Header Page of 16 I HC QUC GIA H NI TRNG I HC KHOA HC T NHIấN - - Ngụ Th Kim Toỏn NGHIấN CU PHN LP TUYN CHN CC CHNG VI SINH VT NG DNG X Lí NC THI GIU NIT, PHOTPHO LUN VN THC S KHOA HC H Ni 2012 Footer Page of 16 Header Page of 16 I HC QUC GIA H NI TRNG I HC KHOA HC T NHIấN - Ngụ Th Kim Toỏn NGHIấN CU PHN LP TUYN CHN CC CHNG VI SINH VT NG DNG X Lí NC THI GIU NIT, PHOTPHO Chuyờn ngnh: Sinh hc thc nghim Mó s: 60 42 30 LUN VN THC S KHOA HC NGI HNG DN KHOA HC: TS NGUYN QUANG HUY H Ni 2012 Footer Page of 16 Header Page of 16 Lời Cảm ơn Lời đầu tiên, em xin gửi lời cảm ơn chân thành đến TS Nguyễn Quang Huy, người Thầy tận tình hướng dẫn, bảo em suốt thời gian học tập thực luận văn Em xin gửi lời cảm ơn chân thành đến thầy cô giáo Khoa Sinh học, Trường Đại học Khoa học Tự Nhiên, ĐHQGHN dành tâm huyết giảng dạy, trang bị kiến thức cho chúng em suốt trình học tập thực luận văn Trong trình học tập thực luận văn, em nhận nhiều giúp đỡ, hỗ trợ Em xin gửi lời cảm ơn chân thành đến thầy cô, cán bộ, học viên, sinh viên Bộ môn Sinh lý thực vật Hóa sinh, Khoa Sinh học; Phòng Enzym học Phân tích hoạt tính sinh học, Phòng thí nghiệm trọng điểm Công nghệ Protein Enzym Đề tài thực có hỗ trợ kinh phí đề tài Nghiên cứu phát triển công nghệ màng sinh học xử lý nước thải giàu nitơ photpho Bộ Công thương Cuối cùng, em xin gửi lời cảm ơn đến gia đình, bạn bè bên động viên, giúp đỡ em suốt thời gian học tập thực luận văn, giúp em trưởng thành bước đường Hà Nội, tháng 12 năm 2012 Học viên Ngô Thị Kim Toán Footer Page of 16 Header Page ofLun 16 thc s MC LC M U CHNG 1: TNG QUAN TI LIU 1.1 Tỡnh trng ụ nhim mụi trng nc hin Vit Nam v th gii 1.2 Cỏc phng phỏp x lý ụ nhim nc thi cú cha hp cht nit, photpho hin 1.2.1 Phng phỏp húa hc 1.2.1.1 X lý cỏc hp cht cha nit bng phng phỏp húa hc 1.2.1.2 X lý cỏc hp cht photpho bng phng phỏp húa hc 1.2.2 Phng phỏp sinh hc 1.3 Cỏc vi sinh vt cú kh nng chuyn húa cỏc hp cht cha nit, photpho x lý ụ nhim nc thi .10 1.3.1 Vi sinh vt cú kh nng chuyn húa cỏc hp cht cha nit 10 1.3.2 Vi sinh vt cú kh nng tớch ly photpho 12 1.4 Mng sinh hc v ng dng ca mng sinh hc vic x lý ụ nhim nc thi giu nit, photpho .14 1.4.1 Mng sinh hc 14 1.4.1.1 nh ngha v mng sinh hc 14 1.4.1.2 Thnh phn v quỏ trỡnh hỡnh thnh mng sinh hc 14 1.4.2 Vai trũ v ng dng ca s hỡnh thnh mng sinh hc 18 1.4.2.1 Vai trũ ca s hỡnh thnh mng sinh hc 18 1.4.2.2 ng dng ca mng sinh hc x lý ụ nhim 20 CHNG 2: NGUYấN LIU V PHNG PHP NGHIấN CU 23 2.1 Nguyờn liu 23 2.2 Húa cht, thit b 23 2.2.1 Mụi trng nuụi cy 23 2.2.2 Mỏy múc thit b 24 2.3 Phng phỏp nghiờn cu .25 Ngụ Th Kim Toỏn Footer Page of 16 i K19 Sinh hc thc nghim Header Page ofLun 16 thc s 2.3.1 Phng phỏp phõn lp vi khun 25 2.3.2 Phng phỏp ỏnh giỏ kh nng hỡnh thnh biofilm 25 2.3.3 Quan sỏt cu trỳc biofilm bng chp nh trờn kớnh hin vi in t quột (SEM) 26 2.3.4 nh hng ca cỏc iu kin mụi trng nuụi cy lờn s hỡnh thnh mng sinh hc 26 2.3.4.1 nh hng ca nhit mụi trng nuụi cy 26 2.3.4.2 nh hng ca pH mụi trng nuụi cy 27 2.3.5 Phng phỏp nhum Gram 27 2.3.6 Phng phỏp s dng kit APi 28 2.3.7 Phng phỏp ỏnh giỏ kh nng chuyn húa cỏc hp cht nit 28 2.3.7.1 Phng phỏp phõn tớch nit tng s 28 2.3.7.2 Phng phỏp phõn tớch hm lng amoni (NH4+) 29 2.3.7.3 Phng phỏp th kh nng chuyn húa nitrite 30 2.3.8 Phng phỏp ỏnh giỏ kh nng tớch ly photpho 31 2.3.8.1 Phng phỏp phõn tớch photpho tng 32 2.3.8.2 Phng phỏp phõn tớch hm lng Ortho photphate (PO43-) 32 2.3.9 Phng phỏp phõn loi vi sinh vt da trờn gen 16S rRNA 33 2.3.10 Phng phỏp thng kờ sinh hc 33 CHNG 3: KT QU V THO LUN 34 Nghiờn cu cỏc chng vi sinh vt cú kh nng chuyn húa nit 34 3.1.1 Phõn lp chng vi sinh vt cú kh nng chuyn húa nit v cú kh nng hỡnh thnh mng sinh hc 34 3.1.1.1 Phõn lp cỏc chng vi sinh vt cú kh nng chuyn húa nit 34 3.1.1.2 Kh nng hỡnh thnh mng sinh hc ca cỏc chng phõn lp 36 3.1.2 Kh nng chuyn húa nit 38 3.1.2.1 Kh nng chuyn húa amoni 38 3.1.2.2 Kh nng chuyn húa nitrite 39 Nghiờn cu cỏc chng vi sinh vt cú kh nng tớch ly photpho .40 Ngụ Th Kim Toỏn Footer Page of 16 ii K19 Sinh hc thc nghim Header Page ofLun 16 thc s 3.2.1 Phõn lp chng vi sinh vt cú kh nng tớch ly photpho v cú kh nng hỡnh thnh mng sinh hc 40 3.2.1.1 Phõn lp cỏc chng vi sinh vt cú kh nng tớch ly photpho 40 3.2.1.2 Kh nng hỡnh thnh mng sinh hc ca cỏc chng phõn lp 41 3.2.2 Kh nng x lý photpho ca cỏc chng nghiờn cu 42 3 Cỏc c im hỡnh thỏi, sinh lý, sinh húa ca cỏc chng nghiờn cu 43 3.3.1 Kh nng to hỡnh thnh mng sinh hc trờn mt s giỏ th 43 3.3.2 c im hỡnh thỏi ca cỏc chng nghiờn cu 45 3.3.3 nh hng ca mt s yu t mụi trng lờn s hỡnh thnh mng sinh hc 46 3.3.4 Kh nng chuyn húa mt s cht b kit APi 48 3.3.5 Trỡnh t 16S rRNA v cõy phỏt sinh chng loi 50 KT LUN 54 KIN NGH 54 TI LIU THAM KHO 55 Ngụ Th Kim Toỏn Footer Page of 16 iii K19 Sinh hc thc nghim Header Page ofLun 16 thc s DANH MC BNG Bng 3.1 Hm lng thnh phn nit v photpho mu phõn tớch 34 Bng 3.2 a im v s lng cỏc chng vi sinh vt cú kh nng chuyn húa nit 35 Bng 3.3 a im v s lng cỏc chng vi sinh vt cú kh nng tớch ly photpho 41 Bng 3.4 Mt s c im sinh hoỏ ca cỏc chng theo kit APi (BioMộrieus) 48 Ngụ Th Kim Toỏn Footer Page of 16 iv K19 Sinh hc thc nghim Header Page ofLun 16 thc s DANH MC HèNH Hỡnh 1.1 Chu trỡnh nit t nhiờn Hỡnh 1.2 Cỏc giai on chớnh ca quỏ trỡnh hỡnh thnh mt biofilm 16 Hỡnh 3.1 Hỡnh nh cỏc khun lc phõn lp trờn mụi trng 35 Hỡnh 3.2 Kh nng hỡnh thnh mng sinh hc ca cỏc chng trờn mụi trng Winogradsky c phõn lp t cỏc mu nc thi thu t b biogas 36 Hỡnh 3.3 Kh nng hỡnh thnh mng sinh hc ca cỏc chng trờn mụi trng Winogradsky c phõn lp t cỏc mu nc thi khu trung rỏc thi 37 Hỡnh 3.4 Kh nng to hỡnh thnh mng sinh hc ca cỏc chng trờn mụi trng Winogradsky c phõn lp b biogas 37 Hỡnh 3.5 Kh nng chuyn húa amoni ca cỏc chng nghiờn cu 38 Hỡnh 3.6 Kh nng chuyn húa nitrite ca cỏc chng nghiờn cu 39 Hỡnh 3.7 Mt s khun lc phõn lp trờn mụi trng AMM 40 Hỡnh 3.8 Kh nng hỡnh thng mng sinh hc ca cỏc chng vi sinh vt cú kh nng tớch ly photpho 41 Hỡnh 3.9 Kh nng tớch ly photpho ca cỏc chng nghiờn cu mụi trng vi hm lng photpho 6mg/l 42 Hỡnh 3.10 Kh nng tớch ly photpho ca cỏc chng nghiờn cu mụi trng hm lng photpho18mg/l 43 Hỡnh 3.11 Kh nng hỡnh thnh biofilm trờn giỏ th nha ng eppendorf 44 Hỡnh 3.12 Mng biofilm ni ca chng B11.11, B21.10, B23.2, A4.2 44 Hỡnh 3.13 nh nhum Gram cỏc chng nghiờn cu phúng i 1000 ln 45 Hỡnh 3.14 Cu trỳc hin vi mng biofilm ca chng nghiờn cu 45 Hỡnh 3.15 nh hng ca nhit lờn kh nng hỡnh thnh mng sinh hc 46 Hỡnh 3.16 nh hng ca pH mụi trng 47 Hỡnh 3.17 S cõy phỏt sinh chng loi ca chng B11.11 50 Hỡnh 3.18 S cõy phỏt sinh chng loi ca chng B21.10 51 Hỡnh 3.19 S cõy phỏt sinh chng loi ca chng B23.2 52 Hỡnh 3.20 S cõy phỏt sinh chng loi ca chng A4.2 53 Ngụ Th Kim Toỏn Footer Page of 16 v K19 Sinh hc thc nghim Header Page ofLun 16 thc s BNG Kí HIU VIT TT ADH L-arginine ADI Axit adipic AMM Acetate mineral medium ARA L-arabinose BOD Biochemical oxygen demand (Nhu cu oxy sinh hc) BTNMT B Ti nguyờn Mụi trng CAP Axit capric CIT Trisodium citrate COD Chemical oxygen demand (Nhu cu oxy húa hc) EPS Mng li cỏc hp cht ngoi bo (Extracellular polymeric substances) ESC Esculin ferric citrate GEL Gelatine GLU D-glucose GNT Potassium gluconate HEPES 4-(2-hydroxyethyl)-1- piperazineethanesulfonic LB Mụi trng Luria betani MAL D-mantose MAN D-mannitol MLT Axit malic MNE D-mannose NAG N- acetyl- glucosamine PAC Axit phenyl acetic PNPG 4-nitrophenyl D galactopyranoside QCVN Quy chun Vit Nam SEM nh vi in t quột (Scanning Electron Microscope) TRP L- tryptophan URE Urea Ngụ Th Kim Toỏn Footer Page of 16 vi K19 Sinh hc thc nghim Header Page 10 ofLun 16 thc s M U Hin nay, ụ nhim mụi trng ang l c quan tõm ca nhiu quc gia trờn th gii ú cú Vit Nam ễ nhim ngun nc khụng ch nh hng n i sng ngi m cũn nh hng n a dng sinh hc Tỡnh trng ụ nhim nc thi ú cú nguyờn nhõn t cỏc hp cht nit v photpho ang cú chiu hng gia tng nhng nm gn õy cựng vi s phỏt trin ca kinh t, xó hi lm gim mc ụ nhim t nc thi giu nit v photpho, nhiu phng phỏp ó v ang nghiờn cu bng cỏch kt hp cỏc bin phỏp vt lý, húa hc v sinh hc X lý nc thi da vo phng phỏp húa hc, vt lý thng cú hiu qu cao, nhanh nhng chi phớ ln, khụng mang tớnh bn vng X lý nc thi bng phng phỏp sinh hc da trờn c s s dng cỏc vi sinh vt l mt phng phỏp c quan tõm nghiờn cu Nhiu nhúm vi sinh vt t nhiờn cú kh nng chuyn húa cỏc hp cht cha nit v photpho nc thi thnh cỏc cht khụng c hi vi mụi trng Mng sinh hc c nh ngha l dng sng tn ti ph bin t nhiờn v khỏc bit vi dng t bo sng t bi mng li cỏc hp cht ngoi bo bao quanh v nhng thay i, bit húa t bo vi sinh vt thớch nghi vi mụi trng sng Vi sinh vt hỡnh thnh mng sinh hc khụng ch giỳp chỳng tn ti v chng chu c vi nhng iu kin bt li, tn dng c ngun dinh dng ca mụi trng m cũn thụng qua mi quan h hp tỏc gia cỏc loi khỏc tng quỏ trỡnh phõn gii cỏc cht c hi mụi trng Vic nghiờn cu x lý ụ nhim ngun nc thi núi chung v nc thi cú hm lng nit, photpho cao bng vic s dng cỏc vi sinh vt to mng sinh hc l hng nghiờn cu mi mang tớnh bn vng Tuy nhiờn, hin cha cú nhiu cụng trỡnh cụng b v kt qu phõn lp cng nh kh nng chuyn húa nit, photpho ca cỏc cỏc chng vi sinh vt cú kh nng to mng sinh hc Chỳng tụi tin hnh thc hin ti :Nghiờn cu phõn lp tuyn chn cỏc chng vi sinh vt ng dng x lý nc thi giu Nit, Photpho nhm mc ớch gúp phn tỡm Ngụ Th Kim Toỏn Footer Page 10 of 16 K19 Sinh hc thc nghim Header Page 62 ofLun 16 thc s 0.01 66 Bacillus methylotrophicus_EU194897 68 Bacillus amyloliquefaciens_NR041455 62 Bacillus siamensis_GQ281299 Bacillus velezensis_AY603658 59 Bacillus nematotocita_AY820954 71 Bacillus vallismortis_AB021198 Bacillus subtilis_AB042061 100 81 Bacillus mojavensis_AB021191 99 77 Bacillus axarquiensis_AY603657 59 Bacillus malacitensis_AY603656 Bacillus atrophaeus_AB021181 Bacillus sonorensis_AF302118 Bacillus aerius_AJ831843 100 Bacillus licheniformis_X68416 100 A4.2 97 Bacillus pumilus_AY876289 100 Bacillus safensis_AF234854 Bacillus stratosphericus_AJ831841 99 Bacillus altitudinis_AJ831842 99 Bacillus aerophilus_AJ831844 Bacillus isabeliae_AM503357 Bacillus idriensis_AY904033 100 Bacillus indicus_AJ583158 100 Bacillus cibi_AY550276 Staphylococcus aureus_X68417 100 89 68 Hỡnh 3.20 S cõy phỏt sinh chng loi ca chng A4.2 Trỡnh t gen 16S rARN ca chng A4.2 tng ng 99.9% (1411/1413 bp) vi on gen 16S rARN ca vi khun Bacillus licheniformis_X68416 so sỏnh vi cỏc trỡnh t gen 16S rARN ca cỏc vi sinh vt ngõn hng gen quc t (Hỡnh 3.20) iu ny cho chỳng tụi kt lun: chng A4.2 thuc chi Bacillus õy l cụng b u tiờn Vit Nam, chng vi khun Bacillus licheniformis cú kh nng x lý photpho nc thi bng vic tớch ly vo c th vi sinh vt, mt phn cung cp nng lng cho cỏc quỏ trỡnh sinh húa v trao i cht Ngụ Th Kim Toỏn Footer Page 62 of 16 53 K19 Sinh hc thc nghim Header Page 63 ofLun 16 thc s KT LUN ó phõn lp v c 65 chng vi sinh vt trờn mụi trng Winogradsky v 21 chng trờn mụi trng AMM t cỏc mu nc thi Thanh Húa v H Ni ó tuyn chn c chng vi sinh vt cú kh nng hỡnh thnh mng sinh hc v cú kh nng x lý nit v photpho tt nht l cỏc chng cú ký hiu B11.11, B21.10, B23.2 v A4.2 Cỏc chng cú kh nng hỡnh thnh mng sinh hc nhit khong t 37 n 50 oC, pH t n ó phõn loi v xỏc nh c chng cú kh nng x lý nit tt nht, ú, chng B11.11 cú kh nng chuyn húa 85.21% NH4+ sau 20 ngy nuụi cy, chng B21.10 chuyn húa 97,28% v B23.2 chuyn húa 97,14% lng NO2sau 20 ngy nuụi cy ó phõn loi v xỏc nh c chng A4.2 cú kh nng x lý photpho tt nht, sau 10 ngy, hm lng photpho mụi trng gim i 39.32% tng ng vi mụi trng cú hm lng photpho l 18 mg/l Da trờn trỡnh t gen 16S rARN, c im khun lc, t bo, cỏc c im sinh lý, sinh húa, chng B11.11 v A4.2 c xỏc nh gn vi loi Bacillus licheniformis, chng B21.10 c xỏc nh gn vi loi Bacillus amyloliquefaciens, chng B23.2 xỏc nh gn vi loi Pseudomonas pseudoalcaligenes KIN NGH Thay i hm lng nit v photpho phự hp vi nghiờn cu Kim tra kh nng chuyn húa cỏc cht mụi trng v s hỡnh thnh mng sinh hc thay i hm lng Nghiờn cu, ti u húa iu kin chuyn húa cỏc hp cht nit v photpho, nghiờn cu v la chn giỏ th, cht mang phự hp cho s hỡnh thnh mng sinh hc ca cỏc chng nghiờn cu Ngụ Th Kim Toỏn Footer Page 63 of 16 54 K19 Sinh hc thc nghim Header Page 64 ofLun 16 thc s TI LIU THAM KHO Ting Vit Kiu Hu nh (2006), Giỏo trỡnh vi sinh vt hc, phn 1, NXB i hc Quc Gia H Ni B Ti Nguyờn v Mụi trng (2010), Bỏo cỏo mụi trng Quc gia 2010, H Ni Lờ Vn Cỏt (2007), X lý nc thi giu hp cht nit v photpho, NXB Khoa hc t nhiờn v Cụng ngh Lng c Phm (2003), Cụng ngh x lý nc thi bng bin phỏp sinh hc, NXB Giỏo dc Nguyn Hoi Hng (2009), Giỏo trỡnh thc hnh vi sinh ng dng, NXB i hc Quc Gia TPHCM Nguyn Vn Phc (2007), X lý nc thi sinh hot v cụng nghip bng phng phỏp sinh hc, NXB Xõy dng Ting Anh Anderson I.C., Poth M., Homstead J., and Burdige D (1993), A comparison of NO and N2O production by the autotrophic nitrifier Nitrosomonas europaea and the heterotrophic nitrifier Alcaligenes faecalis, Applied and Environmental Microbiology, 59 (11), pp 3525-3533 Annachhatre A.P and Bhamidimarri S.M.R (1992), Microbial attachment and growth in fixed-film reactors: Process startup considerations, Biotechnology Advances, 10 (1), pp 69-91 APHA (2001), Standard Methods for the Examination of Water and Wastewate., 20th edition, American Public Health Association, Washington, DC 10 Asgari M.J., Safavi K., and Mortazaeinezahad F (2011), Landfill biogas production process, International Conference on Food Engineering and Biotechnology, 9, pp 208-212 Ngụ Th Kim Toỏn Footer Page 64 of 16 55 K19 Sinh hc thc nghim Header Page 65 ofLun 16 thc s 11 Bao L.-L., Li D., Li X.K., Huang R.X., Zhang J., Yang L., and Xia G.Q (2007), Phosphorus accumulation by bacteria isolated from a continuousflow two-sludge system, Journal of Environmental Sciences, 19 (4), pp 391-395 12 Bernet N., Dangcong P., Delgenốs J., and Moletta R (2001), Nitrification at low oxygen concentration in biofilm reactor, Journal of Environmental Engineering, 127 (3), pp 266-271 13 Boelee N.C., Temmink H., Janssen M., Buisman C.J.N., and Wijffels R.H (2011), Nitrogen and phosphorus removal from municipal wastewater effluent using microalgal biofilms, Water Research, 45 (18), pp 5925-5933 14 Boyd C.E and Tucker C.S (1998), Pond Aquaculture Water Quality Management, Kluwer Acad Publ 15 Broda E (1977), Two kinds of lithotrophs missing in nature, Zeitschrift fỹr allgemeine Mikrobiologie, 17 (6), pp 491-493 16 Cheung K.C., Chu L.M., and Wong M.H (1997), Ammonia stripping as a pretreatment for landfill leachate, Water, Air, and Soil Pollution, 94 (1-2), pp 209-221 17 Cong L.T.N., Huyen H.T., and Minh N.N (2012), Phenol degradation of biofilm formed by mixing - marine bacteria, VNU Journal of Science, 28 (2S), pp 75-81 18 Costerton J.W., Lewandowski Z., Caldwell D.E., Korber D.R., and LappinScott H.M (1995), Microbial biofilms, Annual Review of Microbiology, 49, pp 711-745 19 Czaczyk K and Myszka K (2007), Biosynthesis of extracellular polymeric substances (EPS) and its role in microbial biofilm formation, Polish Journal of Environmental Studies, 16 (6), pp 799-806 20 Di Bonaventura G., Stepanovic S., Picciani C., Pompilio A., and Piccolomini R (2007), Effect of environmental factors on biofilm formation by clinical Ngụ Th Kim Toỏn Footer Page 65 of 16 56 K19 Sinh hc thc nghim Header Page 66 ofLun 16 thc s Stenotrophomonas maltophilia isolates, Folia Microbiologica., 52 (1), pp 86-90 21 Donlan R.M (2002), Biofilms: microbial life on surfaces, Emerging Infectious Diseases Journal, (9), pp 881-890 22 Federation W.E (1998), Biological and chemical systems for nutrient removal, Water Environment Federation, Alexandria, VA 23 Flemming H.-C (1993), Biofilms and Environmental Protection, Water Science & Technology, 27 (7-8), pp 1-10 24 Giaouris E., Chorianopoulos N., and Nychas G.J.E (2005), Effect of temperature, pH, and water activity on biofilm formation by Salmonella enterica enteritidis PT4 on stainless steel surfaces as indicated by the bead vortexing method and conductance measurements, Journal of Food Protection, 68 (10), pp 2149-2154 25 Gilbert P., Das J., and Foley I (1997), Biofilm susceptibility to antimicrobials, Advances in Dental Research, 11 (1), pp 160-167 26 Hang T.T and Huy N.Q (2011), Isolate biofilm forming Bacillus strains 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combined treatment of municipal wastewaters and landfill leachates in coastal areas, Water science and technology : a journal of the International Association on Water Pollution Research, 46 (8), pp 111-118 67 Zhang J., Wu P., Hao B., and Yu Z (2011), Heterotrophic nitrification and aerobic denitrification by the bacterium Pseudomonas stutzeri YZN-001, Bioresource Technology, 102 (21), pp 9866-9869 68 Zhang Q.-L., Liu Y., Ai G.-M., Miao L.-L., Zheng H.-Y., and Liu Z.-P (2012), The characteristics of a novel heterotrophic nitrificationaerobic denitrification bacterium, Bacillus methylotrophicus strain L7, Bioresource Technology, 108 (0), pp 35-44 Website 69 http://en.wikipedia.org/wiki/Nitrogen_cycle 70 http://www.visionlearning.com/library/module_viewer.php?mid=98 71 http://www.thewaterplanetcompany.com/docs/WPC_Nitrification and Denitrification.pdf Ngụ Th Kim Toỏn Footer Page 71 of 16 62 K19 Sinh hc thc nghim Header Page 72 ofLun 16 thc s PH LC Trỡnh t gen mó húa 16S rARN ca vi khun B11.11 c c trờn mỏy c trỡnh t t ng ABI PRISM 3100 Avant (Hoa K) GTGCCTAATACATGCAAGTCGAGCGGACCGACGGGAGCTTGCTCCCTTA GGTCAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGA CTGGGATAACTCCGGAAACCGGGGCTAATACCGGATGCTTGATTGAACC GCATGGTTCAATCATAAAAGGTGGCTTTTAGCTACCACTTACAGATGGA CCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACG ATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACA CGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGG ACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGAT CGTAAAACTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGG CACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCA GCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTA AAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCA ACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAG AGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAAC ACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGCGCGA AAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTA AACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGC AAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACT CAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAA TTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAAC CCTAGAGATAGGGCTTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATG GTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAG CGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTG ACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCA TGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGAACAAAG Ngụ Th Kim Toỏn Footer Page 72 of 16 K19 Sinh hc thc nghim Header Page 73 ofLun 16 thc s GGCAGCGAAGCCGCGAGGCTAAGCCAATCCCACAAATCTGTTCTCAGTT CGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAAT CGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACC GCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACC TTTTGGAGCCAGCCGCCGAAGGTGGGACAGA Trỡnh t gen mó húa 16S rARN ca chng B21.10 c c trờn mỏy c trỡnh t t ng ABI PRISM 3100 Avant (Hoa K) AGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAG ATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTG GGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATA CCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGG CTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAA CGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGC CACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTA GGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGA GTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAG TGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCAC GGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTG TCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGA TGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAA CTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATG CGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGT AACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATAC CCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTC CGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTA CGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGC GGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGT CTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCA GAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTG Ngụ Th Kim Toỏn Footer Page 73 of 16 K19 Sinh hc thc nghim Header Page 74 ofLun 16 thc s GGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTC AGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGG GGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGC TACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATC CCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTG AAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGT TCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACAC CCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGA CAGA Trỡnh t gen mó húa 16S rARN ca chng B23.2 c c trờn mỏy c trỡnh t t ng ABI PRISM 3100 Avant (Hoa K) AGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGATGA AGGGAGCTTGCTCCTGGATTTAGCGGCGGACGGGTGAGTAATGCCTAGG AATCTGCCTGGTAGTGGGGGATAACGTTCCGAAAGGAACGCTAATACCG CGTACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCA GATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAG GCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTG AGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGAC AATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTT CGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGTTGTACGTTAATACC GTGCAATTTTGACGTTACCGACAGAATAAGCACCGGCTAACTTCGTGCC AGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTAATCGGAATTACTGG GCGTAAAGCGCGCGTAGGTGGTTCAGTAAGTTGGAAGTGAAATCCCCGG GCTCAACCTGGGAACTGCTTTCAAAACTGCTGAGCTAGAGTACGGTAGA GGGTAGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGG AACACCAGTGGCGAAGGCGACTACCTGGACTGATACTGACACTGAGGT GCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGC CGTAAACGATGTCAACTAGCCGTTGGGATCCTTGAGATCTTAGTGGCGC AGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAA Ngụ Th Kim Toỏn Footer Page 74 of 16 K19 Sinh hc thc nghim Header Page 75 ofLun 16 thc s ACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTT AATTCGAAGCAACGCGAAGAACCTTACCTGGCCTTGACATGCTGAGAAC TTTCCAGAGATGGATTGGTGCCTTCGGGAACTCAGACACAGGTGCTGCA TGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACG AGCGCAACCCTTGTCCTTAGTTACCAGCACCTCGGGTGGGCACTCTAAG GAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCA TCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGTCGGTACA AAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGT AGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAG TAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACA CACCGCCCGTCACACCATGGGAGTGGGTTGCTCCAGAAGTAGCTAGTCT AACCTTCGGGGGGACGGTACCACGGAG Trỡnh t gen mó húa 16S rARN ca chng A4.2 c c trờn mỏy c trỡnh t t ng ABI PRISM 3100 Avant (Hoa K) CTCTGCTCAGGACGAACGCCGGCGGCGTGCCTAATACATGCAAGTCGAG CGGACCGACGGGAGCTTGCTCCCTTAGGTTAGTGGCGGACGGGTGAGTA ACACGTGGGTAACCTGCCTGTAAGATTGGGATAACTCCGGGAAACCGGG GCTAATACCGGATGCTTGATTGAACCGCATGGTTCAATCATAAAAGGTG GCTTTTAGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGG TGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGG TGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGG CAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACG CCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGGGA AGAACAAGTACCGTTCGAATAGGGCGGCACCTTGACGGTACCTAACCAG AAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGG CAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCT TAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAA CTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCG GTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCT Ngụ Th Kim Toỏn Footer Page 75 of 16 K19 Sinh hc thc nghim Header Page 76 ofLun 16 thc s CTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCGAACAGG ATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTA GAGGGTTTCCGCCCTTTAGTGCTGCAGCAAACGCATTAAGCACTCCGCC TGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCC CGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAAC CTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTCCCCT TCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGT GAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTG CCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAG GAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTAC ACACGTGCTACAATGGGCAGAACAAAGGGCAGCGAAGCCGCGAGGCTA AGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCG ACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGT GAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTT TGTAACACCCGAAGTCGGTGAGGTAACCTTTGGAGCCAGCCGCCGAAGT GGACAGAATGACTCA Ngụ Th Kim Toỏn Footer Page 76 of 16 K19 Sinh hc thc nghim ... lời cảm ơn chân thành đến thầy cô, cán bộ, học vi n, sinh vi n Bộ môn Sinh lý thực vật Hóa sinh, Khoa Sinh học; Phòng Enzym học Phân tích hoạt tính sinh học, Phòng thí nghiệm trọng điểm Công nghệ... kinh phí đề tài Nghiên cứu phát triển công nghệ màng sinh học xử lý nước thải giàu nitơ photpho Bộ Công thương Cuối cùng, em xin gửi lời cảm ơn đến gia đình, bạn bè bên động vi n, giúp đỡ em... phỏp sinh hc 1.3 Cỏc vi sinh vt cú kh nng chuyn húa cỏc hp cht cha nit, photpho x lý ụ nhim nc thi .10 1.3.1 Vi sinh vt cú kh nng chuyn húa cỏc hp cht cha nit 10 1.3.2 Vi sinh