Isolation Of Anaerobes From The Microbial Consortium TMC7 And Study Of The Synergistic Mechanism For Lignocellulose Degradation

Lignocellulose is an important biomass resource and widely used.Due to the complex structure,the degradation of lignocellulose requires the synergistic action of cellulose,hemicellulose and lignin degrading enzymes,and it is difficult for a single strain to fully and completely degrade lignocellulose.TMC7 is an effective lignocellulolytic microbial consortium constructed in our laboratory in previous studies.In order to study the mechanism of its efficient degradation of lignocellulose,metagenomic sequencing analysis was carried out in previous studies,and it was found that most of the key enzymes of lignocellulose degradation were encoded by anaerobic bacteria in TMC7.On this basis,under the guidance of metagenomic information,anaerobes were isolated from TMC7 and characterized.Lignocellulose decomposition characteristics of the isolates were studied.During the experiment,we found that there was a symbiosis between anaerobic bacteria and aerobic bacteria.Furtherly,the symbiosis between anaerobic bacteria and aerobic bacteria were studied by metagenomic sequencing.The anaerobic symbiosis system isolated in this study and the aerobic bacteria obtained in previous studies were used to build engineering microbial consortium that can effectively degrade CMC-Na and xylan,and the synergistic effect between anaerobic and aerobic bacteria was studied.The research results obtained in this study are as follows:(1)In anaerobic culture environment,178 single bacterial colonies were isolated from TMC with the decomposition ability of CMC-Na/ xylan using Congo red plate.16 S r DNA sequencing showed that these isolates were mainly Thermoanaerobacterium and Geobacillus.The decomposition characteristics showed that Thermoanaerobacterium could degrade CMC-Na and had the ability to degrade lignocellulose efficiently.In the process of liquid culture of the single colony,it was found that there was a symbiosis of anaerobic bacteria and aerobic bacteria in the single colony.(2)Four single colony cultures,YT1,YT3,YT7 and YT9 with strong decomposition ability,were selected and 16 S r DNA metagenomic sequencing were performed.The analysis of bacterial structure confirmed that the symbiosis between anaerobes and aerobes.Kruskal-Wallis test showed that there were significant differences on Thermoanaerobacterium,Geobacillus and Pseudoclostridium in the four anaerobic symbiosis systems: YT1,YT3,YT7 and YT9.Correlation analysis showed that p H was negatively correlated with CMC enzyme activity and positively correlated with xylanase activity.(3)Anaerobe symbiotic system mixed culture with aerobic bacteria and symbiotic system YT1 mixed culture with group C consisting of aerobic bacteria T11,T12...T15 obtained an efficient xylan degrading strain with a xylan degradation rate of 76.8%.The commensal system YT7 was cultured with group C mixture,and an effective degradation CMC-Na strain with 47.9% CMC-Na degradation rate.(4)The anaerobic symbiosis system was combined with the aerobic bacteria(including20 aerobic isolates and 2 aerobic consortium),which were isolated from TMC7 in previous study.It was found that the combination of aerobic bacteria and anaerobic bacteria could significantly improve the decomposition ability of xylan and CMC-Na.Among which the combine of anaerobic symbiosis system YT1 and Group C aerobic bacteria(contained T11,T12,T13,T14 and T15)had the highest xylan degradation rate of 76.8%,and the combine of YT7 and group C had the highest CMC-Na degradation rate of 47.9%.