L-lactic acid fermentation by Rhizopus oryzae using dairy manure as a nitrogen source

Experiments were conducted to investigate the possibility of using fresh dairy manure as the nitrogen source in a culture medium to produce L-lactic acid by Rhizopus oryzae NRRL 395. The parameters of the culture medium, including glucose (120 vs. 240 g L ^-1), nitrogen (1.0 vs. 3.0 g L ^-1, from dairy manure), KH ₂PO ₄ (0.65 vs. 1.3 g L ^-1), MgSO ₄ (0.25 vs. 0.5 g L ^-1), ZnSO4 (0.0 vs. 0.05 g L ^-1), spore culture time (3 vs. 6 h), and the inoculum dosage (6% vs. 12%, v/v), were first examined using the Plackett-Burman design to single out the most important ones to reduce the number of experiments in the optimization stage while still maintaining the statistical validity of the experimental results. In the optimization stage, experiments featuring a two-level, four-factor factorial design (2 ⁴) for the four most important components, i.e., glucose, nitrogen, KH ₂PO ₄, and MgSO ₄, were carried out, and the results were subjected to ANOVA to determine the optimum levels for each component, within the ranges used in this study, in the culture medium based on the percent L-lactic acid yield. The results showed that glucose, nitrogen, KH ₂PO ₄, and MgSO ₄ concentrations had the strongest impact on percent L-lactic acid yields by Rhizopus oryzae NRRL 395 (degrees of confidence were 93.08%, 96.11%, 89.59%, and 82.2%, respectively). The ANOVA revealed that glucose, nitrogen, and KH ₂PO ₄ demonstrated a significant main effect on the yield of L-lactic acid (p<0.0004). In addition, the interactions of glucose, nitrogen, KH ₂PO ₄, and MgSO ₄ were also found to be significant at p < 0.05. The main effects, as well as the interactions, were summarized by a polynomial regression equation, which was found to be able to sufficiently describe the L-lactic acid production observed in this study (p < 0.0002). The equation was examined against the experimental data, resulting in a coefficient of determination (R ²) of 0.9436 for the linear regression relationship between the predicted and observed values. Finally, the optimum culture medium composition with respect to the four major components studied using dairy manure as a base substrate for L-lactic acid generation by Rhizopus oryzae was found to be 3 g L ^-1 nitrogen, 120 g L ^-1 glucose, 1.3 g L ^-1KH ₂PO ₄, and 0.5 g L ^-1 MgSO ₄, and the corresponding percent L-lactic acid yield was 29.1% (measured) and 30.76% (predicted), based on the experimental conditions used in this study.