Purification And Cloning The Bacterial Xylanase From The Hindgut Of Macrotermes Barneyi And The Microbial Diversity In Combs Of Fungus-growing Termites

Termites are major group of biomass conversion in ecological system, they utilize the lignocellulose for energy and nutrition, avoiding pollution to the environment of the physical and chemical processing, which is a moderate and high efficient bio-reactor. It is generally known that this efficient digestion of wood is closely linked to the symbiotic microorganism and the surroundings. Termites are generally divided into lower termites and higher termites based on protozoa in their hindguts or not. Many studies have been performed about cellulolytic system of the lower termites:a dual cellulose degradation mechanism--endogenous cellulases (termite origin) and exogenous cellulases (symbiotic microbe origin) cooperatively accomplish the high ability of lignocellulose degradation, while higher termites occupying 75% of the total termites degrading cellulose seem complicated.Fungus-growing termites(Termitidae, Macrotermitinae), named after the symbiotic fungus- Termitomyces, are distributed in tropical and subtropical regions of Asia and Africa. Cultivating and feeding on the Basidiomycete fungi, it forms the symbiotic system among the termite, microbe in the gut and symbiotic fungi in vivo, which intrigues all of us. In order to understand the role that the microbe in the gut and symbiotic fungi in vivo playing in the degradation of wood, we did some work as following:Firstly, Paenibacillus sp.Mbl was isolated in a previous study from the hindgut of the fungus-growing termite Macrotermes barneyi. The strain produced extracellular xylanase named XylMbl. This was purified with ammonium sulfate precipitation and three consecutive purification steps using HiTrapTM QFF, HiTrapTM Butyl FF, and Superdex TM 75. The XylMbl remained stable at neutral to alkaline pH (pH 7.0-pH 11.0). Those confirmed that the microbe in the hindgut of fungus-growing termite have the potential to process lignocellulose, and we got the XylMbl gene successfully.Secondly, microorganism in the different aged fungus comb of Odontotermes formosanus were investigated using high-throughput 16S rRNA sequencing:firstly, there existed less species in the fungus comb, the advantage bacterium group was Bacteroidetes、Firmicutes and Proteobacteria, Bacteroidetes owned a larger proportion than in the gut of O.formosanus, they may work as the the source of nitrogen for the host, we also detected the Spirochaetae which were not found in the gut; secondly, Rikenellaceae and Porphyromonadaceae were the dominant micro flora in the hindgut of Macrotermes barneyi, the percentage they took was similar to that in Fungus Comb 2 which formed shortly; finally, the elder fungus comb had larger Bacteroides, Lachnospiraceae, Rhodospirillaceae and Alphaproteobacteria, the young fungus comb involved many types of bacteria, except 36.32% Rikenellaceae, it also had some species such as Acholeplasmataceae, Caldilineaceae, Nitrospira, Rhodospirillaceae and so on the symbiotic fungus-Termitomyces proved to be an essential part of the fungus-growing termite with the existence in the two combs plentifully, whether as the food or in the digestion of lignin and esters. The study about the fungus microbial diversity of O.formosanus enriched our understanding of the termites’ surroundings.Above all, this article does some research on the fungus-growing termite, through purification and analysis of the XylMbl from Paenibacillus sp.Mbl screened from the hindgut of M. barneyi, we confirmed the microbe in the hindgut of fungus-growing termite had the potential to process lignocellulose, and we got the gene XylMbl succesfully. Meanwhile high-throughput 16S rRNA sequencing was used to identify the microorganism in the different aged fungus comb of O.Formosanus, it enriched our understanding of the termites’surroundings.