Study On The Effects Of Different Carbon Sources On The Diversity Of Thermophilic Bacteria In Tengchong Hot Springs And Screening Strains With Thermostable Enzyme Activities

Xylanase and cellulase are important industrial enzymes in feed production,but they have the limitation due to their poor thermal stability.Therefore,the development of high temperature resistant feed enzyme has been the focus of feed industry.Hot springs are high temperature habitats,under the pressure of high temperature environment,the microorganisms in hot spring habitats have evolved special physiological functions,genetic genes and adaptive mechanisms,and can produce a variety of thermophilic functional enzymes,it is an important source of high temperature enzyme and new enzyme resources.Tengchong,located in the western border of Yunnan Province,China,is famous for its strange and spectacular geothermal landscape.Previous studies have shown that the habitat contains abundant thermophilic microorganisms.Culturable microorganisms only account for less than 1%of the total amount of microorganisms in the world at present.Culturability of uncultured microorganisms is still a scientific problem that scholars need to solve.Biolog ECO technology combines the advantages of natural environment simulated culture and mixed culture,break through the limitations of traditional microorganism separation and cultivation.It provides an opportunity to explore new thermophilic microbial resources,due to the diversity of carbon source types of Biolog ECO microplate substrate.The hot spring group of Tengchong,Yunnan,China was used as the research object in this study,combining Biolog ECO technology,Biolog ECO microplate enrichment culture,culture-dependent method and high-throughput sequencing technology,the purpose of this study is to explore the impact of different carbon sources on the composition of thermophilic bacteria community in Tengchong hot spring,and to screen thermophilic cellulase and xylanase active strains.In this study,18 typical hot spring samples were selected from Tengchong,including Heinitang hot spring in Houqiao town,Ruidian hot spring in Diantan town,and Huanxi hot spring in Zhonghe township.The research results obtained mainly include:(1)Based on Biolog ECO technology,the utilization ability of carbon sources and the diversity of metabolic function of environmental microbial communities in 18 sample sites of Tengchong hot spring were obtained,an effective separation and culture system suitable for enrichment by Biolog eco microplate was established.The results showed that there were three types of microbial communities in 18 hot springs.The microbial communities of HNT-2,HNT-7,HNT-3,HNT-5 and HXQ-WQ have higher utilization of all carbon source substrates;HXQ-HB-1,JZ,GXS-2,HXQ-HB-2,HNT-1,HXQ-HB-3,HNT-6,and GXS-3have general ability to utilize carbon source substrate;JML,XRD,JMW,GXS-1,and HNT-4 have low utilization of carbon source substrate.(2)Based on Illumina Miseq high-throughput sequencing technology,six types of carbon source enrichment cultures of HNT-2 and HXQ-WQ hot spring samples were selected to study the effect of different carbon sources on the community structure of thermophilic bacteria.A total of 1,971,621 original sequences were obtained from 36 samples,with an average sequence length of 417 bp.The OTUs of all sequences were divided into a total of 590 OTUs,which belonging to 48 phyla,86 class,141 order,192family and 268 genera.The results showed that the enrichment of different carbon sources changed the community structure of thermophilic bacteria in the hot springs of Tengchong.Proteobacteria(78.61%)and Firmicutes(17.94%)were the dominant populations,and their abundance was greatly improved.Heatmap shows that the abundance of amino acid and sugar enrichment bacterial communities is higher,while the abundance of alcohols and amines is lower.Venn diagram analysis shows that there are a large number of endemic bacterial groups in enrichment cultures with different carbon sources.At the level of the top 10 genera,the six types of carbon sources have a significant effect on the growth of Pannonibacter,Pseudomonas,Anoxybacillus,Caloramator,Brevundimonas and Thermomonas.Functional prediction of16S r DNA can be found a very rich variety of functional genes.(3)Based on culture-dependent method,six kinds of isolation media were designed according to 31carbon sources of Biolog ECO microplate.In order to study the effect of different carbon sources on the diversity of culturable thermophilic bacteria,the enrichment cultures of 18 hot spring samples were cultured and separated.Results showed that atotal of 920 strains were isolated.After identification,the strains were belong to Proteobacteria(52.0%),Firmicutes(30.3%),Deinococcus-Thermus(6.6%),Actinobacteria(6.4%),Bacteroidetes(2.6%),Chloroflexi(2.1%),comprising 10 classes,22 orders,36families,65 genera and 2 potential new taxa.The results showed that the enrichment and culture of different carbon sources resulted in a great increase in the abundance of Proteobacteria and a large number of Brevundimonas members.The addition of amino acid carbon sources could increase the richness and diversity of thermophilic bacteria.And the addition of carbohydrate carbon sources,more specific groups were expected to be obtained.(4)For the two potential new taxa,the new genus Kallokrene roseus CFH 73958~T gen.nov.,sp.nov.and the new specie Rubellimicrobium sediminis CFH 75288~T sp.nov.were determined the exact taxonomic position by polyphasic taxonomic analyses.The 16S r RNA gene sequence of strains CFH 73958~T and CFH75288~T have been deposited in Gen Bank under the accession number MK395365 and MK433208,respectively.Whole Genome Shotgun project of strains CFH 73958~T and CFH 75288~T have been deposited at Gen Bank/ENA/DDBJ under the accession VIYF0000000000 and WYDP00000000,respectively.(5)The thermophilic enzyme active strains were screened by Congo red staining from 920Thermophilic bacteria strains,96(positive output rate:10.43%)strains with xylanase activity and 28(3.04%)strains with cellulase activity were found,respectively,and of which 28 strains had both xylanase and cellulase activities.The analysis of the results showed that the addition of carbon sources of amino acids can increase the abundance of xylanase strains.The addition of alcohol carbon source can increase the diversity of enzyme active strains.Acid or alcohol carbon source can obtain more strains with xylanase and cellulase activities.The present research provides guidance for the isolation and cultivation of thermophilic bacteria in hot spring habitats,and obtained abundant the thermophilic bacteria and strains with enzyme activity,which will provid potential resources for the production of aquafeeds.