i hc quc gia h ni

VIN VI SINH VT V CNG NGH sinh hc -----o0o-----

BO CO TI C BIT CP HQG ti nghin cu khoa hc 2007-2008

NGHIN CU C IM SINH HC V KH NNG PHN GII C T CU MT S CHNG Microcystis PHN LP H HON KIM, H NI

M s: QG.07.24

Ch tr ti : Nguyn Th Hoi H

H ni - 2008

BO CO TM TT

1. Tn ti Nghin cu c im sinh hc v thm d kh nng phn gii c t ca mt s chng Microcystis phn lp h Hon Kim, H Ni 2. Cc thnh vin tham gia ti Ch tr ti - H v tn: Nguyn Th Hoi H Cc thnh vin: Lu Th Thy Giang Nguyn Quang Huy L Trung Thy Phm Th Bch o Trn Th ip 3. Tm tt tng quan ca ti Trong nhng nm gn y H Ni pht trin vi tc nhanh chng, ko theo l s nhim hng lot thy vc ni thnh v vng ln cn. H Hon Kim mt a danh ni ting ca th cng b nhim. Cht lng nc thay i dn ti s pht trin thng xuyn v khng bnh thng ca vi khun lam, lm bin i cu trc thnh phn loi ca h vi to trong h. S suy gim mt s loi vi to c hu ng thi vi s gia tng thnh phn loi v mt ca vi khun lam gy c h Hon Kim c cnh bo v gy nn nhng lo ngi v mi trng cho khu h ng thc vt sng trong h. Trong s cc loi vi khun lam gy n hoa nc h Microcystis aeruginosa Kutzing l loi b bt gp thng xuyn v ph bin nht M.aeruginosa cha c t thuc nhm hepatotoxin (c t gan) cu to t cc peptid mch vng c tn gi l

microcystin.. Microcystin l cht c ch i vi enzyme protein phosphatases (nhm PP1 v PP2A) [30]. c t cng gy nh hng xu ln hai loi protein serine v threonin phosphatases. c mt s bi bo chng minh hin tng nc n hoa ca vi khun lam gy hi ti sinh vt thy sinh, ng vt v con ngi. Microcystin thng tch lu trong ni quan t bo hoc di dng t do trong nc. c khng t cc cng trnh nghin cu hin tng n hoa nc v gim thiu tc hi ca c t vi khun lam gy c, tuy nhin kt qu vn cn cc mc khc nhau. bo v mi trng nc h, chng ti thc hin ti: Nghin cu c im sinh hc v thm d kh nng phn gii c t ca mt s chng Microcystis phn lp h Hon Kim, H Ni. 4. Mc tiu ca ti * Phn lp, nh tn, nui cy v lu gi c cc chng vi khun lam Microcystis trong Bo tang Vi to ca Phng Cng ngh To v Sinh hc Mi trng - Vin Vi sinh vt v Cng ngh Sinh hc - HQGHN. * Thm d kh nng phn gii c t ca chng bng vi khun. 5. Tm tt ni dung nghin cu ca ti Phn tch thnh phn thu l, thu ho ca nc h Hon Kim ti

thi im thu mu. Phn lp, thun khit v nghin cu cc iu kin nui cy thch hp

ca cc chng Microcystis. Phn loi n loi cc chng Microcystis tuyn chn c. Tch chit v phn loi c t ca cc chng Microcystis trn my sc

k lng cao p (HPLC). Thm d kh nng phn gii c t microcystin ca vi khun lam gy

c bng vi khun bn a (vi khun nc). 6. Kt qu chnh ca ti

- H Hon Kim ang tnh trng nhim, hm lng COD, BOD vt tiu chun Vit Nam, TCVN 4942- 1995 (loi B) 20 ln. Vi gi tr NH4+ t 0,1290,699 (mg/l), NO2- t 0.012- 0,05 (mg/l), NO3- t 0,047- 0,129 (mg/l), PO43- t 0,08- 0.64 (mg/l) chng t h c hm lng dinh dng N, P kh cao. - phn lp thun khit v nui cy 10 chng vi khun lam thuc chi Microcystis theo phng php ca Shirai c ci tin. Mi trng thch agarose B12 vi t l 0,4% l mi trng thch hp nht cho vic phn lp Microcystis. - Cc chng Microcystis u sinh trng tt trn mi trng Bold 3N vi s lng t bo t t 32,94 68,6 106 t bo/ml sau 15 n 19 ngy nui cy. c bit chng LT2 s lng t bo t cao nht l 68,6 106 t bo/ml sau ngy th 19. Trn mi trng J, cc chng Microcystis sinh trng s lng t bo ca cc chng t t 40,42 76,46 106 t bo/ml sau 15 n 19 ngy nui cy. Trong chng LT2 vn pht trin vi s lng t bo t cao nht 76,46 106 t bo/ml sau ngy th 19. Trn mi trng B12, s lng t bo t t 35,36 54,78 106 t bo/ml sau 12 n 15 ngy nui cy. mi trng ny chng LT8 pht trin vi mt t bo t cao nht 54,78 106 t bo/ml sau ngy th 15. - C 7 loi axt bo khng no dng C15, C17 v C19 trong t bo 2 chng Microcystis LT2 v LT8. trong thnh phn axit palmitic (C16H32O2) chim lng ln nht vi 1,302 g/mg trng lng kh chng LT2, chng LT8 l 0,9683 g/mg trng lng kh. Kt qu cho thy c s sai khc nhau v lng ca 7 loi axit bo c trong t bo 2 chng LT2 v LT8. - Da vo phng php phn loi truyn thng theo phn loi hnh thi hc v phng php phn loi hin i da trn phn tch trnh t rADN 16S, 2 chng vi khun LT 2, LT8 c xc nh l Microcystis aeruginosa, 2 chng vi khun nc N4 v N7 c xc nh thuc cc loi Sphingomonas mali v Sphingomonas asaccharolytica - Da vo kt qu phn tch trn HPLC, hm lng c t MC RR t ln nht l 512,8 ng/mg trng lng kh, c t MC YR l 236,8 ng/mg v thp nht l c t MC LR: 114,25 ng/mg.

- Hai chng vi khun Sphingomonas tuyn chn c c kh phn gii microcystin kh nhanh. nng microcystin 3 g/l thi gian bn phn gii l 1,34 gi v vi nng 20g/l, thi gian l 3,43 gi. - Nhit thch hp cho s phn gii microcystin ca cc chng vi khun Sphingomonas mali N4 v Sphingomonas asaccharolytica N7 l 30C. Sau 7 ngy s phn gii gn nh hon ton l 100%. 7. Cc cng trnh cng b v sn phm o to Cng trnh cng b - Phm Th Mai, Phm Tin c, V Th Lan Anh, Nguyn Th Hoi H 2008. nh gi s bin ng s lng theo ma v theo tng nc ca vi khun lam Microcystis aeruginosa h Hon Kim, H Ni, Tp ch Khoa hc i hc Quc gia H Ni. Khoa hc T nhin v Cng ngh 24 s 15, tr 125 128. - Phm Th Mai, Phm Tin c, V Th Lan Anh, Nguyn Th Hoi H 2008. Tm hiu kh nng phn gii c t ca vi khun lam Microcystis trong h Hon kim bng vi khun. Tp ch Khoa hc i hc Quc gia H Ni. Khoa hc T nhin v Cng ngh 24 s 15, tr 119 122. o to 4 C nhn - Bc u phn lp, tuyn chn mt s chng vi khun c kh nng phn gii c t ca vi khun lam Microcystis h Hon Kim, H Ni. 2007. Kho lun tt nghip h i hc chnh quy ca ng Th Kim Anh. - Bc u nh gi kh nng phn gii c t microcystin ca vi khun lam Microcystis aeruginosa bng vi khun Sphingomonas. 2008. Kho lun tt nghip h i hc chnh quy ca Nguyn Th Hoi. - Kho st s bin ng theo ma ca vi khun lam Microcystis aeruginosa h Hon Kim v kh nng c ch sinh trng ca chng bng dch chit v chui, v qut. 2009. Kho lun tt nghip h i hc chnh quy ca L Th Trang. - Phn lp, nghin cu mt s c tnh ca to c Microcystis tch chit c t v thm d bin php x l.2009. n tt nghip ca Nguyn Hu Hiu

 o to 1 Thc s Phn gii t bo Microcystis v s phn hu c t microcystin bng vi khun nc 2007 - 2009. Nhng ng gp ca ti: - L cng trnh u tin tch v lu gi c chng vi khun lam Microcystis. - Phn lp c 2 chng vi khun thuc chi mi Sphingomonas c kh nng phn gii c t microcystin ca vi khun lam Microcystis. - Xy dng c quy trnh tch chit c t microcystin. 8. Tnh hnh s dng kinh ph ca ti - Kinh ph c cp: 100 000 000 ng - Kinh ph thc hin: 100 000 000 ng

H Ni , ngy 18 thng 10 nm 2008 Ch tr ti Th trng n v (k tn, ng du)

TS. Nguyn Th Hoi H

ABSTRACT OF PROJECT

1. Title of the project: Study on biological characteristis and capacity of biodegradation toxin of some Microcystis isolated from Hoan Kiem Lake, Hanoi 2. Coodinator: Nguyen Thi Hoai Ha Member: Luu Thuy Giang Nguyen Quang Huy Le Trung Thuy Pham Thi Bich Dao Tran Thi Diep 3. Introduce Hoan Kiem Lake is a famous place because of its historical and cultural value. This is the habitat of valuable and rare species with endemicity and is also diversifying about the component of microalgae species. In recent years, the socioeconomic developmental process caused a lot of influences on the lake ecosystem especially on water quality. Mesotrophic situation of this lake resulted in the bloom water reducing specific microalgae species and concurrently increasing the species component and density of toxic algae. Among toxic cyanobacterial genera, Microcystis accounts for highest population (approximately 80% of total cyanobacterial strains that cause the bloom water). Microcystis aeruginosa Krutzing is the most popular species. This is a species which produces hepatotoxin, a type of liver toxin, composed from cyclic peptides called microcystin. They have effect on both animals and plants in waters. microcystin usually accumulates in cells or releases to surrounding waters. Thus the research on toxic cyanobacteria Microcystis in Hoan Kiem lake, Hanoi and finding effective methods to diminish their negative effects are essential. In this report, we describe the

isolation, selection and classification location as well as biological characters of some Microcystis strains from Hoan Kiem lake, as a basic for the next researches. 4.The airm of project - Isolation, identification, inoculation and maitainance of Microcystis strains (blue-green, Cyanobacteria) at Microalgal Technology and Enviromental Biology Lab, Institute of Microbiology and Biotechnology, Vietnam National of University, Hanoi. - Capacity of toxin degradation by aquatic bacteria. 5. Brief summary: - Biophysical and biochemical analysis of freshwater in Hoankiem lake at sampling time. - Isolation, screening, study on characteristics of Microcystis isolated. Identification of Microcystis isolated. Determination microcystin of same Microcystis strains by HPLC. Capacity of toxin degradation by fresh bacteria

6. Results obtained - Hoankiem lake is in pollution condition in the aspect of chemical evacuation such as COD and BOD concentration is over the corresponding ones in Vietnamese standard system, TCVN 4942-1995 (type B) 20 times. Concentration of ammonium cation 0,129- 0,699 (mg/l), nitrite 0.012- 0,05 (mg/l), nitrate 0,0470,129 (mg/l), and phosphate 0,08- 0.64 (mg/l) that demonstrate concentration of nitrogen and phosphorus are relatively high. - Based on modified Shirai method, 10 pure strains of Microcystis were isolated and cultured. B12 medium (0.4% agarose) was selected as the most suitable for Microcystis isolation. - All of Microcystis strains were grown well on the Bold 3N medium that the numbers of cell from 68,6 106 cells/ml after 15-19 days of culture. Especially, the

maximal cell number of LT2 strain increased to 68,6 106 cells/ml after 19th culture. For J medium, the cell density was from 40,42 76,46 106 cells/ml after 15-19 days of culture in which LT2 was the best growth strain with the numbers of cell 76,46 106 cells/ml after 19th culture. - Analytical results of unsaturated fatty acid in Microcystis LT2 and LT8 strains showed different 7 types of C15, C17 and C19 in which Palmitic acid occupied as major part with 1,302 g/mg and 0,9683 g/mg (in dry weight) in LT2 and LT8, respectively. The results indicated the diference in the concentration between 7 types of fatty acid. - Based on morphological and molecular taxonomy (for example comparision the sequence of 16S rDNA), LT2 and LT8 cyanobacterial strains were identified as M. aeruginosa; aquatic N4 and N7 bacterial strains were type of Sphingomonas mali v Sphingomonas asaccharolytica. - HPLC analysis showed the maximal concentration of MC-RR of 512,8 ng/mg in dry weight, 236,8 ng/mg of MC-YR and the lowest 114,25 ng/mg MCLR. - Sphingomonas strain was demonstrated to degrade microcystin in relatively good level. Time for degradation of a half of 3 g/l and 20g/l microcystin was 1,34 h and 3,43 h; respectively. - The suitable temperature for microcystin degradation by Sphingomonas mali N4, Sphingomonas asaccharolytica N7 strains was at 30C. The degradation was completely after 7 day.

7. Publications and training activity

Publications - Pham Thi Mai, Pham Tien Duc, Vu Thi Lan Anh, Nguyen Thi Hoai Ha. Investigate water quality and the composition of algae in some lakes in Hanoi. 2008. Vietnam National University, Hanoi. Journal Natural Science. Vol 24, No 18, pp 125 128. - Pham Thi Mai, Pham Tien Duc, Vu Thi Lan Anh, Nguyen Thi Hoai Ha. Study on toxin lysis in blue green algae by some strains bacteria isolated in Hoan Kiem lake. 2008. Vietnam National University, Hanoi. Journal Natural Science. Vol 24, No 18, pp 119 122. Training activity Master thesis completed in 2009 Lysis of Microcystis and degradation of microcystin by aquatic bacteria. 2007-2009. Tran Thi Dieps official undergraduate thesis. Bacheler thesis completed - Isolation and screening of Microcystis toxin-degrading bacterial strains in Hoankiem lake, Hanoi, 2007. Dang Thi Kim Anhs official undergraduate thesis. - Investigation of Microcystis aeruginosa microcystin degradation by Sphingomonas. 2008. Nguyen Thi Hoai s official undergraduate thesis. - Study on the seasonal fluctuation of cyanobacterial Microcystis aeruginosa in Hoankiem lake and growth restriction by banana skin and mandarin peel extracted solution, 2009. Le Thi Trangs official undergraduate thesis. - Isolation and study on Microcystis toxic cyanobacterial characteristics: toxin extraction and treatment investigation, 2009. Nguyen Huu Hieus official undergraduate thesis.

8. Contributions of project:

- Here is the first report that described isolation, maintaince and preservation of Microcystis strain. - Two Sphingomonas bacterial strains were isolated and proved as potent microcystin-degrading agent. - Establishment of toxin microcystin extraction procedure. 9. Research grant - Supplied expenditute: 100 000 000 Vnd - Used expenditute: 100 000 000 Vnd The project is supported by Vietnam, National University, Hanoi Hanoi, 18th October 2008 Project Coodinator Institute of Microbiology and Biotechnology

Nguyen Thi Hoai Ha Vietnam, National University, Hanoi

MC LC M U .....................................................................................................................1 Chng 1. TNG QUAN TI LIU .........................................................................3 1.1. VI NT V H HON KIM .........................................................................3 1.2. VI KHUN LAM (CYANOBACTERIA) GY C .......................................3 1.2.1. Vi khun lam Microcystis................................................................................... 6 1.2.2. c t microcystin ca Microcystis .................................................................. 6 1.2.3. C ch gy c ca microcystin ......................................................................... 8 1.3. S PHN GII C T MICROCYSTIN CA VI KHUN NC Sphingomonas .............................................................................................................9 1.3.1. Vi khun Sphingomonas ...................................................................................... 9 1.3.2. C ch phn gii c t microcystin ca Sphingomonas .............................. 10 1.4. NH HNG CA C T TI NGI V GIA SC ............................11 1.5. NHNG NGHIN CU V VI KHUN LAM TRN TH GII V VIT NAM..........................................................................................................................13 1.5.1. Trn th gii ........................................................................................................ 13 1.5.2. Vit Nam ......................................................................................................... 14 Chng 2. NGUYN LIU V PHNG PHP NGHIN CU ........................16 2.1. I TNG NGHIN CU ...........................................................................16 2.1.1. i tng nghin cu ........................................................................................ 16 2.1.2. a im v thi gian nghin cu .................................................................... 16 2.1.3. My mc, dng c .............................................................................................. 17 2.1.4. Ha cht ............................................................................................................... 17 2.1.5. Mi trng nui cy vi khun lam v vi khun .............................................. 18 2.2. PHNG PHP NGHIN CU ......................................................................18 2.2.1. Phng php iu tra thu mu vi khun lam h Hon Kim v nh gi cht lng nc ............................................................................................................. 18 2.2.2. Phng php phn lp v nui cy vi khun lam Microcystis ..................... 18 2.2.2.1. Lm giu mu .......................................................................................18 2.2.2.2. Phng php tch v thun khit trn thch a ...................................18 2.2.3. Phn loi vi khun lam Microcystis ................................................................. 19 2.2.3.1. Bng phng php hnh thi .................................................................19 2.2.3.2 . Phng php tch ADN .......................................................................19 2.2.3.3. Ha phn loi phn tch thnh phn acid bo .......................................20

2.2.4. Xc nh mt t bo ...................................................................................... 21 2.2.5. Phng php tch chit c t microcystin t t bo ca Microc ystis ....... 22 2.2.6. Phng php xc nh hm lng trn my quang ph ................................ 22 2.2.7. Cc phng php sc k .................................................................................... 22 2.2.7.1. Phng php sc k bn mng (TLC) ..................................................23 2.2.7.2. Phng php sc k lng cao p (HPLC) ............................................23 2.2.8. Phng php nui cy chng vi khun Sphingomonas ................................. 24 2.2.9. Phng php xc nh kh nng phn gii microcystin ................................ 24 Chng 3. KT QU V THO LUN ................................................................26 3.1. PHN TCH CC CH TIU THY L, THY HA NC H HON KIM .........................................................................................................................26 3.1.1. Nhit ............................................................................................................... 26 3.1.2. pH ......................................................................................................................... 26 3.1.3. Cc cht l lng SS ............................................................................................ 26 3.1.4. Hm lng oxy ha tan trong nc (DO- Dissolve oxygen) ........................ 27 3.1.5. Nhu cu oxy ha hc (COD- Chemical oxygen demand) ............................. 27 3.1.6. Nhu cu oxy sinh ha (BOD- Biochemical oxygen demand)....................... 27 3.2. TUYN CHN CC CHNG VI KHUN LAM THUC CHI Microcystis PHN LP C ...................................................................................................27 3.2.1. c im nui cy .............................................................................................. 27 3.2.2. Phn lp cc khun lc ca Microc ystis ......................................................... 29 3.2.3. Kh nng sinh trng ca 10 chng Microcystis LT1, LT2, LT3, LT4, LT5, LT6, LT7, LT8, LT9 v LT10 trn cc mi trng khc nhau ................. 32 3.2.4. Thnh phn acid bo trong t bo ca 2 chng LT2 v LT8 ........................ 35 3.2.5. Xc nh v phn tch trnh t rADN 16S ...................................................... 36 3.3. TCH CHIT C T MICROCYSTIN ....................................................38 3.3.1. Sc k bn mng (TLC) .................................................................................... 38 3.3.2. Xc nh hm lng microcystin trn my quang ph .................................. 38 3.3.3. Hm lng microcystin trn sc k lng cao p (HPLC) ............................. 39 3.4. PHN LP V TUYN CHN CC CHNG VI KHUN NC C KH NNG PHN GII MICROCYSTIN .....................................................................44 3.4.1. Phn lp cc chng vi khun nc .................................................................. 44 3.4.2. Xc nh v phn tch trnh t rADN 16S 2 chng N4 v N7 ..................... 47 3.5. KH NNG PHN GII C T MICROCYSTIN CA 2 CHNG Sphingomonas N4 V N7 .........................................................................................49

3.5.1. Hiu qu phn gii microcystin ca 2 chng N4 v N7 ................................ 49 3.5.2. Xc nh iu kin nhit thch hp cho s phn gii microcystin ca chng Sphingomonas N4 v N7 .................................................................................. 51 KT LUN V KIN NGH...................................................................................54 TI LIU THAM KHO .........................................................................................56 PH LC 1 ...............................................................................................................62 PH LC 2 ...............................................................................................................65 PH LC 3 ...............................................................................................................66 PH LC 4 ...............................................................................................................77 PH LC 5 ...............................................................................................................78 PH LC 6 .................................................................................................................1

DANH MC HNH V BNG Danh mc hnh Hnh 1.1. H Hon Kim H Ni ................................................................................3 Hnh 1.2. Microcystis flos-aquae [62] ........................................................................6 Hnh 1.3. Microcystis aeruginosa [63] .......................................................................6 Hnh 1.4. M hnh cu trc ho hc ca c t MC LR [54] ..................................7 Hnh 1.5. M hnh cu trc ho hc ca c t MC RR [54] ..................................7 Hnh 1.6. M hnh cu trc ho hc ca c t MC YR [54] ..................................8 Hnh 1.7. Hnh dng t bo ca Sphingomonas [57] .................................................9 Hnh 1.8. Con ng phn gii mycrocystin nh Sphingomonas [20] .....................11 Hnh 2.1. S v tr thu mu ...................................................................................16 Hnh 3.1. Hnh thi khun lc ca Microcystis trn mi trng C .........................28 Hnh 3.2. Hnh thi khun lc ca Microcystis trn mi trng Bold 3N ................28 Hnh 3.3. Hnh thi khun lc ca Microcystis trn mi trng B12 .......................28 Hnh 3.4. Hnh thi khun lc ca Microcystis trn mi trng J ...........................28 Hnh 3.5. Hnh dng t bo chng Microcystis sp., LT1 ..........................................29 Hnh 3.6. Hnh dng t bo chng Microcystis sp., LT2 ..........................................29 Hnh 3.7. Hnh dng t bo chng Microcystis sp., LT3 ..........................................30 Hnh 3.8. Hnh dng t bo chng Microcystis sp., LT4 ..........................................30 Hnh 3.9. Hnh dng t bo chng Microcystis sp., LT5 ..........................................30 Hnh 3.10. Hnh dng t bo chng Microcystis sp., LT6 ........................................30 Hnh 3.11. Hnh dng t bo chng Microcystis sp., LT7 ........................................31 Hnh 3.12. Hnh dng t bo chng Microcystis sp., LT8 ........................................31 Hnh 3.13. Hnh dng t bo chng Microcystis sp., LT9 ........................................31 Hnh 3.14. Hnh dng t bo chng Microcystis sp., LT10 ......................................31 Hnh 3.15. Sinh trng ca cc chng Microcystis trong mi trng dch th Bold 3N ..............................................................................................................................33 Hnh 3.16. Cc chng Microcystis pht trin tt trong mi trng dch th Bold 3N ...................................................................................................................................33 Hnh 3.17. Sinh trng ca cc chng Microcystis trong mi trng dch th J .....34 Hnh 3.18. Cc chng Microcystis pht trin tt trong mi trng dch th J ..........34 Hnh 3.19. Sinh trng ca cc chng Microcystis trong mi trng dch th B12 35 Hnh 3.20. Cc chng Microcystis pht trin tt trong mi trng dch th B12.....35 Hnh 3.21. Cy ph h da trn phn tch gii trnh t rADN 16S ca 2 chng LT2, LT8 v cc loi c quan h h hng gn ...................................................................37 Hnh 3.22. Kt qu chy TLC trc khi nhum nihyrin .........................................38

Hnh 3.23. Kt qu chy TLC sau khi nhum nihyrin ............................................38 Hnh 3.24. th ng chun microcystin .............................................................39 Hnh 3.25a. Sc k microcystin RR chun .........................................................40 Hnh 3.25b. Sc k microcystin YR chun .........................................................40 Hnh 3.25c . Sc k microcystin LR chun ..........................................................41 Hnh 3.25d. Sc k microcystin ca chng LT2 .................................................41 Hnh 3.25e. Sc k microcystin ca chng LT8 .................................................42 Hnh 3.26. Nui cy lm giu cc chng vi khun nc trn mi trng NA v M7 ...................................................................................................................................45 Hnh 3.27. Cy pha khun lc vi khun trn mi trng NA ...................................45 Hnh 3.28. Cy ph h da trn phn tch gii trnh t rADN 16S ca chng N4 v cc loi c quan h h hng gn................................................................................48 Hnh 3.29. Cy ph h da trn phn tch gii trnh t rADN 16S ca chng N7 vi cc loi c quan h h hng gn................................................................................49 Hnh 3.30. Thi gian bn phn gii microcystin cc nng khc nhau ca 2 chng N4, N7. ...........................................................................................................50 Hnh 3.31. Kh nng phn gii microcystin ca vi khun Sphingomonas N4 cc nhit khc nhau ....................................................................................................52 Hnh 3.32. Kh nng phn gii ca vi khun Sphingomonas N7 nhit khc nhau ...........................................................................................................................53 Danh mc bng Bng 1.1. Nhng loi vi khun lam gy c v s phn b ca chng [16] ..............4 Bng 3.1. Kh nng sinh trng ca 10 chng Microcystis trn mi trng Bold3N ...................................................................................................................................32 Bng 3.2. Kh nng sinh trng ca 10 chng Microcystis trn mi trng J ........33 Bng 3.3. Kh nng sinh trng ca cc chng Microcystis trn mi trng B12 ..34 Bng 3.4. Thnh phn acid bo c trong t bo ca 2 chng LT2 v LT8 ..............36 Bng 3.5. Hm lng microcystin ca 2 chng LT2 v LT8 ................................43 Bng 3.6. c im hnh thi ca 2 chng vi khun nc N4 v N7 .......................46 Bng 3.7. c im sinh l, sinh ho ca 2 chng N4 v N7 ...................................46 Bng 3.8. Hng s tc phn gii (K) v thi gian bn phn gii microcystin cc nng khc nhau ....................................................................................................50 Bng 3.9. T l % microcystin khng b phn gii do chng Sphingomonas N4 cc nhit khc nhau ..............................................................................................52 Bng 3.10. T l % microcystin khng b phn gii do chng Sphingomonas N7 cc nhit khc nhau ..............................................................................................52

BNG CC CH VIT TT

Cc ch vit tt Adda MC-LR MC-RR MC-YR PCR WHO TLC HPLC bp tb

Tn y 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6dienoic acid Leucine and arginine in the positions of X and Z of microcystin Arginine and arginine in the positions of X and Z of microcystin Tyrosine and arginine in the positions of X and Z of microcystin Phn ng trng hp (Polymerase chain reaction) T chc y t th gii (World Health Organisation) Sc k bn mng (Thin layer chromatography) Sc k lng cao p (High permormance liquid chromatography) Cp baz (Base pair) T bo

M U Trong nhng nm gn y H Ni pht trin vi tc nhanh chng, ko theo l s nhim hng lot thy vc ni thnh v vng ln cn. H Hon Kim mt a danh ni ting ca th cng b nhim. Cht lng nc thay i dn ti s pht trin thng xuyn v khng bnh thng ca vi khun lam, lm bin i cu trc thnh phn loi ca h vi to trong h. S suy gim mt s loi vi to c hu ng thi vi s gia tng thnh phn loi v mt ca vi khun lam gy c h Hon Kim c cnh bo v gy nn nhng lo ngi v mi trng cho khu h ng thc vt sng trong h [1, 2, 5, 9]. Trong s cc loi vi khun lam gy n hoa nc h Microcystis aeruginosa Kutzing l loi b bt gp thng xuyn v ph bin nht M.aeruginosa cha c t thuc nhm hepatotoxin (c t gan) cu to t cc peptid mch vng c tn gi l microcystin (MC) [14, 15, 19, 23, 29]. Microcystin l cht c ch i vi enzyme protein phosphatases (nhm PP1 v PP2A) [30]. c t cng gy nh hng xu ln hai loi protein serine v threonin phosphatases [15, 16, 17, 18, 22]. c mt s bi bo chng minh hin tng nc n hoa ca vi khun lam gy hi ti sinh vt thy sinh, ng vt v con ngi [16,17, 19, 20, 21]. Microcystin thng tch lu trong ni quan t bo hoc di dng t do trong nc [16, 18, 35, 36, 42]. c khng t cc cng trnh nghin cu hin tng n hoa nc v gim thiu tc hi ca c t vi khun lam gy c, tuy nhin kt qu vn cn cc mc khc nhau. bo v mi trng nc h, chng ti thc hin ti: Nghin cu c im sinh hc v thm d kh nng phn gii c t ca mt s chng Microcystis phn lp h Hon Kim, H Ni. Mc tiu ca ti * Phn lp, nh tn, nui cy v lu gi c cc chng vi khun lam Microcystis trong Bo tng Vi to ca Phng Cng ngh To v Sinh hc Mi trng - Vin Vi sinh vt v Cng ngh Sinh hc - HQGHN. * Thm d kh nng phn gii c t ca chng bng vi khun.

Ni dung ca ti Phn tch thnh phn thu l, thu ho ca nc h Hon Kim ti Phn lp, thun khit v nghin cu cc iu kin nui cy thch hp Phn loi n loi cc chng Microcystis tuyn chn c. Tch chit v phn loi c t ca cc chng Microcystis trn my sc Thm d kh nng phn gii c t microcystin ca vi khun lam gy

thi im thu mu. ca cc chng Microcystis. -

k lng cao p (HPLC). c bng vi khun bn a (vi khun nc).

Chng 1. TNG QUAN TI LIU 1.1. VI NT V H HON KIM H Hon Kim nm trung tm th H Ni, xa l mt on ca dng sng hng v cn c tn gi khc l sng Nh. Din tch h c tnh khong 12 hecta, chiu di Nam - Bc l 700m. Chiu rng ng Ty l 200m. Trong vi thp k gn y song song vi qu trnh pht trin kinh t x hi ca H Ni,do khng c bin php ngn chn nhng tc ng gy nhim lm bin i h sinh thi h, nh hng ti cht lng nc h, hm lng N v P tng ln dn ti s pht trin bng n ca vi khun lam. H Hon Kim tr thnh mt Hnh 1.1. H Hon Kim H Ni trong nhng h ph dng H Ni.

Trong h c s pht trin mnh m ca mt s vi khun lam thuc chi Microcystis, to thnh nhng vng ln ni bng bnh che kn mt mt gc h. Kt thc s n hoa, vi khun lam cht hng lot gy mi hi lan xung quanh khu vc h [6]. 1.2. VI KHUN LAM (CYANOBACTERIA) GY C Vi khun lam l nhm c th c xa nht v mang nhiu tn gi khc nhau trong qu trnh nghin cu: thc vt phn ct (Schizophyceae), to nhy (Myxophyceae), to lam (Cyanophyta), vi khun lam (Cyanobacteria). T nm 1854, Kopp coi vi khun v to lam l mt nhm c chung ngun gc pht sinh. Trong phn loi hin i, sinh vt cha c cu trc nhn hon ho (Procaryota) gm vi khun lam v vi khun, cn c vo c im c hai u khng c nhn in hnh, ch c cht nhn v nhim sc th. Vi khun lam v vi khun cn c nhng nt ging nhau nh: mng t bo u cu to t murein, mt loi glucozaminopeptit;

trong t bo cht c nhiu riboxom; nhiu loi vi khun v vi khun lam u c kh nng ng ho c N2 t do; c hai u c th hnh thnh cc pha cyanophase v bacteriophase. Vi khun lam khc vi cc ngnh to v thc vt bc cao cu trc rt c xa, t bo khng c cu trc nhn in hnh v khng c cc giai on phn chia nhn. Vi khun lam phn b khp ni trn tri t, a s sng trong nc ngt, mt s phn b trong thu vc nc mn giu cht hu c hoc trong nc l [7]. c t vi khun lam ph bin nht l c t gan (hepatotoxin) v c t thn kinh (neurotoxin). Mt s loi vi khun lam c cha c hai loi c t trn. Cc qun th n hoa trong thu vc nc ngt ch yu thuc v chi Microcystis thng gy c [8, 9], nhng cng c cc chng thuc chi ny khng gy c [29]. C kin cho rng c tnh khng phi l tnh trng c hiu nhng loi nht nh no , nh vi microcystin (hepatotoxin) th c tnh ca mt chng ph thuc vo iu kin l chng c mang gen m ha cho microcystin hay khng [38, 39]. Th nghim nui cy vi khun lam cng ch ra rng sn sinh microcystin l tnh trng khng i ca mt s chng nht nh, him khi b tc ng bi cc yu t mi trng [16, 49]. Trong khi cc iu kin dn ti s n hoa ca vi khun lam c sng t, th cc yu t dn ti s bng n ca cc chng gy c so vi cc chng khng c vn cha c lm sng t [15, 20, 22, 34]. Bng 1.1. Nhng loi vi khun lam gy c v s phn b ca chng [16] Loi Anabaena spp. (flosaquae, lemmermannii, circinalis) Anabaena circinalis Anabaena flos-aquae Microcystis aeruginosa M. viridis M. botrys Planktothrix agardhii P. agardhii Oscillatoria limosa Loi c t Microcystins Ni pht sinh Anh

Microcystins Microcystins Microcystins Microcystins Microcystins Microcystins Microcystins Microcystins

Php Nauy Worldwide Nht Bn an Mch Trung Quc Nauy Thu S

Loi Nostoc sp. Anabaenopsis millerii Haphalosiphon hibernicus (soil isolate) Nodularia spumigena Aphanizomenon Ovalisporum Cylindrospermopsis raciborskii Umezakia natans Anabaena flos-aquae Anabaena blooms Anabaena sp. Aphanizomenon sp. Aphanizomenon blooms Cylindrospermum sp. Microcystis sp. Planktothrix sp. Planktothrix Formosa Anabaena flos-aquae A. lemmermannii Anabaena circinalis Aphanizomenon flosaquae Cylindrospermopsis raciborskii Lyngbya wollei

Loi c t Microcystins Microcystins Microcystins Nodularins Cylindrospermopsin Cylindrospermopsin Cylindrospermopsin Anatoxin-a Anatoxin-a Anatoxin-a Anatoxin-a Anatoxin-a Anatoxin-a Anatoxin-a Anatoxin-a Homoanatoxin-a Anatoxin-a(S) Anatoxin-a(S) Sacidoxins Sacidoxins Sacidoxins Sacidoxins

Ni pht sinh Anh B M c Isaen c Nht Canada c Nht Phn Lan c Phn Lan Nht Bn Anh Nauy Canada an Mch c M Braxin M

S nghin cu vi khun lam gy c nhiu nc trn th gii cho thy c khong 60% cc mu vi khun lam c iu tra mang c t (bng 1.1). Tuy nhin, c t ca hin tng n hoa nc c th thay i theo thi gian v khng gian. Nu chng minh c qun th vi khun lam trong mt h no c mang c tnh nhng mt ca chng thp th khng nht thit phi bo ng cho cng

ng v nhng tc hi ca chng i vi sc khe con ngi v mi trng [16, 22, 43, 48, 50]. 1.2.1. Vi khun lam Microcystis Trong s cc loi vi khun lam c xc nh gy c cho con ngi th Microcystis aeruginosa c xem l thng gp nht, l mt trong nhng loi vi khun lam tham gia ng k hnh thnh hin tng n hoa ca nc. Vi c im l tp on bao gm hng nghn t bo ring l c t 2- 3 m v mi t bo u cha mt khng bo kh. Chnh s tp hp ca hng nghn khng bo kh nh v trong v s cc t bo ny gip chng ni trn mt nc v thu nhn c ngun nh sng mt tri, thng qua qu trnh quang hp tng hp sc t dip lc to nn vng xanh trn mt nc [25].

Hnh 1.2. Microcystis flos-aquae [62] Hnh 1.3. Microcystis aeruginosa [63] 1.2.2. c t microcystin ca Microcystis Microcystin [16, 40] l nhng c t vi khun lam gp ph bin v rng ri nht. Hp cht ny c tch ra u tin t loi vi khun lam Microcystis aeruginosa do vy c gi l microcystins. Chng l cc heptapeptide mch vng do c cha 1 amino acid c hiu (ADDA) chui bn [24]. Cho n nay, y l nhng dng cu trc ch gp microcystins v nodularin (mt loi pentapeptide vng ca vi khun lam trong nc l). Ngi ta bit khong 70 loi c t microcystins khc nhau. Chng khc nhau cc nhm metyl v 2 amino acid. bn trong chui. T dn n s khc nhau v cu trc bc 4, tnh c cng nh cc

c tnh a nc hoc k nc. Microcystin gn protein phosphataza 1 v 2 l nhng loi protein quan trng trong t bo nhn tht bng 1 lin kt ng ha tr khng thun nghch. Cc knh gn acid mt c tm thy trong t bo gan l con ng cho php microcystin i vo trong t bo. i vi ng vt c xng sng, liu lng gy cht ca microcystin s lm cho gan b hoi t trong thi gian vi gi hoc vi ngy. C l rng cc cu trc k nc c th i xuyn qua 1 vi loi t bo m thm ch khng theo knh acid mt. Ngoi ra, Fitzgeorge cung cp bng chng cho rng cc MC-LR thng gp c th hu hoi cu trc ca m mi. Trong khi , c tnh gy ra do hp th qua ng ming thng t gy nh hng hn nu so vi tim vo di mng bng trong cc th nghim m ngi ta tim s khin chng c tc dng ph hu mng t bo v lm gia tng c tnh ca anatoxin-a (mt loi c t thn kinh) [49].

Hnh 1.4. M hnh cu trc ho hc ca c t MC LR [54]

Hnh 1.5. M hnh cu trc ho hc ca c t MC RR [54]

7

Hnh 1.6. M hnh cu trc ho hc ca c t MC YR [54] Bin th xy ra ch yu ti cc v tr 1 v 2. V d, Microcystin-LR cha leucine amino acid (L) v arginine (R) ti cc v tr 1 v 2 tng ng. MicrocystinRR c arginine c hai v tr. Cc nghin cu c ch c hc t bo cho bit microcystins nh hng n cc cu trc t bo v qu trnh nguyn phn v iu ny gip gii thch kh nng kch thch gy ung th ca chng. 1.2.3. C ch gy c ca microcystin C ch gy c ca microcystin mc phn t l c ch cc hepatocyte protein phosphatases P 1 (PP1) v 2A (PP2A) hai enzyme iu chnh nhiu qu trnh din ra trong t bo ng vt v thc vt. S km hm ny dn ti s siu phosphoryl ha ca t bo cytoskeratin. Trong t bo gan ca ngi v ng vt, iu ny cn lm tng s ph v cc vi si hnh thnh nn khung t bo. Khi cc vi si tr ln tch ri mng t bo s lm xut hin cc bng nc trong mng, ng thi lm mng t bo b co li, dch t bo thot ra. y c xem l nguyn nhn gy ra chy mu gan, ph v cu trc ca gan thng thy ng vt. S tip xc lu di vi nng microcystin thp trong nc ung l nhn t gy ra ung th gan ngi [14]. Cc nghin cu c ch ng hc t bo cho bit microcystin nh hng ln n cu trc t bo v qu trnh nguyn phn, iu ny gip gii thch kh nng kch thch gy ung th ca chng. c t ny cng gy nh hng xu n hai loi enzyme l serin photphatase v threonin photphatase lin quan ti s iu ha pht trin cc t bo nhn tht (Eukaryote), iu khin s hot ng ca t bo ng thc vt trong qu trnh phn chia, sinh trng, trao i cht, sao chp
8

ADN v s biu hin cc tnh trng c lin quan. iu cho thy microcystin c th nh hng n cc chc nng ca t bo nhn tht mc phn t v t c th nh hng ti cc ng vt thy sinh sng trong mi trng c s tip xc vi c t ny. Microcystin thng tch ly trong cc t bo vi khun lam, khi nc n hoa, c t dng tan hoc theo phng thc t nhin hoc do s iu khin ca t bo m gii phng vo nc. Trong trng hp n hoa bi Microcystis th microcystin dng khng ha tan, kh bn vng, s c th tn ti vi tun trc khi b phn gii sinh hc [10]. 1.3. S PHN GII C T MICROCYSTIN CA VI KHUN NC Sphingomonas 1.3.1. Vi khun Sphingomonas Sphingomonas l mt vi khun Gram m, khng bo t, d dng, hon ton hiu kh v sinh sc t xanh hoc vng nht. Chng c kh nng di ng nhanh nh cc lng roi v a s c kh nng pht Hnh 1.7. Hnh dng t bo ca Sphingomonas [57] Sphingomonas l vi khun hnh que c kch thc 0.3-0.8 x 1.0-2.7m, chng c mu vng hoc mu trng. Sphingomonas hon ton khng c lipopolysaccarid (LPS) cha ni c t. Thay vo , n c mt mng t bo bao gm c protein, photphoslipid, quinone h hp v mng ngoi cha glycosphingolipit (GSLs). GSLs gi mt v tr ging nh LPS trong cc vi khun Gram m khc v xut hin vi nhiu chc nng tng t, nh l mt tr ngi i vi cc thc th khng khun. Cng bi phn hydrocarbon ca mt GSL ngn hn ca LPS nn b mt t bo ca Sphingomonas c nhiu hydrophobic hn cc vi khun Gram m khc. y c th l mt l do gii thch cho vic loi vi khun ny c kh nng lm gim cc hydrocarbon thm, a hydrophobic v tnh nhy cm ca n i vi vic ngn cc
9

quang di nh sng cc tm.

khng khun hydrophobic. Sphingomonas c tm thy ph bin trong mi trng t nhin bao gm trong nc (nc ngt v nc mn), trn cn, trong r cy, trong cc mu xt nghim bnh v nhiu ni khc. S phn b rng ri ca chng trong mi trng l do chng c kh nng s dng nhiu hp cht hu c, c th sinh trng v tn ti di cc iu kin dinh dng thp. Nhiu loi Sphingomonas cng c tm thy cc ni c cha cc hp cht c nh PCBs, creozot, pentachlorophenoL, thuc dit cCc nghin cu cho thy trong mi trng b nhim, Sphingomonas c kh nng s dng cht gy nhim lm ngun nng lng c th cnh tranh c vi cc sinh vt khc trong cc mi trng khc nhau [20]. 1.3.2. C ch phn gii c t microcystin ca Sphingomonas Sphingomonas c David G. Bourne chng minh l chng vi khun c cha cc enzyme tham gia vo con ng phn gii c t microcystin. S phn gii c t ny c xc tc bi enzyme ni bo [20]. Vic s dng cc cht c ch protease kinh in cho php: (1) phn loi cc enzyme ny thnh cc h protease c bn v (2) tch ly tc thi trong iu kin invitro cc sn phm trung gian t s phn hy MC - LR. V tr phn ct ban u trn phn t MC - LR bi enzyme c xc nh l lin kt peptides 3 - amino - 9 methoxy - 2,6,8 - trimethyl - deca - 4,6 dienoic acid (Adda) - Arg. Hai sn phm phn gii trung gian c tm thy l MC - LR mch thng: NH2 - Adda - Glu(iso) -methyldehydroalanine - Ala - Leu - - methylaspartate Arg - OH (cht A) v sn phm cn li l tetrapeptide: NH2 - Adda Glu (iso) methyldehydroalanine - Ala -OH (cht B) l kt qu ca s lm gim MC - LR nh hai enzyme trung gian. Chnh hin tng m vng c t MC - LR nh enzyme microcystinase ca vi khun khin cht ny tr nn khng c do gim ng k tng tc vi cc protein phosphatase. Con ng phn hy MC - LR bi Sphingomonas c xut nh sau:

10

Hnh 1.8. Con ng phn gii mycrocystin nh Sphingomonas [20] 1.4. NH HNG CA C T TI NGI V GIA SC C rt nhiu nghin cu cho thy ng vt b ng c do ung nc c cha vi khun lam gy c. Bo co u tin vo nm 1800 v hin trng gia sc b cht do ung phi nc c cha vi khun lam gy c. Cht c ny gy hi ln ng bi tit, gy d ng da, nh hng ti mng nhy rut hoc lm con vt b m. Bo co tip theo v cc phn ng d ng qua da hoc kch thch da ni nga do nc cha cc loi vi khun lam khc nhau nh Anabaena, Aphanizomerzon, Nodularia, Oscillatoria, Gloeotrichia. Cc vn ng vin khi tin hnh bi, li, ngi i bi mc o tm hoc o ln gi li cc t bo vi khun lam, do c st m cc t bo b v ra v gii phng c t, t cht c tc ng ln da [16].
11

Vo nm 1959, Canada mc d c gia sc b cht v c cnh bo v ngun nc s dng song ngi ta vn bi trong mt h c cha nhiu vi khun lam. Kt qu l c 13 ngi b m, au u, au c, a chy nng. Trong dch tit ca mt bnh nhn ngi ta pht hin ra mt s lng ln t bo Microcystis spp., v mt s on si to ca Anabaena circinalis [19]. Nm 1979, c ngn chn s n hoa ca Cylindropermopsis raciborskii trong ngun nc ung, ngi ta x l chng bng ng sunfat. iu ny lm cho c t trong t bo c gii phng gy ra nh hng nghim trng v lm 141 ngi phi nhp vin [34]. Nm 1988, Brazil tip sau trn lt do v con p Itaparica thuc bang Bahia, khong 2000 ca bnh v ng tiu ho c bo co sau 42 ngy v c 88 ngi cht. Cc iu tra nguyn nhn sau cho bit c hm lng c t vi khun lam rt cao [16]. Nm 1989, Anh c 10 trong s 20 lnh b m sau khi bi tp trong nc b nhim bi vi khun lam Microcystis spp., phi nhp vin [17]. Nm 1993, Trung Quc mt s ca bnh ung th gan c chn on l c lin quan n vic s dng nc ung trn b mt b nhim nhiu vi khun lam [16, 18]. Nm 1994, Thu in do qun l khng tt ngun nc cha c x l nh my ng, nn khi chy ra n ho ln vo nc sng. iu ny khin loi vi khun lam Planktothrix agardhii pht trin mnh to nn hin tng n hoa nc. Kt qu phn tch cho thy rng loi ny cha c t microcystin. Trong tng s 304 nng dn s dng ngun nc ny th c 121 ngi, k c ng vt nui ca h b tiu chy, vim c [16, 18]. Nm 1995, c, kt qu iu tra vi 852 bnh nhn tip xc vi nc c cha vi khun lam cho thy rng sau 2 7 ngy nhiu bnh nhn b mc tiu chy, st d ng da. Monte Hermosa (Achentina) vo thng 11/1995 c 45 triu con ngao Mesoderma marcoides b cht do s n hoa ca to gy hi. Cng vo nm ny Vit Nam, loi Noctiluca scintilans n hoa khu vc vnh Vn Phong
12

Khnh Ho vo thng 5 v 6 lm cht 20 tn tm hm vi thit hi ln n 6 t ng [5]. 1.5. NHNG NGHIN CU V VI KHUN LAM TRN TH GII V VIT NAM 1.5.1. Trn th gii Nhng quan st v pht hin sm nht v vi to (trong c vi khun lam) c cc nh thc vt hc m t. Cc nh nghin cu cho rng nhn thc ca con ngi v s n hoa gy c ca vi khun lam c bit n t th k 12 v thi gian gn y h cnh bo cc c dn nhiu quc gia nh Trung quc, Bc Phi, Nam Phi v vic s dng ngun nc ti cc thy vc c vng to mu xanh [2, 47, 48]. Cc bin php phn gii c t microcystin c nghin cu bi Harada, ng a ra cc bin php lm gim hm lng microcystin trong nc t kt qu ca cc qu trnh pha long, hp th cc ht vt cht, s phn gii ph thuc nhit , pH, s quang phn v s phn gii sinh hc [13]. Nm 1997, Takenaka v Watanabe kim tra cc loi vi khun phn lp t mt h Nht Bn v pht hin ra rng thng qua hot ng ca enzyme alkalil protease, Pseudomunas aeruginosa (Sphingomonas) c kh nng lm gim MC LR. l cc loi c kh nng m vng c t v to ra dng mch thng dn ti lng c t gim 160 ln. Nhiu nghin cu v vic s dng ozon gim bt hin tng n hoa ca vi khun lam ch ra: vi tc bm ozon vo trong nc l 1gm O3 /m3 nc/gi, hu ht cc t bo vi khun c th b tiu dit trong 15 pht [8]. Tng t nh vy cc nghin cu v tnh n nh ca microcystin di tc ng ca tia UV cho thy c t b phn gii nhanh bi tia UV bc sng di tng ng vi s hp th ti a lng c t [14]. Trong iu kin t nhin, cc vi sinh vt, c bit l cc vi khun nc ng vai tr quan trong trong vic phn gii c t khng ha tan m c gii phng vo nc. Ngi ta chng minh c rng microcystin c th b phn gii bi cc vi sinh vt c trong h sinh thi thy vc t nhin [12]. c bit, s phn gii

13

sinh hc microcystin cn xy ra nhanh hn cc thy vc c s n hoa bi vi khun lam. Thc t cho thy cc bin php c hc nhm lc, vt b to tiu tn nhiu thi gian, cng sc li khng cho hiu qu cao hoc cc bin php l hc, ha hc (dng CuSO4, vi, Al2(SO4)3, ferric chlorit) thng khng kh thi khi p dng i vi cc thy vc s dng cho mc ch sinh hot bi mt lng ha cht tn d [8]. Nhiu nghin cu khng nh tro ca la mch, g mc v mt s loi l c bit l l si c tc dng c ch mt cch ng k s sinh trng ca vi khun lam gy c [15]. Dch chit t v qut v v chui ln cng c kh nng c ch hin tng nc n hoa bi vi khun lam [3]. 1.5.2. Vit Nam Vit Nam, nghin cu v to c mi ch tin hnh trong 1-2 thp nin va qua. Nm 1987, khi mt l ngao Vit Nam xut sang Cng ng chung Chu u (EU) b pht hin nhim c t to gy ch trong d lun. Nghin cu thu triu do thc vt ph du bin gy ra mi c bt u t nm 1993 vi nhng kt qu ban u v thnh phn loi v s phn b to c hi mt s vng bin ca Vit Nam, trong ng ch l cng b v Thc vt ph du c hi bin Vit Nam ca 2 tc gi Nguyn Ngc Lm v on Nh Hi ti Hi ngh Quc t ln th 7 v To c t chc ti Nht Bn. Tip l cng b ca Nguyn Ngc Lm v cng s nm 1996 ti Hi ngh Khoa hc Bin Asean-Canada v s bng n ca loi Noctiluca scintilans lin quan n hin tng ph dng vnh Vn Phong Khnh Ho. Nguyn Th Minh Huyn (1996) c nhng ghi nhn u tin v mt s lai to c vng bin Bch Long V. Chu Vn Thuc (1998) nghin cu to bin c hi vng ca sng ven bin min bc Vit Nam [2, 3, 4, 5, 6, 9]. Nm 1996-1999, ti quc gia do ng Ngc Thanh lm ch tr c thc hin vi ni dung nghin cu v thnh phn loi bin Vit Nam v mt s vn lin quan n sinh thi to c [6]. Theo Trnh Th Thanh v ng Th Sy ti mt s m ao nui th c Thanh Tr, mc d ngnh vi khun lam (cyanobacteria) c thnh phn loi khng ln (khong 24 loi, chim khong 13%) nhng thng t sinh khi cao vo thng 6 v 7, c bit ti cc ao c s dng phn bc lm ngun thc n cho c [6, 9]. Kt qu iu tra nghin cu cc h cha v mt s thu
14

vc thuc cc tnh pha Bc c tin hnh bi ng nh Kim, ng Hong Phc Hin (2006) cho th nhng loi to c tim nng c gy n hoa nc thuc v cc chi Microcystis, Anabaena, Aphanizomenon, Oscillatoria v Gomphospheria [4]. Theo Dng c Tin (2001-2005) xc nh trong h Hon Kim v mt s thu vc min Nam Vit Nam c cc chi Microcystis, Cylindrospermopsis, Anabaena, Oscillatoria, Lyngbya thuc ngnh vi khun lam (Cyanobacteria) chim a s v thnh phn loi gy n hoa nc. Trong c cc loi c kh nng gy c nh Microcystis aeruginosa, M. viridis, M. botrys, M. wesenbergii v Cylindrospermopsis raciborskii [9].

15

Chng 2. NGUYN LIU V PHNG PHP NGHIN CU 2.1. I TNG NGHIN CU 2.1.1. i tng nghin cu i tng nghin cu bao gm: - Vi khun lam Microcystis c phn lp t h Hon Kim - Vi khun Sphingomonas c phn lp t h Hon Kim 2.1.2. a im v thi gian nghin cu a im thu mu: H Hon Kim Thi gian tin hnh nghin cu: T thng 1/2007 n thng 12/2008 ti phng Cng ngh To v Sinh hc Mi trng, Vin Vi sinh vt v Cng ngh Sinh hc, i hc Quc gia H Ni. Thi im thu mu: Thu mu vo hai t. t 1: 6/3/2007, t 2: 8/5/2007 V tr cc im thu mu: thu mu ti 6 im nh trong hnh sau.

D1: Ca cng hng Khay D2: i din ngn hng Incombank L Thi T D3: Ngn hng ANZ D4: Nh hng thy t D5: Ca cng i din tng cng ty in lc Vit Nam D6: n Ngc Sn

Hnh 2.1. S v tr thu mu

16

2.1.3. My mc, dng c S dng my mc v dng c c ti phng Cng ngh To v Sinh hc mi trng, cc phng th nghim thuc Vin Vi sinh vt v Cng ngh Sinh hc- i hc Quc Gia. Trung tm Cng ngh Mi trng v Pht trin bn vng- i hc Khoa hc T nhin. Knh hin vi quang hc Knh lp quan st khun lc My o pH (Olympus, Nht Bn) (Olympus, Nht Bn) (Osi, Php)

My sc k lng cao p (High Performance Liquid Chromatography, HPLC 1100, c) 2.1.4. Ha cht Ha cht dng cho phn tch v th nghim khi tin hnh ti gm: Ha cht CH3COONa Methanol Isopropanol Xut x Merk, c Merk, c Merk, c Ha cht Nihyrin n- butanol Ethyl axetat Xut x Merk, c Merk, c Merk, c

Ho cht dng trong phn ng PCR: Trnh t cc on mi dng trong nghin cu : 27f 780f 920r 1525r 5 - TTGATCCTGGCAG - 3 5 - GATTAGATACCCTGGTAG - 3 5 - GTCAATTCCTTTGAGTTT - 3 5 - AAAGGAGGTGATCCAGCC - 3 dNTP mix 2.5mM

10 Taq buffer Taq polymeraza (5U).

17

2.1.5. Mi trng nui cy vi khun lam v vi khun Nui vi khun lam Microcystis trn 6 loi mi trng l Bold 3N, B12, J, BG11, C, MA (Ph lc 1) Nui cy vi khun Sphingomonas trn 2 mi trng NA v M7 (Ph lc 2). 2.2. PHNG PHP NGHIN CU 2.2.1. Phng php iu tra thu mu vi khun lam h Hon Kim v nh gi cht lng nc - iu tra thu mu theo quy phm ca B Ti nguyn v Mi trng. - Cc ch tiu c phn tch da theo Standard methods for the examination of water and waste water ca t chc Y t M (Ph lc 3). 2.2.2. Phng php phn lp v nui cy vi khun lam Microcystis 2.2.2.1. Lm giu mu Phng php phn lp Microcystis bc 1 lm giu mu [31]. Quan st nc n hoa h Hon Kim thi im thu mu cho thy loi ch yu trong qun x l Microcystis aeruginosa chim trn 80%. Ht 1000 l mu nc h c cc loi vi khun lam khc nhau, cho vo ng Eppendorf, ly tm 7000vng/pht trong 10 pht v ra 2 ln vi dung dch mui sinh l 0,05% nhm mc ch loi b bt cc loi to khc. Lp dch ni bn trn l vi khun lam, sau ht 100 l dch ni bn trn cho vo nui cy trong cc bnh tam gic dung tch 500 ml cha 200 ml mi trng B12. Nui gi trong iu kin nhit 30oC di nh sng n neon vi cng nh sng l 1500lux theo quang chu k l 12 gi chiu sng v 12 gi ti. Sau thi gian 3 tun nui cy, quan st kh nng sinh trng ca mu vi khun lam c lm giu bng knh hin vi quang hc phng i 400 1000 ln. 2.2.2.2. Phng php tch v thun khit trn thch a Quy trnh phn lp c tin hnh theo phng php ca Shirai c ci tin [31]. Cc tp on vi khun lam sau khi lm giu c xc nh di knh hin vi l

18

cc chng thuc chi Microcystis trong mu nc c ly tm 10.000 vng/pht trong 15 pht nhm tip tc loi b cc loi to khc. Tp on vi khun lam ni pha trn c ht ra, t vo a petri v tch ring r tng tp on ra di knh lp Olympus. Cc tp on c ht ring vo tng ng Eppendorf, sau tip tc s dng my vortex mu nhm mc ch phn tch tp on thnh cc t bo ring r. Pha long mu theo phng php ca Bold v cy trang trn cc mi trng dinh dng agaroza c nhit to gel cc thp (type IX, Sigma) vi nng 0,4%, cc hp a thch c qun kn bng paraphin v t di n neon vi cng nh sng 1000 1500 lux nhit 30oC. Sau thi gian t 07 30 ngy, quan st khun lc bng knh lp Olympus. Nhng khun lc sch sau khi mc trn mi trng thch agaroza c tch sang cc l Penicillin cha 5ml mi trng dch th. Theo di s pht trin ca t bo vi khun lam, quy trnh c tin hnh lp i lp li cho n khi thu c cc chng thun khit. 2.2.3. Phn loi vi khun lam Microcystis 2.2.3.1. Bng phng php hnh thi Cc chng Microcystis sau khi phn lp thun khit c quan st di knh hin vi quang hc Olympus c phng i 400-1000 ln quan st cc c im hnh thi v kch thc t bo ca chng. nh loi, chng ti s dng cc ti liu chnh sau: - Phn loi thc vt hc bc thp . - Phn loi Vi khun lam Vit Nam [07]. - The on-line database of Cyanobacterial genera [27]. 2.2.3.2 . Phng php tch ADN ADN c chit xut v tch t sinh khi t theo phng php [44]. Ly 3 vng que cy sinh khi t bo sau 14 gi nui, ly tm lnh 4oC vi tc 8000 vng trong15 pht v ra 2 ln bng 300l dung dch mui EDTA pH 8,0 (0,5M Na2EDTA, 0,15M NaCl). Sau to dch huyn ph tr li trong 200l m 10 x TE (10mM Tris-HCl, 1mM EDTA, pH 8,0, t l mu:m, 1: 2). B sung 10l ca

19

lyzozym (Sigma, 1mg/ml Lysozym), gi 37oC trong ni cch thu trong 15 pht. B sung dung dch Tris-SDS (0,1M Tris-HCl pH 9,0 v 1% SDS) vi th tch bng th tch mu. Dch mu c gi 60oC trong ni cch thu khong 10-30 pht. S tan t bo c xc nh bng s trong sut ca dung dch cng vi s tng nht tng ng. Dch tan t bo c lm lnh trong trong 10 pht, b sung 150l cloroform-isoamyl alcohol, 24:1, v/v (gi 4oC) vi th tch bng 1/3th tch mu. Ly tm lnh 4oC vi tc 10.000 vng trong 20 pht phn lp. Sau chuyn phn nht phn u sang Eppendorf (C. 1,5ml) mi v ADN c tch ra bng b sung etanol lnh (etanol 95,5% gi -22oC) vi th tch gp i th tch mu v 2l ca 3M Na acetate (pH=4,5). Ngm ADN trong 100l ethanol 70% trong 30 pht v sau ngm lin tip trong ethnol 80, 90, 95% cho mi nng trong 5 pht. Si ADN lm kh bng nhit phng trong 10 pht v c lm tan tr li trong 20l m 0,1 x TE (10 x TE) 4oC qua m. Dch em phn tch bng my quang ph k cc bc sng 230, 260 v 280nm. ADN hp th rt nhanh bc sng 260nm. bc sng 280 chng t s c mt ca protin. Bc sng 230nm biu hin s c mt ca cht m, cc mui nh EDTA v ca ARN. Cc t l chun v hp th ca ADN cc bc sng 230 : 260 : 280 l 1,0 : 0,45 : 0,515. Hm lng ADN c tnh nh sau: 1 n v OD (mt quang, Optical Durity) 260nm tng ng vi 50g/ml ADN. Cch tnh: n v mu x long x ch s/1000 = ADN g/l. Nu ADN cha sch cn kh protein v ARN bng enzym proteaza K v RNazaA 2.2.3.3. Ha phn loi phn tch thnh phn acid bo Ly 50mg mu cho vo ng thy tinh nt vn (c 13mm x 100mm) [32]. Alkal ha bng 1.0ml cht phn ng I (reagent I, gm 15g sodium hydroxide (NaOH), 50ml methanol v 50ml Milli- Q). T bo c thy phn 1000C ti ni nhit kh trong 5 pht. Sau lm huyn ph bng my Vortex trong 5 giy ri tip tc cho thy phn trong 25 pht. Dch thy phn c lm lnh nhit phng.
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Methyl ha dung dch Alkal vi 0.2ml cht phn ng 2 (65ml 6N hydrochloric acid v 55ml methanol). Sau khi metyl ha, gi 800C trong ni cch thy khong 10 pht. Dung dch methyl ha c b sung 1.25ml ca cht th 3 (gm 50ml nhexane-methyl tert butyl ether, 1:1, v/v) v c trn u bng my lc 2000rpm trong 10 pht. Dch nht pha trn c loi b. Dung dch c b sung 3ml thuc th 4 (1.2g sodium hydroxide trong 100ml Milli-Q) v c trn u bng tay trong 5 pht. Ly tm 2500rpm trong 10 pht. Dch trn cng c chuyn sang ng thy tinh mi c nt vn v cho bay hi bng kh nitrogen thng thng ti khi kh. Cui cng, ton b dch mu c lm tan vi 50l ca diethyl ether 300 (hoc acetone 300, n-hexane). Ton b mu c chm vo sc k bn mng (TCL) ri c t trong bn thy tinh cha 100ml n-hexane-diethyl ether (4:1,v/v) trong 30 45 pht. Thnh phn acid bo c hin vt bng iot trong 30 45 pht. Acid bo c cc v khng cc xut hin nh nhng vt xanh. Nhng vt c co v ra gii hai ln bng diethyl ether 300. Trn u dung dch silicagel trn my lc 2000rpm trong 10 pht v ly tm 2500rpm trong 5 hoc 10 pht. Dung dch c bay hi bng kh nitrogen thng thng ti khi kh mu. Mu c ha tan tr li vi 50l cht phn ng 3. Dng pipet nh vo l nh GC c nt y. Sau , em phn tch trn my sc k kh (gas chromatography). 2.2.4. Xc nh mt t bo S lng t bo vi khun lam trong mt th tch nc c xc nh bng cch m s t bo trn 400 nh thuc 25 ln ca bung m Goriaev. Mt ln ca bung Goriaev c din tch 1/25 mm2 v chiu cao 1/10 mm. Nh vy th tch ca 1 l 1/25 1/10 (mm3), th tch ca 25 ln l: 1/25 1/10 x 25 (mm3) Nu a l s lng t bo vi khun lam m c trn bung m th s lng t bo to trong 1 ml l: X = a 104 (t bo).

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2.2.5. Phng php tch chit c t microcystin t t bo ca Microc ystis Trong th nghim ca chng ti, microcystin ca Microcystis c tch chit theo phng php ng tan [44]. Thu sinh khi bng cch ly tm vi tc 7000 vng/20 pht sau lc dch ni bng thit b lc GF/C (Whatman). Dch ni c lc qua mng lc kch thc 0,2 m (Milipore). Sinh khi thu c ng kh nhit 83oC, chit rt c t bng Methanol : nc ct (80:20) v bng Methanol : n.Hexan (85:15). Lc qua ct C18 loi b sc t chit t t bo Microcystis. Dch chit lng c gi trong catrid Sep-Pak (Water) v ra bng dung dch methanol 20%. Dch chit c chy qua sc k bn mng (TLC) lm sch c t, co phn c t trn bn TLC. Microcystin sau c tch ra nh x l vi 3 ml methanol 90%, em sy kh qua dng kh nitrogen v ho tan trong mt lng nh hn hp nc- ethanol 20%. Thnh phn v nng ca dch chit c xc nh bng my HPLC (Hewlett Packard model 1100 LC) qua ct ODS II (4,6 mm; 150 mm) vi methanol cha 0,05 M phosphatase, pH 3 (t l 6:4 theo th tch), tch trong pha ng. 2.2.6. Phng php xc nh hm lng trn my quang ph c t microcystin c phn tch trn my quang ph theo phng php [28 ]. Hm lng microcystin c tnh ton theo phng trnh ng chun thu c ca microcystin chun. Da vo th chun ca microcystin theo mt quang, xc nh hm lng microcystin c trong dch chit. Phng php xy dng ng chun hm lng microcystin theo mt quang: Dch chit MC - LR chun c nng 10ng/ml c pha long gp 1000, 3000, 5000, 7000, 9000, 11000 ln vi hn hp dung mi methanon : nc theo t l 80 : 20. Tin hnh o bc sng 242 nm trn my quang ph ERMA 11 TOKYO. Cn c vo cc gi tr mt quang o ta lp c th biu th mi quan h vi nng dch chit microcystin. 2.2.7. Cc phng php sc k L phng php hiu qu cho vic nh tnh, nh lng v tinh sch cc hp cht hu c. Nguyn tc chung ca phng php sc k l da trn s phn b cc
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cht gia hai pha l pha ng v pha tnh. Pha ng c th l cht kh hoc lng y mu qua vng cha pha tnh. Pha tnh cha cht rn hoc lng gi l cc hp cht ph c kh nng cha cc cht ha tan. Mu phn tch cha mt hay nhiu cu t, khi tip xc vi pha tnh v pha ng cc cu t khc nhau tch nhau ra do chng c p lc khc nhau vi c hai pha. 2.2.7.1. Phng php sc k bn mng (TLC) Vic tch cc cht trong sc k bn mng [49] da trn s phn tch cc cht gia hai pha: - Pha c nh l cht hp ph c tri rng trn mt phin knh to thnh lp mng. - Pha ng l dung mi thch hp ng trong bnh c np y kn. Trong qu trnh sc k dung mi di chuyn lm dch chuyn cc thnh phn trong mu ( c chm thnh tng vt trn phin knh tri cht hp ph) Tin hnh th nghim: - Pha mu sau c quay thnh 1ml bng hn hp dung mi methanol: nc (80:20), chm ton b mu ln trn bn silicagel. t bn mng trong bn thy tinh cha 50ml hn hp dung mi ethyl acetat: isopropanol: nc = 8:5:3 (v/v), trong 45-60 pht. Sau khi lm kh ti nhit phng, nhum TLC bng nihydrin 2% trong butanol bo ha. Sy kh, quan st cc vt xut hin trn nn TLC. Sau nh du, co cc vt ln cng vi microcystin chun. Ti chit rt c t ny bng hn hp dung mi methanol : nc (80:20). em dch ny ly tm tc 8000 vng/ pht, trong 15 pht. Sau , lc dch qua ct C18. 2.2.7.2. Phng php sc k lng cao p (HPLC) Sc k lng cao p [27 ] l phng php chia hn hp cc cht trong ct sc k trong pha ng trang thi lng nh mt p lc ln c y qua pha tnh Trong HPLC, mu phn tch c bm vo ct sc k qua mt van bm su chiu, sau nh bm cao p m pha ng c y qua ct vi tc dng khng i trong sut thi gian chy sc k. y pha ng va ng vai tr l cht mang,

23

mang mu phn tch vo ct tch, va l cht ra gii trong sut qu trnh chy sc k. Pha ng c th l mt dung mi hu c hoc hn hp nhiu dung mi hu c khc nhau. Thit k h thng HPLC gm: Tin hnh th nghim My HPLC trn ct ODS II 4.6mm x 150mm, ng knh ht 5 m. B lc dung mi v b lc mu ng knh l mng 100Ao. Detector UV-VIS, = 242 nm, my vi tnh ghi tn hiu ni vi my in. Pha tnh: Supelcosil LC 18 Pha ng : Methanol cha 0,5M phosphate = 6:4 (v/v) Cc mu c ha tan trong h dung mi ca pha ng, tin hnh chy MC chun v mu nghin cu. Tc dng l 1ml/ pht, pH = 3. 2.2.8. Phng php nui cy chng vi khun Sphingomonas Chng vi khun Sphingomonas [22] c phn lp t mu nc h. Ly 1 ml nc h pha long cc nng pha long thch hp. Nh 1ml dch pha long trn mi trng dch th NA v M7. Sau 24 gi nh 100l dch cy trang u trn mt thch quan st thy cc khun lc, tch v cy ria nhm khun lc cho tch nhau ring r. Tch v cy mt khun lc trn mi trng tng ng. Qu trnh lp li nhiu ln cho n khi thu c cc chng thun khit. Nui gi chng vi khun ny trn mi trng thch nghing, bo qun nhit 4C dng cho cc nghin cu tip theo. 2.2.9. Phng php xc nh kh nng phn gii microcystin S phn gii c bt u bng cch b sung dch chit microcystin th vo cc mu nc v trng thu c cc nng microcystin khc nhau. Cc chng vi khun phn gii vi khun lam c a vo vi mt 2.5 106 t bo/ml. Sau 20 pht kim tra mu mt ln bng cch ly 50ml dch em lc qua mng lc kch thc 0,22m, sau em o bc sng 242nm xc nh nng c t cn li. Mu i chng (khng c vi khun) c tin hnh song song. Cc th nghim

24

c lp li 3 ln. Hiu qu phn gii microcystin c xc inh nh phng php bn thi gian nh m t ca Kenefick [28]: Ct = Co.e(-kt) Trong : Ct nng microcystin ti thi im o (g/ml) Co nng microcystin trc phn hy (g/ml) k hng s phn hy bc u tin (ngy -1) t thi gian bn phn hy (ngy)

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Chng 3. KT QU V THO LUN 3.1. PHN TCH CC CH TIU THY L, THY HA NC H HON KIM c im ca cc ma c nh hng ti cc qun th vi to v vi khun lam. nh vi to silic kt hp vi vic pht trin nhanh roi trong ma ng v ma xun, tip theo l to lc vo cui ma xun v u h. Ti ni ph dng v thu vc ln, vi khun lam thng chim u th trong cc loi thc vt ni vo cui xun u h. S thay i v ma l cc nhn t mi trng thng khng gy ra s thay th vi khun lam bng cc loi thc vt ni. Vi khun lam l hin hu chim u th hu ht trong nm. Do vy, nh gi cht lng nc h, chng ti tin hnh phn tch tng hp cc thnh phn v cc iu kin nh hng n nc. Trong nghin cu ny, chng ti tin hnh hai t thu mu vo thng 03 v 05 nm 2007. Kt qu phn tch chi tit c trnh by ph lc 5. 3.1.1. Nhit Nhit mt t thu mu trong cng mt h li khc nhau ty thuc vo thi gian v a im thu mu. Ti cc cng dn nc thi vo h, nhit nc lun cao hn cc im xa b v gia h t 1- 2C. 3.1.2. pH Trong thi gian chng ti thu mu, pH ca h kh cao. Gi tr pH ti cc im ly mu dao ng t 9,57- 9,84 vo t thu mu thng 06/03/2007 v t 9,7710,37 vo t thu mu thng 08/05/2007. 3.1.3. Cc cht l lng SS Hm lng cc cht l lng trong h tng i cao, dao ng t 230450mg/l trong hai t thu mu.

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3.1.4. Hm lng oxy ha tan trong nc (DO- Dissolve oxygen) T s liu nghin cu cho thy hm lng oxy ha tan trong nc cao, dao ng t 9,42mg/l- 22,4mg/l. iu ny ph hp vi thc t l vo thi im chng ti tin hnh thu mu, trong h c s n hoa rt nhiu ca vi khun lam Microcystis, chnh s quang hp ca nhng vng n hoa v cc loi vi to khc dn ti hm lng oxy ha tan trong h tng ng k. 3.1.5. Nhu cu oxy ha hc (COD- Chemical oxygen demand) S dao ng gi tr COD trong h t 80,85mg/l- 767,20mg/l. Gi tr COD cao nht gp 20 ln so vi tiu chun Vit Nam loi B v cht lng nc mt. 3.1.6. Nhu cu oxy sinh ha (BOD- Biochemical oxygen demand) Kt qu nghin cu ca chng ti cho thy hm lng BOD trong h dao ng thp nht l 55 mg/l, cao nht l 260 mg/l vo thng 3, thng 5 thp nht l 100 mg/l, cao nht l 548 mg/l. Hm lng BOD5 cao gp 20 ln so vi tiu chun Vit Nam loi B v cht lng nc mt. Nhng nhn xt chung v nc h Hon Kim: T cc s liu phn tch c trong bng kt qu, c th nhn xt h ang trong tnh trng nhim. Vi gi tr hm lng NH4+ dao ng 0,129mg/l 0,699mg/l, NO2- khong 0.012mg/l 0,05mg/l, NO3- t 0,047mg/l 0,129mg/l v PO43- l 0,08mg/l 0.64mg/l chng t nc h c hm lng dinh dng kh cao. S pht trin ca vi khun lam l quanh nm nhng thng 5 pht trin nhiu hn thng 3 bi thi tit thng m p hn. Vi khun lam pht trin dy c trn mt h v c bit tp trung khu vc cui hng gi. 3.2. TUYN CHN CC CHNG VI KHUN LAM THUC CHI Microcystis PHN LP C 3.2.1. c im nui cy Ly 1 lng mu t cc l ng mu trong mi trng nui cy lm giu, cho vo Eppendorf sch, ly tm tc 10000 vng/15 pht. Sau , tin hnh pha
27

long mu cc nng thch hp (10-2; 10-3), nh 100l mu v trang u trn 6 loi mi trng thch Bold 3N, B12, BG11, C, MA v J. Do M. aeruginosa c khng bo kh (gas vacuoles) nn chng khng to thnh khun lc trn cc loi agar hoc agarose thng thng. Tuy nhin, iu ny c th c khc phc nh k thut ci tin ca Watanabe [11, 49]. Dch th c cha M. aeruginosa vi mi trng s dng loi agarose c nhit to gel cc thp nng 0,4% (type IX, Sigma) c s dng lm gi th. Cc a thch sau khi cy c qun paraphin, t di cng nh sng 1500 Lux. Sau 10 ngy quan st khun lc. Kt qu c trnh by hnh 3.1 ; 3.2 ; 3.3 ; 3.4

Hnh 3.1. Hnh thi khun lc ca Microcystis trn mi trng C

Hnh 3.2. Hnh thi khun lc ca Microcystis trn mi trng Bold 3N

Hnh 3.3. Hnh thi khun lc ca Microcystis trn mi trng B12

Hnh 3.4. Hnh thi khun lc ca Microcystis trn mi trng J

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Kt qu th nghim cho thy trn 3 mi trng Bold 3N, B12 v J khun lc ca Microcystis c kh nng mc tt hn so vi cc mi trng BG11, C, MA. 3 mi trng sau, khun lc Microcystis sau 2 - 4 tun nui mi thy xut hin khun lc. iu ny cho thy trn cc mi trng c cht khc nhau, dn n kh nng sinh trng ca chng cng khc nhau. 3.2.2. Phn lp cc khun lc ca Microc ystis Phn lp cc khun lc Microcystis trn cc mi trng nui cy, bc u chng ti thu c 10 chng. Cc chng ny c nhng c im hnh thi t bo c nghin cu di knh hin vi quang hc v c pht trin nui cy trn cc loi mi trng khc nhau. Kt qu c trnh by hnh 3.5; 3.6; 3.7; 3.8; 3.9; 3.10; 3.11; 3.12; 3.13; 3.14

Hnh 3.5. Hnh dng t bo chng Microcystis sp., LT1 Tp on vi t bo hnh cu, t bo c mu xanh lam vi nhiu khng bo kh, ng knh t bo 3-7 m. Bao nhy khng mu, m bao quanh t bo.

Hnh 3.6. Hnh dng t bo chng Microcystis sp., LT2 Tp on vi t bo hnh cu,t bo c mu xanh nht, ng knh t bo 2-5 m, tp on hnh dng khng n nh, cc khi nh hnh cu hoc thu knh hi dt.

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Hnh 3.7. Hnh dng t bo chng Microcystis sp., LT3

Hnh 3.8. Hnh dng t bo chng Microcystis sp., LT4

Tp on vi t bo hnh cu,t bo c Tp on vi t bo hnh cu,t bo c mu xanh nht, ng knh t bo 2-5 mu xanh lam vi nhiu khng bo kh, m, tp hp thnh tng tp on nh. ng knh t bo 5-7 m, bao nhy khng mu.

Hnh 3.9. Hnh dng t bo chng Microcystis sp., LT5

Hnh 3.10. Hnh dng t bo chng Microcystis sp., LT6

Tp on vi t bo hnh cu,t bo c Tp on vi t bo hnh cu,t bo c mu xanh lam vi nhiu khng bo kh, mu xanh nht, ng knh t bo 4-7 m, ng knh t bo 3-7 m, bao nhy tp hp thnh tng tp on nh. khng mu.

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Hnh 3.11. Hnh dng t bo chng Microcystis sp., LT7 T bo hnh cu,t bo c mu xanh nht, ng knh t bo 3-6 m, tp hp thnh tng tp on nh.

Hnh 3.12. Hnh dng t bo chng Microcystis sp., LT8 Tp on vi t bo hnh cu,t bo c mu xanh lam vi nhiu khng bo kh, ng knh t bo 5-7 m, thng sp xp dy c.

Hnh 3.13. Hnh dng t bo chng Microcystis sp., LT9

Hnh 3.14. Hnh dng t bo chng Microcystis sp., LT10

Tp on vi t bo hnh cu,t bo c Tp on vi t bo hnh cu,t bo c mu xanh nht, cc khi nh hnh cu mu xanh lam, c ng knh t bo 2-4 ng knh t bo 2-4 m. m, cha khng bo kh Kt qu hnh 3.5; 3.6; 3.7; 3.8; 3.9; 3.10; 3.11; 3.12; 3.13; 3.14 cho thy c im hnh thi ca 10 chng trn c nhiu im tng ng, chng LT2
31

v LT8 u thy tp on vi t bo hnh cu, t bo c mu xanh lam vi nhiu khng bo kh bn trong, ng knh t bo 5-7 m. iu ny c th l do tnh thch ng ca tng chng trong iu kin mi trng nui cy. Miu t trn ph hp vi nhng miu t ca Watanabe [9, 49]. Tip theo chng ti la chn c nhng chng Microcystis mc nhanh, tt trn cc mi trng dch th v tm c thi gian nui cy thch hp cho cc chng nhm thu c lng sinh khi cho cc th nghim tip theo. 3.2.3. Kh nng sinh trng ca 10 chng Microcystis LT1, LT2, LT3, LT4, LT5, LT6, LT7, LT8, LT9 v LT10 trn cc mi trng khc nhau Cc khun lc xut hin trn cc a thch, c tch v kim tra thun khit di knh hin vi. Qu trnh c lp i lp li cho n khi thu c chng thun khit. Cc chng vi khun lam c tin hnh nui trn 3 mi trng dch th c chn lc l Bold3N, J v B12. T l ging ban u a vo l 4 105 tb/ml. C 4 ngy ly mu, kim tra kh nng sinh trng cho ti khi kt thc thi gian sau 23 ngy nui cy. Bng 3.1. Kh nng sinh trng ca 10 chng Microcystis trn mi trng Bold3N Thi gian (ngy) 3 7 11 15 19 23 K hiu chng ging (S lng t bo 106/ml) LT1 1,47 4,63 15,6 36.46 32,9 28,78 LT2 3,09 7,66 18,9 39,8 68,6 54,8 LT3 2,63 6,24 19,59 42,4 56,06 52,42 LT4 3,78 8,38 21,69 41,68 60,48 53,59 LT5 2,32 4,98 14,06 40,82 36,7 31,86 LT6 3,86 8,95 26,75 42,67 62,24 57,42 LT7 1,14 3,9 16,08 32,94 26,78 22,82 LT8 4,46 9,16 29,84 45,62 66,46 58,74 LT9 2,12 4,98 15,18 33,34 30,54 27,86 LT10 3,20 6,38 20,76 48,96 45,54 40,24

32

Hnh 3.15. Sinh trng ca cc chng Microcystis trong mi trng dch th Bold 3N

Hnh 3.16. Cc chng Microcystis pht trin tt trong mi trng dch th Bold 3N

Bng 3.2. Kh nng sinh trng ca 10 chng Microcystis trn mi trng J

Thi gian (ngy)

K hiu chng ging (S lng t bo 106/ml) LT1 2,27 5,36 LT2 4,08 8,42 LT3 3,56 7,42 LT4 3,98 8,26 LT5 2,32 5,18 15,36 39,82 48,7 43,62 LT6 3,92 9,25 24,56 45,79 72,69 67,84 LT7 1,84 4,92 15,28 40,42 36,28 32,86 LT8 4,58 9,46 25,24 46,48 74,76 68,36 LT9 2,32 5,76 17,20 43,56 38,78 34,56 LT10 4,28 8,88 23,62 50,69 69,79 64,44

3 7 11 15 19 23

16,68 21,39 19,98 20,16 43,64 43,86 41,48 40,68 40,29 76,46 66,36 68,48 36,87 64,54 62,75 63,69

33

Hnh 3.17. Sinh trng ca cc chng Microcystis trong mi trng dch th J

Hnh 3.18. Cc chng Microcystis pht trin tt trong mi trng dch th J

Bng 3.3. Kh nng sinh trng ca cc chng Microcystis trn mi trng B12
Thi gian (ngy)

K hiu chng ging (S lng t bo 106/ml) LT1 5,26 19,64 38,82 35,56 33,22 21,4 LT2 6,88 LT3 5,69 LT4 4,98 25,26 52,44 48,32 43,46 32,8 LT5 4,82 15,96 35,36 33,26 28,72 22,0 LT6 5,46 19,72 44,12 50,29 41,36 34,2 LT7 3,89 14,32 35,98 30,52 26,28 18,6 LT8 6,63 18,46 35,56 54,78 44,96 33,4 LT9 3,82 15,57 37,28 33,54 28,26 18,2 LT1 4,36 12,44 33,49 43,48 37,89 22,4

3 7 11 15 19 23

22,48 21,12 42,69 49,66 36,45 28,6 39,88 46,48 42,46 27,0

34

Hnh 3.19. Sinh trng ca cc chng Microcystis trong mi trng dch th B12

Hnh 3.20. Cc chng Microcystis pht trin tt trong mi trng dch th B12

Kt qu bng 3.1 cho thy cc chng Microcystis sinh trng trn mi trng Bold 3N, mt t bo ca cc chng t t 32,94 68,6 106 sau 15 n 19 ngy nui cy. c bit chng LT2 mt t bo t cao nht 68,6 106 /ml sau ngy th 19. bng 3.2 cho thy cc chng Microcystis sinh trng trn mi trng J, mt t bo ca cc chng t t 40,42 76,46 106 /ml sau 15 n 19 ngy nui cy. Trong chng LT2 vn pht trin vi mt t bo t cao nht 76,46 106 /ml sau ngy th 19. bng 3.3 cho thy cc chng Microcystis sinh trng trn mi trng B12, mt t bo ca cc chng t t 35,36 54,78 106/ml sau 12 n 15 ngy nui cy. mi trng ny chng LT8 pht trin vi mt t bo t cao nht 54,78 106/ml sau ngy th 15. T nhng kt qu trn, chng ti nhn thy kh nng sinh trng ca cc chng khc nhau l khc nhau. Hai chng LT2 v LT8 u pht trin tt nht trn cc mi trng dch th v thi gian sinh trng ca chng ch trong 15 ngy, do , chng ti la chn 2 chng ny tip tc nui thu sinh khi cho nhng nghin cu tip theo. 3.2.4. Thnh phn acid bo trong t bo ca 2 chng LT2 v LT8 Bn cht ha hc ca cc cht d tr v thnh t bo ca vi to ng vai tr quan trng trong vic phn bit cc nhm vi to khc nhau. Trong nghin cu ny

35

chng ti tin hnh phn tch thnh phn acid bo ca 2 chng LT2 v LT8 trn my sc k kh. Kt qu c trnh by bng 3.4 Bng 3.4. Thnh phn acid bo c trong t bo ca 2 chng LT2 v LT8 STT Thnh phn cc acid bo Lng acid bo c trong t bo (g/mg mu) LT2 0,01572 0,0133 1,302 0,0906 0,1926 0, 0822 0,1574 LT8 0,01566 0,272 0,9683 0,1458 0,1787 0,0718 0,0577

1 2 3 4 5 6 7

Myristic acid ( C14H28O2) Palmitoleic acid (C16H30O2) Palmitic acid (C16H32O2) 6,9,12- Octadecatrienoic acid ( Linolenic, GLA) (C18H30O2) 9,12- Octadecadienoic acid (Linoleic) (C18H32O2) 12- Octadecenoic acid (Oleic) (C18H34O2) Octadecanoic acid (Stearic) (C18H36O2)

Bng 3.4 cho thy kt qu phn tch thnh phn acid bo ca 2 chng Microcystis LT2 v LT8 c 7 loi acid bo no v khng no t C14 n C18, trong thnh phn acid palmitic (C16H32O2) chim lng ln nht vi 1,302 g/mg trng lng kh chng LT2 (74% tng s acid bo) v 0,9683 g/mg chng LT8 (56%). Cc acid bo khng no ch chim t l thp. y c th l c im ca chi Microcystis. Nhn chung hai chng LT2 v LT8 c hm lng acid bo tng i ging nhau, ch khc nhau c bn hm lng acid palmitoleic v acid stearic. 3.2.5. Xc nh v phn tch trnh t rADN 16S y ngoi vic phn loi 2 chng LT2 v LT8 bng phng php truyn thng da trn cc c im hnh thi, nui cy, c im sinh l, c hai chng LT2 v LT8 ny u thuc chi Microcystis. xc nh r 2 chng thuc loi no ca chi Microcystis chng ti tin hnh phn loi theo phng php sinh hc phn t da trn phn tch gii trnh t rADN 16S. y khng phi l mt cng vic n gin v my tnh s gip ta hon ton cc tnh ton cn thit ti thiu ho s khong trng v cc li bt cp sai ca cc trt t c so snh. S phn loi da vo qu trnh phn tch mi quan h da trn ADN trong tin ho ca phn loi
36

chng loi pht sinh hoc da vo nhng c im tng t nhau trong phn loi hnh thi u c th hin nhng s dng cy gi l cy ph h. Cy ph h cho php nhn bit mi quan h di truyn gia cc loi v gia cc chng cng loi. S dng Cluxtal 18.1 v chng trnh in ton MEGA 2.1 cng cc thng s tin ho cn thit cho thy mi quan h c thit lp trn c s phn tch phn on, so snh gia cc vng bo ton ca nhng on gen rADN 16S.
100 87 100 100 100 73 Microcystis aeruginosa EU078505
Microcystis aeruginosa EU078504

Microcystis aeruginosa EU078503

LT2 LT8
Microcystis wesenbergii AB038538 Microcystis viridis AY121357 Microcystis viridis AY065976 100 88 Microcystis ichthyoblabe AB012338 Microcystis aeruginosa AB023261 Anabaena circinalis EU078518 Anabaena circinalis EU780161 Anabaena circinalis EU780160 Lyngbya majuscula AF510965 Lyngbya majuscula AF510964

100

100 96 100 0.005

Hnh 3.21. Cy ph h da trn phn tch gii trnh t rADN 16S ca 2 chng LT2, LT8 v cc loi c quan h h hng gn Cy ph h (hnh 3.21) cho thy mi quan h gn gia chng LT2 (on gen 456bp) v LT8 (on gen 457bp) vi 13 trt t nucleotit ca cc chng thuc Cyanobacteria trong ngn hng d liu gen. Chng nm trn cng mt nhnh v cng nhm vi loi Microcystis aeruginosa EU078503 vi tng ng l 100% (chng LT2) v 99% (chng LT8). Cy ph h v theo phng php neighbour joining thc o = 0,05 Knor trong trnh t nucleotid. Cc gi tr hin th ti cc nhnh cy l kt qu phn tch boostrap vi lp li 1000 (vi nhng gi tr >50% mi c th hin trn cy). S khc bit gia 2 chng l khng ng k v nm trong phm vi khc bit cng loi (conspecific variation). Da vo cc kt qu phn tch c im sinh l,
37

ho v cy ph h cho thy chng c nhiu c im chung v vy 2 chng LT2 v LT8 u thuc loi Microcystis aeruginosa. 3.3. TCH CHIT C T MICROCYSTIN Chng ti tin hnh tch chit c t microcystin theo phng php nh miu t phn phng php (mc 2.2.5). 3.3.1. Sc k bn mng (TLC)

Microcystin chun

Hnh 3.22. Kt qu chy TLC trc khi nhum nihyrin

Hnh 3.23. Kt qu chy TLC sau khi nhum nihyrin

T kt qu hnh 3.22 v 3.23 ta thy trn bn silicagel chy mu tch chit microcystin xut hin cc bng tng ng vi bng MC chun. iu chng t s c mt ca c t trong mu chit. Da vo kt qu ny, chng ti tin hnh phn tch v xc nh hm lng microcystin trong dch chit trn my quang ph v sc k lng cao o HPLC. 3.3.2. Xc nh hm lng microcystin trn my quang ph Cc bng cha microcystin ca dch chit chy trn sc k bn mng c co v ti chit rt bng methanol 80%. Sau lc qua ct C18, ly 1ml dch lc, pha long 7000, 8000, 9000 ln. em cc dung dch o trn my quang ph bc sng 242 nm. Kt qu c trnh by hnh 3.24

38

1.2 1 y = 117.17x R = 0.967

G i tr OD (242nm )

0.8 0.6 0.4 0.2 0 0 0.002 0.004 0.006 0.008 0.01 Nng m ic roc ystin

Hnh 3.24. th ng chun microcystin Da vo phng trnh ng chun microcystins y = 117,17x v gi tr mt quang o c ca dch pha long trn my quang ph, xc nh c hm lng microcystin trong dch chit chng LT2 l 102,4ng/ml v chng LT8 l 108,8ng/ml. 3.3.3. Hm lng microcystin trn sc k lng cao p (HPLC) Dung dch sau khi lc qua ct C18, tip tc c lc qua mng lc. Sau , cho bay hi bng dng kh nitrogen ti 50l. Mu c phn tch trn my sc k lng cao p ti bc sng 242 nm. Dng microcystin ca hng Sigma, lm cht chun. Kt qu c trnh by hnh 3.25. a, b, c, d, e

39

Hnh 3.25a. Sc k microcystin RR chun

Hnh 3.25b. Sc k microcystin YR chun

40

Hnh 3.25c . Sc k microcystin LR chun

Hnh 3.25d. Sc k microcystin ca chng LT2

41

Hnh 3.25e. Sc k microcystin ca chng LT8 Nhn vo kt qu sc k ca dch chit trn HPLC, trn sc k thy xut hin cc ct biu hin c trng ca c t microcystin. Trong biu , c s xut hin ca cc ct vi thi gian lu l 4,776 pht, 7,409 pht, 10,422 pht gn tng ng vi thi gian lu ca MC - RR, YR, LR chun, chng t trong dch chit c cha c ba loi c t. Ngoi cc ct c trng ny cn xut hin cc ng ct khc l cc ct ca dung mi. Da vo din tch ct xc nh c hm lng ca cc thnh phn c t nh sau

42

Bng 3.5. Hm lng microcystin ca 2 chng LT2 v LT8 K hiu chng LT2 Lng MC (ng/mg trng lng kh) LT8 Lng MC (ng/mg trng lng kh) Lng MC- RR Thi Vng xut gian hin ct 4 715 29,705 Lng MC YR Thi gian Vng xut hin ct 7 378 26,570 Lng MC - LR Thi gian Vng xut hin ct 10 27 30,460

474,3 476 7488

132,6

114,1 10422

31,450

27,140

30,450

512,8

236,8

114,25

Bng 3.5 cho thy kt qu sc k trn HPLC cho ra cc ct biu hin c trng c t t mu vi khun lam, microcystin c thm d vi nhiu gii hn. Tuy nhin, trong biu c s xut hin ca cc ct khng mong mun l cc ct ca dung mi. Kt qu phn tch cho thy c 2 chng LT2 v LT8 t bo u cha c t vi cc loi microcystin trong t bo nh MC - LR, -YR v -RR. Hm lng MCLR trong chng LT2 l 114,1 ng, LT8 l 114,25 ng/mg trng lng kh. Hm lng MC-YR trong chng LT2 l 132,6 ng, LT8 l 236,8 ng/mg trng lng kh. Hm lng MC - RR trong chng LT2 l 474,3 ng, LT8 l 512,8 ng/mg trng lng kh. T kt qu bng 3.5 cho thy trong dch chit microcystin t t bo kh Microcystis, hm lng c t MC RR t ln nht 512,8 ng/mg trng lng kh so vi lng c t MC - LR. Trong khi , hm lng MC - LR cho php trong nc ung ca WHO l 1 g/L [48]. Nh vy nng MC - LR trong hai chng ny 1,141g/L vi LT2 v 1,1425 g/L vi LT8 vt qu ngng cho php ca WHO, s l nguy c e da cho ngun nc nu sau khi t bo phn gii, ton b lng c t ni bo gii phng vo trong nc.

43

3.4. PHN LP V TUYN CHN CC CHNG VI KHUN NC C KH NNG PHN GII MICROCYSTIN 3.4.1. Phn lp cc chng vi khun nc Do tm quan trng v mc gy c ca cc vi khun lam n hoa trong cc thu vc ngy cng gia tng nn cc nghin cu v bn vng v phn gii c t ca chng tr nn rt cn thit. Trong iu kin t nhin, cc vi sinh vt, c bit cc vi khun nc ng vai tr quan trng trong vic phn gii c t khng ho tan c gii phng vo trong nc. Ngi ta chng minh rng microcystin c th b phn gii bi cc vi sinh vt c trong h sinh thi thu vc t nhin [28]. Ngoi ra c cc bng chng cho rng s phn gii sinh hc microcystin xy ra nhanh hn cc thu vc thng b to lam c n hoa [49, 51]. Ngy nay khi hin tng n hoa ca to lam c trong cc thu vc tr nn nghim trng th cc nghin cu tp trung vo vn vi khun nc l cch gim thiu tc hi ca vi khun lam v c t ca chng. Do vy, cc chng vi khun nc c tuyn chn v phn lp t 6 a im vi s xut hin Microcystis theo t l 1:1 (tnh theo ml) c nui cy lm giu trong cc ng nghim dung tch 10 ml cha 3 ml mi trng NA, M7 [44]. Sau em nui gi trong iu kin nhit 30oC di nh sng n neon yu vi cng nh sng l 1000 lux theo quang chu k l 12 gi chiu sng : 12 gi ti. Sau 24 nui cy, em ly tm dch th vi tc 7000 vng/10 pht thu sinh khi. Ra sinh khi 3 ln bng dung dch NA, M7 v trng. Sau em nui cy li trong thi gian 24 gi di cc iu kin m t trn ( kch thch cc chng vi khun thch ng vi s phn gii t bo M. aeruginosa v phn gii c t microcystin).

44

Hnh 3.26. Nui cy lm giu cc chng vi khun nc trn mi trng NA v M7

Hnh 3.27. Cy pha khun lc vi khun trn mi trng NA

Hnh 3.26 v 3.27 cho thy sau 24 gi nui cy lm giu trn 2 mi trng. Chng ti nhn thy mu ca dch nui cy xanh trn mi trng MA v cn trn mi trng M7 khng thy xut hin mu xanh. iu ny cho thy mi trng. MA thch hp cho vic phn lp v nui cy cc chng vi khun nc. Cc mu tip tc c cy ria trn mi trng thch NA. Sau 12 gi quan st s xut hin cc khun lc c tit sc t xanh vo trong mi trng. Tch cc khun lc n cy ria trn mi trng thch nhiu ln cho n khi tch c chng thun khit. Trong th nghim ny chng ti chn c 2 chng vi khun c k hiu N4 v N7 c kh nng sinh trng tt ch sau 12 gi tit sc t xanh vo mi trng. Nui gi ging gc vi khun trn mi trng thch nghing (mi trng NA) v bo qun nhit 4oC, dng cho cc nghin cu tip theo.

45

Bng 3.6. c im hnh thi ca 2 chng vi khun nc N4 v N7 c im Hnh thi khun lc sau 30 gi Hnh dng t bo Nhum Gram Menaquinon Kh nng di ng Phn ng lng trng Oxygen Kh nng pht quang di n UV K hiu chng N4 Khun lc tit sc t xanh vo trong mi trng, b mt t v nhn. T bo hnh que nh, c roi Q10 C kh nng di ng nhanh K kh khng bt buc C kh nng pht quang N7 Khun lc tit sc t xanh vo trong mi trng, b mt t v nhn. T bo hnh que nh, c roi Q10 C kh nng di ng nhanh K kh khng bt buc C kh nng pht quang

Ghi ch : - (khng c phn ng) Bng 3.6 cho thy c 2 chng u c kh nng di ng v c kh nng pht quang di tia cc tm v l vi khun Gram m. c bit c 2 chng u cha Q10 c trung ca cc vi khun nc Sphingomonas. Bng 3.7. c im sinh l, sinh ho ca 2 chng N4 v N7 K hiu chng c im Kh Tinh bt nng thy phn Protenase Catalase Ln men glucose Phn ng VP 0,5% 1% Kh 1,5% nng chu 2% mui 3% 5% Ghi ch: N4 + ++ ++ + ++ ++ ++ ++ ++ ++ N7 + + ++ ++ ++ ++ ++ ++ ++ Kh nng ng ha Fructose Rgamnose Ribose Sucrose Glucose Cellulose Dextrin Mantose Xylose Galactose Trehalose Lactose K hiu chng N4 ++ ++ ++ + ++ + ++ ++ ++ ++ ++ ++ N7 ++ ++ ++ + ++ + ++ ++ ++ ++ ++ ++

+ C phn ng

- Khng xy ra phn ng
46

Bng 3.7 cho thy 2 chng vi khun N4 v N7 u khng kh nng phn gii tinh bt. C 2 chng u c kh nng phn hy protein, ngha l c hot tnh protease. Ngoi ra c 2 chng ny u cho hot tnh catalase rt mnh. Chng cng c kh nng chu mui tt t 0,5- 5% v ng ha c hu ht cc loi ng. iu ny hon ton ph hp vi nghin cu ca David m t cc chng vi khun Sphingomonas phn gii c t MLR [20]. 3.4.2. Xc nh v phn tch trnh t rADN 16S 2 chng N4 v N7 c c nhiu hn na thng tin xc nh v v tr phn loi v pht sinh chng loi ca 2 chng N4, N7. ADN genom ca 2 chng N4 v N7 c dng lm khun khuych i on ADN bng phn ng PCR, s dng cp mi 27f v 1252r. Chng ti xc nh trnh t rADN 16S ca n v so snh vi trnh t c sn trong Ribosomal Data Project. Tm kim trnh t tng ng trc tuyn ch ra rng, trong s cc loi cng b, trnh t rADN 16S (on 700bp) ca N4 ging trnh t ca Sphingomonas mali Y09638 (99%), Sphingomonas asaccharolytica Y09639 (99%). Vi chng N7 (on 564bp) ging Sphingomonas asaccharolytica Y09639 (100%) v Sphingomonas mali Y09638 (99%). Khong cch tin ha c tnh ton thit lp c s d liu bao gm trnh t ca chng N4, N7 v 27 trnh t khc ca nhm Sphingomonas phn lp alpha ca Proteobacteria vi Rhodospirillum rubrum l outgroup (ngoi nhm). Cy pht sinh chng loi c xy dng li trn c s cc s liu ma trn v khong cch thu c nh (hnh 3.28, 3.29). Chng N4, N7 phn nhnh su t 1 nhm chnh, nhm ny bao gm mt s thnh vin ca chi Sphingomonas. Cc loi cng b ca chi Sphingomonas khc nhau v pht sinh chng loi Hai cy ph h v theo phng php neighbour joining thc o = 0,02 Knor trong trnh t nucleotid. Cc gi tr hin th ti cc nhnh cy l kt qu phn tch boostrap vi lp li 1000 (vi nhng gi tr >50% mi c th hin trn cy ph h).

47

0.02 Sphingomonas suberifaciens D13737 Sphingomonas yanoikuyae SPP16SRR7 Sphingomonas chlorophenolica X87162 50 Sphingomonas flava 100 68 Sphingomonas agrestis Y12803 X87164 Erythromicrobium ramosum AB013 100 91 Erythrobacter litoralis AB013354 Erythrobacter longus M59062 Porphyrobacter neustonensis L0 78 55 67 Porphyrobacter tepidarius D84429 Sphingomonas subarctica X94102 86 Novosphingobium capsulatum D16147 Sphingomonas subterraneae U20773 99 99 Novosphingobium aromaticivorans U20756 67 71 Erythr ursincola AB024289 100 Sphingomonas natatoria AB024288 Sphingomonas macrogoltabida D13728 Sphingomonas terrae D13727 92 58 88 Sphingomonas adhaesiva D13722 N4 Sphingomonas mali Y09638 56 100 Sphingomonas asaccharolytica Y09639 Sphingomonas trueperi X97776 Sphingomonas paucimobilis D13725 86 Sphingomonas sanguinis D13726 98 62 Sphingomonas parapaucimobilis D13724 Sphingomonas sibiricus Y10678 Rhodospirillum rubrum D30778

Hnh 3.28. Cy ph h da trn phn tch gii trnh t rADN 16S ca chng N4 v cc loi c quan h h hng gn

48

0.02 Sphingomonas macrogoltabida SPP16SRR Sphingomonas terrae D13727 54 Sphingomonas adhaesiva D13722 Sphingomonas suberifaciens D13737 Erythr ursincola AB024289 100 Sphingomonas natatoria AB024288 Sphingomonas yanoikuyae D13728 Sphingomonas flava X87164 75 100 Sphingomonas chlorophenolica X87162 93 96 Sphingomonas agrestis Y12803 Sphingomonas subarctica X94102 Novosphi capsulatum D16147 78 Sphingomonas subterraneae U20773 96 87 Novosph aromaticivorans U20756 56 Erythrobacter litoralis AB0133 Erythrobacter longus M59062 69 100 68 Porphyrobacter neustonensis L0 Erythromicrobium ramosum AB013 99 Porphyrobacter tepidarius D844 N7 58 Sphingomonas asaccharolytica Y09639 61 Sphingomonas mali Y09638 96 Sphingomonas trueperi X97776 Sphingomonas paucimobilis D13725 80 Sphingomonas sanguinis D13726 94 67 Sphingomonas parapaucimobilis D13724 Sphingomonas sibiricus Y10678 Rhodospirillum rubrum D30778 99

Hnh 3.29. Cy ph h da trn phn tch gii trnh t rADN 16S ca chng N7 vi cc loi c quan h h hng gn 3.5. KH NNG PHN GII C T MICROCYSTIN CA 2 CHNG Sphingomonas N4 V N7 3.5.1. Hiu qu phn gii microcystin ca 2 chng N4 v N7 Hm lng microcystin trong dch chit chng LT2 l 102,4ng/ml v chng LT8 l 108,8ng/ml. S phn hy c bt u bng cch b sung dch chit microcystin th vo cc mu nc v trng (dng bnh c th tch 1000ml b sung 600 ml) thu c nng microcystin tng s l 10 g/l. Cc chng vi khun phn gii vi khun lam c a vo bnh vi mt t bo nui cy l 2,5 106 t bo/ml. Cc bnh sau c nui tnh trong iu kin phng th nghim (30oC). Kim tra mu sau 1 gi bng cch ly 50 ml dch em lc qua mng lc kch thc

49

0,2 m. Dch thu c bo qun trong catrit Sep-Pak ODS trc khi tin hnh phn tch. Th nghim i chng (khng c vi khun) c tin hnh song song. Hai chng vi khun N4, N7 c tuyn chn dng lm phn gii microcystin cho cc th nghim c nng microcystin cao t 2, 5 v 10 g/l theo nh phng php trnh by. Hiu qu phn gii microcystin c xc nh nh phng php bn thi gian nh m t ca Kenefick. Kt qu c trnh by bng 3.8 v hnh 3.30. Bng 3.8. Hng s tc phn gii (K) v thi gian bn phn gii microcystin cc nng khc nhau Chng vi khun N4 N7 0,076 0,094
4 3.5

Nng microcystin ban u 2 g/l (1) K(1/ngy) t0,5(ngy) 0,074 0,092 5 g/l (2) K(1/ngy) 0,045 0,052 t0,5(ngy) 0,102 0,156 10 g/l (3) K(1/ngy) 0,043 0,056 t0,5(ngy) 0,140 0,153

Hm lng MC phn gii

N4 N7

3 2.5 2 1.5 1 0.5 0 2 5 10

Nng microcystin ban u

Hnh 3.30. Thi gian bn phn gii microcystin cc nng khc nhau ca 2 chng N4, N7.

50

Kh nng phn gii c t ca chng kh nhanh, c bit nng c t thp 2 g/l, thi gian bn phn gii microcystin tng i ngn, i vi chng LT2 l 0,074 ngy (1 gi 47 pht), i vi LT8 l 0,092 ngy (2 gi 12 pht). Vi nng microcystin cao gp 5 ln (2 g/l), thi gian bn phn gii ca 2 chng LT2, LT8 ln lt l 0,14 ngy (3 gi 21 pht) v 0,153 ngy (3 gi 40 pht). Hng s tc ban u trong cc nghin cu ca chng ti l t 0,43-0,56 /ngy, so snh vi hng s tc trong bo co ca Jones [28] t 0,05-0,3/ngy, Jones o mc phn gii microcystin bng cc th tch khc nhau ca t bo cn li khng b tch chit t mi trng nui cy vi khun phn gii microcystin. Hng s tc theo Kenefick [28 ] l 0,25/ngy cng phn nh s phn gii chm hn. Cc kt qu trn cho php chng ti s b kt lun 2 chng vi khun Sphingomonas N4, N7 phn lp t nc h c hot tnh phn gii c t microcystin. Kh nng phn gii i vi tng loi c t microcystin c th s c tin hnh trong cc nghin cu tip theo. 3.5.2. Xc nh iu kin nhit thch hp cho s phn gii microcystin ca chng Sphingomonas N4 v N7 Mt trong cc yu t gy nh hng nhiu nht ti tc phn gii c t microcystin ca vi khun Sphingomonas c xc nh l nhit . Chnh v vy, trong nghin cu ny chng ti tin hnh cc th nghim kh nng phn gii c t microcystin bi vi khun Sphingomonas cc nhit khc nhau l 20, 25, 30C, di nhit ny cho ph hp vi iu kin t nhin ca h vi nng microcystin ban u l 40 g/l. Kt qu c trnh by trong bng 3.9, 3.10 v hnh 3.31, 3.32.

51

Bng 3.9. T l % microcystin khng b phn gii do chng Sphingomonas N4 cc nhit khc nhau Nhit (C) 20 25 30 0 100 100 100 T l % microcystin khngb phn gii sau cc khong thi gian (ngy) 1 82 74 56 2 80 52 28 3 75 46 16 4 68 38 6 5 56 31 0 6 49 23 7 33 0

120

T l phn % microcystin khng b phn gii

20 25 30

100 80 60 40 20 0 0 1 2 3 4 5

Thi gian (ngy)

Hnh 3.31. Kh nng phn gii microcystin ca vi khun Sphingomonas N4 cc nhit khc nhau Bng 3.10. T l % microcystin khng b phn gii do chng Sphingomonas N7 cc nhit khc nhau Nhit (C) 20 25 30 T l % microcystin b phn gii sau cc khong thi gian (ngy) 0 100 100 100 1 88 81 58 2 79 63 43 3 70 46 31 4 65 35 19 5 58 28 11 6 47 22 0 7 31 0

52

120

T l % microcystin khng b phn gii

100 80 60 40 20 0 0 1 2 3 4 5 Thi gian (ngy) 6 7 20 25 30

Hnh 3.32. Kh nng phn gii ca vi khun Sphingomonas N7 nhit khc nhau Hnh 3.31 v 3.32 cho thy tc phn gii microcystin ca hai chng vi khun Sphingomonas N4 v N7 ph thuc nhiu vo nhit . Tc phn gii c s khc bit r rng cc nhit t 20-30oC. Trong khong nhit th nghim ny, tc phn gii t l thun vi s gia tng nhit . Ti nhit 20C t l phn gii t 44% v 42% sau 5 ngy, ln lt vi N4 v N7. Tc phn gii ln nht t c khi tin hnh kho st 30C, sau 5 ngy (N4) v 6 ngy (N7), hm lng c t microcystin cn li trong dch chit nm di ngng pht hin. S phn gii microcystin bt u trong vng 1 ngy khi nhit nm trong khong 20 - 30oC. Trong thc t, thi gian n hoa cc i h Hon kim ri vo khong cui thng 3, u thng 5, khi m cc vng xanh dy ni trn b mt h nh mt lp sn, dt vo bng rm cc tn l r trn mt h. Lc ny ngng nhit dao ng trong khong 25 - 30oC, ti thch cho s pht trin ca Microcystis. Mi quan h dinh dng mt thit gia Microcystis v Sphingomonas ko theo hot ng mnh m ca loi vi khun ny vo cng thi im. Th nghim ny l c s cho nhng nghin cu tip theo v iu kin tuyn chn v phn lp thm cc chng vi khun c kh nng phn gii cc loi c t microcystin.

53

KT LUN V KIN NGH Kt lun 1. H Hon Kim ang tnh trng nhim, hm lng COD, BOD vt tiu chun Vit Nam, TCVN 4942- 1995 (loi B) 20 ln. Vi gi tr NH4+ t 0,1290,699 (mg/l), NO2- t 0.012- 0,05 (mg/l), NO3- t 0,047- 0,129 (mg/l), PO43- t 0,08- 0.64 (mg/l) chng t h c hm lng dinh dng N, P kh cao. 2. phn lp thun khit v nui cy 10 chng vi khun lam thuc chi Microcystis theo phng php ca Shirai c ci tin. Mi trng thch agarose B12 vi t l 0,4% l mi trng thch hp nht cho vic phn lp Microcystis. 3. Cc chng Microcystis u sinh trng tt trn mi trng Bold 3N vi s lng t bo t t 32,94 68,6 106 tb/ml sau 15 n 19 ngy nui cy. c bit chng LT2 s lng t bo t cao nht l 68,6 106 tb/ml sau ngy th 19. Trn mi trng J, cc chng Microcystis sinh trng s lng t bo ca cc chng t t 40,42 76,46 106 tb/ml sau 15 n 19 ngy nui cy. Trong chng LT2 vn pht trin vi s lng t bo t cao nht 76,46 106 tb/ml sau ngy th 19. Trn mi trng B12, s lng t bo t t 35,36 54,78 106 tb/ml sau 12 n 15 ngy nui cy. mi trng ny chng LT8 pht trin vi mt t bo t cao nht 54,78 106 tb/ml sau ngy th 15. 4. C 7 loi axt bo khng no dng C15, C17 v C19 trong t bo 2 chng Microcystis LT2 v LT8. trong thnh phn acid palmitic (C16H32O2) chim lng ln nht vi 1,302 g/mg trng lng kh chng LT2, chng LT8 l 0,9683 g/mg trng lng kh. Kt qu cho thy c s sai khc nhau v lng ca 7 loi acid bo c trong t bo 2 chng LT2 v LT8. 5. Da vo phng php phn loi truyn thng theo phn loi hnh thi hc v phng php phn loi hin i da trn phn tch trnh t rADN 16S, 2 chng vi khun LT 2, LT8 c xc nh l Microcystis aeruginosa, 2 chng vi khun nc N4 v N7 c xc nh thuc cc loi Sphingomonas mali v Sphingomonas asaccharolytica.

54

6. Phn tch trn TLC cho thy dch chit t sinh khi Microcystis kh c cha c t, hm lng microcystin th phn tch trn quang ph vi chng LT2 l 102,4ng/ml v chng LT8 l 108,8ng/ml 7. Da vo kt qu phn tch trn HPLC, hm lng c t MC RR t ln nht l 512,8 ng/mg trng lng kh, c t MC YR l 236,8 ng/mg v thp nht l c t MC LR: 114,25 ng/mg. 8. Nhit thch hp cho s phn gii microcystin ca cc 2 chng vi khun Sphingomonas sp., N4, N7 l 30C. Sau 7 ngy s phn gii gn nh hon ton l 100%. .Kin ngh - Cn tip tc gim st s n hoa ca cc chng vi khun lam gy c h Hon Kim - H Ni v mi lin quan ca chng vi cc iu kin mi trng. - Cn tip tc tin hnh nhng nghin cu v cc bin php gim thiu v ngn nga tc hi do vi khun lam gy c gy ra, trong ch n cc bin php sinh hc va cho chi ph r m li thn thin vi mi trng.

55

TI LIU THAM KHO Ti liu ting Vit 1. ng nh Kim, ng Hong Phc Hin (2006), iu tra pht hin to c ti cc thu vc trng im ca H Ni v mt s tnh pha Bc lm c s cho vic xy dng ch tiu quan trc sinh hc trong gim st cht lng ngun nc, Bo co Tng kt nhim v khoa hc cng ngh phc v qun l nh nc v bo v mi trng, Vin Cng ngh Mi trng Vin Khoa hc v Cng ngh Vit Nam. 2. ng nh Kim, ng Hong Phc Hin (1999), Cng ngh Sinh hc vi to. Nh xut bn Nng nghip.Tr 120 134. 3. ng nh Kim, ng Hong Phc Hin, Dng Th Thu, Dng c Tin (1997), Pht hin c mt s vi to c trong cc ao h H Ni. K yu Vin Cng ngh Sinh hc, tr.408-412. 4. Chu Vn Thuc & cs (1998), Bc u nghin cu v to bin c hi vng ca sng ven bin min bc Vit Nam, Ti nguyn v Mi trng bin, tp 5, tr. 155-163, Nh xut bn Khao hc v K thut. 5. Dng c Tin & cs (2002), Bo v ngun gen vi to (microalgae) c hu qu him h Hon Kim H Ni, Bo co khoa hc trong chng trnh ti tr bo v mi trng v gn gi di sn vn ho - Cng ty Ford Motor. 6. Dng c Tin (1995), Phn loi Vi khun lam Vit Nam. Nh xut bn Nng nghip. 7. Dng c Tin, V Vn Chi (1978), Phn loi Thc vt hc bc thp. Nh xut bn i hc v Trung hc chuyn nghip.Tr 87-95. 8. Trnh Th Thanh (2000), c hc Mi trng v Sc kho con ngi. Nh xut bn i Hc Quc gia H Ni. tr 39-45. 9. Don oan Trinh (2003), H Ni- a ch du lch vn ho. Nh xut bn Vn ho thng tin.tr 36-40. Ti liu ting Anh

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10. A.Munkhong, A.Mahakhant, P.Thavipoke, V.Arunpairojana (2005), Lysis of Microcystis cells and degration of microcystins by soil bacteria, Algal Culture Collection and the Environment, pp. 109-119. 11. American public health association. (1975), Standard methods for the examination of water and wastewater, 4th edition. 12. Ball As, Williams M, Vincent D and Robinson J (2001), Algal growth control by a barley straw extract. Bioresour. Technol. 77, pp.177-181. 13. Barrett PRF, Curnow J and Little John JW (1996), The control of diatom and cyanobacterial blooms in reservoirs using barley straw. Hydrobiol. 340, pp. 307-311. 14. Carmichael W (1995), Toxic Microcystis and the environment. In: Watanabe M, Harada K.pp. 230-234. 15. Carmichael, W.W. (1992), A Status Report on Planktonic Cyanobacteria (Blue-Green Algae) and their Toxins. EPA/600/R-92/079, United States Environmental Protection Agency, Cincinnati, Ohio. 16. Chorus I, Bartram J, ed. (1999), Toxic cyanobacteria in water. A guide to their public health consequences, monitoring and management. Published by E & FN Spon on behalf of the World Health Organization. 17. Codd, G.A, Ward, C.J. and Bell, S.G. (1997), Cyanobacterial toxins: occurrence, modes of action, health effects and exposure routes. In: J.P. Seiler and E. Vilanova [Eds], Applied Toxicology: Approaches through Basic Science. Archives of Toxicology Supplement 19, Springer-Verlag, Berlin, pp. 399-410. 18. Codd, G.A. (1983), Cyanobacterial poisoning hazard in British freshwaters. Vet. Rec., 113. pp. 223-224. 19. Dillenberg HO, Dehnel MK (1960), Toxic waterbloom in Saskatchewan, Canadian Medical Association Journal, 83: pp. 11511154. 20. Dvid G. Bourne (1996), Enzymatic pathway for the bacteria degradation of the Cyanobacteria cyclic peptide toxin Microcystin LR. Pp.1086-4094 21. Falconer I (1996), Potential impact on human health of toxic cyanobacteria. Phycologia, 35(Suppl. 6): pp. 611.

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22. Falconer I, Buckley TH (1989), Tumor promotion by Microcystis sp., a blue-green algae occurring in water supplies. Medical Journal of Australia, 150: pp. 351352. 23. Falconer I, ed. (1993), Algal toxins in seafood and drinking water. New York, NY, Academic Press, pp. 224. 24. Fastner J, Neumann U, Wirsing B, Weckesser J, Wiedner C, Nixdorf B, Chorus I (1999), Microcystins (heptapeptides) in German fresh water bodies. Environmental Toxicology, 14(1):pp. 1322. 25. Gibson MT, Welch IM, Barrett PRF and Ridge I (1990), Barley straw as an inhibitor of algal growth II: Laboratory studies. J. Appl. Phycol. 2. pp. 241-248. 26. Jianzhong Chen, Zhili Liu, Guanju Ren, Pengfu Li and Yiwen Jiang (2004), Control of Microcystis aeruginosa TH01109 with batangas mandarin skin and dwarf banana peel, Technical note in School of Life Sciences, Nanjing University, Nanjing 210093, China.pp. 1200 1211. 27. Jones, G. J., and P. T. Orr. (1994), Release and degradation of microcystin following algicide treatment of a Microcystis aeruginosa bloom in a recre- ational lake, as determined by HPLC and protein phosphatase inhibition assay. Water Res. 28:pp. 871876.
28. Kenefick, S.L., S.E. Hrudey, H.G. Peterson and E.E. Prepas. (1993) Toxin release from Microcystis aeruginosa after chemical treatment. Water Sci. Tech. 27:pp. 433-440.

29. L.R. Song, N.Q.Gan, H.Pan (2005), Toxic Microcystis blooms and microcystin composition in eutrophic lakes in China, Algal Culture Collection and the Environment, pp. 89-108.
30. MacKintosh C, Beattie KA, Klumpp S, Cohen P, Codd GA (1990), Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2a from both mammals and higher plants. Federation of the European Biochemical Society Letters, 264:pp. 187192.

31. Makoto Shirai, Katsumi Matumaru, Akio Ohotake, Yoshichika Takamura, Tokujiro Aida and Masayasu Nakano (1989), Development of a Solid

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Medium for Growth and Isolation of Axenic Microcystis Strains (Cyanobacteria), Appl Environ Microbiol. 1989 October; 55(10): pp. 2569-2571.

32. Miller L.T (1982), Single derivatization method for routine analysis of bacterial whole-cell fatty acid methyl esters, including hydroxy acids. J.Clin. Microbiol.,16. pp. 584 586. 33. Nakagawa, M., Y. Takamura, and 0. Yagi. (1987), Isolation of the slime from a cyanobacterium, Microcystis aeruginosa K-3A. Agric. Biol. Chem. 51:pp. 329-337. 34. Padisork J (1997), Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba Raju, an expanding, highly adaptive blue-green algal species: worldwide distribution and review of its ecology. Archiv fr Hydrobiologie, Suppl., 107:pp. 563593. 35. Pillinger JM, Cooper JA and Ridge I (1994), Role of phenolic compounds in the antialgal activity of barley straw. J. Chem. Ecol. 20, pp. 15571569. 36. Ridge I, Pillinger JM and Walters J (1995), Alleviating the problems of excessive algal growth. In: DM Harper and AJD Ferguson (eds.) The Ecological Basis for River Management. John Wiley. Chichester,pp. 211-218. 37. Ridge I, Walters J and Street M (1999), Algal growth control by terrestrial leaf litter: a realistic tool, Hydrobiol. 395/396,pp. 173-180. 38. Rinehart KL, Namikoshi M, Choi BW (1994), Structure and biosynthesis of toxins from bluegreen algae (cyanobacteria). Journal of Applied Phycology, 6: pp. 159176. 39. Rouhiainen L, Buikema W, Paulin L, Haselkorn R (1995), Cloning and characterization of peptide synthetase genes from a hepatotoxic Anabaena strain. 40.Oral presentation at the 1st International Congress on Toxic Cyanobacteria, Bornholm. pp. 127 -130.. 40. Sivonen K, Jones J (1999), Cyanobacterial toxins. In: Chorus I, Bartram J, ed. Toxic cyanobacteria in water. A guide to their public health on sequences,

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monitoring and management. Published by E & FN Spon on behalf of the World Health Organization, pp. 41112. 41. Staley, J.T., Bryant, M.P., Pfennig, N. and Holt, J.G. [Eds] 1989 Bergey's Manual of Systematic Bacteriology. Volume 3, Williams & Wilkins,

Baltimore.pp.58-62. 42. Tisdale ES (1931), Epidemic of intestinal disorders in Charleston, W. Va., occurring simultaneously with unprecedented water supply conditions. American Journal of Public Health, 21: pp. 198200. 43. Turner PC, Gammie AJ, Hollinrake K, Codd GA (1990), Pneumonic associated with cyanobacteria. British Medical Journal, 300: pp. 14401441. 44. Tzong-Huei Lee (2000), Isolation and identification of seven microcystins from a culture M.TN-2 strain of Microcystis aeruginosa, Bot.Bull.Acad.Sin., 41: pp. 197-202. 45. Vollenweider, R.A. (1968), Scientific Fundamentals of the

Eutrophication of Lakes and Flowing Waters with Particular Reference to Nitrogen and Phosphorus as Factors in Eutrophication. Organisation for Economic Cooperation and Development, Paris. pp. 120 -123. 46. WHO (1993), Guidelines for Drinking-water Quality, Second edition. Volume 1, Recommendations. World Health Organization, Geneva. Pp. 46- 59. 47. WHO (1996), Guidelines for Drinking-water Quality, Second edition. Volume 2, Health Criteria and Other Supporting Information. World Health Organization, Geneva.pp. 267 268. 48. WHO (1998), Guidelines for Drinking-water Quality. Second edition, Addendum to Volume 2, Health Criteria and Other Supporting Information. World Health Organization, Geneva.pp. 245 -249. 49. Watanabe, M. M., and F. Kasai. (1985), NIES-collection list of strains, 1st ed., The National Institute for Environmental Studies, The Environmental Agency, Tsukuba, Japan. pp. 97.

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50. WRc (1996), The Fate of Intracellular Microcystin-LR During Water Treatment. Report Ref. 96/DW/07/4, UK Water Industry Research Ltd., London.pp 135- 168. 51. Yoo S, Carmichael W, Hoehn R, Hrudey S (1995), Cyanobacterial (bluegreen algal) toxins: A resource guide. Denver, CO, American Water Works Association Research Foundation, pp. 229. 52. Yu SZ (1995), Primary prevention of hepatocellular carcinoma. Journal of Gastroenterology and Hepatology, 10: pp. 674682. Trang web tham kho 53. http://en.wikipedia.org/wiki/Microcystin 54. http://www-cyanosite.bio.purdue.edu/cyanotox/toxins/microcystins.html 55. http://www.glerl.noaa.gov/pubs/brochures/mcystisflyer/mcystis.html 56. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1287722 57. http://microbewiki.kenyon.edu/index.php/Sphingomonas 58. http://elearning.hueuni.edu.vn/file.php/81/phanloaithuvat/htm/c2.htm 59. http://www.rimf.org.vn/bantin/tapchinewsdetail.asp?TapChiID=1&news 60. http://www.vnn.vn/khoahoc/2003/4/8715/ 61. http://dnr.metrokc.gov/wlr/waterres/lakes/ToxicAlgae.htm 62. http://who.int/water_sanitation_health/resourcesquality/toxcyanchap1 63. http://etd.lib.ttu.edu/theses/available/etd-04272006-122817

61

PH LC 1 1. Cc mi trng nui cy dng trong nghin cu 1.1. Mi trng Bold3N* ml/l Dung dch gc 30 10 10 10 10 10 1 6 40 NaNO3 CaCl2.2H2O MgSO4.7H2O K2HPO4 KH2PO4 NaCl Vitamin B12 PIV metal Nc chit t 1.2. Mi trng B12 (pH=9,0)* g/400ml H2 O 10.0 1.0 3.0 3.0 7.0 1.0 60.0 x 10-6 FeCl3.6H2O MnCl2.4H2O ZnCl2 CoCl2.6H2O Na2MoO4.2H2O Na2EDTA Vi lng PIV ca B3N Ho cht Khi lng (mg/l) 97 41 5 2 4 750

Ho cht NaNO3 K2HPO4 MgSO4.7H2O CaCl2.2H2O Na2CO3 Ferric citrate Disodium EDTA. 2H2O Vitamin B12

Khi lng 100 mg 10 mg 75 mg 40 mg 20 mg 6 mg 1 mg 0,1 ml

62

1.3. Mi trng C (pH=7,5) *

Ho cht Ca(NO3)2.4H2O KNO3 -Na2glycerophosphate.5 H2O MgSO4.7H2O Vitamin B12 Biotin Thiamine HCl PIV metals Tris (hydroxymethyl) aminomethane Nc ct 1.4. Mi trng J*

Khi lng 15 mg 10 mg 5 mg 4 mg 0.01g 0.01g 1g 0,3 ml 50mg

99,7ml

c to ra bi s phi trn 3 loi dung dch gc C, D v G9 metal. Dung dch gc C, D v G9 metal c chun b ring trong 1000ml nc ct 2 ln vi cc thnh phn nh trong bng di v bo qun trong lnh 4oC Dung dch gc C, pH=9.0 Dung dch gc D, khng iu chnh pH Ho cht Khi lng (g/l) 10.0 g/l 5.0 g/l 0.5 g/l 4.2 g/l 4.4 g/l

Ho cht

Khi lng (g/l) 67.0 0.67 3.8 25.0

NaNO3 Na2HPO4 K2HPO4 Tricine buffer

MgSO4.7H2O MgCl2.6H2O FeCl3.6H2O CaCl2.2H2O Na2EDTA

63

Sau khi lm lnh, phi trn 2 dung dch trn theo t l 1:1 to thnh mi trng J. Dung dch gc G9 metal Ho cht H3BO3 MnSO4.H2O ZnSO4.7H2O (NH4)6Mo7O24.4H2O CuSO4.5H2O Co(NO3)2.6H2O KI Khi lng (g/l) 3.25 1.5 0.3 0.08 0.05 0.15 0.01

(*) Nies Collection List of strains seventh edition 2004 microalgae and protozoa

64

PH LC 2 Cc mi trng nui cy vi khun [39] 2.1. Mi trng NA Ha cht Pepton Cao tht NaCl Thch Nc ct Khi lng (g) 10,0 1,0 1,5 15,0 1 lt 2.2. Mi trng M7 Ha cht Peptone Yeast extract Agar Glucose Nc ct Khi lng (g) 1,0 1,0 15,0 1,0 1 lt

Kh trng 121oC trong 15 pht, pH=7

Kh trng 121oC trong 15 pht, pH=7

65

PH LC 3 Phng php phn tch mt s thng s thu ho 3.1. Xc nh hm lng oxy ho tan (DO) bng phng php Winkler Nguyn tc: phng php xc nh DO da vo kh nng ca oxy ho tan oxy ho Mn2+ thnh Mn4+ trong mi trng kim. Mn4+ li c kh nng oxy ho Ithnh I2 trong mi trng axt. Do vy lng I2 c to ra c quan h nh lng vi DO v c xc nh bng Thiosulfat Natri. Phng trnh phn ng: MnSO4 + 2KOH = Mn(OH)2 + K2SO4 2Mn(OH)2 + O2 = MnO2 + H2O MnO2 + 2KI + 4H+ = Mn2+ + 2K+ + I2 + 2H2O 2Na2S2O3 + I2 = Na2S4O6 + 2NaI (chun ) Ho cht cn dng: 1) Dung dch MnSO4: cn 480g MnSO4.4H2O khng cha st, dng nc ct pha thnh 1 lt 2) Dung dch i--dua kali kim: cn 350g KOH v 75g KI, dng nc ct pha thnh 500ml 3) Acid HCl c 4) Dung dch Thiosulfat Natri 0,025N: Ho tan 0,2g Na2S2O3.5H2O trong 1 lt nc ct un si ngui. Dung dch c ng trong bnh mu nu, cho thm 0,2g Na2CO3 5) Dung dch tinh bt Cch tin hnh: Ly mu v c nh mu: dng bnh sch ly y mu nc. Mu cn phi c nh ngay, ht 1ml MnSO4, u pipet dng vo trong mt nc xong, ri t t cho thuc th vo bnh. Sau ht 1ml dung dch kim i-o-dua kali. Sau khi c nh xong, y nt, lc u mu nc n khi thy sinh ra kt ta vng nu. Mu nc c nh nh th c th gi c vi gi khng thay i, em v phng th nghim ri tin hnh cng tc phn tch.

66

Phn tch: trong mu nc m oxy ho tan b c nh v kt ta hon ton lng xung, dng pipet ht cho vo 1ml acid H2SO4 m c. Sau y nt li, lc u bnh cho kt ta vng nu ho tan ht. Ly 100ml dung dch ny vo bnh tam gic 250ml. Dng dung dch Na2S2O3 chun cho n mu vng rt nht, cho thm 3 git dung dch tinh bt, dung dch c mu xanh lam. Tip tc dng dung dch Na2S2O3 chun cho ti khi mu lam va vn bin mt. Ghi lng Na2S2O3 dng. Cng thc tnh ton kt qu:
DO mg / l = 8.W.N 1000 V

Trong : N - nng dung dch Na2S2O3 (N) V - th tch mu dng chun (ml) W - th tch dung dch chun Na2S2O3 (ml) 3.2. Xc nh nhu cu oxy sinh hc BOD (Biology Oxygen Demand) BOD l lng oxy cn thit oxi ha hon ton cc hp cht hu c c trong nc bng vi sinh vt. Nguyn tc xc nh: nh lng nhu cu oxi sinh ha (BOD) bng thit b Oxitop (c gn sensor BOD) da trn s gim p do kh CO 2 sinh ra trong qu trnh oxi ha, c hp th li bi cc ht kim mnh . S gim p sut c b vi x l trong sensor chuyn thnh gi tr BOD tng ng. Dng c v ha cht: - Dung dch m pht pht pH = 7,2 gm: + KH2PO4 : 8,5 g/l + Na2HPO4.7H2O : 33,4 g/l - Dung dch MgSO4.7H2O : 22,5 g/l - Dung dch CaCl2 : 27,5 g/l - Dung dch FeCl3.6H2O : 0,25 g/l - Dung dch H2SO4 0,1N - Dung dch NaOH 0,1 N - Dung dch Na2SO3 0,025 N
67

+ K2HPO4 : 21,75 g/l + NH4Cl : 1,7 g/l

- Nc ct c ct vi kalipemanganat - KOH tinh th X l mu: - Loi b cc cht ty ra bng dung dch Na2SO3 0,025N. - Loi b kim loi c bng cch iu chnh pH ca mu n 8,5 khi hu ht cc kim loi s kt ta dng oxit v hydroxit. em lc b kt ta - Trung ha mu: iu chnh pH ca mu t 6,5 7,5 bng dung dch NaOH hoc H2SO4. - B sung vi sinh vt nu lng vi sinh vt trong mu khng bng cch ly nc cng trn b mt, lng t 1- 2 gi. Nc thu c c hm lng vi sinh vt trong nc khong 103 106 vi khun/ ml . Th tch nc b sung c th dao ng t 3 30% th tch mu, thng thng bng 10% th tch mu. - Nu mu nc khng dinh dng cn b sung thm 1ml cc dung dch m photphat, dung dch MgSO4.7H2O, dung dch CaCl2, dung dch FeCl3.6H2O vo mt lt nc pha long. - Chun b nc pha long cy vi sinh vt: Nc ct b sung cht dinh dng, c sc n bo ha oxi pha vi nc c cha vi sinh vt theo t l 10/1. Cc bc tin hnh: - Mu c trong cc iu kin tiu chun: nhit l 200C, khng c nh sng, pH mu khong 6,5 7,5. - Cho mu pha long, ph hp vi thang o la chn trong bng di y vo chai th nghim. Th tch mu (ml) (mg/l) 432 365 250 0 40 0-80 0-200 1 2 5 Khong BOD H s pha long (f)

68

164 43.5

0-400 0-2000

10 50

- Cho con t vo mi chai , t lng cao su vo ming chai v cho ha cht hp th CO2 (KOH ht hoc Soda ht) vo lng cao su. - t v vn cht sensor BOD vo mi chai, ci t v bt u qu trnh o. - Tin hnh lm vi mu trng bng nc pha long cy vi sinh vt theo trnh t tng t - Xp chai vo gi khuy t v t thit b khuy t vo t iu nhit v ni vi ngun in trong t Tnh ton kt qu:

BOD5 = D - D f
Trong : BOD5 l gi tr BOD ca mu cn xc nh sau 5 ngy. D l gi tr hin trn sensor BOD sau 5 ngy ly mu cn xc nh. D l gi tr hin trn sensor BOD sau 5 ngy ca mu trng. f l h s ph thuc vo th tch mu. 3.3. Nhu cu oxy ho hc ( COD) (Chemical Oxygen Demand) COD la lng oxy cn thit dung cho qua tri nh oxy hoa hoan to cht hu c co trong ngun nc bng cac cht oxy hoa manh nh K KMnO4. Xc nh bng phng php bicromat n cc hp 2Cr2O7 hay

Nguyn tc: Khi cho dung dch bicromat vo mu nc, di tc dng ca nhit v H2SO4 c th phn ln cc cht hu c v v c trong nc b oxy ho. phn ng Oxy ho xy ra hon ton ta cho thm xc tc Ag2SO4. Sau khi oxy ho, lng Bicromat d s c xc nh bng phng php chun vi dung dch mui Morh. Lng cht hu c b oxy ho s tnh ra bng lng oxy tng ng qua Cr2O72- b kh, lng oxy tng ng ny chnh l COD. Phn ng oxy ca Bicromat c din ra theo phng trnh sau: Cr2O72- + 14 H+ + 6e = 2 Cr3+ + 7 H2O
69

Dng c v ho cht: Bnh nn 50ml sch v kh Pipet, nt bng sch Dung dch :

+ K2Cr2O7 sy kh nhit 1050C trong 2 gi, ho tan trong nc ct, a th tch n 1 lt; + Dung dch H2SO4 m c; + Tinh th Ag2SO4; + Vn thu tinh; + Ch th mu Diphenylamin: 1g Diphenylamin trong 100ml H2SO4; + Dung dch mui Morh 0,25N : Ho tan 98g mui Fe (NH4)2(SO4)2 .6 H2O trong nc ct. Thm 20ml H2SO4 m c, ngui thm nc ct n 1 lt. Mi ln dng mui Morh phi kim tra li nng bng cch ht 25ml dung dch K2Cr2O7 0,25N cho vo bnh tam gic, thm nc ct n th tch 200ml, thm 15ml dung dch H2SO4 1:1, thm 5 git ch th diphenylamin. Dng dung dch mui Morh chun n khi mu dung dch chuyn sang xanh l cy. Ghi th tch dung dch mui Morh tiu tn (V ml). Nng ca mui Morh c xc nh theo cng thc sau: N= Cch tin hnh : - Ly 10ml nc mu cho vo bnh nn; - Thm 5ml K2Cr2O7 0,25N + 1ml H2SO4 m c + 200mg Ag2SO4 - Mt t vn thu tinh - Lc u, y nt bng - Lm tng t vi mu nc ct - un hi lu trong 2 gi, sau 4 gi em ra lm lnh - Thm 50ml nc ct

25 x0,25 V

70

- Thm 2 git ch th mu Diphenylamin, chun bng dung dch mui Morh 0,25N n khi c mu xanh l cy. Ghi li lng mui Morh dng. o Cng thc tnh lng COD: COD = Trong : V1 - l th tch dung dch mui Morh chun mu trng (m); V2 - l th tch dung dch mui Morh chun mu nc cn xc nh (ml); N - l ng lng gam mui Morh (N). 3.4. Xc nh hm lng NH4+ bng phng php so mu vi thuc th Nessler Nguyn tc: Ion NH4+ trong nc tc dng vi thuc th Nessler cho hp cht vng nu. NH4+ + 2K2Hg2I4 + 3KOH NH2Hg2OI + 7KI + 2H2O. Cng mu ca dung dch cha NH2Hg2OI t l vi hm lng NH4+ tham gia to thnh hp cht ny. Cc yu t nh hng: cc amin, cloramin, axeton, cn v mt s cht hu c ho tan gy cn tr php o do chng tc dng vi thuc th Nessler. Ngoi ra, cc cht gy nn cng, st sunfua, c nh hng n qu trnh xc nh. Loi nh hng cn tr bng cch thm mui Seignet. Dng c v ho cht: - Bnh nh mc 1 lt, 500 ml (1 ci), 50 ml (12 ci). - Pipet cc loi. - Dung dch mui Seignet 50%: ho tan 50 g mui seignet trong 100 ml nc ct 2 ln. - Dung dch thuc th Nessler: cn 15 g HgI2 v 10 g KI ho vo 500 ml nc ct, cho vo 40 g NaOH khuy u cho tan, lng vi ngy, gn dung dch vo bnh nu. Phng php lp ng chun:
(V 1 V 2) xNx8 x1000 V

71

Dung dch NH4Cl tiu chun: cn 0,382 g NH4Cl tinh khit sy kh 105 C trong 2 gi, pha vo bnh nh mc 1 lt. Dung dch c nng l 0,1 mg NNH4+ trong 1 ml. Ly 10 bnh nh mc c s th t t 1- 10. Dng pipet ht dung dch chun cho vo bnh nh mc vi cc th tch l: 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml sau cho thm 1 ml dung dch seignet v 1 ml dung dch nessler, thm nc ct n vch nh mc. Tin hnh so mu bc sng 420 nm trn my so mu quang in ERMA 11 TOKYO. Cn c vo cc gi tr mt quang o c ta lp ng chun. Cch tin hnh: Ly 5 ml nc mu cn phn tch cho vo bnh nh mc 50 ml, pha long bng nc ct ti 25 ml. Cho vo bnh 1 ml mui seignet, tip tc cho vo 1 ml thuc th nessler, thm nc ct n vch nh mc. Lm tng t i vi mu trng v so mu bc sng 420 nm. Tra ng chun xc nh hm lng N- NH+ 4 trong mu. Cng thc tnh kt qu: N-NH4+ =
W 0,1 1000 V
o

Trong : W- th tch NH4Cl tiu chun tra trn ng chun. V- th tch mu nc dng phn tch (5 ml). 3.5. Xc nh hm lng NO3- bng acid phenoldisunfonic Nguyn tc: Ion NO3- phn ng vi acid phenoldisunfonic to ra acid nitrophenoldisunfonic. Acid ny tc dng vi dung dch kim cho mui mu vng, cng mu t l vi hm lng NO3- trong dung dch. C6H3OH(SO3H)2 + HNO3 + 3NH4OH C6H2ONH4(SO3NH4)2NO2 + 4H2O. (acid phenoldisunfonic) Dng c, ho cht: - Bt s - Bnh nh mc 50 ml. - Dung dch NaOH 20%. - Pipet cc loi. - Dung dch NH4OH 25%. (mu vng)

72

- Acid phenoldisunfonic: cn 3 g phenol nguyn cht kt tinh mu trng cho vo bnh tam gic, thm 20 ml H2SO4 c (d=1,84). y bnh bng nt c ng thy tinh lm lnh bng khng kh. un si cch thu trong 6 gi c dung dch mu vng nht. Bo qun trong bnh sm mu. Phng php lp ng chun: Cn 0,7216 g KNO3 sy kh 105oC trong 2h, cho vo bnh nh mc 1 lit, dung nc ct 2 ln ho tan v thm n vch nh mc. 1 ml dung dch ny cha 0,1 mg N-NO3-. Ly 50 ml dung dch ny cho vo bt s c cn, ngui ri thm 1 ml thuc th disunfonic, dng a thu tinh co nh thnh bt trn u thuc th, yn trong 10 pht thuc th ngm u. Sau thm 15 ml nc ct, trung ho bng NaOH 20% n pH kim, cho ton b dung dch mu vng ny vo bnh nh mc 200 ml (dung dch A). Ly 10 bnh nh mc 50 ml, cho dung dch A vo bnh nh mc ln lt vi cc th tch 1 ml, 3 ml, 5 ml, 7 ml, 9 ml ri nh mc n vch. So mu bc sng 420 nm trn my so mu quang in ERMA 11 TOKYO, xy dng ng chun. Cch tin hnh: Ly 50 ml nc mu cn phn tch cho vo bt s, chng cn, ngui, cho vo 1 ml dung dch acid phenoldisunfonic, dng a thu tinh co thnh bt ho tan ht cn, yn trong 10 pht acid ngm u. Cho thm mt t nc ct, cho vo 7 ml dung dch NH4OH 25%, tt c sang bnh nh mc 50 ml, trng bt s bng nc ct ri sang bnh nh mc. Dng nc ct nh mc n vch. So mu bc sng 420 nm trn my so mu quang in ERMA 11 TOKYO. Cn c vo mt quang o c v ng chun tnh hm lng NO3- trong mu cn phn tch. Cng thc tnh kt qu: N-NO3- (mg/l) =
W 0,025 1000 V

Trong : W- th tch tra trn ng chun. V- th tch mu dng phn tch (50 ml). 3.6. Phng php xc nh hm lng NO2Nguyn tc:
73

Phng php ny da vo phn ng Diajo ha ca nitrit trong mi trng acid vi acid sunphanilamide to thnh mui aro c mu hng. Phng php ny thch hp vi vic xc nh NO2- c hm lng 1 mg/l N dng NO2- tr xung. Cc yu t nh hng: Cc ion sau y c th gy cn tr: Fe3+, Pb2+, Hg2+, Ag+, cc ion c mu, khi chng c mt trong dung dch lm thay i mu ca h. Cc cht rn cng nh hng n vic xc nh NO2-. Dng c v ho cht: - Pipet cc loi - Bnh nh mc 50 ml.

- Thuc th sunphanilamide: ho tan 5g sunphanilamide trong hn hp 50 ml acid HCl c v 300 ml nc ct. Pha long n 500 ml bng nc ct. - Dung dch NED dihydroclorua: ho tan 500 mg NED dihydroclorua trong 500 ml nc, ng trong chai ti mu, pha ch li hng thng hoc ngay tc khc khi thy dung dch chuyn sang mu nu. Cc lp ng chun: Dung dch NO2- tiu chun: cn 0,4927 g NaNO2 khan vo trong bnh nh mc 1 lt, thm nc ct n vch, dung dch ny cha 0,1 mg N-NO2- trong 1 ml. Ly 10 ml dung dch ny cho vo bnh nh mc 1 lt, thm nc ct n vch, dung dch ny l dung dch tiu chun c nng 0,001 mg N-NO2- trong 1 ml. Cho vo bnh nh mc 50 ml c th t t 1- 10 dung dch chun vi cc th tch 1 ml, 2ml, 3ml, 4ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml. Sau thm nc ct n vch, so mu bc sng 530 nm trn my so mu quang in ERMA 11 TOKYO. Cn c vo gi tr mt quang lp ng chun. Cch tin hnh: - Ly 5 ml nc mu cn phn tch cho vo bnh nh mc 50 ml, i vi nc thi th pha long thnh 25 ml. - Dng pipet cho vo bnh 1ml dung dch sunphanilamide, lc trong 2 pht, cho thm 1ml dung dch NED dihydroclorua, lc trong 10 pht. - Lm tng t vi mu trng. Tin hnh so mu trn my so mu quang in ERMA 11 TOKYO bc sng 530 nm. Cng thc tnh kt qu:

74

Hm lng N-NO2- c tnh theo cng thc: N-NO2- (mg/l) =

W 0,001 1000 V

Trong : W- th tch dung dch N-NO2- tra trn ng chun. V- th tch mu nc ly phn tch (5 ml). 3.7. Phng php xc nh hm lng photphat PO43Nguyn tc: Khi un nng dung dch PO43- vi molipdat amon c mt H2SO4 c s to thnh acid photpho molipdat, acid ny tc dng vi acid ascorbic trong mi trng pH= 4 s cho hp cht photpho molipdat mu xanh lam. Cn c vo cng mu o c v ng chun xc nh hm lng PO43- trong dung dch cn phn tch. Ho cht v dng c: - Pipet cc loi - Bnh nh mc 50 ml.

- Dung dch (NH4)6Mo7O24.4H2O 2,5%: cn 25 g molipdat amon, ho tan trong bnh nh mc 1000 ml. Thn trng cho thm 175ml dung dch H2SO4 c, ngui dung dch ri thm nc ct n vch nh mc. - Dung dch acid ascorbic 1%: 1g acid ascorbic ho tan trong 100ml dung dch CuSO4 0,05M (dung dch ny ch chun b trc khi dng). Cch lp ng chun: - Dung dch chun photphat: cn 0,22131 g KH2PO4 sy kh nhit 105oC trong 2h, ho tan n 1 lt bng nc ct 2 ln ta c dung dch chun c hm lng 0,05 mg P-PO43- trong 1 ml. - Cho vo 6 bnh nh mc 50 ml ln lt cc th tch dung dch chun photphat nh sau: 0,1 ml; 0,3 ml; 0,5 ml; 0,7 ml; 0,9 ml; 1,1 ml. Thm 2 ml dung dch molipdat amon; 0,5 ml dung dch acid ascorbic, thm nc ct n vch nh mc. un cch thu nhit 30oC trong 10 pht. So mu trn my so mu quang in ERMA 11 TOKYO bc sng 660 nm. T kt qu o mt quang lp ng chun. Cch tin hnh phn tch: Ly 10 ml nc mu cho vo bnh nh mc 50 ml, dng pipet cho 2 ml dung dch thuc th molipdat amon v 0,5 ml dung dch acid ascorbic, thm nc ct n
75

vch nh mc. un cch thu nhit 30oC trong 10 pht. So mu bc sng 660 nm trn my so mu quang in ERMA 11 TOKYO. Cn c vo gi tr mt quang o c v ng chun ta xc nh c hm lng PO43- trong mu. Cng thc tnh kt qu: P- PO43- =
W 0,05 1000 V

Trong : W- th tch tng ng vi mt quang o c. V- th tch mu dng phn tch (10 ml). 3.8. Xc nh hm lng cn l lng SS Nguyn tc: Nc mu c lc u em lc qua giy lc c bit trc khi lng. Cn cn li trn giy lc c sy n trng lng khng i nhit 1050C trong 2 gi. Khi lng chnh lch trc v sau khi sy chnh l cn l lng SS hay cn gi l cht rn huyn ph Cc bc tin hnh: Giy lc c sy kh ti trng lng khng i 1050C trong 2 gi, ngui trong bnh ht m sau cn chnh xc trng lng ca giy lc. Ly 100 ml nc mu lc qua giy lc. em sy giy lc v lp cn bn trn, ngui trong bnh ht m v cn chnh xc trng lng giy sau khi lc. Cng thc tnh kt qu:
SS mg / l =

A - B 1000
V

Trong :

A l trng lng ca giy lc v lp cn sau khi sy (mg) B l trng lng ca giy lc ban u (mg) V l th tch mu nc lc qua giy lc (ml)

76

PH LC 4 4.1. Phng php nhun Gram Nguyn tc: Nhum Gram (phn ng Gram) c vai tr c bit trong vic phn loi vi khun. Trong qu trnh nhum Gram, t bo bc u c x l vi tm kt tinh v iot nn c s to thnh phc cht tm kt tinh iot bn trong t bo. Khi vi khun Gram m b ty cn, lipit ca lp mng ngoi b ho tan lm tng tnh thm ca mng dn n s ra tri phc cht tm iot v lm cho vi khun mt mu. Khi nhum b sung chng s bt mu vi thuc ny ( vng vi Safranin hay ta vi Fuchsin) . vi khun Gram dng, cn lm cho cc l trong peptidoglycan co li do phc cht tim iot b gi li bn trong t bo. Cch tin hnh: Dng que cy v trng ly dch huyn ph t bo vi khun t ln phin knh. C nh tiu bn bng ngn la ri nhun thuc u bng dung dch tm kt tinh (crystal violet) trong khong 1 pht. Ra bng nc. Nhum tip bng dung dch it (dung dch Lugol) trong 1 pht. Ra bng nc. Ph ln vt bi dung dch etanol 95%:axeton (1:1) trong khong 1 pht. Li ra bng nc. Sau nhum tip bng thuc nhum mu (nh Safranin hay Fuchsin Ziehl). Ra qua nc, kh, soi knh. 4.2. Oxidaza Th bng kit (strip), chng dng tnh cho mu tm, chng m tnh khng mu. 4.3. Xc nh kh nng di ng (Motility test) Phn ln cc vi khun Bacillus v sphingomonas u c kh nng di ng, ch 1 s t loi l khng c kh nng ny. Nguyn tc: Tin mao (lng roi) l nhng si lng di, un khc, mc mt ngoi mt s vi khun v c tc dng gip chng c th chuyn ng. Kh nng ny c th c quan st da vo s sinh trng v di dng ca vi khun vo bn trong mi trng thch mm. Mi trng: S dng mi trng MRS bn lng (0,15 - 0,4% thch). Mi ng nghim cha 5ml mi trng. Kh trng 121oC/15 pht. Lm ngui trng thi thng ng.

77

Cch tin hnh: m su u que cy thng c cha t bo vi khun lactic xuyn vo gia mi trng trong ng nghim n su khong 2ml. Ch a que cy vo chnh gia ng nghim. Nui cy nhit 30 - 370C trong 48 - 56 gi. Quan st v tr sinh trng ca vi khun trong ng nghim. Nu t bo mc thng theo vch que cy th khng c kh nng di ng, ngc li nu t bo mc loang xung quanh vch que cy th c kh nng di ng. 4.4. Xc nh kh nng phn gii tinh bt Cy chm hoc cy vch vi khun trn mi trng thch thng c b sung 1% tinh bt tan, 370C trong 2 ngy. thuc nhum Lugol. Chng no c hot tnh s to vng trong xung quanh hoc ngay di chn khun lc. 4.5. Pht hin vi khun Sphingomonas Ngoi kh nng pht hin vi khun Sphingomonas da vo kh nng di ng rt nhanh ca chng khi soi di knh hin vi cn c th phn bit c chng khi cy trn mi trng Kings B: Proteose peptone - 20g; Glycerol - 10ml; K2HPO4 1,5g; MgSO4 -1,5g; thch - 18g; nc - 1000ml. Trn mi trng ny, a s cc chng vi khun Sphingomonas u pht quang di n cc tm (tuy nhin vn c 1 s chng khng pht quang).

78

79

PH LC 5 Kt qu phn tch thy l, thy ha

t thu mu ln 1 (6/3/2007) TT Ch tiu D1 1. 2. 3. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14 Mu n-c Mi n-c T0 n-c trong pH SS BOD5 COD DO NH4NO2NO3PO43Xanh lc D2 Xanh lc D3 Xanh en D4 Xanh en D5 Xanh lam Tanh 16.7 19 9.57 380 460 932 7.75 0.50 D6 Xanh lam Tanh 16.7 21 9.48 390 440 932 7.67 0.20 D1 Xanh lc D2 Xanh lc D3 Xanh en D4 Xanh en D5 Xanh lam Tanh 29.4 22 9.95 360 180 300 19.6 1.30 D6 Xanh lam Tanh 29.3 25 10.37 6-8.5 5.5-9 380 90 160 13.4 2.00 20 <4 <10 >6 0.05 0.01 10 80 <25 <35 >2 1.00 0.05 15 t thu mu ln 2 (8/ 5/2007) TCVN 5942-1995 A B

Hi nh Hi nh Rt thi Rt thi 16.6 29 9.80 250 120 160 9.42 0.24 0.012 0.170 0.60 16.6 28 9.82 320 65 80 80.80 0.36 0.080 0.130 0.50 16.7 12 9.77 480 520 960 9.65 1.24 0.090 0.060 1.00 16.9 14 9.79 440 310 520 9.30 1.57 0.070 0.140 0.43

Hi nh Hi nh Rt thi Rt thi 29.8 27 10.52 230 220 360 22.0 1.22 0.008 0.035 0.87 30.8 25 10.50 290 150 240 23.4 1.70 0.050 0.020 0.15 31.8 13 10.48 510 270 500 17.4 1.26 0.050 0.012 0.14 31.3 17 10.47 420 260 400 18.8 1.82 0.025 0.012 0.16

0.075 0.014 0.150 0.030 0.82 0.82

0.025 0.007 0.090 0.019 0.07 0.14

PH LC 6 Quy trnh tch chit c t Sinh khi ng kh nhit -830C Sinh khi ng kh nhit -83C Chit rt bng methanol:nc (80:20)

Lc qua giy lc

C quay mu

Chy TLC (ethyl acetat:isopropanol:nc-8:5:3)

Nhum TLC bng nihyrin (2% trong n-butanol bo ho)

Co phn mu trn TLC sang ng Eppendorf

Ti chit rt c t bng methanol:nc (80:20)

Lc qua ct C18 Xc nh hm lng trn my quang ph Phn tch trn HPLC

Phiu ng k kt qu nghin cu cc ti khcn

Tn ti: Nghin cu c im sinh hc v thm d kh nng phn gii sinh hc c t ca mt s chng Microcystis phn lp h Hon Kim, H Ni.

M s: QG 07 - 24

C quan qun l ti: i hc Quc gia H Ni a ch: 144 Xun Thu, Cu Giy, H Ni in thoi: 7548664 C quan ch tr ti: Vin vi sinh vt v Cng ngh Sinh hc a ch: 144 Xun Thu, Cu Giy, H Ni in thoi: 7547907 Tng kinh ph thc chi: 100.000.000 Trong : - T ngn sch Nh n-c: 100.000.000 - Ngun khc: 0 Thi gian nghin cu: Thi gian bt u: 05/2007 Thi gian kt thc: 05/ 2009 Tn cc cn b phi hp nghin cu:

- Ch tr ti: TS. Nguyn Th Hoi H - Nhng ng-i tham gia: CN. L-u Th Thu Giang, CN. Nguyn Quang Huy, CN. L Trung Thu, CN. Phm Th Bch o, Thc s. Trn Th ip S ng k ti : S chng nhn ng k KQNC A. Ph bin rng ri B. Ph bin hn ch C. Bo mt Ngy Ngy Tm tt ngha, kt qu nghin cu (k c kt qu o to): ngha: Thng qua cc hot ng ca ti s gip cho ng-i dn xung quanh h ni ring v ng-i dn H Ni ni chung nhn thc -c rng mi tr-ng ca h cn -c bo v, tr-c ht l v li ch ca chnh h, ca Th nghn nm vn hin, ng thi cng chnh l ti sn chung m ai cng phi c trch nhim cn phi bo v v gi gn. Cc loi vi khun lam -c phn lp, vi l lch c th s lm tin quan trng cho nhng nghin cu tip theo, nhm gp phn xy dng mi tr-ng h sch, an ton v bn vng. Mt khc s b sung vo danh mc cc loi vi khun lam gy c n-c ngt cho Vit Nam. ti -c thc hin s gip nhm cn b nghin cu hiu su hn v c im sinh hc ca vi khun lam gy c Microcystis, t tm cc kh nng c th ng dng hn ch tc ng xu gy ra bi Microcystis tnh min Bc ni chung. Kt qu: - H Hon Kim ang tnh trng nhim, hm lng COD, BOD vt tiu chun Vit Nam, TCVN 4942- 1995 (loi B) 20 ln. Vi gi tr NH4+ t v c t microcystin ca chng trong cc h, thu vc nuc ngt trn a bn H Ni v cc Bo mt:

0,129- 0,699 (mg/l), NO2- t 0.012- 0,05 (mg/l), NO3- t 0,047- 0,129 (mg/l), PO43t 0,08- 0.64 (mg/l) chng t h c hm lng dinh dng N, P kh cao. - phn lp thun khit v nui cy 10 chng vi khun lam thuc chi Microcystis theo phng php ca Shirai c ci tin. Mi trng thch agarose B12 vi t l 0,4% l mi trng thch hp nht cho vic phn lp Microcystis. - Cc chng Microcystis u sinh trng tt trn mi trng Bold 3N vi s lng t bo t t 32,94 68,6 106 tb/ml sau 15 n 19 ngy nui cy. c bit chng LT2 s lng t bo t cao nht l 68,6 106 tb/ml sau ngy th 19. Trn mi trng J, cc chng Microcystis sinh trng s lng t bo ca cc chng t t 40,42 76,46 106 tb/ml sau 15 n 19 ngy nui cy. Trong chng LT2 vn pht trin vi s lng t bo t cao nht 76,46 106 tb/ml sau ngy th 19. Trn mi trng B12, s lng t bo t t 35,36 54,78 106 tb/ml sau 12 n 15 ngy nui cy. mi trng ny chng LT8 pht trin vi mt t bo t cao nht 54,78 106 tb/ml sau ngy th 15. - C 7 loi axt bo khng no dng C15, C17 v C19 trong t bo 2 chng Microcystis LT2 v LT8. trong thnh phn axit Palmitic acid (C17H34O2) chim lng ln nht vi 1,302 g/mg trng lng kh chng LT2, chng LT8 l 0,9683 g/mg trng lng kh. Kt qu cho thy c s sai khc nhau v lng ca 7 loi axit bo c trong t bo 2 chng LT2 v LT8. - Da vo phng php phn loi truyn thng theo phn loi hnh thi hc v phng php phn loi hin i da trn phn tch trnh t rADN 16S, 2 chng vi khun LT 2, LT8 c xc nh l Microcystis aeruginosa, 2 chng vi khun nc N4 v N7 c xc nh thuc loi Sphingomonas asaccharolytica - Da vo kt qu phn tch trn HPLC, hm lng c t MC RR t ln nht l 512,8 ng/mg trng lng kh, c t MC YR l 236,8 ng/mg v thp nht l c t MC LR: 114,25 ng/mg. Chng vi khun Sphingomonas tuyn chn c c kh phn gii Sphin Sphingomonas mali v

microcystin kh nhanh. nng microcystin 3 g/l thi gian bn phn gii l 1,34 gi v vi nng 20g/l, thi gian l 3,43 gi.

- Nhit thch hp cho s phn gii microcystin ca cc chng vi khun Sphingomonas mali N4 v Sphingomonas asaccharolytica N7. Sau 7 gi s phn gii gn nh hon ton l 100%. Cc cng trnh cng b v sn phm o to Cng trnh cng b - Phm Th Mai, Phm Tin c, V Th Lan Anh, Nguyn Th Hoi H 2008. nh gi s bin ng s lng theo ma v theo tng nc ca vi khun lam Microcystis aeruginosa h Hon Kim, H Ni, Tp ch Khoa hc i hc Quc gia H Ni. Khoa hc T nhin v Cng ngh 24 s 15. Tr 125 128. - Phm Th Mai, Phm Tin c, V Th Lan Anh, Nguyn Th Hoi H 2008. Tm hiu kh nng phn gii c t ca vi khun lam Microcystis trong h Hon kim bng vi khun. Tp ch Khoa hc i hc Quc gia H Ni. Khoa hc T nhin v Cng ngh 24 s 15. Tr 119 122. o to 4 C nhn - Bc u phn lp, tuyn chn mt s chng vi khun c kh nng phn gii c t ca vi khun lam Microcystis h Hon Kim, H Ni. 2007. Kho lun tt nghip h i hc chnh quy ca ng Th Kim Anh. - Bc u nh gi kh nng phn gii c t microcystin ca vi khun lam Microcystis aeruginosa bng vi khun Sphingomonas. 2008. Kho lun tt nghip h i hc chnh quy ca Nguyn Th Hoi. - Kho st s bin ng theo ma ca vi khun lam Microcystis aeruginosa h Hon Kim v kh nng c ch sinh trng ca chng bng dch chit v chui, v qut. 2009. Kho lun tt nghip h i hc chnh quy ca L Th Trang. - Phn lp, nghin cu mt s c tnh ca to c Microcystis tch chit c t v thm d bin php x l.2009. n tt nghip ca Nguyn Hu Hiu o to 1 Thc s Phn gii t bo Microcystis v s phn hu c t microcystin bng vi khun nc. 2007 - 2009. Nhng ng gp ca ti: - L cng trnh u tin tch v lu gi c chng vi khun lam

Microcystis. - Phn lp c 2 chng vi khun thuc chi mi Sphingomonas c kh nng phn gii c t microcystin ca vi khun lam Microcystis. - Xy dng c quy trnh tch chit c t microcystin.

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