Seawater Systems for Sustainable Development: Evaluation of a Marine Microalgal Strain as Biomass Feedstock for Hypersaline Bioethanol Production

Abstract

The potential of microalgal biomass as a feedstock for bioethanol fermentation has been widely considered alongside the mix of other bioenergy streams. Yet only a modest level of research has been reported in this area compared to other renewable feedstock for bioethanol. Use of marine microalgae from seawater systems provides greater sustainability at the scale required for biofuels to circumvent reliance on fresh water, but presents processing challenges associated with fermentation of hypersaline biomass. The marine microalgae Tetraselmis sp. strain MUR-233 was selected based on biomass productivity as part of a broader Australian Research Council Linkage Project (ARCLP 100200616) to assess the diversification of energy streams from microalgae. Detailed carbohydrate analysis was conducted on monoculture isolates of this strain to assess its potential as a bioethanol feedstock. MUR-233 when cultivated for biomass productivity had a total carbohydrate content that ranged from 6.8 % to 11 % ash free dry weight (afdw). The cell wall carbohydrates of MUR-233 were composed primarily of 3-deoxy-manno-2-octulosonic acid and galactose, present at respective quantities of up to 63.1 % and 19.3 % molar ratio of cell wall carbohydrates. Accumulated intracellular starch was the key variant with biomass total carbohydrate composition, and could be enriched to an average of 47 % afdw when MUR-233 was cultured under continuous illumination at 7 % salinity in hypersaline seawater. Glucose from starch was determined to be the primary substrate from MUR-233 biomass for ethanologenic conversion. For utilising the starch-enriched hypersaline MUR-233 biomass, the filamentous fungus Rhizopus oryzae NRRL 1526 was found to possess sufficient facultative halotolerance for simultaneous saccharification and fermentation of the material in its undiluted hypersaline state. Performance tests on NRRL 1526 had determined that in nutrient rich control medium it was able to survive, grow and produce ethanol in hypersaline submersed culture at up to 10.5 % salinity in sea salt, although increasing salt concentrations had a negative impact on fungal growth and ethanol production. Starch enzymatic hydrolysis by the fungus was not impacted up to 7 % salinity, indicating that the native starch degrading enzymes of NRRL 1526 were halotolerant. It was found that although this fungal strain was capable of producing up to 26.1 g⋅L-1 ethanol with an 84.8 % conversion efficiency (or percent yield), when fed with hypersaline MUR-233 biomass there was a shift in carbon flux towards other metabolic products. At 7 % salinity ethanol was still the major product of NRRL 1526 from assimilation of MUR-233 biomass, ranging between 9.62 g⋅L-1 to 11.24 g⋅L-1, but the percent ethanol yield was reduced to as low as 44.8 %. Under these conditions, lactic acid was also produced. The studies conducted herein addressed a knowledge gap about whether hypersaline Tetraselmis marine microalgal biomass produced in seawater could be fermented to ethanol through selective use of a halotolerant microorganism, confirming that such an approach was possible. However, the dilute aqueous nature of MUR-233 biomass and its fermentable substrates presents significant challenges to the feasible use of this alternative biomass feedstock for microbial bioethanol production.