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Abstract
BackgroundThermophilic fungi have attracted increased interest for their ability to secrete enzymes that deconstruct biomass at high temperatures. However, development of thermophilic fungi as enzyme producers for biomass deconstruction has not been thoroughly investigated. Comparing the enzymatic activities of thermophilic fungal strains that grow on targeted biomass feedstocks has the potential to identify promising candidates for strain development. Thielavia terrestris and Thermoascus aurantiacus were chosen for characterization based on literature precedents.ResultsThermoascus aurantiacus and Thielavia terrestris were cultivated on various biomass substrates and culture supernatants assayed for glycoside hydrolase activities. Supernatants from both cultures possessed comparable glycoside hydrolase activities when incubated with artificial biomass substrates. In contrast, saccharifications of ionic liquid pretreated switchgrass (Panicum virgatum) revealed that T. aurantiacus enzymes released more glucose than T. terrestris enzymes over a range of protein mass loadings and temperatures. Temperature-dependent saccharifications demonstrated that the T. aurantiacus proteins retained higher levels of activity compared to a commercial enzyme mixture sold by Novozymes, Cellic CTec2, at elevated temperatures. Enzymes secreted by T. aurantiacus released glucose at similar protein loadings to CTec2 on dilute acid, ammonia fiber expansion, or ionic liquid pretreated switchgrass. Proteomic analysis of the T. aurantiacus culture supernatant revealed dominant glycoside hydrolases from families 5, 7, 10, and 61, proteins that are key enzymes in commercial cocktails.ConclusionsT. aurantiacus produces a complement of secreted proteins capable of higher levels of saccharification of pretreated switchgrass than T. terrestris enzymes. The T. aurantiacus enzymatic cocktail performs at the same level as commercially available enzymatic cocktail for biomass deconstruction, without strain development or genetic modifications. Therefore, T. aurantiacus provides an excellent platform to develop a thermophilic fungal system for enzyme production for the conversion of biomass to biofuels.
Highlights
Thermophilic fungi have attracted increased interest for their ability to secrete enzymes that deconstruct biomass at high temperatures
The majority of Iionic Liquid pretreatment (IL) that are effective biomass solvents are toxic to microbes, fungi have been shown to grow in the presence of high concentrations of some ILs [8]
Growth of thermophilic fungi on biomass substrates Thielavia terrestis NRRL 8126 and Thermoascus aurantiacus ATCC 26904 were cultured on ethanol-extracted switchgrass (SG), ammonia fiber expansion treated switchgrass (AFEX-SG), 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) treated switchgrass (IL-SG), and microcrystalline cellulose (MCC, Sigma #435244) for 3 days at 50°C
Summary
Thermophilic fungi have attracted increased interest for their ability to secrete enzymes that deconstruct biomass at high temperatures. Thermophilic fungi may provide new enzymatic cocktails that are optimized for the industrial biochemical conversion of biomass to sugars for fermentation to biofuels. A strain of the thermotolerant fungus Aspergillus fumigatus was grown on switchgrass in the presence of 5% 1-butyl-3-methylimidazolium chloride, abbreviated as [C4mim]Cl, secreting high amounts of cellulase and hemicellulase enzymes. These GHs were shown to retain residual activity in up to 20% [C4mim]Cl [9]. Thermophilic fungal enzymes may express enzymes that are tolerant to residual IL present in pretreated biomass, enabling a more costeffective IL conversion process with minimal washing required after pretreatment
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