Analysis Of Microbial Community Structure In Tengchong Hot Spring And Screening Of Thermophilic Strains With Xylanase Activity

Thermophiles are important extremophiles,and the main source of natural thermostable enzymes.By using the culture-dependent method and high-throughput sequencing technology,we explored the diversity and community composition of thermophilic microorganisms in Tengchong hot spring.Using birch sawdust and corncob of waste agricultural lignocellulosic materials as substrates,we have established the efficient screening system for directional screening xylanase active strains in the habitat of high-temperature hot springs by in-situ enrichment and re-enrichment in laboratory.Furthermore,we obtained thermostable xylanases through heterologous expression and compared the enzymatic properties and degradation capabilities with four commercial xylanases to analyze it's market application potential,aiming to achieve the goal of providing high-temperature enzyme resources for the aquafeed industry.The sediments of Tengchong hot springs and enrichments samples were collected as objects, major findings are as follows:(1)We isolated thermophilic bacteria from sediments of several hot springs in Tengchong,Yunnan and enrichment samples by 12 isolation media and 50°C/55°C culture temperatures,analyzing the diversity,community structure and isolation effect of thermophilic bacteria.The results showed that a total of 824 pure strains from this batch of samples were isolated and purified,which belong to Firmicutes(71.1%),Deinococcus-Thermus(13.2%),Proteobacteria(12.5%),Actinobacteria(1.8%),Chloroflexi(1.8%)and Bacteroidetes(0.1%).Anoxybacillus(44.66%)?Bacillus(8.25%)?Brevibacillus(7.77%)and Thermus(6.31%)were the dominant groups of the samples at the genus level.The results showed that factors such as sample processing methods and separation medium have a significant impact on the diversity of isolated thermophilic strains.(2)The diversity and community structure of thermophilic bacteria in the 6 in-situ enrichment samples were studied by high-throughput sequencing technology and bioinformatics analysis,and the functional genes(especially the xylanase genes),were tapped.A total of 1,053,114 sequences were detected in this sequencing,with an average sequence length of 417 bp,and a total of 1027 OTUs were obtained by clustering.At the phylum level,the bacterial communities of the in-situ enrichment samples belong to 65phyla,and the dominant groups were Chloroflexi(29.42%),Acetothermia(11.62%),Proteobacteria(9.20%)and Cyanobacteri(8.32%).Based on the COG database,a total of 24 types of homologous protein clusters were annotated,indicating that the bacterial communities of the in-situ enrichment samples contained abundant functional genes.A total of 6456 pieces of KO information were annotated by KEGG function prediction analysis,including 3 xylanase-related genes.(3)The isolated thermophilic strains were screened with xylanase activity by the Congo red staining method(transparent circle method).The xylanase active strains and positive rates obtained from the three types of samples were 17 strains(5.1%),38 strains(11.0%)and 33 strains(22.3%),respectively.At the genus level,these xylanase-active strains were distributed in Anoxybacillus,Bacillus,Thermus,Calidithermus,Meiothermus,Cohnella,Exiguobacterium,Paenisporosarcina,Geobacillus,Rubrobacter and Tepidimonas,of which Anoxybacillus and Bacillus are the dominant groups.The results indicated that the targeted,efficient screening and separation of xylanase functional strains can be achieved through enrichment.(4)The taxonomic status of the two new candidate species CFH 77666^T and CFH 77786^T isolated from the in-situ enrichment were determined by multiple classifications,and they were named as Meiothermus venustus CFH 77666^T sp.nov.and Cohnella xylansis CFH 77786^T sp.nov.CAZy annotation results showed that the genomes of these two new candidate strains contain a large number of genes related to xylanase,cellulase and chitinase,indicating that these two strains are capable of lignocellulose degradation.(5)A stable thermostable xylanase Thrxyn X1 was obtained by heterologous expression,and compared its enzymatic properties and degradation ability with four commercial xylanases.The results showed that Thrxyn X1 could withstand high temperatures above 90?,and had a wide range of p H tolerance,and could maintain more than 80%of the enzyme activity at p H 5.0-p H 8.0.In addition,it has a strong ability to degrade corn cobs,wheat stalks and corn stalks,which proved to be an excellent candidate enzyme resource for aquatic animal feed production and agricultural waste treatment and reuse.The present study explored systematically the diversity and community composition of thermophilic bacteria in Tengchong hot spring sediments and enrichments systematically,and tapped the thermostable xylanase resources.The targeted and efficient screening of xylanase functional strains have been achieved through the enrichment treatment and a large number of xylanase active strains were obtained.And we got a stable and efficient high temperature resistant xylanase Thrxyn X1 by heterologous expression,which provides excellent candidate high temperature enzyme resources for aquatic feed industries.