Microbiome metatranscriptomics of the black soldier fly larvae gut for identification and functional characterization of lignocellulosic biomass-degrading microbes

Abstract

Second-generation biofuel production has emerged as a sustainable and alternative energy option to the fast depleting, ecologically unfriendly petroleum-based fuels. Second-generation biofuels are produced from lignocellulosic industrial and agricultural wastes, energy crops, and crop residues, and therefore, are not a threat to food security. Despite second-generation biofuels being a promising technology, obstacles in their production prevent the bioconversion process from attaining optimal performance under minimal capital investment. The Black soldier fly (BSF) (Hermetia illucens) larvae have been demonstrated as a key source of alternative protein in the animal feeds industry, the production of chitin and essential oils, and as a useful tool in the valorization of organic biomass and other biodegradable wastes, mainly attributed to their potent larval gut microbiome. The BSF larval gut microbiome is an active area of study due to the rapid growth and broad degradation capabilities of the larvae, and their non-competence as a vector for any known human diseases. This presents the BSF larvae as a potential source of lignocellulolytic microorganisms and enzymes capable of the breakdown of recalcitrant organic biomass and potential applications in the second-generation biofuel industry. Members of the genera Dysgonomonas, Bacteriodes, and Actinomycetes were identified in high abundance from preliminary BSF microbiota studies. These genera have shown lignocellulolytic abilities and thus have the potential to be implemented in the production of different value-added products such as biofuels and animal feeds. To investigate the effects of dietary intervention and the presence of microorganisms and enzymes associated with lignocellulolytic activities in the BSF larval microbiome, a randomized control trial design was employed in this study where BSF larvae were bred under different diets, selected based on their increasing lignin contents i.e., processed chicken feed (CF), chicken manure (CM), brewers’ spent grain (BSG), water hyacinth (WH) and feed mix (FM) that contained all diets mixed in the ratio 1:4. mRNA libraries from 16 samples (n=16) were prepared and RNA-Sequencing was conducted using the PCR-cDNA approach with the MinION sequencing platform. The gut microbiome and functional profiles were compared between the samples while mainly screening for the lignocellulolytic microbial genera reported from preliminary studies. Metatranscriptome samples from diets BSG and WH possessed in high abundance two of the three main genera identified in preliminary 16S BSF microbiota studies and hypothesized to be involved in lignocellulolytic functions - Bacteroides and Dysgonomonas. Additionally, lignocellulolytic CAZyme families GH43_16 and GH51 containing enzyme α-L-arabinofuranosidase (abf, EC3.2.1.55), known for degrading arabinoxylan and arabinogalactan hemicellulose fractions, were identified in the BSG and WH metatranscriptome samples, respectively. Polysaccharide Utilization Loci (PUL) screening further revealed PUL0013 and PUL0395 gene clusters that encode for the hemicellulolytic enzyme abfB in CAZy family GH51. We conclude that the BSF larvae gut microbiome profiles were significantly altered with dietary intervention and that the BSF gut microbiome could not only be used as a source of lignocellulolytic microbes and CAZymes, but could also guide the identification of degradative pathways involved that could be applied in, but not limited to, enzyme hydrolysis in second-generation biofuel production.