Biological Carbon Recovery from Sugar Refinery Washing Water into Microalgal DHA: Medium Optimization and Stress Induction

Abstract

Sugar refinery washing water (SRWW) contains abundant levels of carbon sources and lower levels of contaminants than other types of wastewater, which makes it ideal for heterotrophic cultivation of microalgae. Here, carbon sources in SRWW were utilized for conversion into the form of value-added docosahexaenoic acid (DHA) using Aurantiochytrium sp. KRS101. Since SRWW is not a defined medium, serial optimizations were performed to maximize the biomass, lipid, and DHA yields by adjusting the nutrient (carbon, nitrogen, and phosphorus) concentrations as well as the application of salt stress. Optimum growth performance was achieved with 30% dilution of SRWW containing a total organic carbon of 95,488 mg L^-1. Increasing the nutrient level in the medium by supplementation of 9 g L^-1 KH₂PO₄ and 20 g L^-1 yeast extract further improved the biomass yield by an additional 14%, albeit at the expense of a decrease in the lipid content. Maximum biomass, lipid, and DHA yields (22.9, 6.33, and 2.03 g L^-1, respectively) were achieved when 35 g L^-1 sea salt was applied on a stationary phase for osmotic stress. These results demonstrate the potential of carbon-rich sugar refinery washing water for DHA production using Aurantiochytrium sp. KRS101 and proper cultivation strategy.

Figure 1

Effect of various concentrations of sugar refinery washing waster (SRWW) on the: (a) biomass yield (dry cell weight), (b) glucose concentration, and (c) lipid content of Aurantiochytrium sp. KRS101 cultured under heterotrophic conditions. Error bars indicate mean ± standard deviation (n = 6 for (a), and 4 for (b) and (c)). Statistical analysis was conducted with ANOVA tests (significant, P < 001) for (a) and (c) at day 5, and Student t-test, where ***P < 0.001, **P < 0.01, *P < 0.05 for all at day 5.

Figure 2

Changes in biomass yield (dry cell weight) (gray bars) and lipid content (black squares) of Aurantiochytrium sp. KRS101 using 30% SRWW supplemented with various concentrations of yeast extract and KH₂PO₄. The control involved the culture grown in conventional modified basal media containing 30 g L⁻¹ glucose. Two concentrations of KH₂PO₄ (9 and 18 g L⁻¹) and four concentrations of yeast extract (5, 10, 15, and 20 g L⁻¹) were tested. Error bars indicate mean ± standard deviation (n = 3 and 6). ANOVA tests were conducted for biomass yield and lipid content, and both showed significant differences (P < 0.001). ANOVA test with post-hoc Tukey Honestly Significant Difference showed that groups having same concentration of phosphate ((9.5)–(18, 5), (9, 10)–(18, 10), (9, 15)–(18, 15), and (9, 20)–(18, 20)) have no significant differences (P > 0.05) for biomass yield and lipid content. Student t-test was also conducted and showed significant differences with control as ***P < 0.001, **P < 0.01, *P < 0.05.

Figure 3

Effect of various concentrations of sea salt on the: (a) biomass yield (dry cell weight), (b) DHA yield (bar), and DHA content (line) of Aurantiochytrium sp. KRS101 cells in exponential phase. Error bars indicate mean ± standard deviation (n = 3). Statistical analysis were conducted with ANOVA tests for (a) and (b) at 48 h, and (a) at 48 h has no significant differences (P > 0.05) and DHA yield and DHA content at 48 h have significant differences as P < 0.001 and P < 0.01, respectively. Also, Student t-test was conducted and showed significant differences with no treatment as ***P < 0.001, **P < 0.01, *P < 0.05.

Figure 4

Effect of various concentrations of sea salt on the: (a) biomass yield (dry cell weight), (b) DHA yield (bar), and DHA content (line) of Aurantiochytrium sp. KRS101 cells in stationary phase. Error bars indicate mean ± standard deviation (n = 3). ANOVA tests were conducted for (a) and (b) at 24 h, and (a) at 24 h showed no significant differences (P ≈ 0.05) and DHA yield and DHA content have significant differences as P < 0.001 and P < 0.05, respectively. Also, Student t-test was conducted and showed significant differences with no treatment as ***P < 0.001, **P < 0.01, *P < 0.05.

Figure 5

Changes in the: (a) biomass yield (dry cell weight, gray bars), lipid yield (blue squares), DHA yield (red circles), and (b) fatty acid profiles of Aurantiochytrium sp. KRS101 cultured under various heterotrophic conditions of sea salt stress. Error bars indicate mean ± standard deviation (n = 3). ANOVA tests were conducted for (a) and it showed significant differences (P < 0.001) for all biomass yield (dry cell weight), lipid yield, and DHA yield. Also, Student t-test was conducted and showed significant differences with no treatment as ***P < 0.001, **P < 0.01, *P < 0.05.