Development of marine biomass-derived biofuel producing strains for improvement of biomass utilization

Abstract

Renewable energy source has attracted worldwide attention because of the oil crisis and climate change. Biomass is the only renewable energy source capable of producing liquid fuels (biofuels) for storage and as a transport fuel. In contrast to land plant biomass, marine biomass has emerged as the next-generation biomass holding several benefits: high growth rate, low land usage, high CO2 absorption and no competition for food resources. Therefore, the use of lignin-free marcoalgae as a raw material is arising as a third generation biomass for bioethanol production which is the most produced biofuel in the world. To establish the sustainable biorefinery platform, the effective feedstock utilization is required by developing conversion technologies into value-added biochemical and biofuels. The present study describes a developed process system towards a marine macroalgae; two particular species of brown seaweed, Laminaria japonica which is the most abundant species in korea and red seaweed, Gelidium amansii which is composing the most carbohydrates contents biorefinery for sustainable complete utilization of feedstock for biofuel and a variety of natural products by solving some of problems in macroalgae utilization. In this study, to maximize utilization of marine biomass-feed stock for bioethanol production, it is highlighted to isolate CBP microorganism and construct engineered Saccharomyces cerevisiae , which have the ability to produce saccha-rolytic enzyme and utilize the sugars present in brown algae, alginate and red algae, 3,6-anhydro-L-galactose, respectively. Firstly, in case of brown algae, Brown seaweed contains up to 67% of carbohydrates by dry weight and presents high potential as a polysaccharide feedstock for biofuel production. To effectively use brown seaweed as a biomass, degradation of alginate is the major challenge due to its complicated structure and low solubility in water. This study focuses on the isolation of alginate degrading bacteria, determining of the optimum fermentation conditions, as well as comparing the conventional single fermentation system with the two-phase fermentation system which is separately using alginate and mannitol extracted from Laminaria japonica. Maximum yield of organic acids production and volatile solids reduction obtained were 0.516 g/g and 79.7%, respectively, using the two-phase fermentation system in which alginate fermentation was carried out at pH 7 and mannitol fermentation at pH 8. The two-phase fermentation system increased the yield of organic acids production by 1.14 times and led to a 1.45-times reduction of VS when compared to the conventional single fermentation system at pH 8. The results show that the two-phase fermentation system improved the utilization of alginate by separating alginate from mannitol leading to enhanced alginate lyase activity. Secondly, despite an increasing potential of red algal biomass as a feedstock due to highest contents of carbohydrates among three macroalgae, biological conversion of red algal biomass has been limited by lack of feasible microorganisms which can convert structured AHG, which is a main component of red algal carbohydrate, into a common metabolite. In the AHG uptake pathway, AHG dehydrogenase (AHGD) is known to be a key step, therefore it is important to find an efficient dehydrogenase to break down 3,6-anhydro-L-galactose (AHG) for practical use of red macroalgae biomass in biorefineries requires. In this study, we isolate a novel AHG dehydrogenase (AHGD) with high activity produced by a newly isolated bacteria strain, Roultella ornithinolytica B6–JMP12. The stability and compatibility of the enzyme were evaluated under various conditions to achieve high enzyme production. The AHGD was partially purified using conventional protein purification techniques such as ammonium sulfate precipitation and ion exchange followed by gel filtration chromatography, 37.24 fold with a final specific activity of 5.47 U/mg of protein with 32% yield recovery. SDS-PAGE was used to determine the molecular weight of the partially purified AHGD and its molecular weight was found to be around ~34 kDa. The optimal pH and temperature for the partially purified AHGD were 7.0 and 35°C, respectively. The Km and Vmax for 3,6-anhydro-L-galactose are 0.63 mg/ml and 0.38 µM/ml/min, respectively. Additionally, to access these limitations of AHG for utilization of red macroalgae as a biomass, this study engineered Saccharomyces cerevisiae as an efficient red algae fermentation microorganism for ethanol production. S.cerevisiae is known for having high ethanol productivity, high tolerant to toxic compounds and robust growth. However, S.cerevisiae has several limitations for utilization of red macroalgae as a feedstock due to 1) low efficient saccharification to convert polymeric-agarose into its monomeric sugars such as D-galactose and 3,6-anhydro-L-galatose (AHG) and 2) incapability to metabolize AHG. These limitations largely decrease the efficiency of utilization on red macroalgae as a feedstock for the production of bioethanol. To access these limitations, we firstly introduced several AHG metabolism pathways as well as agarase in S.cerevisiae. When recombinant S.cerevisiae harboring pYES-Aga-AHGD-ACI ferment pre-hydrolysate of agarose which is not over decomposed by acid is suitable for more efficient ethanol productivity from agarose fermentation, simultaneous fermentation of galactose and AHG exhibited improved cell growth (7.3%) and ethanol productivity (9.1%) compared to that of strong acid hydrolysate fermentation. The successful integration of agarase and AHG fermentation pathways in yeast is a critical step towards enabling economic biofuel production. This study proposed the possibility of improving the usage efficiency of seaweed biomass in biofuel production.