Multi-omics and taxonomic analyses of empty fruit bunch adapted mangrove microbial communities with lignocellulolytic abilities

Abstract

Current demand for energy drives the rapid progress of second-generation biofuel development. Use of lignocellulosic biomass, such as oil palm empty fruit bunch (EFB) in second-generation biofuels production resolved the limitation of firstgeneration biofuels which compete with food source. Lignocellulosic pre-treatment and saccharification are two crucial steps in second-generation biofuel production. These steps require synergistic action of lignocellulolytic enzymes. The use of large volume of freshwater in biofuel industry is a major concern as it creates competition between biofuel industry and human consumption. Seawater, which cover 96.5% of the biosphere could be an alternative to freshwater in biological pre-treatment and saccharification of lignocellulosic biomass. Therefore, the discovery of novel salttolerant microorganisms and their halophilic enzymes is an important aspect of lignocellulosic waste deconstruction using seawater. In this study, halophilic microbial community was collected from mangrove soil at Tanjung Piai National Park, Johor. Their ability to degrade lignocellulose was explored using culture independent and culture dependent approaches. The mangrove soil was used as inoculum and incubated with EFB in artificial seawater medium for 10 weeks. Total DNA, RNA and proteins were extracted (culture independent). 16S rRNA and 18S rRNA gene fragments were amplified from total DNA and composition of microbial community was analyzed based on amplicon metagenome sequencing. Taxonomic analysis showed that phyla Proteobacteria and Bacteroidetes were predominant prokaryotic population. Metatranscriptomic analysis revealed a total of 9,953 open reading frames (ORFs) related to lignocellulose degradation: 3,867 glycosyl hydrolases (GHs), 2,485 carbohydrate binding modules (CBMs), 2,156 carbohydrate esterases (CEs), 947 auxiliary activities (AAs) and 498 polysaccharide lyases (PLs). The highly expressed enzyme families were GH74, CE1, GH5, AA2, GH43, CE3, GH3, CE15, GH10 and GH6. Metaproteomic analysis identified a total of 87 lignocellulolytic enzymes in bound fraction of EFB and culture supernatant. Synergistic action of different lignocellulolytic enzymes from diverse microbial origin was observed with mostly affiliated to phyla Proteobacteria and Bacteroidetes. In addition, bacteria from the mangrove microbial community were isolated and their lignocellulolytic abilities were assessed (culture dependent). Two halophilic bacteria from the phylum Bacteroidetes, namely Meridianimaribacter sp. CL38 and Robertkochia sp. CL23 were selected for genomic analyses. A total of 30 and 89 lignocellulolytic enzymes were encoded in the genomes of strain CL38 and CL23, respectively. Furthermore, both strains demonstrated their abilities to degrade EFB. Genomic analyses of these two strains are the first genomic information from their respective genera. Due to the low similarity of 16S rRNA gene with closely related member, strain CL23 was further taxonomically characterized via polyphasic approach. Based on phenotypic, chemotaxonomic and genomic evidences, the strain CL23 is proposed as a new species with the name Robertkochia solimangrovi sp. nov. Multi-omics and taxonomic analyses in this study identified new halophilic microorganisms from mangrove with a wide array of new lignocellulolytic enzymes that are able to degrade EFB. These enzymes could be further investigated for development of enzyme cocktails which will be useful for seawater based lignocellulosic biorefining.