Research On Cellulolytic Microorganisms, Microbial Consortia And Relative Cellulases In Mangrove Ecosystems

Mangrove ecosystems are diverse intertidal wetlands commonly situated in tropical, sub-tropical and temperate coastal systems. The degree of bacterial diversity in these systems is expected to be high due to the combination of the mixing of seawater and freshwater and the resuspension of sediments and particles from many sources. The unique swampy, saline and partially anaerobic environment makes it a special source for obtaining microorganisms as well as enzyme and gene resource. Some reports have shown that a large number of bacteria and fungi participate in the cellulolytic processing of the abundant mangrove lignocellulose. In modern industries, cellulases are widely used in textile industry, laundry detergents, pulp and paper industry, and pharmaceutical applications. Cellulase is also used in the fermentation of biomass into biofuels, although this process is relatively experimental now. Thus, exploration of the cellulase-producing bacteria and relative enzymes in the specific mangrove ecosystem is important for further research and industrial applications.In this study, we isolated a cellulase-producing bacterium G21 from the mangrove soil samples.16S rRNA analysis indicated that it belonged to genus Vibrio, and polyphasic taxonomic approach was used to characterize it. Meanwhile, a cellulase gene was cloned from strain G21 and expressed in E. coli BL21, and the recombinant enzyme was characterized. Besides, a high-stable cellulolytic consortium SQD-1.1 was obtained by enrichment from mangrove soil samples and successive subcultivation. The consortium was further analyzed by PCR-DGGE,16S rRNA gene library construction and plate cultivation and metagenomic sequencing.1. Isolation and characterization of strain G21Strain G21 was isolated from mangrove soil samples collected from Xiamen Fujian province of China, when cellulas-producing bacteria were screened using CMC-Na as substrate. The cells of G21 are Gram-negative, slightly curved rods (0.7-0.8μm×1.4-1.55μm) and motile with a single polar flagellum. Its colonies on marine 2216E agar plate are circular to a little fusiform, white and half-translucent after incubation at 37℃for 24 h. The strain grows in the range of 15-40℃and 0.5-10% NaCl(w/v).Growth of G21 in anaerobic condition is detected in the presence of 10 mM glucose, but no gas was produced. Positive for oxidase and catalase. Negative for Voges Proskauer. Nitrate is reduced to nitrite. Starch, CMC and Tween 80 are hydrolyzed. Gelatine, casein, alginate and DNA are not hydrolyzed. The major cellular fatty acids of G21 were sum in feature 3 (C16:1ω7c and/or iso-C15:0 2-OH), C16:0 and C18:1ω7c.Phylogenetic analysis based on the 16S rRNA gene sequences showed that strain G21 belonged to the genus Vibrio and formed a cluster with V. furnissii ATCC 350116T (sequence similarity,97.4%) and V.fluvialis LMG 7894T (97.1%). However, multilocus sequence analysis (rpoA, recA, ftsZ, mreB and gapA) and DNA-DNA hybridization experiments indicated that it was distinct from related Vibrio species. The DNA G+C content was 46.0 mol%.Based on the phylogenetic, phenotypic, chemotaxonomic and DNA-DNA hybridization analysis, it is concluded that strain G21 represents a novel species of the genus Vibrio, for which the name Vibrio xiamenensis sp. nov. is proposed. The type train is G21 (=DSM 22851T=CGMCC 1.10228T).2. Gene cloning and characterization of cellulase Cel5AAlthough cellulases have been isolated from various microorganisms, no functional cellulase gene has been reported from the Vibrio genus until now. In this study, a novel endo-β-1,4-glucanase gene, cel5A,1362 bp in length, was cloned from the newly-identified bacterium, Vibrio sp. G21. The deduced protein of cel5A contains a catalytic domain of glycosyl hydrolase family 5 (GH5), followed by a cellulose binding domain (CBM2). The catalytic domain of Cel5A shows the highest sequence similarity (69%) to the bifunctional beta 1,4-endoglucanase/cellobiohydrolase from Teredinibacter turnerae T7902.The gene cel5A was overexpressed in Escherichia coli and the mature Cel5A enzyme was purified to homogeneity. The optimal pH and temperature of the recombinant enzyme were determined to be 6.5-7.5 and 50℃, respectively. Cel5A was stable over a wide range of pH and retained more than 90% of its total activity even after treatment in pH 5.5-10.5 for 1 h, indicating its high alkali resistance. Moreover, the enzyme was activated after pretreatment with mild alkali, a novel characteristic that has not been previously reported in other cellulases. Cel5A also showed a high level of salt tolerance. Its activity rose to 1.6-fold in 0.5 M NaCl and remained elevated even in 4 M NaCl. Further experimentation demonstrated that the thermostability of Cel5A was improved in 0.4 M NaCl. In addition, Cel5A showed specific activity towardsβ-1,4-linkage of amorphous region of lignocellulose, and the main final hydrolysis product of carboxymethylcellulose sodium and cellooligosaccharides was cellobiose.Considering the salt-tolerant and alkali-resistant properties of the enzyme, Cel5A of Vibrio sp. G21 has the potential to be used in industrial applications. Additionally, the characteristics of the enzyme, including thermostability improvement in salt solution and activation by alkaline, make it a good candidate for further research on the structure-function relationship and enzyme directed evolution.3. Enrichment and characterization of anaeraobic cellulolytic microbial consortiumIn nature, lignocellulose is partly decomposed by microbial communities inhabiting in anaerobic environments. These communities can keep stable and show high cellulolytic efficiency in native conditions. In this study, several mangrove soil samples from Hainan province were used to enrichment anaerobic cellulolytic microbial consortia. Eight consortia were transferred continually for 27 times and still could keep stable for efficient cellulose degradation.Consortium SQD-1.1, which can efficiently degrade filter paper in three days, utilize cellulose as the sole carbon source, and degrade xylan efficiently, was further characterized by PCR-DGGE,16S rRNA gene library and plate cultivation methods. Time-course analysis of the community composition within 10 days by PCR-DGGE showed that the members in SQD-1.1 were stable and no obvious change in consortium structure was found. The compostion of different generations was also stable.16S rRNA gene library analysis showed the presence of 27 OUT sequences, belonging to 21 genera of 7 phyla, and a potential cellulolytic bacterium M117 was found. Isolates using plate cultivation method both under anaerobic and aerobic conditions were belonged to Clostridium, Trichococcus and Proteiniclasticum and so on, of which five were potential new species and/or new species of new genus. Further analysis showed that strain P2 showed endoglucanase activity and should played an important role in the consortium in cellulose degradation.The study of SQD-1.1 can provide a candidate for investigating the mechanism of anaerobic cellulose degradation in nature environments, which is in favor of the industrial lignocellulose utilization for ethanol production.4. Metagenomic sequencing and analysis of consortium SQD-1.1In order to mine the gene and enzyme resources of consortium SQD-1.1, the metagenome DNA of consortium SQD-1.1 was sequenced.1.2 Gbp of DNA sequences was obtained and 1240 contigs longer than 1 kbp were assembled. According to the analysis result of EGTs database, the consortium mainly contains microorganisms belonging to Proteobacteria, Spirochaetes, Bacteroidetes and Firmicutes, and the potential anaerobic cellulolytic genus Clostrium of phylum Firmicutes was one of the main members. Annotation result showed that there were all of three types of cellulase, including endoglucanase, exoglucanase andβ-glucanase in the consortium, many of which are novel in sequence. Further analysis using the Blastall tool indicated that the consortium contained gene fragments belonging to dockerin and cohesion domains, the main components of cellulosome in anaerobic cellulolytic bacteria.As the cellulase genes from metagenomic sequences of consortium SQD-1.1 shared low similarity with proteins in GenBank database, the enzymes should be novel. Expression and further study of these novel cellulose genes can expand the source of cellulases, and may reveal new cellulose-degradation mechanism. Meanwhile, our study indicated that there are still many cellulolytic microorganism and enzyme resources in mangrove ecosystems to explore.