Study On Prokaryotic Microbial Diversity Of Ruidian Hot Spring And Exploitation Of Cellulase And Xylanase Resources

Thermophiles in hot spring were recognized as precious resources of thermophilic enzymes for their unique physiological functions and metabolic mechanisms.In the present study,the diversity of prokaryotic thermophiles from Ruidian hot spring was systematically studied with culture-dependent method as well as metagenomic technology.The isolated strains with cellulase and xylanase activities were screenned,and metagenomic function genes with cellulase and xylanase activities were marked and analysed by using COG,KEGG and CAZy databases.Furthermore,the microbial metagenomic Fosmid library of Jinze hot spring was constructed and thermophilic cellulose and xylanase genes were screened for heterologous gene expression.All these isolated strains with thermophilic enzyme activities and the acquired themophilic enzyme genes were expected to apply abundant and effective enzyme preparation for the process and production of aquafeeds.Sediment,water and enrichment samples were selected as research objects from three hot springs of Ruidian in Tengchong county,Yunnan province of south-west China.The main results are as follows:(1)Seven isolation media and three temperature gradients(45,53 and 60?)were employed for the isolation of prokaryotic thermophiles.A total of 1042 strains were isolated from 19 different samples.All these isolated strains were affiliated to seven phyla of Deinococus-Thermus(39.8%),Firmicutes(28.7%),Proteobacteria(22.0%),Actinobacteria(6.8%),Chloroflexi(2.3%),Bacteroidetes(0.3%)and Rhodothermaeota(0.1%),included 46 different genera of 30 families,24 orders and 12 classes,among which 41 potential novel taxonomies existed.The dominant genera were Thermus(27.7%),Anoxybacillus(13.8%),Meiothermus(11.7%),Bacillus(7.3%)and Caldimonas(7.2%).Results of metagenomic sequencing showed the dominant phyla in Ruidian hot spring were Chloroflexi(13.5%),Acidobacteria(12.3%),Proteobacteria(10.7%),Armatimonadetes(7.9%),Firmicutes(6.4%)and Bathyarchaeota(5.1%).In details,the dominant genera were unclassified Armatimonadetes(7.5%)and unclassified Bathyarchaeota(5.1%).Thermus(with 2.4%abundance in metagenomic data)exhibited high abundance both in culture-dependent and metagenomic results,indicating that the genus Thermus was easy to be isolated by culture-dependent method in laboratory and was the dominant host in Ruidian hot spring.The results showed that different samples,temperatures and isolated media had significant effects on the diversity of isolated strains.(2)The taxonomic of novel species CFH 72773T and novel genus CFH 75059T were determined by the polyphasic taxonomy method,including experiments of molecular taxonomy,morphology,physiological,biochemical characteristics,enzymatic properties and chemical composition.The two strains were named as Thermus thermoamylovorans sp.nov.and Crenbacterium sediminis gen.nov.sp.nov.,respectively.(3)Thermophilic strains with cellulase and xylanase activities were screened by Congo red staining method.Meanwhile,the cellulase and xylanase gene resources were explored by metagenomic technology.The results showed that strains with cellulase activity(46 strains)were mainly affiliated to genera of Thermus(39.1%),Meiothermus(23.9%),Caldimonas(13.0%),Bacillus(10.9%)and Anoxybacillus(6.5%).Strains with xylanase activity(91 strains)were mainly affiliated to genera Thermus(27.5%),Anoxybacillus(19.8%),Meiothermus(17.6%),Caldimonas(9.9%)and Bacillus(8.8%).The rates of strains with cellulase and xylanase activities were 4.6%and 8.7%,respectively.The results also indicated that the genus of Thermus was the impotant resource for producing thermophilic enzymes with cellulase and xylanase activities.Analysis of thermophilic strains with enzyme activities indicated that the number of significant functional strains can be increased by enrichment The functional gene annotation analysis of Jiemingli and Jinze samples showed high abundance of carbohydrate metabolism and amino acid metabolic pathway linked to cellulose degradation and xylan degradation was detected from COG database,KEGG database and CAZy database.Therefore,it can be predicted that abundant thermophilic cellulase and xylanase gene resources existed in these samples,especially in the sediment sample of Jinze hot spring.(4)Metagenomic Fosmid library was constructed from Jinze hot spring,which was demonstrated abundant microbial diversity and potential enzymes.A total of 4100 clones were obtained from the library,and the insert length was 36 Kb-50 Kb averagely.Based on the method of functional screen,sodium carboxymethylcellulose and xylan were added as the solely substrate to the growth medium.16 positive clones with cellulase activity and 12 positive clones with xylanase activity were selected by Congo red staining method.The present research explored microbial diversity,thermophilic cellulase and xylanase resources from hot springs systematically,obtained abundant thermophilic strains and genes with thermophilic enzyme activities,which will provid potential resources for the production of aquafeeds.