Isolation, Identification And Characteristics Of A Microcystin-degrading Bacterium

Toxic blooms consisting of cyanobacteria belonging to the genera Microcystis,Anabaena, Planktothrix, and Nostoc occur in lakes and reservoirs as a result ofeutrophication. In recent years, toxic cyanobacterial blooms have occurred frequentlyin eutrophic lakes, rivers and reservoirs in China. Microcystins are cyclicheptapeptide hepatotoxins predominantly produced by freshwater cyanobacteria，which impose a health risk to animals and human populations when surface water isused for drinking water. The stable cyclic structure of these peptide toxins havepresented many challenges to water treatment facilities where conventional processeshave limited effect on the removal of microcystins. At present, the biodegradation ofmicrocystins in water has been proven to be very effective as they can be used ascarbon source by heterotrophic bacteria. The majority of these studies has focused onthe isolation and purification of MC degradation bacterium and degradationcharacteristics. To date, the only reported mechanism of MC biodegradation wasinvestigated by Bourne. In order to futher study the biodegradation pathways forMCLR, we need to isolate more different MC-degrading bacteria from different waterbodies. In this thesis, we isolated a strain Paucibacter sp. CH from Lake Chaohu,which showed high ability in degrading MCLR. We identified the strain based onmorphological as well as16S rDNA Gene sequencing. We preliminary investigatedthe MCLR degradation mechanism by studying the enzyme degradation pathway anddetecting the degrading gene cluster. Main research contents and results are reportedas follows:(1) The morphology identification results show that the strain CH wasgram-negative and short rod-shaped, bacteria cells were1~2μm in length and0.1~0.3μm in width,16S rDNA sequence homology analysis result show that the bacteriaCH was close to Paucibacter sp. The phylogenetic tree indicate that CH was belongsto the Paucibacter genera under β-proteobacteria.(2) Experiments conducted to verify the capability of strain CH to grow using MCLR (11.6μg·mL-1) as the sole carbon and energy source, demonstrated that theMCLR concentration had decrease to an undetectable level within10h incubation.This confirming its capability to use MCLR as sole carbon and energy for growthunder aerobic conditions. The degradation rate of MCLR was1.16μg·mL-1·h-1. Thebiomass of strain CH has significantly increased. The optimum condition ofdegrading bacterium CH was at25~35℃、pH7~9. CH consumed10%of MCLR incultures at pH5.0. At the alkaline conditions (pH=10,11), the degradation of MCLRby CH was significantly inhibited, MCLR was completely degraded within50h.Carbon source glucose significantly inhibited the degradation rate of MCLR, butadding NaNO3in cultures had no significant effect on the degradation of MCLR.Strain CH can rapidly degrade MCLR under aerobic conditions, but do not haveMCLR degradation activity at anaerobic conditions.(3) The enzymatic activity test shows that extracellular enzyme has no degradingactivity of MCLR, but intracellular enzyme of CH can degrade94.4%MCLRwithin3h. HPLC chromatograms revealed that the peak corresponding to MCLRdecreased gradually. There were two intermediate products that could be clearly seenas separate peaks A and Adda in time sequence HPLC chromatograms as well. Thepeak of MCLR had completely disappeared within10h incubation, while peaks Adisappeared and Adda will be significantly accumulated as the final product. We inferthat CH cannot produce protease to degrade Adda. The UV spectra of biodegradationproducts A and Adda are very similar to that of MCLR. The λmaxof A, Adda andMCLR are all238nm. This indicate that Adda side chain remains intact in allproducts.(4) Using three different protease inhibitors to suspend the degradation of MCLRby the intracellular enzyme of CH. We found inhibitory effect with1,10-phenanthroline which caused scarcely MCLR degradation. It inhibits the firststep reaction during the degradation process of MCLR. PMSF and EDTA did notsignificantly inhibit the primary degradation reaction, but slow down the rate ofreaction. The results indicate that the product of MCLR catalyzed by the crudeenzymes of CH is product A, then product A is converted to product B, finallyproduct Adda is produced from B and is not biodegraded further. The degradation of MCLR process will produce three kinds of intermediate. We infer that there are atleast three kinds of enzymes involved in the strain CH for the degradation of MCLR.(5) HPLC chromatograms revealed the retention time are different between theintermediate product during the degradation of MCLR by Strain CH are differentfrom ACM-3962. Combining with the result of degradation mechanism anddegradation products, We draw the conclusion that the degradation mechanism andpathway of MCLR by strain CH are quite different from ACM-3962.