Studies On Diversity And Polyphasic Identification Of Macroalgal Epiphytic Bacteria And Enteromorpha Prolifera Polysaccharide Degrading Enzymes

Algae are very important resources in the marine environment.As primary producers,they play an important role in the marine ecosystem.There are complex interrelationships between algae and their epiphytic microorganisms,and studies of the diversity of algal epiphytic microorganisms are beneficial to understanding the complex interrelationships between epiphytic microorganisms and algae and the action mechanisms.The resources of Enteromorpha prolifera near the coast of Qingdao are rich,but they have not been fully utilized.Studies on Enteromorpha prolifera polysaccharide-degrading enzymes will facilitate the utilization and development of Enteromorpha prolifera resources.In this thesis,macroalgal epiphytic bacteria were firstly isolated from macroalgae samples collected from Qingdao and Weihai offshore areas and the diversity of the isolated macroalgal epiphytic bacterial stains were further analyzed.Secondly,two novel macroalgal epiphytic bacterial stains isolated were identified using a polyphasic approach to determine their taxonomic positions.Finally,three Enteromorpha prolifera polysaccharide degrading bacterial strains were screened from Enteromorpha prolifera samples,six polysaccharide degrading enzymes were mined from their genomes and the enzymatic properties of the enzymes mined were further studied.1.Isolation and diversity analysis of epiphytic bacteria from macroalgal samplesEight macroalgae samples were collected from different sites in Qingdao and Weihai coastal areas and epiphytic bacteria were isolated from the macroalgal samples using the dilution-plating method.A total of 96 epiphytic bacteria were obtained and they belonged to 45 genera in.the Proteobacteria,Bacteroides,Firmicutes and Actinobacteria phyla.Most of strains isolated belonged to the phylum Proteobacteria(61.5%),followed by those belonging to the phylum Bacteroides(25.0%),the phylum Firmicutes(11.5%)and the phylum Actinobacteria(2.0%).Among them,29 strains were isolated from Laminaria japonica samples,which belonged to 16 genera with Pseudoalteromonas as the dominant genus;28 strains were isolated from Enteromorpha prolifera samples,which belonged to 21 genera with Pseudoalteromonas as the dominant genus;18 strains were isolated from the coralline algae samples,which belonged to 12 genera with Aquimarina as the dominant genus;11 strains were isolated from the Gracilaria samples,which belonged to 4 genera with Bacillus as the dominant genus;7 strains were isolated from Ulva sample which belonged to 5 genera with Psychrobacter as the dominant genus.Totally 9 agar-degrading strains,22 alginate-degrading strains and 15 carrageenan-degrading strains were further screened from the 96 epiphytic bacterial strains isolated.2.Polyphasic taxonomy of strain SM1977TStrain SM1977T was isolated from the surface of a coralline algae sample collected from the coast of Qingdao city,Shandong.Strain SM1977T is a Gram-negative,heterotrophic,facultative aerobic bacterium having non-flagellated and short rod-shaped cells.Strain SM1977T grew at 10-35?,pH 4.5-8.5 and with 1-8.5%(w/v)NaCl.The genomic G+C content of strain SM1977T was 42.3%.Strain SM1977T showed the highest 16S rRNA gene sequence similarity(97.6%)with type strain of Vibrio casei.In the phylogenetic trees strain SM1977T clustered with members of the Rumoiensis clade and alone or formed a separate branch together with Vibrio casei.Strain Sv1977T represents a new species in the genus Vibrio,for which the name Vibrio algicola is proposed.The type strain of Vibrio algicola is SM1977T(=MCCC 1k04351T=KCTC 72847T).3.Polyphasic taxonomy of strain SM1960TStrain SM1960T was isolated from the surface of a kelp(Laminaria japonica)sample collected from the offshore of Rongcheng city,Shandong.Strain SM1960T was a Gram-negative,aerobic,non-flagellated,short rod-shaped heterotrophic bacterium.Strain SM1960T grew at 10-30? pH 6-10 and with 1-12%NaCl.Strain SM1960T showed the highest 16S rRNA sequence similarity(97.5%)with type strain of Winogradskyella litorriviva KMM 6491T and Winogradskyella sediminis S5-23-3T The phylogenetic trees based on the 16S rRNA gene sequences showed that strain SM1960T clustered with known species in the genus Winogradskyella and formed separate branches together with Winogradskyella profunda or with other three Winogradskyella species.Strain SM1960T represents a novel species of the genus Winogradskyella,for which the name Winogradskyella rongchengensis sp.nov.is proposed.The type strain of Winogradskyella rongchengensis is strain SM1960T(=MCCC 1K04389T=KCTC 72849T).4.Study on the Enteromorpha prolifera polysaccharide degrading enzymesThree Enteromorpha prolifera polysaccharide degrading bacterial strains,named HT1,HT2 and HT3,were screened from the surface of Enteromorpha prolifera samples collected from the coast of Qingdao city.The genomes of the three strains were sequenced,and six ulvan lyases with the potential to degrade Enteromorpha prolifera polysaccharide were mined from the genomes.Through the heterologous expression,purification and enzyme activity determination,it was found that enzyme U-3282 showed the highest enzyme activity toward?Enteromorpha prolifera polysaccharide(more than 10000 U/mg).U-3282 belongs to the PL24 family of polysaccharide lyases with a molecular weight of 60 kDa.The optimum temperature,pH and NaCl concentration for enzymatic reaction toward Enteromorpha prolifera polysaccharide were 35?,pH 8.0 and 0.25 M NaCl,respectively.The enzyme activities of U-3282 toward Enteromorpha prolifera polysaccharide and Ulva lactuca polysaccharide were 10467±13 U/mg and 8472±389 U/mg,respectively.High performance liquid chromatography(HPLC)and negative electrospray ionization mass spectrometry(ESI-MS)analysis showed that the main degradation products of U-3282 to the Enteromorpha prolifera polysaccharide were GlcA1Rha1(SO3H)1,GlcA1Rha2(SO3H)2 and GlcA1Xyl1Rha3(SO3H)2.