Insights Into The Diversity Of The Microbial Community And Glycohydrolase Genes In Mushroom Compost And Luxi Cattle Rumen

In nature, decomposition of cellulosic biomass, the most abundant organic component of the biosphere, is a key step in the global carbon cycle. It has been also widely recognized that efficient bioconversion of cellulosic biomass is of extreme importance for the production of sustainable biochemicals. Increasing R&D projects have thus been focused on exploring the natural potential of diverse bioecosystems for efficient hydrolysis of cellulosic feedstocks.Among the decay ecosystems, composting represents an important solution for more sustainable management of organic waste. As a self-heating, aerobic and solid-phase process, composting is driven by the resident microbial community to decompose organic waste to stable materials. It is well established that composting features a succession of microbial communities continuously adapting to the changing nutrient and environmental conditions. Unlike other waste management composting, the substrate preparation using agricultural feedstocks for large-scale mushroom production is a more regulated strategy based on partial composting (phase Ⅰ) followed by pasteurization and conditioning (phase Ⅱ). Even so, this mushroom composting process also involves a complex driving the decomposition of cellulosic substrates. However, microbial communities found in mushroom compost have not been well characterized and the few available culture-independent studies have focused on bacteria from the different mushroom composting stages.In addition to composting, rumen also attracts wide attention of researchers due to its efficient cellulose degradation. As a typical anaerobic habitat, rumen is a good experimental system to study the lignocellulosic degradation process under anaerobic conditions. Cellulose, hemicellulose and pectin polysaccharides can be degraded into simple sugars and then converted to volatile fatty acids and carbon dioxide by different kinds of microorganisms in rumen. Rumen microorganisms are essential to the host, as changes in the microbial community structure have a direct impact on the digestive function and health status of the host. Therefore, research on rumen microorganisms has important implications for the development of animal husbandry. However, rumen microbial communities have not been well characterized to date, and most studies have focused on rumen bacteria or archaea communities.In this article, the diversity of the microbial community and glycohydrolase genes in mushroom compost and Luxi cattle rumen was studied based on culture-independent methods.I. Diversity and dynamics of the microbial community on decomposing wheat straw during mushroom compost productionMushroom compost production represents a specific organic waste handling process. However, knowledge of the complex microbiota involved in this composting process is still incomplete. Here, we explored the development of communities of three important composting players including actinobacteria, fungi and clostridia during the composting of wheat straw for mushroom production. The results revealed the presence of a highly diversified actinobacteria community all across the composting process. The diversity of the fungal community was higher in the thermophilic phase but sharply decreased in the mature compost. Species belonging to the cellulolytic clostridia were otherwise detected only during the thermophilic phase and in the mature compost. Furthermore, we demonstrated an apparent succession of both actinobacteria and fungi with intensive changes in the composition of communities during composting. Notably, cellulolytic actinomycetal and fungal genera represented by Thermopolyspora, Microbispora and Humicola were highly enriched in the mature compost.II. Diversity and dynamics of the cellulose-degrading microbial community on decomposing wheat straw during mushroom compost productionCellobiohydrolases represented by Cel7A and GH48family members have been developed as suitable markers for the study of relevant cellulolytic microorganisms. We showed that a diverse exocellulase-producing actinomycetes were present across all stages of the composting, especially in the mature stage, and they changed during decomposition. Besides, a significant proportion of GH48gene sequences could not be assigned to any known taxa and thus formed novel lineages in the actinomycetal GH48cluster. In contrast with the cellulolytic actinobacteria, exocellulase producing clostridia and fungi were detected only at specific stages of composting. In accordance with the presence of clostridia cluster III species only in phase I and the mature composts, clostridial GH48genes were detectable also in these two phases. Like the case for cellulolytic actinobacteria, there was a significant increase in the diversity in the the cellulolytic clostridia in mature compost. The diversity of cellulolytic fungal community was the highest at the initial stage of composting although the community is dominated by species closely related to the endophyte Bipolaris sorokiniana. No cellulolytic fungi were detected during the thermophilic phase probably due to the high temperature at this stage. In contrast with cellulolytic actinomycetes and clostridia, the diversity of cellulolytic fungi sharply decreased in the mature compost. The two OTUs detected in the mature compost were both closely related to the Ascomycota Humicola grisea Cel7A, highlighting an important role this species may play in the following growth of mushroom. Taken together, although cellulose represents a substrate that is continously present during the entire composting process, the community of cellulolytic microorganisms showed similar successive changes to those of the total microbial community and an efficient cellulose-degrading community may develop during the maturation of compost to exert a beneficial effect on the mushroom growth. Furthermore, the prevalence of these diversified cellulolytic microorganisms holds the great potential of mining novel and efficient lignocellulose decomposing enzymes by digging up this unique resource.III. Insights into the abundance of microbial community in rumen samples.The rumen digesta was sampled at different time points after the cattle was fed. The soluble sugar content in rumen samples was found to decrease gradually along with the cattle feeding time, and was relatively low at12h. Quantification of bacterial rDNA and copy numbers at the genomic level showed that both communities increased continually and were relatively high at12h. The copy numbers of bacterial rDNA were10to20times more than those of anaerobic fungal ITS in all samples. Anaerobic fungi-specific GH6family genes (GH6-AF) group was selected as a representative to get a general knowledge of the transcription level of cellulase genes. The results showed that the dynamics of the two sub-groups GH6-AFa and GH6-AFb were similar, with a low level before8h, and increased significantly between8h and12h. The transcription level of GH6-AFa was30-250times more than that of GH6-AFb at each time point. It is worth noting that there was an apparent delay of the transcription of GH6-AF group genes compared to the anaerobic fungal community, which may be due to the soluble sugar content in the forage. In addition, we also tried to enrich the fiber-adherent microorganisms. The quantification results showed that anaerobic fungi and bacteria were significantly enriched in the fiber fraction of rumen sample, while protozoa was the opposite. These results and the related technical methods established laid the foundation for subsequent high-throughput sequencing analysis.IV. Insights into the diversity of microbial community in Luxi cattle rumen based on pyrosequencing.To investigate the microbial community structure in cattle rumen, bacterial16S rDNA and fungal ITS sequences were amplified from the DNA of the total and the fiber fraction of rumen12h sample and sequenced using Roche454high-throughput sequencing platform. In total,85024bacterial16S rDNA sequences represented by18781OTUs and47186fungal ITS sequences represented by4190OTUs were obtained. Despite that the sequence numbers varied greatly between the samples, all rarefaction curves appeared to be reaching asymptotes, indicating that the current sequencing capacity was sufficient for all the libraries.(1) Most bacterial taxa were present in both the total and the fiber fraction of rumen sample. Most of the abundant bacterial genera in both samples were unclassified, distributing in the orders Clostridiales, Bacteroidales, Spirochaetales and Fibrobacterales. Phylogenetic analysis showed that bacteria in the two samples distributed mainly in the Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria phyla, in which Proteobacteria contained the most orders but their abundances were relatively low.(2) Similar to the bacteria, most fungal taxa were present in both the total and the fiber fraction of rumen sample. Fungal species of the genera Asteromassaria, Pandora, Gibellula and Sphaerulina were abundant in both samples, in which Asteromassaria was predominant, representing21.0%and29.5%of the total and the fiber libraries respectively. The two samples had a similar fungal taxa distribution on the order level, with the most abundant order Pleosporales, followed by Hypocreales and Entomophthorales. The total proportion of these three orders was more than50%in both of the two samples. Phylogenetic analysis showed that a considerable portion of fungi in the two samples belonged to the classes Dothideomycetes and Sordariomycetes within the Ascomycota phylum. Within the anaerobic fungi, the two most abundant genera were Cyllamyces and Piromyces. Besides, there were a lot of unassigned anaerobic fungi.V. Insights into the diversity of microbial function in Luxi cattle rumen based on metatranscriptomic sequencing.To get a comprehensive insight into the functional diversity of microorganisms including anaerobic eukaryotes and bacteria within cattle rumen, mRNA from both communities adherent to plant fiber and in whole digesta were isolated, reverse-transcribed and sequenced. In total,27.31gigabases of sequences were obtained and348442contigs assembled. From these data, a total of358773ORFs were predicted. Despite that the number of genes predicted varied greatly between the samples, all rarefaction curves appeared to be reaching asymptotes, indicating that the current sequencing capacity was sufficient for all the libraries.To get more information about cellulose degradation in cattle rumen, plant wall-degrading related genes were annotated and classified based on relevant databases. In total,743glycoside hydrolase family genes,893carbohydrate binding domain sequences and16142transporter genes were identified. Moreover, lignocellulose-degrading transcripts were significantly enriched in the fiber adherent microbiota. Further data mining is needed to obtain more informations about the respective contribution of eukaryotes and bacteria in the actual degradation of lignocellulose in cattle rumen.