vdi Methanosarcina semeisiae (97%),
377
Nguyen Thu Hoai el ai chung 23b (KC57I193) vdi Methanolobus profundi
(96%) va chimg 25a (KC571194) vdi Methanosarcina vacuolala (96%). So sanh vdi cac bang chinh tren gel dien di DGGE cua quan xa methanogen lam giau, co the thiy chiing 21 co
ngu6n goc tu nhom methanogen tuang img vdi bang dien di b2 {Methanosarcina spp), trong khi dd chiing 23a xuat phat tu nhdm methanogen tuong dng vdt bang dien di bl {Methanolobus spp.) (Hinh 5).
Hinh 4 Tinh da dgng cua cSc chiing methanogen phSn lap flucrc tu' c^c m i u \hm gi^u the hien qua phuang phap phan tich PCR-DGGE dogn gen 16S rDNA
-Melhanococciis moripaliidis (U38486) Melhanosarcina iiiazei (JQ346757)
V~Methanosarcina sicihae (FR~33698) - ^ 2 5 a ( K C 5 7 1 1 9 4 )
^Melhauosarcniavacitolafa (FR733661) L21(KC57I195)
^^^^^Melhanosaixiiin semesiae (NR028182) -Melhanohhiiszmdeh O^K0AA55Z)
—•Methmiolobiisoj-egonensis (U20152) Bl -23a(KC5"1193)
^Melhanolobiisprofitudn {^K0M6&5)
Hinh 5. Cay phSn logi dya tren trinh tu doan 16S rDNA cua cac chung methanogen dai dien (21, 23a, 25a) va c^c bang di^n dl bien tinh (bl, b2) so sanh vdi cac loai gin gui thu6c hai chi Methanosarcina va Mettianococcus Don vi - 0,02 Kmc trong trinh tu nucleotide. Cac so hi^n th| d cac v\ tri phan nhanh la ket quA phan tich bootstrap doi vdi 1000 phep so sSnh (chi c6 cSc gia tn tren 500 dugc trinh bay tren hinh). Methanococcus maripaludis dugc chgn lam outgroup.
Methanosarcina va Methanolobus 'a hai chi cimg nam trong hp Methanosarcinaceae. Trong khi chi Methanosarcina chii yeu gom cac loai methanogen sd dung acetate de tao methane (CHjCOOH -> CH4 + CO,) (Whitman el al., 1992),
thi cac loai Methanolobus dugc phan lap diu thupc
nhdm su dyng cac hpp chat cd nhom methyl (nhu
methanol hay methylamine) lam ca chit dl sinh
methane (4CH3OH -^ 3CH4 -1- CO2 + 2H2O)
(Mochimaru et ai, 2009; Doerfert el ai, 2009), thgm
Tap chi Cong ngh? Sinh hoc 12(1): 373-380, 2014 chi nhifiu loai hoan toan khong co kha nang sinh methane tir H2/CO2 hay acetate (Mochimani et al., 2009). Nhu vay trong nghien cdu nay cac chung raethanogen phan 13p dupc dai dien cho hai nhdm co dac diem sinli ly chuyen biet U Methanosarcina va Methanolobus, hinh thanh qua qua trinh lam giau tit cac miu tram tich bien vdi hai ngudn co chat acetate va methanol.
KET LUAN
Tir hai mau tram tich bien Nha Trang va Cat Ba, cac quan xa methanogen cd hoat tinh sinh khi cao va 6n dinh dS duoc thifit ISp thdng qua lam giau su dung hai nguon ca chat quan trpng ddi vdi methanogen la acetate va methanol. Tdng so 10 chung raethanogen thuan khiet da duoc phan lap tir cdc mau Iam giau nay. Dyra tren vi tri cua doan gen 16S rDNA tren gel dien di bi6n tinh, cac chung nay duoc xep vao 3 nhom, dgi dien cua mdi nhom cd dp tuong dong cao vk trinh tu cua gen 16S rDNA l4n \ugt vdi Methanosarcina semeisiae (97%), Methanoiobus profiindi (96%) va Methanosarcina vacuolala (96%).
Day la cdc chung methanogen dau tien phSn lap tu mdi trudng biSn d Viet Nam dupc cdng bo.
Ldi cam un: Cong trinh diroc hoan thdnh nhd sif ho tra kinh phi cua de tai ma so QG.11.27 Cac tdc gia xin trdn trong cdm an Vien Vi sinh vat vd Cong nghe sinh hoc da tgo dieu Iden lien hdnh cdc thi nghiim trong nghien cuu.
T A I LIEU THAM KHAO
Colwell FS, Boyd S, Delwiche ME, Reed DW, Phelps TJ, Newby DT (2008) Estimates of biogenic methane production rates in deep marine sediments at Hydrate Ridge, Cascadia Margin. AppI Eviron Microbiol 74: 3444- 3452
Doerfert SN, Reichlen M, Iyer P, Wang M, Ferry JG (2009) Methanoiobus zindert sp. nov., a methylotrophic methanogen isolated from a deep subsurface coal seam. Int J Syst Evol Microbiol 59: 1064-1069.
Dolfing J, Mulder JW (1985) Companson of methane production rate and coenzyme F42o content of methanogenic consortia in anaerobic granular sludge. Appl Environ Microbiol 49: 1142-1145.
Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783-791 Ganzerl L, Jurgens G, Muenster U, Wagner D (2007) Methanogenic communities m permafrost-affected soils of
the Laptev Sea coast, Siberian Arctic, characterized byl6S rRNAgene fingerprints. FEMSMicrobiolEcol 59: 476-488, Grosskopf R, Janssen P, Liesack W (1998) Diversity and structure of the methanogenic community in anoxic nee paddy soil microcosms as examined by cultivation and direct 16S rRNA gene sequence retrieval. Appl Environ Microbiol 64- 960-969.
Kuivila KM, Murray JW, Devol AH (1990) Methane production in the sulfate-depleted sediments of two marine basins. Geochim Cosmochim Acta 54; 403-411.
Lazar CS, Parkes RJ, Cragg BA, L'Haridon S, Toffin L (201!) Methanogenic diversity and activity in hypersaline sediments of the centre of the Napoli mud volcano. Eastern Mediterranean Sea Environ Microbiol 13-2078-2091.
Mikucki A J, Liu Y, Delwiche M, Colwell F.S, Boone D R (2003) Isolation of a methanogen from deep marine sediments that contain methane hydrates and description of Methanoculleus submannus sp.nov. Appl Environ Mcroifo/69: 3311-3316
Mochimaru H, Tamaki H, Hanada S, Imachi H, Nakaraura K, Sakata S, Kamagata Y (2009) Methanoiobus profiindi sp. nov., a methylotrophic methanogen isolated from deep subsurface sediments in a natural gas field. Inl J Syst Evol Microbiol 59:714-718.
Marmur J (1961) A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Moi Biol 3 • 208-218
Muyzer G, De Waal EC, Utteriinden AG (1993) Profiling of complex microbial population by denaturing gradient gel electrophoresis analysis of polymerase chain reaction amplified genes coding for 16S rRNA. Appl Environ Microbiol 59: 695-700
Saitou N, Nei M (1987). The neighbor-joming method: a new method for reconstructing phylogenedc trees. Moi Biol Evol 4: 406-425
Smgh N, Kendall MM, Liu Y, Boone PR (2005) Isolation and characterization of methylotrophic methanogens from anoxic manne sediments in Skan Bay, Alaska, description of Melhanococcoides alskense sp nov., and emended description of Methanosarcina baltica. Int J System Evol Microbiol 55:25l\-25l%.
Watanabe T, Asakawa S, Nakamura A, Nagaoka K, Kiraura M (2004) DGGE method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil.
FEMS Microbiol Lett 2^2\\5^-\6'^.
Whitman Vffi, Bowen TL, Boone OR (1992) The methanogenic bacteria. In Balows A, Triiper HG, Dworkin M, Harder W, Schleifer KH eds. The Prokaryotes, 2"'' ed.
Springer, Berim Heidelberg New York: 165-207.
Widdel F, Bak F (1992) Gram-negative mesophiltc sulfate-reducing bacteria. In Balows A, TrUper HG,
Nguyen Thu Hoai et al.
Dworkm M, Harder W, Schleifer KH eds. The sediments from the eastern Nankai Trough, off central Prokaryotes, 2"'' ed. Springer, Berlin Heidelberg New Japan, Geoc/ie/«J43. 315-321.
York: 3352-3378, Yoshioka H, Saka
Microbial methane production rates in gas hydrate-bearing 316-322.
Zhou J, Bruns MA, Tiedje JM (1996) DNA recovery from Yoshioka H, Sakata S, Cragg B, Parkes RJ, Fujii T (2009). soils of diverse composition Appl Environ Microbiol 62:
E N R I C H M E N T A N D I S O L A T I O N O F M E T H A N O G E N S F R O M M A R I N E S E D I M E N T S IN V I E T N A M
Nguyen Thu Hoai', Nguyen Thi Tuyen^, Nguyen L a n Dung^, Dinh Thuy Hang '"
' Vietnam-Russia Tropical Center, Ministry of Defence of Vietnam
^Institute of Microbiology and Biotechnology, Vietnam National University Hanoi
SUMMARY
Methanogens play important roles in the cycling processes of organic carbon, giving methane as the final product In Viemam, anaerobic technologies such as biogas, UASB (upflow anaerobic sludge blanket) or AF (anaerobic filter) have been applied effectively under freshwater condition, however cannot be succesfiill under brackish and seawater conditions due to low activity of microbial groups incuding methanogens. In order to support anaerobic technologies under salted water conditions, in the present study effective methanogen sources were eshtablished via enrichment and isolation of pure cultures from marine sediment samples in Vietnam. Four active methanogenic consortia were obtained via enrichment procedure using manne sediment samples collected from Nhatrang and Catba with acetate or methanol as organic substrates. The enrichments were carried out in brackish water and seawater environments PCR-DGGE analyses of the I6S rDNA gene showed that the acetate-feeded enrichment cultures were dominated by Methanosarcina species, whereas the methanol-feeded enrichment cultures were dominated by Methanoiobus species. In total, ten methanogenic strains were isolated from these consortia. Based on the PCR-DGGE analyses of the 16S rDNA gene they could be arranged into three groups, the representative strain of each 21, 23a, and 25a showed the highest identities (> 96%) of the I6S rDNA gene with Meliianosarcina semeisiae, Methanoiobus profundi and Methanosarcina vacuolata, respectively. These strains represented the first methanogenes isolated from marine environment in Vietnam
Keywords: Marine sediment, enrichment, methanogen, Methanosarcina, Methanoiobus
Authors for correspondence: Tel: +H4- 4-3754 7488: E-mail, hansdinh cbhn(a)vahoo.com
