Production of ethanol and DagA by sequential utilization of sugars in microalgal hydrolysate

Abstract

Microalgal hydrolysate, an alternative carbon source, could be used as a feedstock for the production of valuable bioproducts such as biofuel and biochemicals. The acid hydrolysate of Nannochloropsis oceanica contains 7 different sugars of glucose, galactose, xylose, rhamnose, ribose, mannose and fucose. At first, DagA, a β-agarase was produced by cultivating a recombinant Streptomyces lividans in a glucose medium or a mixed-sugar medium simulating microalgae hydrolysate. The optimum composition of glucose medium was identified as 25 g/L glucose, 10 g/L yeast extract, and 5 $g/L MgCl_2·6H_2O$. With this, a DagA activity of 7.26 U/mL was obtained. It was tried to produce DagA in mixed-sugar medium mimicking N. oceanica hydrolysate. When a mixed-sugar medium containing 25 g/L of sugars was used, a DagA activity of 4.81 U/mL was obtained with very low substrate utilization efficiency due to the catabolic repression of glucose against the other sugars. When glucose and galactose were removed from the medium, an unexpectedly high DagA activity of about 8.7 U/mL was obtained even though a smaller amount of sugars were used. It is recommended for better substrate utilization and process economics that glucose and galactose should be eliminated from the medium being consumed for some other useful applications before the production of DagA. A novel two-step fermentation process based on sequential utilization of sugars in the mixed-sugar medium has been proposed. In the first step, glucose was consumed by Saccharomyces cerevisiae together with galactose and mannose producing ethanol. In the second step, DagA was produced by the recombinant S. lividans from the residual sugars of xylose, rhamnose and ribose. Fucose was not consumed. By adopting this two-step process, the overall substrate utilization efficiency was increased approximately 3-fold with a nearly 2-fold improvement of DagA production, let alone the additional benefit of ethanol production. Afore-mentioned two-step fermentation process was studied by using real microalgal hydrolysate. To prepare microalgal hydrolysate, 100 g/L lipid-extracted N. oceanica biomass was treated with 1.5 N $HNO_3$ or $H_2SO_4$ at 120 ℃ for 60 min. The N. oceanica hydrolysate treated by nitric acid was desalted by electrodialysis. When a membrane with a molecular weight cut-off of 300, about 98 % salts were removed from N. oceanica hydrolysate. Although, the total salt concentration was low enough, yeast cells in the first stage did not show a substantial level of growth probably due to still remaining inhibitory compounds. The N. oceanica hydrolysate treated by sulfuric acid was neutralized with $CaCO_3$. In this step, sulfate was removed in the form of gypsum. The treated hydrolysate containing total 7.01 g/L of 7 different monosugars: 2.19 g/L glucose, 1.12 g/L galactose, 1.32 g/L mannose, 0.47 g/L xylose, 0.50 g/L rhamnose, 0.86 g/L ribose and 0.55 g/L fucose was used for the sequential production of ethanol and DagA. In the first step, 1.30 g/L of ethanol was produced by S. cerevisiae with complete utilization of glucose, galactose and mannose. In the second step, 3.6 U/mL of DagA was produced by S. lividans with complete utilization of the residual sugars except fucose. Consequently, an overall substrate utilization of as high as 92.2 % was obtained. The unconsumed fucose has a potential to be a high-value-added product with many applications once recovered.