Bioprospecting halotolerant lignocellulolytic enzymes from salt marsh ecosystems

Lignocellulose is an abundant agricultural waste product presenting an attractive renewable feedstock for ethanol production. Currently, lignocellulosic bioprocessing is economically constrained requiring expensive pretreatments, high enzyme loadings and large volumes of freshwater imposing water security concerns. Seawater is an abundant and inexpensive prospective replacement solvent. The discovery of highly active halotolerant enzymes with novel tolerances may enable seawater incorporation in bioprocessing; sustainably improving process economics and mitigating water security concerns. Salt marshes are intertidal ecosystems where lignocellulose is remineralised within the predominantly marine organic rich sediments; representing model ecosystems for targeted bioprospecting of highly active, halotolerant and inhibitor resistant enzymes. In this thesis, salt marsh carbon cycling is explored using an in situ degradation experiment. Metatranscriptomics in conjunction with metasecretome proteomics enabled the identification of the lignocellulose-associated enzymatic profile at the ecosystem level and their most likely taxonomic origin. Amplicon sequencing was conducted to reveal the abundance profile for key taxa. Investigations revealed 11,268 identifiable biomass associated proteins, 410 of which were annotated as putative carbohydrate active domains within 307 proteins. The ecosystem level profile displayed a preference for cellulose over matrix polysaccharide deconstruction. Carbohydrate esterase family 1 enzymes were highly abundant; however, lignin modifying enzymes were not, suggesting a decoupling mechanism for accessibility in lieu of oxidative modification. Fungi made no contribution to the lignocellulolytic library; the dominant lignocellulolytic producing taxa were bacteria, identified as the Proteobacteria and Bacteroidetes families Flavobacteriaceae, Alteromonadaceae, Cytophagaceae, Saccharospirillaceae, Marinilabiaceae, Flammeovirgaceae, Bacteroidaceae and Vibrionaceae and Sphingobacteriaceae. A spectrum of potential enzymes were selected for expression in a heterologous system; two enzymes were characterised in depth and both demonstrated halotolerance. This included a putative GH6; identified as an exo-ß-1,4-glucanase with significant activity on crystalline cellulose. A second protein displayed a product profile representative of a ß-1,4-glucosidase with significant transglycosylase activity on oligosaccharide substrates.