| Microbial oils, which are similar to vegetable oils in fatty acid composition and synthesizedand accumulated by oleaginous microorganisms under certain conditions, have beenconsidered as a promising feedstock for biodiesel due to shorter production time and nolimitation by season or location. Using abundant lignocellulosic biomass for microbial oilproduction can reduce the cost for lipid production and promote its industrializationapplication. Despite Trichosporon fermentans, an oleaginous yeast strain screened by our lab，can grow and accumulate lipid on detoxified sulfuric acid-treated rice straw hydrolysate(SARSH), the growth and lipid accumulation of T. fermantans would negatively affected bythe inhibitors in SARSH. Acetic acid is one of the major inhibitors with the highestconcentration in SARSH generally. However, little is known regarding the inhibitorymechanism of acetic acid on the growth and lipid synthesis of oleaginous microorganisms. Tobetter utilize lignocellulosic hydrolysate for lipid production, the effect of acetic acid on thegrowth and lipid accumulation of T. fermantans was examined, and the impact mechanismwas investigated as well. Based on these studies, an appropriate fermentation strategy wasadopted to alleviate the inhibitory effect caused by acetic acid and increase lipid yield ofT.fermantans.The influence of acetic acid and pH on the cell growth and lipid accumulation of T.fermentans was investigated firstly. At pH6.5, acetic acid can be utilized by T. fermantanswithout glucose repression. The initial utilization rate was0.1g/L h approximately, whichwas not associated with the concentrations of acetic acid. The inhibitory effect on growth andlipid production was related to culture pH, with certain concentration of acetic acid in lowerpH condition led to more severe inhibition. Essentially, the inhibitoy effect of acetic acid andpH on the cell growth of T. fermantans depended on the concentrations of undissociated acidin media. And0.26,0.52and0.96g/L undissociated form of acetic acid caused approximately12,24and48hours’lag phase respectively. At pH4.5,3g/L acetic acid in medium resulted inthe cessation of yeast cells’ growth. At pH6.5, low concentrations of acid contributed to ahigher maximum lipid content and lipid yield in comparison to the control. The specificglucose consumption rate decreased with increasing acetic acid concentrations. The inhibitoryimpact of acetic acid on xylose consumption was not pronounced. The effect of acetic acidand pH on fatty acid composition of obtained lipid was not significant.Fluorescent dye BCECF-AM and Rodamine123were applied to examine the effect ofacetic acid on intracellular pH and membrane potential of T. fermentans, respectively. The results indicated that3g/L acetic acid (pH4.5) in culture medium gave rise to an intracellularpH reduction of0.4pH units approximately, while its impact on membrane potential wasindistinctive. Additionally, the impact of acetic acid (1.5g/L under pH6.5) on the formationof lipid droplets of T. fermentans was investigated with fluorescent dye Nile red. The resultsrevealed that the size of lipid droplets was not changed with the addition of acetic acid, butthe fluorescence intensity of acid-treated cells was marginally higher than that of the control,which was in accordance with the former experimental phenomena.High cell-density cultivation of oleaginous yeast Trichosporon fermentans in fed-batchculture was adopted to accelerate the utilization of acetic acid in lignocellulosic hydrolysateand obtain a higher biomass. The effect of initial sugar concentration on the growth and lipidaccumulation of T. fermentans was firstly investigated and the initial sugar concentration wasdetermined as100g/L. Under which T. fermentans gave higher biomass and lipid yield, andno substrate inhibition was observed. Based on this, fed-batch cultivation of T. fermentans onrice straw hydrolysate was performed. After fermentation156h, the biomass, lipid contentand lipid productivity of T. fermentans were68.3g/L,48.3%(w/w), and0.198g/L·h. GCanalysis showed that the main fatty acid composition of obtained lipid was oleic acid, palmiticacid, stearic acid, and linoleic acid, and unsaturated fatty acid content was over65%, thus thelipid is suitable for biodiesel production.This study is of great significance to realize the high-value utilization of biomass resourcesand reduce the cost of microbial oil production as well. It helps to cope with the bottleneckproblem of oil raw material which hinders the large-scale production and wide application ofbiodiesel. |
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