Description: Highlights: • Characterization of enzymes from bone-degrading marine microbiomes. • Enzymes degrade sialo/glyco-proteins at multiple conditions of pH and temperatures. • Enzyme cocktails are useful for valorising bone residues in biorefinery industry. Abstract: Many microorganisms feed on the tissue and recalcitrant bone materials from dead animals, however little is known about the collaborative effort and characteristics of their enzymes. In this study, microbial metagenomes from symbionts of the marine bone-dwelling worm Osedax mucofloris, and from microbial biofilms growing on experimentally deployed bone surfaces were screened for specialized bone-degrading enzymes. A total of 2,043 taxonomically (closest match within 40 phyla) and functionally (1 proteolytic and 9 glycohydrolytic activities) diverse and non-redundant sequences (median pairwise identity of 23.6%) encoding such enzymes were retrieved. The taxonomic assignation and the median identity of 72.2% to homologous proteins reflect microbial and functional novelty associated to a specialized bone-degrading marine community. Binning suggests that only one generalist hosting all ten targeted activities, working in synergy with multiple specialists hosting a few or individual activities. Collagenases were the most abundant enzyme class, representing 48% of the total hits. A total of 47 diverse enzymes, representing 8 hydrolytic activities, were produced in Escherichia coli, whereof 13 were soluble and active. The biochemical analyses revealed a wide range of optimal pH (4.0–7.0), optimal temperature (5–65 °C), and of accepted substrates, specific to each microbial enzyme. This versatility may contribute to a high environmental plasticity of bone-degrading marine consortia that can be confronted to diverse habitats and bone materials. Through bone-meal degradation tests, we further demonstrated that some of these enzymes, particularly those from Flavobacteriaceae and Marinifilaceae, may be an asset for development of new value chains in the biorefinery industry.

Description: The marine bone biome is a complex assemblage of macro- and microorganisms; however, the enzymatic repertoire to access bone-derived nutrients remains unknown. The bone matrix is a composite material made up mainly of organic collagen and inorganic hydroxyapatite. We conducted field experiments to study microbial assemblages that can use organic bone components as nutrient source. Bovine and turkey bones were deposited at 69 m depth in a Norwegian fjord (Byfjorden, Bergen). Metagenomic sequence analysis was used to assess the functional potential of microbial assemblages from bone surface and the bone-eating worm Osedax mucofloris, which is a frequent colonizer of whale falls and known to degrade bone. The bone microbiome displayed a surprising taxonomic diversity revealed by the examination of 59 high-quality metagenome-assembled genomes from at least 23 bacterial families. Over 700 genes encoding enzymes from 12 relevant enzymatic families pertaining to collagenases, peptidases, and glycosidases putatively involved in bone degradation were identified. Metagenome-assembled genomes (MAGs) of the class Bacteroidia contained the most diverse gene repertoires. We postulate that demineralization of inorganic bone components is achieved by a timely succession of a closed sulfur biogeochemical cycle between sulfur-oxidizing and sulfur-reducing bacteria, causing a drop in pH and subsequent enzymatic processing of organic components in the bone surface communities. An unusually large and novel collagen utilization gene cluster was retrieved from one genome belonging to the gammaproteobacterial genus Colwellia.