Investigation Of Microbial Diversity And Metagenomic Analysis Of Ligninolytic Genes For A Tropical Rain Forest Soil From Xishuangbanna

Ethanol made from lignocellulosic biomass sources, instead of corn, provides a promising way against expensive corn ethanol or fossil energy to produce in a commercial scale. Understanding of the microbial lignocellulose utilization strategy in natural environments will drive a development of commercial technologies for lignocellulosic convertion. In this study, constructing rRNA gene clone libraries and RFLP technologies were used to investigate the diversity of bacterial and fungal communities in forest soil from Xishuangbanna Tropical Forest. A total of 75 bacterial clones and 26 fungal clones were identified and sequenced, the results of sequencing were checked and blast, and then phylogenetic trees were constructed. The bacterial community was grouped into Acidobacteria, Proteobacteria, Actinobacteria, and Unclassified bacteria. Among them, Acidobacteria were the predominant group, about 89.4%, is subgrouped into Gp1, Gp2, Gp3 and Gp5. Community structure in fungi is corresponded to the Fungi incertae sedis, included Mucoromycotina and unclassified Zgomycetes, followed by Ascomycota and Basidiomycota. Most of these fungi were related to the reported lignocellulolytic fungi, such as Cryptococcus and Panaeolus of Basidiomycota, Tricladium, Aspergillus, Penicillium and Neurospora of Ascomycota etc. It suggested diversity in the fungi communities involved in lignocellulose degradation.These diverse fungal communities degrade lignocellulose materials regularly. So Successional regulations of the fungal Communities during the process of microbial lignocellulose degradation were further studied. The lignocellulolytic microorganisms were enriched using the sugar cane bagasse and wood chips as sole carbon source to for 8 days ang 15 days. The 18S rRNA gene clone libraries for each of samles were constructed and analyzed, respectively. Then both of the RFLP phylotypes were detailedly added up and compared, and new phylotypes were sequenced again (16 fungal clones for 8 days and 11 fungal clones for 15 days). The results indicated that the fungal diversity is reduced during sucessional enrichment, and the community structure is distinct in the different stages. In addition, the predominant group also changed. With the succession degradation of lignocellulose, the diversity and number of Ascomycota gradually reduced, but Basidiomycota become the dominant group account for lignocellulose-degrading. The microorganisms produce a battery of enzymes and work synergically to biodegrade lignocellulose, So, laccase gene from the wood-rotting fungi Basidiomycota were selected to explore the miceobial lignocellulose utilization strategy at enzyme gene level. We first cloned a 140bp fungal laccase gene fragment targeted the genomic DNA as as template, labelled probe using DIQ and screened from the constructed soil metagenomic library by colony hybridization. Six positive clones were obtained with hybridization signals. Among them, a clone was subcloned sequenced and analyzed. The study provided a solid basis for metagenomic library screening and acquire more lignocellulose-degrading enzyme from soil environment, has important ecological significance.