Nghiên cứu ứng dụng vi khuẩn tía quang hợp để xử lý sulfide trong các nguồn nước ô nhiễm

viện hàn lâm khoa học công nghệ việt nam viện công nghệ sinh học TH LIấN NGHIÊN CứU ứNG DụNG VI KHUẩN TíA QUANG HợP Để Xử Lý SULFIDE TRONG CáC NGUồN NƯớC Ô NHIễM luận án tiến sĩ sinh học Hà nội - 2016 viện hàn lâm khoa học công nghệ việt nam viện công nghệ sinh học TH LIấN NGHIÊN CứU ứNG DụNG VI KHUẩN TíA QUANG HợP Để Xử Lý SULFIDE TRONG CáC NGUồN NƯớC Ô NHIễM Chuyên ngành : Vi sinh vật học Mã số : 62 42 01 07 luận án tiến sĩ sinh học Ngi hng dn khoa hc: PGS.TS inh Duy Khỏng Vin Cụng ngh sinh hc TS Th T Uyờn Vin Cụng ngh sinh hc Hà nội - 2016 i Li cm n Vi tt c tm lũng, tụi xin by t lũng kớnh trng v bit n sõu sc nht ti PGS.TS inh Duy Khỏng, phũng vi sinh vt hc phõn t v TS Th T Uyờn, phũng Cụng ngh sinh hc mụi trng, Vin Cụng ngh sinh hc, Vin Hn lõm Khoa hc v Cụng ngh Vit Nam l nhng ngi thy ó dnh cho tụi nhng ý tng quý bỏu cng nh s hng dn tn tỡnh, to mi iu kin thun li v ng viờn tụi sut quỏ trỡnh thc hin lun ỏn Tụi xin by t lũng bit n sõu sc ti GS.TS Trng Nam Hi, nguyờn Vin trng Vin Cụng ngh sinh hc ó to iu kin giỳp tụi thc hin lun ỏn ny Tụi cng xin by t lũng bit n ti PGS.TS ng Cm H, PGS.TSKH Trn Vn Nh ó giỳp tụi giai on u ca khúa hc NCS Tụi xin chõn thnh cm n TS ng Vn Quyn ó to iu kin giỳp tụi thc hin cỏc nghiờn cu ti phũng vi sinh vt hc phõn t Tụi cng chõn thnh cm n ThS Nguyn Th Hoa v th cỏn b nghiờn cu phũng thớ nghim vi sinh vt hc phõn t ó giỳp tụi cú c nhng hiu bit v k thut v sinh hc phõn t Tụi xin chõn thnh cm n ThS Nguyn Th Diu Phng v th nghiờn cu ti Trung tõm nghiờn cu a dng sinh hc v ngun li thy sn, Vin nghiờn cu nuụi trng thy sn I, ỡnh Bng - T Sn - Bc Ninh ó giỳp tụi th nghim ch phm vi khun tớa quang hp x lý ỏy ao nuụi cỏ rụ phi thõm canh Tụi xin gi li cm n chõn thnh n Ban lónh o Vin Cụng ngh sinh hc Vin Hn lõm Khoa hc v Cụng ngh Vit Nam ó to mi iu kin cho tụi c hc v nghiờn cu ti Vin sut nhng nm qua Tụi cng xin chõn thnh cm n ch Bựi Th Hi H ó giỳp tụi hon thnh mi th tc cn thit sut quỏ trỡnh nghiờn cu sinh v bo v lun ỏn Trong thi gian qua, tụi ó nhn c s h tr nhit tỡnh v to mi iu kin thun li t phũng thớ nghim Cụng ngh sinh hc mụi trng ni tụi ang cụng tỏc, v s giỳp nhit tỡnh v úng gúp nhng ý kin quý bỏu ca cỏc anh, ch, em ng nghip, nhõn dp ny tụi xin chõn thnh cm n s giỳp quớ bỏu ú Cui cựng, tụi xin by t lũng bit n sõu sc n nhng ngi thõn gia ỡnh v nhng ngi bn thõn thit ó luụn bờn cnh, ng viờn v khớch l tụi sut quỏ trỡnh hc v nghiờn cu Nghiờn cu sinh Th Liờn ii LI CAM OAN Tụi xin cam oan: õy l cụng trỡnh nghiờn cu ca tụi v mt s kt qu cựng cng tỏc vi cỏc cng s khỏc; Cỏc s liu v kt qu trỡnh by lun ỏn l trung thc, mt phn ó c cụng b trờn cỏc khoa hc chuyờn ngnh vi s ng ý v cho phộp ca cỏc ng tỏc gi; Phn cũn li cha c cụng b bt k cụng trỡnh no khỏc Tỏc gi Th Liờn iii MC LC Trang M U Chng 1: TNG QUAN TI LIU 1.1 Mt s c im sinh hc c bn ca vi khun tớa quang hp 1.1.1 Gii thiu chung v vi khun tớa quang hp 1.1.2 Sinh thỏi hc ca vi khun tớa quang hp 1.1.3 Hỡnh thỏi v phõn loi hc ca vi khun tớa quang hp 1.1.4 Mt s c im sinh lý, sinh húa ca vi khun tớa quang hp 1.2 Sulfide quinone reductase - enzyme chỡa khúa ca quỏ trỡnh oxy húa sulfide vi khun tớa quang hp 24 1.2.1 Vai trũ ca sulfide quinone reductase quỏ trỡnh oxy húa sulfide 24 1.2.2 Tỏch dũng gen sqr t R.capsulatus 28 1.3 Nghiờn cu ng dng vi khun tớa quang hp trờn th gii 28 1.3.1 ng dng vi khun tớa quang hp x lý sulfide nc thi 28 1.3.2 ng dng vi khun tớa quang hp sn xut ch phm x lý sulfide cỏc ỏy ao nuụi trng thy sn 35 1.3.3 ng dng vi khun tớa quang hp x lý nc thi 36 1.3.4 Mt s ng dng khỏc ca vi khun tớa quang hp 37 1.4 Tỡnh hỡnh nghiờn cu ng dng vi khun tớa quang hp Vit Nam 39 Chng 2: VT LIU V PHNG PHP NGHIấN CU 42 2.1 Vt liu, húa cht v thit b mỏy múc 42 2.1.1 Vt liu 42 2.1.2 Cỏc thit b mỏy múc 43 2.1.3 Húa cht 44 2.1.4 Thnh phn mụi trng nuụi cy 44 2.1.5 Cỏc dung dch ó s dng 45 2.2 Phng phỏp nghiờn cu 45 2.2.1 Phõn lp vi khun tớa quang hp khụng lu hunh 45 iv 2.2.2 Phng phỏp nuụi cy vi khun tớa quang hp khụng lu hunh 2.2.3 ỏnh giỏ sinh trng v nghiờn cu hỡnh thỏi cỏc vi khun tớa 46 quang hp 46 2.2.4 Phng phỏp tuyn chn vi khun tớa quang hp 47 2.2.5 Phng phỏp nghiờn cu h sc t ca vi khun tớa quang hp 47 2.2.6 Phng phỏp nhum Gram 48 2.2.7 Tỏch chit DNA genom ca vi khun tớa quang hp 48 2.2.8 Phng phỏp PCR 48 2.2.9 Phng phỏp in di DNA trờn gel agarose 49 2.2.10 Phn ng ni ghộp gen 49 2.2.11 Phng phỏp bin np DNA plasmid vo t bo kh bin E.coli bng phng phỏp sc nhit 50 2.2.12 Phng phỏp tỏch chit DNA plasmid 50 2.2.13 Ct plasmid bng enzyme gii hn 51 2.2.14 Tinh sch phõn on DNA 51 2.2.11 Xỏc nh trỡnh t axit nucleic 51 2.2.16 51 Phng phỏp xỏc nh cỏc ch tiờu thy húa 2.2.17 Phng phỏp ỏnh giỏ nh hng ca nhit , pH, mui, ỏnh sỏng 52 2.2.18 Phng phỏp c nh t bo vi khun vo cht mang 52 2.2.19 ỏnh giỏ kh nng x lý sulfide ca ch phm dng dch cỏc ngun nc thi ụ nhim qui mụ phũng thớ nghim 53 2.2.20 Th nghim ch phm i vi nc nuụi cỏ rụ phi quy mụ pilot 53 2.2.21 Phng phỏp kho nghim trờn ao 54 2.2.22 Phng phỏp th tớnh i khỏng ca vi khun tớa quang hp 55 2.2.23 Phng phỏp x lý thng kờ sinh hc 55 Chng 3: KT QU NGHIấN CU 56 3.1 Kt qu phõn lp v tuyn chn cỏc chng vi khun tớa quang hp cú kh nng loi b sulfide 56 3.1.1 Kt qu phõn lp vi khun tớa quang hp 56 3.1.2 Kt qu tuyn chn vi khun tớa quang hp cú kh nng sinh trng v hot tớnh loi b sulfide cao 58 v 3.2 Nhng c im sinh hc c bn ca cỏc chng vi khun tớa quang hp khụng lu hunh c la chn 60 3.2.1 c im hỡnh thỏi 60 3.2.2 c im sc t quang hp 62 3.2.3 Kh nng s dng cỏc ngun carbon 65 3.2.4 Kh nng s dng sulfide ca cỏc chng ó la chn 66 3.2.5 Xỏc nh trỡnh t gen 16S - rDNA ca cỏc chng la chn 68 3.2.6 Xỏc nh trỡnh t gen pufM ca cỏc chng nghiờn cu 70 3.2.7 Tỏch dũng gen sqr mó húa sulfide quinone reductase ca ba chng la chn 3.3 73 Nghiờn cu mt s yu t mụi trng nh hng n sinh trng v kh nng loi b sulfide ca cỏc chng la chn nhm ng dng x lý sulfide 77 3.3.1 nh hng ca nhit 78 3.3.2 nh hng ca pH n sinh trng v hot tớnh kh sulfide 79 3.3.3 nh hng ca nng NaCl n sinh trng v hot tớnh loi b sulfide 81 3.3.4 nh hng ca cng chiu sỏng 82 3.4 Nghiờn cu xõy dng quy trỡnh sn xut sinh vi khun tớa quang hp khụng lu hunh 83 3.4.1 Kh nng s dng mt s ngun c cht n gin 83 3.4.2 Xõy dng quy trỡnh sn xut sinh vi khun tớa quang hp khụng lu hunh 85 3.4.3 Quy trỡnh sn xut ging vi khun tớa quang hp khụng lu hunh 86 3.4.4 Nghiờn cu la chn cht mang c nh vi khun 88 3.5 Th nghim ch phm dch vi khun tớa quang hp x lý sulfide quy mụ phũng thớ nghim, pilot v iu kin t nhiờn 3.5.1 3.5.2 90 Kh nng x lý sulfide ca ch phm dng dch cỏc ngun nc thi ụ nhim quy mụ phũng thớ nghim 90 Th nghim ch phm dch i vi nc nuụi cỏ rụ phi quy mụ pilot 92 vi 3.5.3 Th nghim ch phm x lý sulfide v hu c ỏy ao nuụi thc t 3.5.4 94 ỏnh giỏ vai trũ ca vi khun tớa quang hp ch phm thụng qua gen pufM v gen sqr 97 Chng 4: BN LUN 99 4.1 Kt qu phõn lp v tuyn chn 99 4.2 c im sinh hc ca mt s i din thuc nhúm vi khun tớa quang hp khụng lu hunh 4.3 nh hng ca mt s yu t mụi trng n sinh trng v hot tớnh loi b sulfide ca ba chng la chn 4.4 100 104 Kh nng x lý sulfide mt s ngun nc thi v ỏy ao nuụi cỏ rụ phi thõm cnh 107 KT LUN V KIN NGH 112 DANH MC CễNG TRèNH KHOA HC CA TC GI LIấN QUAN N LUN N 114 SUMMARY 115 TI LIU THAM KHO 120 PH LC vii DANH MC CC Kí HIU V CC CH VIT TT Blast : Basic local alignment search tool Bchl : Bacteriochlorophyll BOD : Biochemical Oxygen Demand Bp : Base pair (Cp baz) cs : Cng s DNA : Deoxyribonucleic acid dNTP : Deoxyribonucleotide triphosphate DSMZ : Doutch samlung microorganisms zentrum ED : Entner - Doudoroff EMP : Embden - Meyerhof FCC : Flavocytochrome c GOGAT : Glutamine -2- oxoglutarate aminotransferase hay glutamate synthase GS : Glutamine synthetase kb : Kilo base kDa : Kilo Dalton M : Marker OD : Optical Density (Mt quang) PCR : Polymerase Chain Reaction (Phn ng chui trựng hp) SQR : Sulfide quinone reductase sqr : Gen mó húa cho enzyme sulfide quinone reductase Tag : Thermus aquaticus TCA : Tri carboxylic acid VKTQH : Vi khun tớa quang hp viii DANH MC CC BNG Trang Bng 1.1 So sỏnh cc i hp th ca cỏc dng bacteriochlorophyll vi khun quang hp (trong t bo nguyờn v dch chit ete) Bng 1.2 Cỏc gc R1 R7 v cc i hp th chớnh ca cỏc loi chlorophyll v bacteriochlorophyll 10 Bng 1.3 Cỏc nhúm carotenoid cú mt t bo vi khun quang hp 10 Bng 1.4 Kh nng oxy húa cỏc hp cht kh lu hunh ca VKTQH 23 Bng 1.5 Cỏc nghiờn cu ng dng nhúm vi khun quang hp h thng x lý nc thi cha sulfide 34 Bng 2.1 Cỏc cp mi ó s dng 43 Bng 3.1 Kt qu phõn lp vi khun VKTQH khụng lu hunh 56 Bng 3.2 Kh nng sinh trng (theo OD800) v hm lng sulfide cũn li bỡnh nuụi cỏc chng VKTQH Bng 3.3 So sỏnh kh nng s dng mt s ngun C ca cỏc chng la chn vi mt s loi thuc chi Rhodobacter Bng 3.4 80 nh hng ca NaCl n kh nng sinh trng v hot tớnh kh sulfide ca cỏc chng chn la Bng 3.9 79 Hm lng sulfide cũn li mụi trng sau ngy nuụi cy ca ba chng VKTQH cỏc pH khỏc Bng 3.8 72 Hm lng sulfide cũn li mụi trng sau ngy nuụi cy VKTQH cỏc nhit khỏc Bng 3.7 66 Kt qu so sỏnh trỡnh t gen pufM ca ba chng VKTQH vi d liu trờn GenBank Bng 3.6 65 nh hng ca sulfide n mc tớch ly sinh ( OD800) v hot tớnh loi b sulfide ca cỏc chng chn la Bng 3.5 59 81 Hm lng sulfide cũn li mụi trng sau ngy nuụi cy ca ba chng la chn cỏc cng sỏng khỏc 83 Bng 3.10 Mc tớch ly sinh (OD800) v hm lng sulfide cũn li cỏc chng la chn trờn cỏc mụi trng khỏc 84 137 174 Visser JM, Stefess GC, Robertson LA, Kuenen JG (1997) Thiobacillus sp W5, the dominant autotroph oxidizing sulfide to sulfur in a reactor for aerobic treatment of sulfidic wastes Antonie Van Leeuwenhoek, 72: 127-134 175 Vừ Th Hnh, Lờ Th Bớch Phng, Lờ Tn Hng, Trng Th Hng Võn, Trn Thch Phong (2004) Nghiờn cu sn xut ch phm VEM dung nuụi trng thy sn Hi tho ton quc v nghiờn cu v ng dng khoa hc cụng ngh nuụi trng thu sn Vng Tu 12/2004 NXB Nụng nghip: 911-917 176 V Th Minh c, Ninh Hong Oanh (2000) Mt s c tớnh ca vi khun quang hp tớa khụng lu hunh phõn lp t nc thi ca vựng H Ni bỏo cỏo tuyn hi ngh sinh hc quc gia v Nhng nghiờn cu c bn sinh hc NXB i hc Quc gia H Ni: 54 - 57 177 V Vn V, V Thanh Tõm, Hong Minh Tn (2001) Sinh lý hc thc vt NXB Giỏo dc: 252 trang 178 Wani AH, Lau AK and Branion MR (1999) Biofiltration control of pulping odors - hydrogen sulphide: Performance, microkinetics and coexistence effects of organo-sulfur species Journal of Chemical Technology and Biotechnology 74: 9-16 179 Weaver PF, Wall DJ, Gest H (1975) Characterization of Rhodopseudomonas capsulata, Arch Microbiol 105: 207-216 180 Willison JC (1988) Pyruvate and acetate metabolism in the photosynthetic bacterium Rhodobacter capsulatus", J Gen Microbiol 134: 2429-2439 181 Wraight CA (1982) Current researchs on photosynthesis, In Godvindjee (ed), Photosynthesis, vol.I, Academic Press New York, London 182 Zhu H, Ueda S, Asada Y, Miyaka J (2002) Hydrogen production as a novel process of wastewater treatment studies on Tofu wastewater with entrapped R sphearoides and mutangenesis Int.J hydrogen Energ, 27: 1349 - 1357 PH LC Ph lc Trỡnh t gen sqr chng QN52 ID SQ QN52 SEQUENCE CAATGGCGCA TGCAGCAGCA ACTTCGTGCC CCGATCTGGC GCCTCGTGCC ACCTCGTGGT AGGGCCACAC AGCGGTTCTG TCGGCCCGGC GCGACCGGGT ACGGAGTGGG GGATCACCCC CCGAGGGCGG CCTTCCGGGG CCCTCGTCGA GCGTGGCGAT TCATGATCGA AGGAGCCCGA GCATCGCCTT GCCGCTGGGT TCGGCACCGC TCAAGGCCGC PRELIMINARY; DNA; 1296 BP 1296 BP; 204 A; 461 C; 420 TGTCGTCGTT CTCGGGGCCG GTCTGGGCGG ACTCCGCAGC GAGGACCGGA TCACCGTCGT CTCCAACCCG TGGGTGGCGG TGGGTTGGCG ACCCACGATG CAGCGGCTCG GGATCGCCTT GGCCGAGAAC CGGGTGGAGC TCGCCGACGG GGCGACCGGT CCCGAGCTTG CCTTCGACGA CCAGTCCATC TGCCATATCG ACCATGCCGA CGAGACCCCG GGCCCCATCG TGGTGGGGGC CTACGAATTC GCCTTCATCC TCGATGCGGC GCCCATGACC TTCGTCACCT CCGAACCCTA CGACACCAAG GGGCTGCTCG AATCCGCGCT GGCCCGGATC TCGCGGGTCG AGGCGGGGAT CGCCACGAAG CCCCATGCCC TGCCCTTCGG GGTGAAGGCC GTCTCGGGCA TCGAGGGGCT TCCTCATCAG CGCAACCCCG CCTTCCCGAA CCCGCCGATG GGGGCCACGC CGGTGCCGGT ATCGATGGTC ACCGCCACGG CGCACAACAT CGAGGTCGCC ACCTGGAACG CGGTCTGTCT CATCGCCCAG CCGCAGATCC CGCCGCGCAA CCATTTCGCC AAGATCGCCT TCGAGAAATA CGAGCCGCTT TACGAATGGG CGGCGATGAA GCGCAAGGGA TGACCCCTTG CCAATC G; 211 T; CTCGATCATG GCCTACGAGT GACCCGCGAT CCCACCTATC GACGCGCAGC CATATCAGCG TCGGACGGTG CCCGCCACGC CAGCGCGCTC GATTACGATT GATCGAGGGC TTCGGCCCCG GGCCGCCCAC GCGGCCTTCG CGCGCAGGGC GCGAGCTGCT CCTGCGTCGG CGCAAGATCC TATCGGCCAT CTCGGCCTCG GCGCAGCCGC CATATCAAAT GATGCATGTC GAGGAAGTGG CTATGCGATG ATGCTGCCCG CTCGAACCCG CGCGGCTTCA CATCTTCGCC GTGGGCGTCT GGGCGTGCCC AAGACCGGCT CGGCGCGCTG GTCCGCGGCA CGCCGATTTC GGCGACCGCG CGTGAACTGG TCGGCCGAGG CTTCCTGCGC AAGGTGAAGC GATGCTCGGC ATCGACAAGC Ph lc Trỡnh t gen sqr chng QN71 ID SQ QN71 SEQUENCE CAATGGCGCA TGCAGCAGCA ATTTCGTGCC CCGATCTGGC GCCTCGTGCT ACCTCGTGGT AGGGCCACAC AGCGGTTCTG TCGGGCCCGC GCGACCGGGT ACGGGGTGGA GGATCACCTC CCGAGGGCGG CCTTCCGGGG CCCTCGTCGA GCGTGGCAAT TCATGATCGA AGGAACCCGA GCATCGCCTT GTCGCTGGGT TCGGCACGGC PRELIMINARY; DNA; 1296 BP 1296 BP; 210 A; 453 C; 417 TGTCGTCGTT CTCGGGGCCG GTCTGGGCGG ACTCCGCAGC GAAGACCGGA TCACTGTCGT CTCCAACCCG TGGGTGGCGG TGGGCTGGCG GCCGACGATG CAGCGGCTCG GGTTCGCCTT GGCCGAGAAC CGGGTGGAGC TCGCCGACGG GGCGACCGGC CCCGAGCTTG CCTTCGACGA CCAGTCCATC TGCCACGTCG ACCATGCCGA CGAAAACCCC GGCCCCATCG TGGTGGGGGC CTACGAATTC GCCTTCATCC TCGATGCAGC GCCCATGACC TTCGTCACCT CCGAACCCTA CGACACGAAG GGGCTGCTCG AATCCGCGCT GGCCCGGATC TCGCGGGTCG AGGCGGGGAT CGCCACGAAG CCCCATGCCC TGCCCTTCGG GGTGAAGGCT GTCTCGGGCA TCGAGGGGCT TCCTCACCAG CGCAACCCCA CCTTCCCCAA CCCGCCGATG GGGGTCACGC CGGTGCCGGT ATCGATGGTC ACCGCCACGG CGCACAACAT CGAGGTCGCC ACCTGGAACG CGGTCTGCCT CATCGCCCAG CCGCAGATCC CGCCGCGCGA GCATTTCGCC AAGATCGCCT TCGAGAAATA CGAGCCGCTT TACGAATGGG CGGCAATGAA TCAAGGCCGC CCGCAAGGGA TGACCCCTTG CCAATC G; 216 T; CTCGATCATG GCCTACGAGT GACCCGCGAT CCCACCTATC GACGCGCAGC CATATCAGCG TCGGACGGTG CCCGCCACGC CACTGCGCTC GATTACGATT GATCGAGGGC TTCGGACCCG GGCCGCCCAC GCGGCCTTCG GGCGCAGGGC GCGAGCTGCT CCTGCGCCGA CGCAGGATCC TATCGGCCAT CTCGGCCTCG GCGCAGCCGC CATATCAAAT GATGCATGTC GAAGAAGTGT CTATGCGATG ATGCTGCCTG CTCGAACCCG CGCGGCTTCA CATCTTCGCG GTGGGCGTCT GGGCGTGCCC AAGACCGGCT CGGCGCGCTG GTCCGCGGCA CGCCGATTTC GGCGACCGCG CGTGAACTGG TCGGCCGAGG CTTCCTGCGC AAGGTGAAGC GATGCTCGGG ATCGACAAGC Ph lc Trỡnh t gen sqr chng TH21 ID SQ TH21 SEQUENCE CAATGGCGCA TGCAGCAGCA ATTTCGTGCC CCGATCTGGC GCCTCGTGCT ACCTCGTGGT AGGGCCACAC AGCGGTTCTG TCGGGCCCGC GCGACCGGGT ACGGGGTGGA GGATCACCTC CCGAGGGCGG CCTTCCGGGG CACCCTCGTC TCTGCGTGGC GCTTCATGAT GCAAGGAACC GCGGCATCGC AGGGCCGCTG AGCTCGGCAC AGCTCAAGGC PRELIMINARY; DNA; 1299 BP 1299 BP; 210 A; 454 C; 420 TGTCGTCGTT CTCGGGGCCG GTCTGGGCGG ACTCCGCAGC GAAGACCGGA TCACTGTCGT CTCCAACCCG TGGGTGGCGG TGGGCTGGCG GCCGACGATG CAGCGGCTCG GGTTCGCCTT GGCCGAGAAC CGGGTGGAGC TCGCCGACGG GGCGACCGGC CCCGAGCTTG CCTTCGACGA CCAGTCCATC TGCCACGTCG ACCATGCCGA CGAAAACCCC GGCCCCATCG TGGTGGGGGC CTACGAATTC GCCTTCATCC TCGATGCAGC GCCCATGACC TTCGTCACCT CCGAACCCTA CGACACGAAG GGGCTGCTCG AATCCGCGCT GGCCCGGATC TCGCGGGTCG AGGCGGGGAT CGCCACGAAG CCCCATGCCC TGCCCTTCGG GGGTGAAGGG CTGTCTCGGG CATCGAGGGG GATCCTCACC AGCGCAACCC CACCTTCCCC AATCCCGCCG ATGGGGGTCA CGCCGGTGCC CGAATCGATG GTCACCGCCA CGGCGCACAA CGACGAGGTC GCCACCTGGA AAGCGGTCTG CTTCATCGCC CAGCCGCAGA TCCCGCCGCG GGTGCATTTC GCCAAGATCG CCTTCGAGAA GGCCGAGCCG CTTTACGAAT GGGCGGCGAT CGCCCGCAAG GGATGACCCC TTGCCAATC G; 215 T; CTCGATCATG GCCTACGAGT GACCCGCGAT CCCACCTATC GACGCGCAGC CATATCAGCG TCGGACGGTG CCCGCCACGC CACTGCGCTC GATTACGATT GATCGAGGGC TTCGGACCCG GGCCGCCCAC GCGGCCTTCG GGCGCAGGGC GCGAGCTGCT CCTGCGCCGA CGCAGGATCC TATCGGCCAT CTCGGCCTCG GCGCAGCCGC CATATCAAAT GATGCATGTC GAAGAAGTGT CTATGCGATG ATGCTGCCTG CTCTCGGACC CGCGCGGCTT AACATCTTCG CCGGTGGGCG GGTGGGCGTG CCCAAGACCG CATCGGCGCG CTGGTCCGCG CCTCGCCGAT TTCGGCGACC CAACGTGAAC TGGTCGGCCG ATACTTCCTG CGCAAGGTGA GAAGATGCTC GGGATCGACA Ph lc Dch mó gen sqr chng QN52 31 61 91 121 151 181 211 241 271 301 331 361 391 421 Amino 10 | | M A H V V V L G A G T V V T R D P T Y H D L A P T M Q R L G A D G S A L D Y D Y G H T Q S I C H I D V G A A Q G A S C F D R V P M T F V T S S A L R S R H I K W E G G A T K P H A L E G L S N P R G F T V A I P P M G A T P H N I G A L V R G K I A F I A Q P Q I P E K Y F L R K V K L K A A R K G acid composition L F I L H G E I P L V E P G G V A V A P P T F V P P R T G P F V E A Y P G D V D N A 15 | S I S N R T A T A A Y E I G A R Y A P H G V E V V N E P M P V G H F H I M Q P A W L A W P P A A L S M R K T S Y Y V A E A F G R L N T W A E 20 | E L A V T R L A F E I L L D V E P A P A G F N A E G W A Q G L F R D G A F F M V R A Q W V D F A V G R P I C W M Q R P E C A G M G N E L V K 25 | L R T R A E I E E T L R D T M H V K I F S M A D H F M L S S N G P R K V A A V F A G E H R F G R G E V V T G K I D I V G P K L E S G A D I D 54 A C 14 H 13 M 22 T 28 R Q 24 I 21 F W N 26 E 29 L 31 P Y 17 D 40 G 15 K 16 S 37 V Number of residues: 426 Molecular weight (MW): 46225 ; Checking number (CN): 901656 This sequence is a translation of sequence QN52 From base to base 1280 30 | R I S A E L P E I V I R L E V A G I V C T A R G A F K L Ph lc Dch mó gen sqr chng QN71 10 15 20 25 | | | | | M A H V V V L G A G L G G S I M A Y E L Q Q Q L R S E D 31 T V V T R D P T Y H F V P S N P W V A V G W R T R S H I 61 D L A P T M Q R L G F A F R T V P A T R L V L A E N R V 91 A D G T A L D Y D Y L V V A T G P E L A F D E I E G F G 121 G H T Q S I C H V D H A E A A H A A F E R F C E N P G P 151 V G A A Q G A S C F G P A Y E F A F I L D A A L R R R R 181 D R V P M T F V T S E P Y I G H L G L D G V D D T K G L 211 S A L R S R H I K W I T S A R I S R V E A G M M H V E E 241 E G G A T K P H A L P F G Y A M M L P A F R G V K A V S 271 E G L S N P R G F T L V D P H Q R N P T F P N I F A V G 301 V A I P P M G V T P V P V G V P K T G F M I E S M V T A 331 H N I G A L V R G K E P D E V A T W N A V C L A D F G D 361 I A F I A Q P Q I P P R D V N W S A E G R W V H F A K I 391 E K Y F L R K V K L G T A E P L Y E W A A M K M L G I D 421 K A A R K G Amino acid composition 51 A C 14 H 13 M 23 T 29 R Q 22 I 22 F W N 26 E 30 L 29 P Y 19 D 39 G 14 K 17 S 39 V Number of residues: 426 Molecular weight (MW): 46439 ; Checking number (CN): 913058 This sequence is a translation of sequence QN71 From base to base 1280 30 | R I S A E L P E I V I R L E V S G I V C T A R G A F K L Ph lc Dch mó gen sqr chng TH21 10 15 20 25 | | | | | M A H V V V L G A G L G G S I M A Y E L Q Q Q L R S E D 31 T V V T R D P T Y H F V P S N P W V A V G W R T R S H I 61 D L A P T M Q R L G F A F R T V P A T R L V L A E N R V 91 A D G T A L D Y D Y L V V A T G P E L A F D E I E G F G 121 G H T Q S I C H V D H A E A A H A A F E R F C E N P G P 151 V G A A Q G A S C F G P A Y E F A F I L D A A L R R R R 181 D R V P M T F V T S E P Y I G H L G L D G V D D T K G L 211 S A L R S R H I K W I T S A R I S R V E A G M M H V E E 241 E G G A T K P H A L P F G Y A M M L P A F R G G E G L S 271 S R G S R T R A A S P S S I L T S A T P P S P T S S P V 301 C V A I P P M G V T P V P V G V P K T G F M I E S M V T 331 A H N I G A L V R G K E P D E V A T W K A V C L A D F G 361 G I A F I A Q P Q I P P R N V N W S A E G R W V H F A K 391 F E K Y F L R K V K L G T A E P L Y E W A A M K M L G I 421 L K A A R K G Amino acid composition 53 A C 13 H 13 M 25 T 31 R Q 21 I 19 F W N 26 E 30 L 30 P Y 17 D 39 G 14 K 25 S 36 V Number of residues: 427 Molecular weight (MW): 46200 ; Checking number (CN): 927281 This sequence is a translation of sequence TH21 From base to base 1283 30 | R I S A E L P E I V I R L E V S R A G V A T D R I A D K Ph lc So sỏnh trỡnh t protein SQR hai chng QN52v TH21 * ALIGNMENT OF TWO PROTEIN SEQUENCES * The two sequences to be aligned are: QN52_PROT Total number of residues: 426 TH21_PROT Total number of residues: 427 Comparison matrix : Structure-genetic matrix Open gap cost : Unit gap cost : The character to show that two aligned residues are identical is '|' QN52_PROT - MAHVVVLGAGLGGSIMAYELQQQLRSEDRITVVTRDPTYHFVPSNPWVAV -50 |||||||||||||||||||||||||||||||||||||||||||||||||| TH21_PROT - MAHVVVLGAGLGGSIMAYELQQQLRSEDRITVVTRDPTYHFVPSNPWVAV -50 QN52_PROT - GWRTRSHISADLAPTMQRLGIAFRTVPATRLVPAENRVELADGSALDYDY -100 |||||||||||||||||||| ||||||||||| |||||||||| |||||| TH21_PROT - GWRTRSHISADLAPTMQRLGFAFRTVPATRLVLAENRVELADGTALDYDY -100 QN52_PROT - LVVATGPELAFDEIEGFGPEGHTQSICHIDHAEAAHAAFERFCETPGPIV -150 |||||||||||||||||||||||||||| ||||||||||||||| ||||| TH21_PROT - LVVATGPELAFDEIEGFGPEGHTQSICHVDHAEAAHAAFERFCENPGPIV -150 QN52_PROT - VGAAQGASCFGPAYEFAFILDAALRRRKIRDRVPMTFVTSEPYIGHLGLD -200 ||||||||||||||||||||||||||| |||||||||||||||||||||| TH21_PROT - VGAAQGASCFGPAYEFAFILDAALRRRRIRDRVPMTFVTSEPYIGHLGLD -200 QN52_PROT - GVGDTKGLLESALRSRHIKWITPARISRVEAGMMHVEEVAEGGATKPHAL -250 || ||||||||||||||||||| |||||||||||||||| |||||||||| TH21_PROT - GVDDTKGLLESALRSRHIKWITSARISRVEAGMMHVEEVSEGGATKPHAL -250 QN52_PROT - PFGYAMMLPAFRGVKAVSGIEGLSNPRGFTLVDPHQR - -287 ||||||||||||| | | TH21_PROT - PFGYAMMLPAFRGGEGLS RASRGSRTRAASPS -282 QN52_PROT - NPAFPNIFAVGVCVAIPPMGATPVPVGVPKTGFMIESMVTATAH -331 | | ||||||||||| ||||||||||||||||||||||| TH21_PROT - SILTSATPPSPTSSPVGVCVAIPPMGVTPVPVGVPKTGFMIESMVTATAH -332 QN52_PROT - NIGALVRGKEPDEVATWNAVCLADFGDRGIAFIAQPQIPPRNVNWSAEGR -381 ||||||||||||||||| |||||||||||||||||||||||||||||||| TH21_PROT - NIGALVRGKEPDEVATWKAVCLADFGDRGIAFIAQPQIPPRNVNWSAEGR -382 QN52_PROT - WVHFAKIAFEKYFLRKVKLGTAEPLYEWAAMKMLGIDKLKAARKG -426 ||||||||||||||||||||||||||||||||||||||||||||| TH21_PROT - WVHFAKIAFEKYFLRKVKLGTAEPLYEWAAMKMLGIDKLKAARKG -427 Identity : 386 (90.61%) Number of gaps inserted in QN52_PROT: Number of gaps inserted in TH21_PROT: Ph lc Trỡnh t gen pufM chng QN52 TGGACCTGTTCTACAACCCCTTCCACGGTCTCTCGATCGCCTTCCTCTACGGTTCGGCCCTGCTCTTCGCGAT GCACGGTGCGACCATCCTCGCGGTCTCCCGCTTCGGCGGCGAGCGCGAGCTGGAGCAGATCGCCGACCGCGGG ACGGCGGCGGAGCGGGCGGCCCTCTTCTGGCGCTGGACCATGGGA Quer E y Ident value cover Max score Total score Rhodobacter sphaeroides ATCC 17025, complete genome 346 346 97% 8e-92 100% CP000661.1 Rhodobacter azotoformans pufL, pufM genes for photoreaction center L subunit, photoreaction center M subunit, partial cds 340 340 97% 4e-90 99% AB062783.1 Rhodobacter johrii partial pufM gene for photosynthetic reaction centre M subunit, type strain JA192T 331 331 98% 2e-87 98% HE966449.1 Rhodobacter sphaeroides 2.4.1 chromosome 1, complete sequence 329 329 97% 8e-87 98% CP000143.2 Rhodobacter sphaeroides ATCC 17029 chromosome 1, complete sequence 329 329 97% 8e-87 98% CP000577.1 Rhodobacter sphaeroides photosynthetic gene cluster 329 329 97% 8e-87 98% AJ010302.1 R.sphaeroides reaction center protein m subunit gene and 5' flank 329 329 97% 8e-87 98% K00827.1 Rhodobacter megalophilus partial pufM gene for photosynthetic reaction centre M subunit, type strain JA194T 326 326 98% 1e-85 98% HE966451.1 R.sphaeroides Y pufM gene for polypeptide M of reaction centre 324 324 97% 4e-85 98% X63405.1 Rhodobacter sphaeroides strain 2.4.1 PufM (pufM) gene, partial cds 322 322 91% 1e-84 100% DQ017878.1 Description Accession Ph lc Trỡnh t gen pufM chng QN71 AGAGTGAGCTCGTCCCGGCACCTGTTCTACACCCCTTCCACGGTCTCTCGATCGCCTTCCTCTACGGGTCGGC CCTGCTCTTCGCGATGCACGGCGCGACCATCCTCGCGGTCTCCCGCTTCGGCGGCGAGCGCGAGCTGGAGCAG ATCGCCGACCGCGGGACGGCAGCGGAGCGGGCCGCCCTCTTCTGGCGCTGGACCATGGGA Max score Total score Query cover E value Ident Accession Rhodobacter johrii partial pufM gene for photosynthetic reaction centre M subunit, type strain JA192T 348 348 96% 3e-92 98% HE966449.1 Rhodobacter sphaeroides 2.4.1 chromosome 1, complete sequence 346 346 96% 9e-92 98% CP000143.2 Rhodobacter sphaeroides ATCC 17029 chromosome 1, complete sequence 346 346 96% 9e-92 98% CP000577.1 Rhodobacter sphaeroides photosynthetic gene cluster 346 346 96% 9e-92 98% AJ010302.1 R.sphaeroides reaction center protein m subunit gene and 5' flank 346 346 96% 9e-92 98% K00827.1 Rhodobacter megalophilus partial pufM gene for photosynthetic reaction centre M subunit, type strain JA194T 342 342 96% 1e-90 98% HE966451.1 R.sphaeroides Y pufM gene for polypeptide M of reaction centre 340 340 96% 4e-90 98% X63405.1 Rhodobacter sphaeroides KD131 chromosome 1, complete sequence 335 335 96% 2e-88 97% CP001150.1 Rhodobacter sphaeroides photosynthetic gene cluster, partial sequence 335 335 96% 2e-88 97% AF195122.1 Rhodobacter sphaeroides ATCC 17025, complete genome 324 324 96% 4e-85 96% CP000661.1 Rhodobacter azotoformans pufL, pufM genes for photoreaction center L subunit, photoreaction center M subunit, partial cds 318 318 96% 2e-83 96% AB062783.1 Rhodobacter sphaeroides strain 2.4.1 PufM (pufM) gene, partial cds 300 300 89% 7e-78 96% DQ017878.1 Description Ph lc 10 Trỡnh t gen pufM chng TH21 GTTCATTTTCAACCGGGGTGAACCTGGCTCTACCGGCCCTTTCACGGATCTCTCGATCGCCTTCCTCTACCGT TCGGCCCTGCTCTTCGCGATGCACGGTGCGACCATCCTCGCGGTCTCCCGCTTCGGCGGCGAGCGCGAGCTGG AGCAGATCGCCGACCGCGGGACGGCGGCGGAGCGGGCGGCCCTCTTCTGGCGCTGGACCATGGGA Max Total Query E value score score cover Ident Accession 307 307 90% 4e-80 96% CP000661.1 302 302 90% 2e-78 95% AB062783.1 291 291 90% 4e-75 94% HE966449.1 Rhodobacter sphaeroides 2.4.1 chromosome 1, complete sequence 289 289 90% 2e-74 94% CP000143.2 Rhodobacter sphaeroides ATCC 17029 chromosome 1, complete sequence 289 289 90% 2e-74 94% CP000577.1 Rhodobacter sphaeroides photosynthetic gene cluster 289 289 90% 2e-74 94% AJ010302.1 R.sphaeroides reaction center protein m subunit gene and 5' flank 289 289 90% 2e-74 94% K00827.1 285 285 90% 2e-73 94% HE966451.1 Rhodobacter sphaeroides KD131 chromosome 1, complete sequence 283 283 90% 7e-73 94% CP001150.1 Rhodobacter sphaeroides strain 2.4.1 PufM (pufM) gene, partial cds 283 283 84% 7e-73 96% R.sphaeroides Y pufM gene for polypeptide M of reaction centre 283 283 90% 7e-73 94% Description Rhodobacter sphaeroides ATCC 17025, complete genome Rhodobacter azotoformans pufL, pufM genes for photoreaction center L subunit, photoreaction center M subunit, partial cds Rhodobacter johrii partial pufM gene for photosynthetic reaction centre M subunit, type strain JA192T Rhodobacter megalophilus partial pufM gene for photosynthetic reaction centre M subunit, type strain JA194T Rhodobacter sphaeroides photosynthetic gene cluster, partial sequence DQ017878 X63405.1 Ph lc 11 Phng phỏp xỏc nh hm lng sulfide bng phng phỏp so mu * Phm vi ỏp dng Cú th s dng phng phỏp ny xỏc nh sulfide hũa tan v sulfide tng s nc n ung, nc mt, nc bin, nc thi cụng nghip Phng phỏp ny thớch hp cho khong nng 20 mgS/l * Nguyờn tc ca phng phỏp Sulfide phn ng vi dimethyl-p-phenylenediamin (p-aminodimethylanilin) cú mt FeCl3 to xanh methylen, mt cht nhum c xỏc nh ti bc súng cc i 625nm * Húa cht Dung dch gc amino-sulfuric acid: hũa tan 27g p-aminodimethylalanin mỏy trn lnh vi 50 ml acid H2SO4 m c v 20ml nc ct bỡnh nh mc cú th tớch 100 ml Gi mỏt v pha loóng n vch du, thuc th mi pha cn c gi bỡnh ti mu - Dung dch A (thuc th acid amino-sulfuric): Hũa tan 25 ml dung dch gc acid amino-sulfuric acid vi 975ml dung dch (1+1) H2SO4, dung dch ny c gi l dung dch A Dung dch ny phi sut v gi bỡnh ti mu - Dung dch B (dung dch Fecl3): hũa tan 100g FeCl3.6H2O 40ml nc ct - Dung dch C: (dung dch diamonium hydrogen phosphat): hũa tan 400g (NH4)2HPO4 800 ml nc ct * Xõy dng dóy nng chun Dung dch chun gc: Hũa tan 0,375g Na2S.9H2O vo 10ml nc ct ó c sc khớ N2 hm lng khớ oxy nc = mg/l Dung dch ny cú nng 5000 mgS2-/l v c gi l dung dch gc Dung dch chun gc pha loóng: dung dch chun gc pha loóng100 ln cho dung dch chun gc pha loóng t nng 50 mgS2-/l Xõy dng ng chun: T l dung dch chun gc pha loóng vi nc ct (ml+ml) Hm lng sulfide dung dch pha loóng (mgS2-/l) 0,075+7,425 0.5 0,15+7,35 0,30+7,20 0,45+7,05 0,60+6,90 0,75 +6,75 * Cỏch tin hnh Chuyn 7.5 ml mu cú cha sulfide vo ng nghim thy tinh, sau ú thờm 0.5ml dung dch A v 0.15ml dung dch B ri lc u ng ch ln i sau 3ữ5 phỳt b sung 0.8ml dung dch C lc u v i tip 3ữ5 phỳt v tin hnh o mu trờn mỏy quang ph bc súng 625nm Mu i chng l nc ct v tin hnh tng t i vi mu thớ nghim Mt hp th ca dung dch mu c so sỏnh vi dch cha sulfide chun mt s nng ó lm ng chun trờn v cn c vo ú xỏc nh hm lng sulfide cú mu * Cỏch tớnh Hm lng sulfide cú dch nghiờn cu c tớnh theo cụng thc: (mgS2-/l) = OD mu OD chun x nng chun(mg/l) x pha loóng mu Ph lc 12 Phõn tớch sulfide bng phng phỏp iodine * Phng phỏp ny ỏp dng cho phõn tớch hm lng sulfide nc v bựn cha nhiu hn 0,5 mgS/l * Phng phỏp ly mu: Th tớch mu ly xỏc nh sulfide khụng nh hn 200ml Mu nc v mu bựn ly xỏc nh sulfide cn phi ly riờng v nu khụng phõn tớch phi c nh sulfide bng dung dch chỡ axetat hoc dung dch cadmi axetat 10% * Xỏc nh sulfide da trờn nguyờn tc xỏc nh H2S v mui ca nú to thnh kt ta CdS, PbS Hũa tan kt ta bng dung dch iot Sau ú chun lng iot d bng thiosunfat * Dng c v thuc th: Bỡnh nún, buret, pipet; Axitclohidric tinh khit, dung dch 1:1; Cadmi axetat dung dch 10%; Natri thiosunfat, dung dch 0,01N; Iot, dung dch 0,01 N; Tinh bt, dung dch 0,5 % * Cỏch tin hnh - Xỏc nh s b Nu phõn tớch sau ly mu, khụng cn c nh mu Ly 20 ml nc th, axit húa bng axit clohidric, thờm mt lng nh dung dch iot, 0,01 N cho n xut hin mu vng chun ngc lng iot d bng dung dch natri thiosunfat 0,01 N - Xỏc nh chớnh xỏc Da trờn kt qu phõn tớch s b, ta ly mt lng nc cú cha t ữ 20 mg sunfua Thờm vo y mt lng cadmi axetat tnh cho ti kt ta lng xung Lc kt ta v ta cn thn bng nc núng Kt ta sau lc chuyn vo bỡnh nún, dung tớch 250 ml Thờm vo ú 25 ữ 50 ml dung dch iot 0,01 N v axit húa dung dch ú bng ml axit clohidric Chun lng iot d bng natri thiosunfat 0,01 N (ghi s ml) * Tớnh kt qu Hm lng H2S (x) tớnh bng mg/l theo cụng thc: x= (a b).0,17 1000 V Trong ú: a - lng dung dch iot 0,01 N, ml; b - Lng dung dch natri thiosunfat, ml; V - th tớch nc ly phõn tớch, ml; 0,17 - s mg H2S tng ng vi ml dung dch iot 0,01 N Ph lc 13 Phng phỏp xỏc nh BOD3 Xỏc nh nhu cu sinh húa oxy (c vit tc l BOD) (TCVN 4566 88) theo phng phỏp Winkler * Phng phỏp ly mu Ly mu theo TCVN 4556-88 Nc cha phõn tớch phi bo qun iu kin nh hn 4o C Chai cha mu xỏc nh nhu cu sinh húa oxy phi sy tit trựng 150o C * Phng phỏp xỏc nh Nguyờn tc nhu cu sinh húa oxy l lng oxy cn thit vi sinh vt phõn hu cỏc cht hu c mt n v th tớch nc nht nh (1000 ml) mt n v thi gian nht nh, iu kin nhit l 20o C v khụng cú ỏnh sỏng xỏc nh lng oxy ú cn phi cung cp cho nc thi mt lng oxy tha cho quỏ trỡnh phõn hu cỏc cht hu c cỏc vi sinh vt (quỏ trỡnh ú cú th l: 3; 5; 10; 15 ngy tựy theo yờu cu nghiờn cu) Lng oxy nc gim i so vi ngy u cho bit s mg oxy m cỏc vi sinh vt ó tiờu th Yu t cn tr: Kim hoc axit nh hng n kt qu xỏc nh, ú phi thc hin mụi trng trung tớnh Cú th dựng axit sunfuric H2SO4 0,05M hay natri hidroxit NaOH 0,1 M xỏc nh iu chnh Nc c, cú nhiu cn phi lng ri ly phn nc xỏc nh Nc cha clo hot ng cn tr phi loi tr clo nh sau: Trong 100 ml nc nghiờn cu cho vo 10 ml kali iodua KI 10%, 10 ml axit axetic 5%, nh vi git h tinh bt nu xut hin mu tớm xanh, nh natri thiosunfat Na2S2O3 0,0125 M cho ti mt mu * Dng c v thuc th Dng c Chai Winkler cú th tớch bit sn hay chai 250 ml nỳt mi, buret, pipet, bỡnh nún, t iu nhit Thuc th Dung dch dinh dng dựng bóo ho oxy gm: Kali dihidrophotphat KH2PO4 2,785 g; Dinatri hidrophotphat Na2HPO4.2H2O 8,493g; Magiờ sunfat MgSO4.7H2O 4,5g; Canxi clorua khan CaCl2 khan 5,5 mg; Amoniclrua NH4Cl 0,4gc th pha riờng tng th mi th 1.000 ml Khi dựng ly mi th 1ml ri pha chung vo 1000 ml hoc pha tt c cỏc th trờn vo mt bỡnh thờm nc ct n 1000 ml Cỏc thuc th khỏc theo TCVN 4564-88 v xỏc nh oxy ho tan Nc ct c sc khớ cú hm lng t 10 mg oxy mt lớt dựng pha vi nc thi Dựng dung dch dinh dng (phn thuc th) pha vo nc ct Ly ml dung dch dinh dng pha 1000 ml nc ct, khuy u, em nh lng, nu lng oxy nc cú t 10 mg mt lớt l c * Cỏch tin hnh nh lng oxy ca nc dựng pha loóng Ly nc ó bo ho oxy v hai chai nỳt nhỏm 250 ml (dựng ng xi phụng a nc vo ỏy chai, khụng c bt khớ) Chai th nht em nh lng oxy, kt qu nh lng chai th nht tớnh mg/l s l 0d1 Chai th hai gi li iu kin nhit 20o C ữ 1o C v trỏnh ỏnh sỏng Sau ngy (5, 10, 15, ngy tu yờu cu nghiờn cu) em nh lng oxy ca chai th hai cho kt qu 0d3 Hiu s gia 0d1 v 0d3 cho bit lng oxy tiờu th sau ba ngy ca nc dựng pha loóng Lng oxy ny khụng vt quỏ 0,5 mg/l nh lng oxy ca nc thi ó pha loóng Ly nc thi ó c pha loóng bng nc bóo ho oxy vo hai chai nỳt nhỏm dung tớch 250ml Chai th nht nh lng Kt qu tớnh mgO2/l ghi l OD1 Chai th nht sau 3, 5, 10, 15, ngy (cựng iu kin nhit v trỏnh ỏnh sỏng), em nh lng oxy Kt qu tớnh mg O2/l ghi l OD3 Hiu s gia OD1 v OD3 cho bit lng oxy ó tiờu th sau ngy i vi nc thi pha loóng Tớnh kt qu lng oxy tiờu th sau ngy hay nhu cu sinh húa oxy tớnh mg/l s l: BOD3 = [(OD1 OD3) (Od1 Od3)] x pha loóng mu Cng tớnh nh vy vi BOD5, BOD10, BOD15 Chỳ thớch: Lng oxy ho tan cũn li ngy cui cựng phi cũn li t mg/l Ph lc 14 nh lng amoni nc ngt theo phng phỏp Nessler Hm lng amoni mu nghiờn cu c xỏc nh thụng qua phn ng mu vng vi thuc th Nessler, cú kh nng hp th ỏnh sỏng bc súng 420 nm, cng mu t l thun vi nng NH4+ cú dung dch Thuc th Nessler: Pha 10g KI 10ml nc ct, thờm t t dung dch HgCl2 bóo hũa n xut hin ta bn B sung 30 g KOH Thờm 10ml dung dch HgCl2 bóo hũa, thờm nc ct n th tớch cui cựng l 200ml dung dch chai ti mu qua ờm, sau ú lc ly phn dung dch trong, bo qun chai ti mu cú nỳt nhỏm Tin hnh: 2ml dung dch Nessler vo 25ml dung dch mu nghiờn cu (hoc pha loóng mu c vi nng thớch hp) v khuy u, yờn 15 phỳt Sau ú nh lng amoni bng cỏch o hp th A420 trờn mỏy quang ph k Dng th chun biu din s tng quan gia hp th ỏnh sỏng v nng amoni t dung dch chun N-NH4 1g/l (Sigma) vi cỏc nng : 1; 2; 3; 4; 5; mg/l Da trờn th chun xỏc nh hm lng amoni mu Ph lc 15 nh lng NO2- theo phng phỏp Griss Hm lng NO2- c xỏc nh da trờn phn ng to mu hng ca NO2vi thuc th Griss Thuc th: Griss I: Ho tan 0,5g axit sunfanilic vo 150 ml axit acetic 5N Griss II: Ho tan 0,5g -Naphtylamin vo 50ml nc ct, b sung 150 ml axit acetic 5N Bo qun t lnh Tin hnh: Thờm 0,05ml Griss I v 0,05ml Griss II vo ml dung dch mu nghiờn cu, sau 10 phỳt phn ng kt thỳc o hp th A520 Cng mu t l thun vi nng nitrit Lng nitrit cú mu nghiờn cu tớnh c da vo th chun s dng dung dch N-NO2 5mg/l [...]... trũ x lý sulfide ca cỏc chng vi khun tớa quang hp 4 Chng 1 TNG QUAN TI LIU 1.1 MT S C IM SINH HC C BN CA VI KHUN TA QUANG HP 1.1.1 Gii thiu chung v vi khun tớa quang hp Vi khun tớa quang hp thuc nhúm vi khun thy sinh cú kh nng sinh trng trong iu kin k khớ bng cỏch quang hp nhng khụng thi oxy nh nhng i tng quang dng khỏc Khi c chiu sỏng, rt nhiu loi trong nhúm ny cú kh nng sinh trng quang t dng vi CO2... giỏ tr, lm thc n ti sng trong nuụi trng thy sn v.v tip tc ng dng nhúm vi khun ny trong lnh vc x lý ụ nhim, bo v mụi trng, chỳng tụi xut ti: "Nghiờn cu ng dng vi khun tớa quang hp x lý sulfide trong cỏc ngun nc ụ nhim" Mc tiờu ca ti - Cú c chng ging VKTQH phõn lp Vit Nam cú kh nng x lý sulfide - Cú c ch phm x lý mụi trng ụ nhim sulfide v cỏc hp cht hu c t cỏc chng chn la, xỏc nh c s tn ti v vai trũ... trỳc phõn t v cc i ca ph hp th trong vựng ỏnh sỏng S khỏc nhau v cc i hp th cỏc loi Bchl c trỡnh by Bng 1.1 Bng 1.1 So sỏnh cc i hp th ca cỏc dng bacteriochlorophyll vi khun quang hp (trong t bo nguyờn v trong dch chit ete) Dng bacteriochlorophyll a b c d e Nhúm vi khun Vi khun tớa Vi khun tớa Vi khun xanh Vi khun xanh S Vi khun xanh dng si Cc i hp th (nm) Chit trong ete Trong t bo 770-775 830-890 790... 1989 11 * nh v ca sc t quang hp trong t bo vi khun tớa quang hp Sc t quang hp nm n v quang hp n v ny phõn b trờn h mng ca b mỏy quang hp (chromotophor) (Hỡnh 1.3) Mng quang hp Hỡnh 1.3 S cu to ca mt dng Chromotophor ca vi khun tớa quang hp Ngun: Trn Vn Nh, 2005 * n v quang hp n v quang hp gm cú cỏc thnh phn ch yu sau õy: Sc t anten thu nhn nng lng ỏnh sỏng Tõm phn ng ca quang h (ký hiu l P) õy... hunh, hydro sulfide, thiosulfide v cỏc hp cht kh ca lu hunh (Brune, 1989; 1995) Theo khúa phõn loi ca Bergey (1989), chỳng c chia thnh 2 nhúm: vi khun tớa quang hp lu hunh - cú kh nng tớch ly git lu hunh bờn trong t bo v vi khun tớa quang hp khụng lu hunh - khụng cú kh nng tớch ly git lu hunh bờn trong t bo (Imhoff and Truper, 1989; 2001) 1.1.2 Sinh thỏi hc ca vi khun tớa quang hp Vi khun tớa quang hp... nng loi b sulfide trong nc v bựn ỏy Nhng úng gúp mi ca lun ỏn 1 ó phõn lp c cỏc VKTQH ti Vit Nam v bc u x lý sulfide iu kin t nhiờn trong cỏc ao nuụi cỏ rụ phi 2 Ln u tiờn s dng cỏc gen pufM (mó húa cho tiu phn M ca protein liờn kt vi sc t nm trong tõm phn ng quang hp) v gen sqr (mó húa sulfide quinone reductase) cỏc chng vi khun Rhodobacter sphaeroides, Rhodovullum sulphidophilum phõn lp ti Vit Nam... t bo VKTQH trong cỏc loi cht mang theo thi gian bo qun 89 Bng 3.12 Kh nng phc hi sinh trng v hot tớnh x lý sulfide ca VKTQH trong cỏc dng ch phm theo thi gian bo qun Bng 3.13 Mc ụ nhim hu c v sulfide trong cỏc ngun thi 89 90 Bng 3.14 Mc tớch ly sinh khi (OD800) hm lng sulfide cũn li trong mụi trng nuụi sau 7 ngy 91 Bng 3.15 Hm lng amoni, nitrite trong nc v hm lng sulfide trong bựn ỏy ao trong cỏc... hin ra cỏc vi khun cú sc t mu v cú kh nng quang hp nờn ụng gi chung nhúm vi khun ny l Rhodobacteria Nhúm vi khun ny bao gm hai h Thiorhodaceae (l nhng vi khun tớa cú kh nng hỡnh thnh git "S" bờn trong t bo) v Athiorhodaceae (l nhng vi khun tớa khụng cú kh nng hỡnh thnh git "S" trong t bo (Imhoff, 2001) Nhúm VKTQH lu hunh l cỏc loi cú th chng chu c hm lng sulfide trong mụi trng mc cao v trong quỏ... trng cha sulfide thỡ cú th d dng phõn bit c nhúm vi khun tớa lu hunh v nhúm vi khun tớa khụng lu hunh nh s quan sỏt git lu hunh tớch ly trong hay ngoi t bo di kớnh hin vi in t phn pha (Imhoff and Truper, 1989; Michael et al., 2008) Theo khúa phõn loi ca Bergey (1989) vi khun quang hp c chia lm ba nhúm: vi khun tớa quang hp, vi khun xanh quang hp v nhúm vi khun hiu khớ cha bacteriochlorophyll (nhng khụng... sulfide) 5 Nghiờn cu cỏc yu t mụi trng nh hng n sinh trng v kh nng loi b sulfide ca cỏc chng la chn nhm ng dng trong x lý sulfide (nhit , pH, ỏnh sỏng, nng sulfide, nng mui) 6 Nghiờn cu xõy dng quy trỡnh sn xut sinh khi to ch phm ng dng 7 ng dng ch phm VKTQH x lý sulfide - Trong iu kin phũng thớ nghim: ỏnh giỏ kh nng x lý sulfide trong cỏc ngun nc thi - ỏnh giỏ kh nng ng dng ca ch phm iu kin pilot