Tgp chi Cdng nghe Sinh hgc 11(2): 303-309, 2013
N U O l T A N G SIIVH VI K H U A N N I T R A T E H O A D A T M A T D O T E B A O C A O T R E N T H I E T BI L E N M E N
Dinh Thuy H^ng, Nguyen Thi HSi, N g u y i n Thi H^ng, Hoang Van Thai Vifrt Vi sinh vgt vd Cdng nghi sinh hgc, Dgi hgc Qudc gia Hd Ngi
TOM TAT
Vi khuan nitrate hoa sii dung oxy phan tii de oxy hoa ammonia thanh nitrate, la mat xich quan trong trong chu trinh chuyen hoa niio trong tir nhien. Do smh trucmg tir duong vo co, nhom vi khuan nay thirong khong tao nhieu sinh khoi, tham chi tren cac thiet bi len men voi cac thong so dirge kiem soat chat che trong phong thi nghiem. Trong nghien ciiu nay, hai chiing vi khuan nitrate hoa gom vi khuin oxy hoa ammonia Niirosomonas sp, DVAl va VI khuan oxy hoa nitrite Nitrobacier sp. DVNl duoc sir dung de lam doi tuong nghien ciiu thu sinh khoi trSn thiet bj len men the tich 10 lit (Satonus, CHLB Diic) Ket qua thi nghiem cho thay nuoi neng re hai chiing nay tren thiet bi len men chi dat mat do te bao cao nhat la 2,5 - 3,8 x iO' te bao ml"' do iic che ciia nitnte irong moi trucmg nuoi cay Trong khi do, nuoi dong thai hai chung tren thiel bi len men cho phep tang m^tdg tibao ien miic 1,2-1,5 x 10* te bao ml"' do nitnte sinh ra boi chiing oxy hoa ammomafM/z-wwrnomij sp, DVAl) ngay lap tiic dupc chuyen hoa boi chiing oxy hoa nitrite {Nitrobacter sp. DVN I), qua do anh huong lie che ciia chat nay toi qua trinh sinh tnrong dugc giam thieu Quy trinh nuoi tang sinh tren ihiet bi len men de thu sinh khoi cho vi khuan nitrate hoa qua nghien ciiu nay duoc thiet lap, lam ca so cho cac nghien ciiu va img dung can sir dung sinh khoi ciia nh6m vi khuan nay.
Tirkhoa: Nitrate hoa. Nurosomanas sp. DVAl, Nurobacler sp. DVNl, sinh truang n MO DAU
Nita la nguyen to dong vai tro quan trpng Irong te bao song, Mac dii co mat vai mot luong lon tren trai dSt a dang khi Nj (79% khong khi) nhung nguon nita trong nuac lai chi duoc giai han 6 miJrc rat han che, Theo quy chuan hien nay ciia Vi?t Nam, ham luang ammonia trong nuac thai ra ngoai moi truang duoc giai han 6 mirc 10 mg/l va ham lupng nita tong so a mirc 40 mg/l (kenh B, QCVN 40:2011). N u a c thai co ham luang nita cao neu khong duoc xu ly truac khi xa ra ngoai moi truong CO th§ gay anh huang nghiem trpng cho he sinh thai cung nhu sire khoe ciia con nguai (Bouchard e r a / . , l992;Bitton, 1999),
Tii lau, vai tro ciia vi khuan trong viec chuyen hoa nito 6 dang hoa tan trong nuac (NH3) thanh d^ng khi (N2) da d u a c iing dung de loai nito trong nu6c thai (Bitton, 1999), Qua trinh loai nita thong dyng nh4t duac thuc hien qua hai buoc nitrate hoa (oxy hoa ammonia thanh nitrate) va khii nitrate (khij nitrate thanh N2), trong do buac nitrate hoa 1^ mat xich quan trpng nhat cua toan bp qua trinh,
Ciing nhu nhi^u loai vi khuan sinh truong tu du&ng vo ca khac, vi khuan nitrate hoa (gom vi
khuan oxy hoa ammonia, AOB NH^' + O2 —>
N O ; " + 2 H ' + H2O va vi khuan oxy hoa nitrite, NOB NO2" + V2 O2 -> NOj" + H2O) thuang kem canh tranh voi cac loai vi khuan di duiing va khong lao nhieu sinh khoi (Abeliovich, 2006;
Koops et al. 2006) Khi nuoi tang sinh trong thiet bl len men, loai vi khuan nay ciJng khong dat mat do le bao cao mac du luong ca chat (ammonia va nitrite) dupc chuyen hoa la khong nho (Gueven, Schmidt, 2009). Dac tinh nay mpt mat rat co loi trong viec xii ly 6 nhiem nita (do VI khuan co hoat tinh trao dot chat cao nhung it tao sinh kh6i phu), nhung dong thai lai la tro ngai cho cac nghien ciru hay irng dung can thu sinh khdi tir nhom vi khuan nay (Bitton. 1999;
Koops et ai, 2006).
0 Viet Nam, viec irng dung vi khuan nitrate hoa 6 dang ch^ phSm sinh hpc con han che, mot ph5n do kho khan trong viec nuoi tang sinh nhom vi khuan nay iren cac thiel bi len men de dat mat do tg bao cao. Nghien ciiu nay do vay dugc tien hanh nhSm tim ra mpt quy trinh toi uu de nuoi tang sinh vi khudn nitrate hoa tren thiet bi len men, lam co sa cho cac nghien ciiu irng dung doi voi nhom M khuan nay.
Nitrate hoa N G i ' Nitrobacter
N O 3 "
'"y^
N O , K h u Nitrat Pseudomonas, Alcaligenes
Bacillus, Agrobactenum
\
N O N2ODinh Thijy Hang etai
Co djnh Nitrogen
Co djnh Nitrogen Azotobacter, Rhizobium
Hinh 1. Chu trinh chuyen hoa nitrogen trong lu nhien (Kuepers et at., 2003)
'H
VAT LIEU v A PHU'ONG PHAP Nguon vi sinh vat
Vl khuan nitrate hoa gom chiing vi khuan oxy _ hoa ammonia (AOB) Niirosomonas sp. DVAl va chung VI khuan oxy hoa nitrite (NOB) Nitrobacter sp DVNl dup'c phan lap tir dat vucm va bao quan tai phong Sinh thai Vi sinh vat, Vien Vi sinh vat va Cong nghe sinh hpc. Hai chiing vi khuan nay the hien hoal tinh trao doi chat cao a dieu kien nuoi cay trong phong thi nghiem.
Moi trtrimg nuoi cay vi khuan nitrate hoa M61 truang nuoi cay vi khuan oxy hoa ammonia (Steinbuchen, Oppermann-Samo, 2003) Niirosomonas sp. DVAl gom nhung thanh phan nhu sau' 1 g NaCI;
0,5 g (NH4)2S04: 0,07 g KH2PO4; 0.05 g MgSOj.yn.O; 0,05 g CaCl2.2H20; 1.5 mg phenol do trong I lit nuoc cat. pH ban dau ciia moi trucmg la 8 va lam moi truang co mau hong Trong qua trinh nuoi cay vi khuan AOB se tao ra nhieu ion H"^ va lam giam pH ciia moi truong, keo theo chuyen mau moi truong tir hong sang vang.
M61 truang nuoi cay vi khuan oxy hoa nitnte (Abeliovich. 2006) Nitrobacter sp. DVNl g6m nhCnig thanh phan nhu sau 1 g NaCI, 0,07 g KH2PO4. 0.05 g MgS04.7H20, 0,05 g CaCl2.2H20;
0,69 g NaN02, 1.5 mg phenol do trong 1 lit nuoc cdt.
Xac dinh ham lurnig ammonium
Ham Iugng ammonium duac xac dinh Iheo phuang phap sir dung Na-nitropruside va phenol trong moi Iruong co mat chat oxy hoa manh la NaCIO (Le 304
Dire, 2004). Cac buac tien hanh co the mo ta torn tat nhu sau: dung dich lam viec gom co dung dich A (3 g phenol va 0,003 g Na-nitropruside hoa tan trong 100 ml nuoc cat) va dung dich B (2 g NaOH hoa trong 98 ml nuac cat, sau do Ihem 2 ml NaCIO 13%), Cac buac phan tich lan lugt gom; lay 40 (il mau hoa trong 9,96 ml nuac cat, sau do them 1 ml dung dich A va trpn deu bang vortex Tiep theo bo sung I ml dung dich B, Uon deu bang vortex roi u 1 gio tai nhiet dp phong trong buong toi, sau do do dp hap phu anh sang tai buac song 635 nm. Ham luong ammonium dupc linh dya tren duong chuan thuc hien voi NH4CI Irong khoang nong dp 1 - 100 |lM.
Xac dinh ham lirtfng nitrate
Ham lupng nitrate duoc xac dinh theo phuong phap sir dung axit disulfofemic (Le Diic, 2004), iheo do dung dich axit dugc chuan bi bang each trpn deu 25 g phenol tinh khiet vai 150 ml H1SO4 dac (tinh khik) va 75 ml H2SO4 b6c khoi (oleum) trong binh sinh han va dun each thiiy trong 2 gia.
Cac buoc tien hanh lan lugt nhu sau: lay 5 ml mau nuac (< 5 mg NO3") vao ong nghiem, trung hoa den pH 7, sau do chuyen mau sang chen su va co can bang dun each thuy. Them 2 ml dung dich axit disulfofemic va hoa tan can bang dua thuy tinh, tiep theo them 20 ml nuac ck va 6 ml KOH 12 N rfii chuyen loan bo the tich phan img sang binh djnh mirc. Them nuac cat cho du 50 ml trong binh djnh miic, tien hanh do OD tai buoc song 410 nm. Him lugng nitrate dugc tinh dua tren duang chuan thgc hien vai dung dich chu5n KNO^ (163,1 mg/lit) trong khoang 0 - 3 0 ml.
Tgp chi Cdng nghe Sinh hgc 11(2); 303-309, 2013 Kiem tra nhanh sir co mat cua nitrite b i n g sir dvng t h u ^ thir Griess
Test dugc thitc hien vai dung djch thiJ Gness (Steinbuchen, Opperniann-Sanio, 2003) Dung djch thir dugc pha tir dung dich sunfanil axit (0,8 g sunfanil axit hoa trong 100 ml 5N acetic acid) va dung dich a-Naphthylamine (0,5 g a-Naphthylamine hoa trong 100 ml 5N acetic acid) theo ty le the tich 1:1, Phan iing test dugc thuc hien trong ong nghiem chita 2 ml mau va 1 ml dung dich thu, quan sat sau 5 phut. Neu dung dich chuyen mau hong la trong mau CO nitrite, neu dung dich khong co mau hong nhung lai chuyen mau sau khi bo sung bot kem thi chirng to trong miu co nitrate (nitrate bj oxy hoa thanh nitnte do oxy dugc tao ra tir kem).
Xac dinh so luwng vi khuan trong dich len men bjing phuwng phap MPN
Thi nghiem dugc thuc hien tren dia 48 gieng chua mot truang dich the tuong irng doi vai moi nhom vi khuan AOB hoac NOB. Truac tien mau dupc pha loang theo cap dp 1/10 tai 10'^ trong moi truang tuong tmg doi voi moi nhom vi khuan. Sau do cay 50 (ll giong tir cac ong pha loang vao cac gieng chira 450 |il moi trucmg tuong irng, 3 gieng cho moi cap dp pha loang va nuoi o 30°C, trong toi.
Kel qua dupc dpc sau 1 tuan dua tren sy co mat cila VI khuan trong cac gieng thong qua cdc phep thir nhanh voi thuoc thii Griess. Xac dinh he so va cap so mu cho so lugng te bao theo bang MPN ciia American Public Health Association (1969),
Phan tich q u i n t h i vi k h u i n trong djch len men bSng PCR-DGGE
DNA tong so tit cac mau dich len men duoc tach chiet theo phuong phap do Zhou va cs. (! 996) mo ta Quan the vi khuan trong dich len men dugc phan tich bing phuang phap PCR-DGGE d6i vai gen 16S rDNA va gen amoA ma hoa cho enzyme ammonia- monooxygenase sii dung cac doan moi dac hieu cho timg gen (Muyzer et ai, 1993, Shimomura et ai, 2012). Dien di bi6n tinh (DGGE) dugc thuc hien Iren gel 8% polyacrylamide trong dem Ix TAE voi dp bicn tinh ciia gel (urea/formamide) tir 25 den 60%, che dp dien di la 200 V 6 60°C trong 3,5 gio. Gel di?n di dugc nhuom voi edithidium bromide (5 mg/ml) trong 15 phiit, nia bang nuac cat trong 5 phiil vk quan sat dudi den UV tren may soi gel GelDoc (Biorad, USA)
Cic bang dien di dugc clt, thu DNA trong nuac qua dem, sau do khuech dai trong phan ung PCR sii
dung cac cap moi dac hieu cua timg gen San pham PCR tu moi bang sau do duoc tinh sach bang kit {Bioneer, Han Quoc) va thye hien phan img giai trinh ty vai ABI Prism BigDye Terminator cycle sequencing kit, sau do dgc trinh tu tren may tu dpng 3110 Avant Appied Biosystems. Trinh tu gen sau do duoc phan tich so sanh voi trinh tu gen I6S rDNA va amoA ciia cac loai c6 lien quan hien da cong bo tren Database GenBank sii dung cong cu BLAST Search.
KET QUA VA THAO LUAN
Nuoi tang sinh rieng biet Nitrosomonas sp. DVAl va Nitrobacter sp. DVNl tren thiet bi ien men
Hai chiing vi khuan Nitrosomonas sp DVAl va Nitrobacter sp. DVNl deu thupc nhom ua am dien hinh, phat trien loi uu o nhiet dp 28 - 30°C. Cac chiing nay hoan toan khong sinh truang tren moi truang giau nhu LB va kem phat tnen tren moi truong thach voi thanh phan khoang dac hieu (xem phan vat lieu va phucmg phap), chiing thuang chi tao cac khuan lac rat nho, kho nhan biel bang mSI thuong. Voi ly do tren, nguon giong cho Ihi nghiem len men la te bao song nuoi 6 moi truong djch the bao quan 6 nhiet dp phong (trong thai gian duoi 1 thang) hoac tai 4°C (trong thai gian duai 3 thang), hoac trong dung dich glycerol 10% tai -20''C (trong then gian duai 6 thang).
Giong cap 1 sii dung cho thi nghiem len men dugc chuan bi trong cac binh tam giac the tich 1000 ml chira 500 ml moi truong khoang dac hieu cho AOB hoac NOB. Te bao vi khuan Nitrosomonci'. sp.
DVAl va Nitrobacter 5p. DVNl sau khi da hoal hoa dugc bo sung vao binh nuoi theo ty le 5 - 10% the lich. Qua Irinh nhan giong cap I keo dai tir 3 - 4 ngay va sinh truang ciia vi khuan 6 buoc nay chi dua vao thay doi mau ciia moi truong do giam pH. Do qua Irinh oxy hoa ammonia hay nitrite doi hoi mirc oxy hoa tan rat cao {> 2 mg/L), oxy duoc cung cap thong qua may nen khi voi toe dp dong la 100 ml/phiit, Ngu6n khi dua vao binh nuoi dugc lpc qua mang 0,2 \im
Thi nghiem nuoi tang sinh sau do dugc tien hanh tren thi^I bi len men the tich 10 lit (Satonus, CHLB Dire) voi th4 tich thuc len men la 8.5 lit, bao gi)m 8 lit moi trucmg va 0,5 lit giflng goc cap I, Cac yeu tci moi truong nhu nhiel dp, pH, nguon cu chal va bien dong CLia mat dp te bao cung nhu hoat linh irao doi chal duac theo doi trong qua trinh len men (Bang 1)
305
Dinh Thiiy Hang et ai Bang 1. DiSu kien nuoi tang smh (56i vai cac chiing Nitrosomonas sp DVAl va /V/trobacfersp. DVNl tren thiet bi len men
Dieu kien thi nghiem M6i truang
Giong gflc Do man pH khdi diem Nhiet 36 Oxy hoa tan
Thoi gian Cac chi tieu theo doi va phan tich
Nitrosomonas 5 p.
DVA1
Khoang AOB (NH4' 10 mM)
5 - 1 0 7 o t h e t i c h 0 , 1 % 8 30°C - 2 mg/L
96 h Mat dp t& bao Hoat tinh trao doi chat pH
Nitrobacter sp.
DVN1 Khoang 10 mM)
NOB
5 - 1 0 % the tich 0 , 1 % 7,2 SO^C
~ 2 mg/L 96 h
(NO2"
Ghi chu
NHa* d u g c bo sung trong qua trinh len men khi ham l u a n g xuong t h i p d u o i 1 mM.
Phenol do lam chat chi thi pH
Sue khi (100 ml/phiit, qua mang !pc 0,2 (im) va khuay.
ODeoo va s6 luang Iheo MPN Dmh luong NH4* hoac NO3"
Chinh b§ng NasCOj 1M khi pH xuong difui 6
Co the thay rang ca hai chiing AOB Nilrosomcmas sp DVAl va NOB Nitrobacter sp DVNl deu chi dat mat dp Ie bao duoi 10^/mi, tuong img la 2,5 x 10^/mI va 3.8 x lOVml (tuang duong vol ODsoo o mirc 0,1 - 0,12) sau qua trinh len men ( H i n h l )
Ket qua nay cung thong nhat voi nghien cuu da cong bo doi vai chimg vi khuan Nitrosomonas europaea (Gueven, Schmidt, 2009), Iheo do mat do Ie bao Irong dich len men dat mirc 2 x lO'* te bao/ml Tac gia ciia nghien ciru tren ciing cho thay viec nuoi tang sinh A' europaea theo che do len men hen tuc CO the tang mat dp te bao ien 2 5 % so vcii len men theo me. Tuy nhien trong thi nghiem cua minh, Gueven va Schmidt chi bo sung co chat ammonia mot lan duy nhat va khong dieu chinh pH trong qua trinh len men O nghien ciru nay, NHj* dugc bo sung Ihem khi ham lugng Irong moi truong xuong thap, dong thin pH dugc hieu chinh ve Irung tinh khi bi giam xuong duoi mirc 6 de dam bao dieu kien sinh Iruong toi uu cho vi khuan, do vay mat dp te bao dat cr miic kha cao,
Nuoi tang sinh dong thoi hai chiing Nitrosomonas sp. DVAl va Nitrobacter sp. DVNl tren thiSt bj len men
Trong qua trinh sinh truang ciia vi khuan nitrate hoa, nitrite la san pham trao doi chat ciia nhom AOB va nguon dien hi ban dau cua nhom NOB Nitrite duoc chirng minh la co tac dung uc che ro ret len hoat tinh oxy hoa ammonia 0 nong dp nitrite > 10 mM, lugng mRNA ciia gen amoA ma hoa cho 306
enzyme ammonia monooxygenase 6 vi khuan AOB giam dang ke (Cua, Stein, 2011) Day cung la ly do khien cho viec nuoi tang smh vi khuan AOB trong thi^t bl len men bj iic che o giai doan sau, khi Irong moi truong da co mot lugng dang ke nitrite duac lao thanh. Nham muc dich keo dai qua trinh sinh trucmg ciia vi khuan AOB trong thiet bi len men, trong nghien ciiu nay vi khuan N O B co mat dong thai trong dich len men de lap tire tieu thu san pham nitnte do vi khuan AOB tao ra.
Thi nghiem len men cung dugc thiel ke tuong tu nhu trong thi nghiem vai timg chiing vi khuan rieng biet cr Iren bang 1, sii dung moi trucmg khoang c6 chiia NH4* la nguon dien tu va khac a Ihanh phan giong cap 1 dua vao binh len men la 500 ml la hon hgp ciia Niirosomonas sp, DVAl va Nitrobacter sp. D V N I .
Co the thay rang khi ca hai chiing vi khuan dong thai CO mat trong djch len men, ngay sau 45 gio 75%
lugng ammonia co trong mot truong da duoc oxy hoa va mat do quang hpc da dal mirc 0,12, lucmg ducmg VOI thoi diem 120 gia khi ten men rieng biej timg chiing (Hinh 2) Ti^p theo, khi N H / dugc bit sung vao moi truang, mat do quang hpc lai tiep Iiic lang, dat 0,17 sau 120 gia (Hinh 2) Mat dp te bao AOB va NOB xac djnh bing phucmg phap MPN tren moi truang djch the dac hieu tuang iing la 1,2 x 10 va 1,5 X 10^ Ie bao/ml. Nhu vay, nuoi tang sinh dong thai hai chiing Niirosomonas sp, DVAl vi Nilrobacler sp. DVN 1 cho phep tang mat dp te bao moi loai tang tuang img len 4,8 va 3,9 ISn trong ciing thai gian.
Tgp chi Cong nghe Sinh hoc 11(2); 303-309, 2013 Nitrosomonas sp. DVA1
100 120 140
W/(rof)ac(ersp. D V N l
Thai gian (h) Thoi gian (h)
Hinh 1. Nuoi tang smh neng biet vi khuan oxy hoa ammonia Nitrosomonas sp DVA1 va ui khuan oxy hoa nitnl Nitrobacter sp DVNl trSn thiet bi len men A, B - Tang mat do t§ bao (OD, so luang tg bao) cung voi tieu Ihu cc chat NH,,* trong thi nghiem len men Nitrosomonas sp, D V A l , C, D - Tang mat 36 te bao (OD, s6 luang te bao) cung voi tao san pham NO3 trong thi nghiem len men Nitrobacter sp DVN 1.
0 20 40 60 80 100 120
Thai gian (h)
Hinh 2. N1161 tang sinh d i n g thin vi khuan oxy hoa ammonia Nitrosomonas sp D V A l va vi khuan oxy hoa nitrite Nitrobacter sp DVNl tr&n Ehi^t bj len men Tang m§t dp t l bao (ODeoo) cung vai tieu thu ca chk N H /
Dinh Thuy Hang et ai Thanh phan va dp tinh sach cua vi khuan nitrate
hoa trong dich len men con dugc kiem tra dua tren cac phan tu chi thi la gen 163 rDNA va gen amoA ma hoa cho tieu phan a ciia enzyme ammonia- monooxygenase thong qua phuang phap dien di bien tinh (DGGE) (Hinh 3),
Phan tich gen 16S rDNA bang phugng phap
DGGE (Hinh 3A) cho thay mau dich len men dong Ihoi hai chCing vi khuan D V A l va DVNl chi co 2 bang, tuong iing vai cac nhom Nitrosomonas spp va Nitrobacter spp. Ket qua phan tich gen amoA cung chi ra co mot nhom Nitrosomonas duy nhat trong dich len men, Duoi kinh hien vi co the quan sat Ihjy mat do DVN 1 (te bao hinh cau hoac oval) cao han so vai DVAl (td bao true khu^n) (Hmh 3B).
/ „>
' . • i « -
W (B) Hinh 3. Kiem tra thanh ph^n va do tinh sach cua cljch len men dong thoi hai chung Nitrosomonas sp. DVA1 va Nitrobacter sp D V N l A - DGGE gen 16S rDNA va gen amoA: B - M i u dich ien men quan sat true liep d u a i kinh h i l n vi phSn pha (400 x)
KET L U A N
Nuoi tang sinh vi khuan oxy hoa ammonia (AOB) Nitrosomonas sp. DVAl tren thiet b) len men CO kiem soat pH va bo sung nguon dien tii theo giai doan sinh truong cho phep thu dich tk bao co mat do o miic 2.5 X 10** te bao/ml. Tuy nhien, khi giS nguyen che dp len men nhung bo sung vi khuin nitnte hoa Nitrobacter sp. DVNl thi mdc sinh truong cua vi khuan tang va mat dp I^ bao cung tang 4,8 lan Irong ciing thoi gian len men. Dong then, mat dp te bao ciia vi khuan nitnte hoa trong dich len men cung tang so vai khi dugc len men rieng biel.
Loi c a m oti; Cdng trinh duac hodn thdnh nha su hd trg kinh pid tir di tdi md sd KC.04. TN07/II-15.
TAI LIEU THAM KHAO
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Gueven D, Schmidt! (2009) Specific activity and viability of Nitrosomonas europaea during discontinuous and continuous fermentation. Procs Biochem 44, 516-520 Koops H-P, Purkhold U, Pommerening-Roese A Timmermann G, Wagner M (2006) The lithoautotrophic ammonia oxidizing bacteria The Prokaryoies 5: 778-811 Kuypers MM, Shekers AO, Lavik G, Schmid M, Jergensen BB, Kuenen JG, Strous M, Jetten MS (2003) Anaerobic ammonium oxidaUon by anammox bacteria m the Black Sea, Nature 422: 608-611,
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Muyzer G, De Waal EC, Utteriinden AG (1993) Profiling of complex microbial population by denaturuig gradient gel electrophoresis analysis of polymerase chain reaction
Tgp chi Cdng nghe Sinh hgc 11(2): 303-309, 2013
amplified genes codmg for 163 rARN. Appl Environ Stembuchen A, Oppermann-Sanio FB (2003) Microbiol 59: 695-700. Mikrobiologisches Praknkum - Versuche und Theone.
Shimomura Y, Monmoto S, Hoshuio YT, Uchida Y, Akyiama ^ P " " ^ ' ' ^ " ' ' " S , Berlm, Heildenberg, New York H,HayatsuM (2012) Con^ranson among amoA primers suited Zhou J, Bruns, MA, TiedjeJM (1996) DNA recovery from for quantification and diversity analyses of ammoma-oxidizing soils of diverse composinon. Appl Ennviron Microbiol 62 bacteria in soil. Microbes Environ 27- 94-98. 316-322.
HIGH C E L L D E N S I T Y C U L T I V A T I O N O F N I T R I F Y I N G B A C T E R I A IN F E R M E N T E R Dinh Thuy Hang', Nguyen Thi Hai, Nguyen Thi Hang, Hoang Van Thai
Inslilule of Microbiology and Biotech njlogy. National University Hanoi
Nitrifying bacteria utilize molecular oxygen lo oxidize ammonia lo nitrate and occupy a key position in the niuogen cycle in nature Being litho-auiouophic organisms, these bacteria tend to produce Imle biomass, even in well controlled fermenters in the laboratory. In the present study, two nitrifying strains, including ammonia- oxydizing Nitrosomonas sp DVAl and nitrite-oxydizing Nilrobacler sp. DVNl, were used for demonstrating mass cukivation in a 10 liter fermenter (Satonus, Germany). It has been shown that cultivation of each strain separately could only reach cell densities of 2,5 - 3,8 x lO' cells ml"', mainly due to the inhibitory etTect of nitrite in the medium At ihe same time, co-cuhivation of these two strains allowed to increase cell density to 1,2-1,5 X lO' cell-mi"' since the nitnte produced by the ammonia-oxydizing strain was immediately utilized by the nitnte-oxydizing strain, and by that way the inhibitory effeci of nitrite was minimized. The mass cultivation procedure for the nitrifying bacteria demonstrated in this study coud be a good reference for other studies and applications that require biomass from this litho-autotrophic bactenal group
Keywords: Nunfymg, Nilnnomonas sp DVAl, Nitrobacter sp DVNl. Iilho-aiilolrophic growth, fermenlalion
Author for correspondence. E-mail: hansdinh [email protected]
