Diversity Of Bacteria And Activity Of Extracellular Enzymes In The Surface Water Of Chinese Marginal Seas And The Arctic Ocean

The ocean is the largest ecosystem on Earth,about 71%of the Earth's surface is covered by the ocean.The ocean harbors large amount of biological resources,especially microbial resources,but there still exist many marine microorganisms that have not been discovered.Microorganisms,as important components of marine ecosystem,have high species diversity and strong environmental adaptability.Meanwhile,microorganisms also can secrete corresponding extracellular enzymes.Extracellular enzymes could participate in and influence the recycling process of marine organic matter by degrading high molecular weight organics into small molecules that can be absorbed and utilized by microorganisms.However,there is no detailed report on the in situ extracellular enzyme activities in the East China Sea and the Yellow Sea,especially the enzyme activities towards high molecular weight substrate.The distribution pattern,type and influencing factors of extracellular enzyme are still unclear.In addition,there are relatively few reports on the extracellular enzyme activities of culturable bacteria in the Arctic ocean.In this thesis,the bacterial diversities and extracellular enzyme activities in the surface waters of Chinese marginal seas and Arctic were studied.The multi-phase classification study of a novel bacterium isolated from the surface water of the South China Sea was carried out.1.Diversity of culturable strains in the surface water of South China Sea and the identification of Parvularcula marina SM1705TThe diversity of culturable strains in the surface seawater samples of South China Sea was studied.The results showed that the culturable strains in this sample mainly belonged to Actinobacteria and Proteobacteria,among which Microbacterium,Erythrobacter and Parvularcula were dominant,accounting for 23.7%,7.9%and 6.2%of all culturable strains,respectively.In addition,a novel strain named SM1705 was isolated.SM1705 is a heterotrophic and aerobic gram-negative bacterium with short rod-shaped cells and flagella.Cells grow at 10-40?(optimum at 35?),at pH 6.0-9.0(optimum at 8.0)and with 0.5-13%NaCl(w/v,optimum with 1.0%).The major cellular fatty acids are summed feature 8(C18:1 ?7c and/or C18:1?6c),C16:0 and C18:0.Polar lipids included three unidentified glycolipids and one unidentified lipid.The genomic DNA G+C content of the type strain is 59.3%.Strain SM1705 was affiliated with the genus Parvularcula,sharing the highest sequence similarity(96.0%)with type strain of P.bermudensis.The phylogenetic tree based on 16S rRNA gene sequence showed that strain SM1705 was clustered with P.bermudensis,P.oceanius,P.lutaonensis,P.dongshanensis and P.flava,and meanwhile formed an independent evolutionary branch.Based on the polyphasic evidence presented in this paper,strain SM1705 represents a novel Parvularcula species,for which the name Parvularcula marina sp.nov.is proposed.The type strain is SM1705T(=KCTC 26795T=MCCC1K03505T=CCTCC AB 2018345T).2.In situ extracellular enzyme activities in surface waters of the Yellow Sea and East China SeaThrough the study of the activities of in situ extracellular enzymes in the surface seawter of Yellow Sea and East China Sea,the distribution patterns and key factors affecting various enzyme activities were analyzed.Phosphatases and casease showed higher activity among total enzymatic activity,indicating that they played an important role in the cycling of phosphorus and nitrogen.Although both?-glucosidase and ?-glucosidase can hydrolyze oligosaccharides and release monosaccharides,the hydrolysis rate of ?-glucosidase was much higher than?-glucosidase.This may because ?-glucosidase plays a key role in the global carbon cycling.Among the 0.22?m-filtered water samples,the activity of casease was the highest,followed by chitinase,cellulase and alginate lyase,while phosphatase,?-glucosidase and N-acetyl-?-glucosidase activities decreased,indicating that most casease and polysaccharase could be free enzymes,other corresponding enzymes may be cell or particle attachment enzymes.Compared to offshore waters,inshore waters,especially in the estuary,obviously presented higher enzyme activities.The enzyme activities from inshore stations clustered together and were driven by latitude,Chl-a,salinity and temperature.In addition,the activity of extracellular enzymes was also related to the structure of marine microbial population.The higher enzymatic activities were associated with Roseobacter,Alteromonas and Pseudoalteromonas.3.Diversity and enzymes productive ability of cultivatable bacteria in the surface water of the ArcticBacterial diversity was studied by isolating and identifying culturable strains in surface sea samples from the Arctic.Culturable strains in the sample mainly belong to Bacteroidetes and Proteobacteria,and Cellulophaga and Pseudoalteromonas were dominant.In addition,through degradation experiments on different high molecule weight substrates,63%of the strains have the ability to degrade a range of substrates.Among these bacteria,65%could degrade casein,and most of them belong to Pseudoalteromonas;35%of the bacteria have the ability to degrade cellulose,Lacinutrix is the main part;71%of the strains can degrade alginate,and the strains are mainly Pseudoalteromonas and Lacinutrix;29%of strains have the ability to degrade starch,and most of them are Pseudoalteromonas;and these strains producing chitinase and esterase all belong to Aeromonas and Cellulophaga.According to the extracellular enzyme annotation of the genome,these bacteria which could degrade various substrates,mostly belong to Aeromonas,Cellulophaga and Pseudoalteromonas.4.Study on extracellular enzyme activity of Cellulophaga sp.SM-W46 at different culture temperaturesCellulophaga sp.SM-W46,isolated from the surface water of the Arctic,is a bacterium that can degrade various substrates such as casein,cellulose and chitin.The strain SM-W46 was cultured using the lysates of Synechococcus sp.PCC7002,and activities of the extracellular enzymes generated by strain SM-W46 cultured at different temperatures were determined.Five kinds of extracellular enzyme activities were detected by using fluorescent molecule substrates.Leucine aminopeptidase activity was the highest,and a small amount of phosphatase,glucosidase and chitinase activities were also detected.By detecting the activities of free enzymes,the activity of phosphatase and aminopeptidase changed little,the activity of chitinase decreased,and the activity of?-glucosidase could not be detected,indicating that most aminopeptidase and phosphatase could be free enzymes,?-glucosidase and chitinase may be cell or particle-attached enzymes.Different culture temperatures had a certain effect on the activity of aminopeptidase,but had little effect on the activities of phosphatase,chitinase and glucosidase,which may be related to the temperature gradient.Although different culture temperatures may cause some changes in the bacterial extracellular enzyme activity,the specific regulation has not been clarified.In conclusion,studying the diversity of marine bacteria and activity of extracellular enzymes in the surface water of Chinese marginal seas and the Arctic ocean is crucial for understanding the cycles of marine organic matter.Meanwhile,this study can provide important information for finding and developing new microbial and enzymatic resources.