(577c) Investigation of Ammonia Inhibition On Oleaginous Yeast Cryptococcus Curvatus Growth

Microbial oil as an alternative feedstock for biodiesel production has recently gained more attention, but suffers the high cost of raw material. Culture of oleaginous yeast Cryptococcus curvatus for biodiesel production has been proved to be practical in our previous research by using the effluent from anaerobic digestion of food waste for hydrogen production as feedstock, which is enriched with volatile fatty acids (VFA). However, high concentration of ammonia in the effluent caused inhibition of yeast growth. Thus the mechanism of ammonia inhibition for the yeast growth was investigated in this study. The yeast C. curvatus was cultured on VFA (including acetic, propionic and butyric acids), glucose and glycerol containing medium respectively with different concentrations of ammonia (from 0.2 g N/L to 4.0 g N/L), and the same concentrations of nitrate were used as the control. The results showed the growth was inhibited by ammonia when the yeast was cultured on VFA. The dry weight significantly reduced with the increasing concentration of ammonia. However, there was no inhibition caused by nitrate. Compared to VFA, ammonia had no negative impact on yeast growth when glucose and glycerol were used as carbon source. The metabolic pathway analysis shows VFA and pyruvate generating substrates (PGS, i.e. glucose and glycerol) use different pathways to produce acetyl-CoA, an important basic molecule for metabolism. The enzyme catalyzing the reaction with VFA as substrate is acetyl-CoA synthetase (ACS) while the counterpart for PGS is pyruvate dehydrogenase. The enzymatic analysis showed ammonia inhibited the ACS significant and the activity lost 39.9% at 2.0 g N/L of ammonia. It is concluded that the high concentration of ammonia is toxic for the oleaginous yeast C. curvatus growth when VFA as the substrate but not PGS, and the mechanism was determined as the ammonia inhibition for the activity of ACS. The clarification of the ammonia inhibition to a specific enzyme provides a potential target for genetic modification to overcome this problem. Moreover, the demonstrated mechanism in this study may help the resolution of ammonia inhibition problems in the pathway with VFA as starting substrate, such as anaerobic digestion for methane production, phototrophic hydrogen production, and electricity production with microbial fuel cell, etc.