Abstract
The rumen wharves conglomerate of symbiotic fibrolytic microflora with the potential to produce repertoire of carbohydrate-active enzymes to elicit the degradation of recalcitrant plant lignocellulosic complex into simpler forms that can be utilized by the host. A metatranscriptomics approach was adopted to explore the metabolically active microbiota and the transcripts involved in the deconstruction of lignocellulose in Indian crossbred cattle rumen. The experimental animals were fed with mixed diet for a period of twenty-one days and the total RNA was isolated from the rumen digesta using a modified total RNA extraction protocol. After mRNA enrichment, the cDNA libraries were subjected to sequencing under Illumina HiSeq platform and generated about 2.89 GB of raw sequences. The contig assembly was accomplished using two different tools and the redundant sequences were removed before further processing. A total of 133,930 orfs were predicted from the assembled contigs using MetaGeneMark tool and the taxonomic affiliation of orfs were achieved using MEGAN 6.0 Community Edition. A total of 17 bacterial, 5 eukaryotic and 1 archaeal phylum were identified from rumen metatranscriptomic dataset. Phylum Bacteroidetes were acknowledged as the most abundant and metabolically active rumen bacterial populations in cattle rumen with 36,414 orfs corresponding to it, followed by Firmicutes with 6,349 orfs. Further, MEGAN annotation of metatranscriptomic data at species level revealed that Prevotella brevis and Prevotella ruminicola as the most metabolically active bacterial populations representing 4,022 and 2,725 orfs each. The functional annotation of metatranscriptomic data using COG database revealed that a large number of transcripts (~16%) were corresponding to translation, ribosomal structure and biogenesis and carbohydrate transport and metabolism (15%). dbCAN annotation of rumen metatranscriptomic data identified and classified 5,267 transcripts belonging to 168 CAZyme families (GH-52%, GT-26%, CBM-14%, CE-6%, PL-2% and AA-0%). The microbial community analysis of the CAZyme encoding transcripts using M5NR database revealed that a significant proportion of CAZymes were contributed by genera Prevotella (37%) and Bacteroides (21%). The cattle rumen microbiome metatranscriptome analysis presented in this study facilitated the detection of large number of transcripts encoding diverse CAZymes that actively take part in the hydrolysis of plant lignocellulose complex that may be useful for improving livestock and biofuel generation.
Highlights
Rumen-the foremost chamber in ruminant’s stomach-encompasses a plethora of obligate anaerobic microorganisms including bacteria, archaea, fungi and protozoa that are essential for the bioconversion of plant lignocellulosic biomass into simpler forms that can be utilized by the host (Deng et al, 2008)
The highly complex and diverse microbiota present in the rumen has evolved into a symbiotic association with the host and can produce a battery of lignocellulolytic enzymes (CAZymes) that in turn hydrolyse the complex plant polysaccharides and release volatile fatty acids and microbial proteins which are the major sources of carbon, nitrogen and energy for the host (Purushe et al, 2010; Youssef et al, 2013)
The highly complex and diverse microbiota present in the rumen has evolved into a symbiotic association with the host and can produce a battery of lingocellulolytic enzymes (CAZymes) that in turn hydrolyze the complex plant polysaccharides and release volatile fatty acids and microbial proteins which are the major sources of carbon, nitrogen, and energy for the host (Youssef et al, 2013)
Summary
Rumen-the foremost chamber in ruminant’s stomach-encompasses a plethora of obligate anaerobic microorganisms including bacteria, archaea, fungi and protozoa that are essential for the bioconversion of plant lignocellulosic biomass into simpler forms that can be utilized by the host (Deng et al, 2008). The DNA based culture independent techniques form the majority of rumen microbial community studies, as traditional culture dependent methods can symbolise approximately 11% of total microbiota (Fernando et al, 2010). Metagenomics, mainly aimed at understanding the rumen microbial community structure and its enzymatic machinery involved in the degradation of plant structural polysaccharides brings new insights by allowing access to the total community and overcoming the limitations imposed by cultivation and other PCR based assays (Li 2015; Pitta et al, 2016; Jose et al, 2017). While metagenomics brings an insight into the overall microbial and fibrolytic potential of rumen microbial ecosystem, metatranscriptomics approach is considered to be more reliable in scrutinizing the metabolically dynamic microbial communities and its functional transcripts encoding fibrolytic enzymes. Since metatranscriptomic approach focuses on the transcripts that are getting
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