Diversity Of Soil Microorganisms And Bacterial Laccases From A Straw-amended Field

Straw amendment is an important farming management practice in the world.It can not only reduce the use of chemical fertilizer,improve the content of soil organic matter,thus maintain soil fertility,but also eradicate straw burning and alleviate environmental pollution.Soil microoganisms represent the major driving force of soil organic matter and nutrient cycling.And soil microbial diversity reflects soil fertility and the changes of ecological environment.Bacterial laccase plays an important role in lignin degradation and the turnover of soil organic matter,which is a potential indicator of soil quality and function.Compared with fungal laccases,bacterial laccases are shown to have advantageous properties,e.g.,high thermo resistance,chloride tolerance and broad pH stability,making them more suitable for textile industry,pulp and paper production and pollutant bioremediation.Moreover,they are easy to be heterologously expressed without glycosylation.In order to understand the turnover of soil organic matter and the corresponding microbial driving mechanism,and to exploit novel bacterial laccases,diversity of soil microorganisms and bacterial laccases from a straw-amended field was investigated.Firstly,microbial community structures in the plough layer and the sub-layer of the straw-amended soil were analyzed by 16S rRNA gene and ITS amplicon sequencing.There was high diversity of bacteria and fungi in the straw-amended soil.And their communities had unique compositions and structures.Though there was no significant difference in bacterial abundance between the two soil layers,the bacterial community structure changed much.But the fungal diversity was not affected.Seven laccase-producing bacterial strains were isolated from the plough layer.Two were spore laccases and the rest were located intracellular.The microorganisms in the plough layer of the straw-amended soil were comprehensively analyzed through metagenomic sequencing.It was found that the amino acid,carbohydrate and energy metabolism related genes were most abundant,which was similar to the metabolism patterns of lignocellulosic-degrading consortia from bovine rumen,beetle gut and compost,indicating that a lot of straw-degrading microorganisms were present in the straw-amended soil.While the composition and distribution of carbohydrate active enzyme coding genes was different from other lignocellulosic-degrading consortia,suggesting its unique lignocellulosic-degrading pattern.The amino acid sequences deduced from the predicted genes in the straw-amended soil metagenome were searched uing the custom profile Hidden Markov Models and 322 potential bacterial laccases were obtained.These bacterial laccases had certain novelty and high diverisity.They were mainly distributed in Proteobacteria,Actinobacteria and Gemmatimonadetes,revealing that the predominant Proteobacteria cooperates with Actinobacteria and Gemmatimonadetes to synergistically act on the processing and metabolizing of lignin in the straw-amended soil.The phylogenetic tree constructed by bacterial laccases from the straw-amended soil and reference sequences further indicates the diversity of the bacterial laccases,many of which may have excellent enzymatic properties.Some bacterial laccase genes were selected for PCR amplification and sequencing.The results suggest that most of the predicted genes from the metagenomic data represent authentic genes in the straw-amended soil microorganisms.The flanking sequences of five laccase genes without complete open reading frames were amplified by thermal asymmetric interlaced PCR.And the full-length sequences were obtained after merging and open reading frame prediction.The complete open reading frames of the seven bacterial laccase genes were translated into amino acid sequences,and sequence alignment showed their high novelty.Although the whole sequences of these seven bacterial laccases were quite different,their copper binding sites were highly conserved.And the predicted three-dimensional structures were similar.All of them were typical three-domain laccases.Four of them contained the signal peptides of the twin-arginine transport system,indicating that they can pass through the cell membrane.The bacterial laccase gene,lacS1,was heterologously expressed in E.coli cultivated under micro-aerobic conditions.And recombinant LacS1 was purified by Ni~+-NTA metal affinity chromatography.The pH range of rLacS1 was wide,and the optimum pH was 5.0.rLacS1 had good stability in pH 5.0-9.0.In addition,the optimum temperature of the recombinant enzyme was 50?,and after treatment at 50?for 1 h,about 25%activity remained.In additon,rLacS1 had high Cl~-tolerance.These properties indicate its application potential.In this thesis,the diversity of soil microorganisms and bacterial laccases from the straw-amended field was studied.The microbial community structure characteristics of the straw-amended soil were comprehensively analyzed.A large number of specific microorganisms were found to play important roles in the straw degradation.And the bacterial laccases showed certain novelty and high diversity,many of which may have excellent enzymatic properties.These provide guidance for understanding lignin degradation and the turnover of soil organic matter,and also provide materials for the development and utilization of bacterial laccase resource.