Fungal Enzymes for the Saccharification of Microalgal Biomass

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Abstract

The global demand for energy is increasing, with currently consumed fossil fuels being non-sustainable and the emission of CO2 and other pollutants due to their use exacerbating global warming. Therefore, there is a recent shift towards utilizing alternative sources of energy. Biofuel from microalgae is considered sustainable, eco-friendly, and does not present a conflict with food supply. Additionally, some microalgal strains can grow efficiently in wastewater, and therefore they satisfy the dual role of bioremediation and biomass production. To produce an efficient and affordable microalgae-based biofuel, the generated microalgal biomass should contain high amounts of lipids and carbohydrates used to produce biodiesel and bioethanol, respectively. The effects of nitrogen stress on the accumulation of lipids, starch and cellulose were assessed in five microalgal strains from wastewater. No significant effect was observed on the accumulation of lipids and starch. However, a significant increase was recorded for cellulose content under certain levels of nitrogen stress for Parachlorella hussii and Hindakia tetrachotoma. Chlorella luteoviridis and H. tetrachotoma acclimated better to stress conditions compared to other microalgal strains, maintaining stable concentrations of lipids. Therefore, P. hussii, H. tetrachotoma and C. luteoviridis were selected as potential biofuel feedstock.Liquid and solid-based (in soil or compost) substrate fermentation approaches were used to degrade intact microalgal biomass by residential fungal species. Potential degrading fungi were classified by morphology and identified by rDNA sequencing. C. luteoviridis, H. tetrachotoma and P. hussii biomass were buried in environmentally-controlled soil and successfully degraded by resident fungi at 25°C; Fusarium solani represented 60-70% of identified fungi. Similarly, C. vulgaris, Chlamydomons reinhadatii CW15 and Scenedesmus sp. microalgal biomass in minimal media were utilized by Trichosporon sp. at 25°C and Galactomyces pseudocandidum at 45°C, both originating from compost and accounting for >60% of fungal species identified at 25°C. However, apart from G. pseudocandidum, no morphologically-distinct fungal species were identified at 45°C for all minimal media cultivations. For C. vulgaris compost cultivation, identified fungi were Penicillium chrysogenum at 25°C and Aspergillus tubingensis at 37°C, accounting for 60-70% of fungi, while Thermomyces lanuginosus accounted for >80% at 45°C. In addition, Aspergillus fumigatus accounted for 20-30% of identified fungi at 37 and 45°C. Screening the efficiencies of identified fungal isolates in saccharifying C. vulgaris biomass showed that the most efficient saccharification was recorded for crude enzymes from A. fumigatus at 37°C, where 66.5 and 93.8% of carbohydrates were released from intact and lipid-extracted C. vulgaris, respectively, with no pre-treatment. Elevating the temperature to 50°C resulted in release of all carbohydrates from intact C. vulgaris by A. fumigatus crude enzymes. Secretome analysis of A. fumigatus crude enzymes, extracted after cultivation with lipid-extracted C. vulgaris, showed the existence of various degrading enzymes, including carbohydrate-active enzymes (CAZy). This approach can be applied to design custom-made enzyme cocktails with potential use in efficient industrial-scale saccharification.

Keyword(s)

Biofuel, Bioethanol, Third generation biofuel, Microalgal biofuel,Microalgae; Saccharification, Fungal enzymes, A. fumigatus secretome

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