Sustainable bioethanol production from enzymatically hydrolyzed second-generation Posidonia oceanica waste using stable Microbacterium metallidurans carbohydrate-active enzymes as biocatalysts

The plant cell wall degradation by extremophilic bacteria is an interesting biological process that is highly relevant for biofuel generation from agrowastes. The halotolerant actinobacterium Microbacterium metallidurans TL13, previously isolated from a tannery wastewater, was studied for its capacity to degrade cellulose and hemicellulose based on genomic and experimental enzymatic analyses. Genome annotation of TL13 strain in dbCAN 2 meta server revealed a total of 158 genes encoding CAZymes including 83 glycosidic hydrolases (GH), 6 carbohydrate esterases, and 25 carbohydrate-binding proteins. Cooperative hydrolytic enzymes required for cellulose degradation such as GH1 and GH3 beta-glucosidases and GH6 endo-beta-1,4-glucanase were found in TL13 genome. For hemicellulases involved in xylan backbone degradation, the related genes were annotated as endo-1,4-beta-xylanase, beta-xylosidase, alpha-arabinofuranosidase, acetyl xylan esterase, and alpha-galactosidase. In TL13 strain, osmoprotectants are taken up by the ProU (ProVWX) transport systems. The synthesis of the powerful compatible solute glycine betaine, which protects the actinobacterium against the high-osmolarity stress, proceeds by the action of three enzymes consisting of choline high-affinity transport system (BetT), choline dehydrogenase (BetA), and betaine aldehyde dehydrogenase (BetB). The presence of genes encoding enzymes involved in osmoadaptation and cellulose/hemicellulose degradation in the TL13 genome is well synchronized with the enzymatic assays, in which CMCase, beta-glucosidase, and xylanase activities were slightly higher under saline conditions compared to controls. Furthermore, the polysaccharide-degrading enzymes of TL13 strain revealed an efficient hydrolysis of untreated Posidonia oceanica waste into fermentable sugars compared to commercial enzymes. The bioethanol production reached 33.35% of the theoretical maximum yield. These genomic and experimental findings make Microbacterium biocatalysts promising green tools for seagrass-based biorefinery.