Oleaginous yeast Cryptococcus curvatus culture with dark fermentation hydrogen production effluent as feedstock for microbial lipid production
Highlights
► Preliminary evidence for integrating lipid production with hydrogen production. ► pH and acetate concentration optimized for C. curvatus' growth with acetate as feedstock. ► VFA produced in biohydrogen production used as feedstock to grow oleaginous yeast. ► No inhibitor to C. curvatus' growth produced in dark fermentation hydrogen production. ► Inhibitors in food waste can be avoided by using continuous culture strategy.
Introduction
Biodiesel can be produced from a multitude of oil sources such as soybeans, canola, animal fat, palm, corn, waste cooking oil, and jatropha, but these sources suffer from production limitations. Production of single cell oil (SCO) or microbial lipid by oleaginous yeast [1], fungus [2], heterotrophic microalgae [3], and phototrophic microalgae [4] to be used as biodiesel feedstocks have recently gained considerable attention because of potential advantages, namely, high productivity and the ability to use organic waste as carbon and other nutritional sources. Compared with phototrophic algae growth, the heterotrophic growth process has the advantage of growing cells to high density without light limitation. Its main disadvantage, however, is the high cost of feedstock. Thus, exploiting an inexpensive feedstock is absolutely necessary for commercial microbial lipid biofuel production.
A variety of organic wastes and wastewater have been investigated as feedstock for dark fermentative hydrogen production [5]. In this process, bacteria first produce sugars by hydrolyzing the organic wastes, and then convert the sugars to hydrogen [6]. Theoretically, one-third of the carbon in glucose is converted to CO2 along with hydrogen production, while the other two-thirds are converted to acetate or butyrate [7]. These volatile fatty acids (VFA) have to be removed in order to avoid potential downstream pollution. To solve this problem, some researchers have converted residue VFAs to valuable products such as methane [8], hydrogen [9], [10], or electricity [6], [11]. Recently, Chang et al. [12] proposed a VFA platform to utilize organic waste biomass to produce biofuel such as alcohol and ester, or other biochemicals. This research group also did pioneer work on converting VFA to microbial lipids with oleaginous yeast fermentation [13]. In comparison to former processes, oleaginous yeast culture is a mature technology, and lipid biofuel is in surging demand. If the VFA are converted into oil-enriched yeast biomass in an efficient method, this process will reduce biological oxygen demand (BOD) in the effluent and provide feedstock for biofuel production. Such a strategy will greatly improve the economical viability of single cell oil production.
Cryptococcus curvatus has been used in large scale cocoa butter substitute production from cheese whey [1]. It can grow on a variety of carbon sources such as glucose, xylose, lactose, glycerol, and ethanol. When ethanol is used as the carbon source, it is usually first converted into acetate. Thus, strains that can grow on ethanol usually can also grow on acetate. In this pathway, acetyl-CoA is synthesized from acetate, and feed into the glyoxylate and tricarboxylic acid cycles to produce oxaloacetate [14].
Culture condition optimization of C. curvatus has been reported in previous research [1], [15], [16], [17]. However, our preliminary research found that optimal conditions for C. curvatus’ growth with acetate as carbon source was significantly different from that with glucose. For example, pH 5.5 is usually used in glucose culture, but pH 5.5 with acetate resulted in poor growth. Therefore, culture conditions with VFA as carbon sources need to be investigated and optimized. Furthermore, the hydrogen fermentation process which uses bacteria produces many metabolites, and some of them may inhibit yeast growth. Thus, the presence of potential inhibitors in HPE should be examined. Finally, an actual waste stream should be tested to prove the feasibility of this entire waste-to-biofuel process. In this study, optimal pH and acetate concentration was first determined. To test if inhibitors were produced in the fermentative hydrogen production process, sucrose, instead of the actual waste stream, was tested as surrogate for raw waste material, since pure sucrose will not introduce any inhibitor into the hydrogen production process. Finally, food waste was used as an actual example of potential commercial raw material for dark fermentation hydrogen production, and the effluent was tested as a feedstock for lipid production with C. curvatus culture.
Section snippets
Cell strain and medium
C. curvatus (ATCC 20509), also known as Candida curvata, was used in this research. The seed cells were pre-cultured with a medium composed of 10 g/L dextrose, 1 g/L of yeast extract (Sigma, St. Louis, MO), and 1 g/L peptone (Sigma, St. Louis, MO). The cells were grown in 250-mL Erlenmeyer flasks, each containing 50 mL of medium and incubated at 25 °C in an orbital shaker set to 170 rpm. Before inoculating into experiment cultures, the seed cells were sub-cultured for 24 h in potassium acetate
The effect of potassium acetate concentration on cell growth
Fig. 1 shows the effect of potassium acetate concentration on C. curvatus’ growth, as well as corresponding pH variation during the culture process. Cultures with 10 g/L potassium acetate obtained the highest OD between the 12th and the 24th hour. Compared to 10 g/L, there was a slight inhibition in the 20 and 40 g/L cultures during the first 24 h, but these cultures resulted in much higher OD thereafter. The cultures with 60 g/L and 100 g/L potassium acetate resulted in lower ODs, and were
Discussion
This study investigated oleaginous yeast culture with hydrogen production effluent (HPE) as the feedstock and demonstrated the feasibility of such a process. It was also shown that VFA (using acetic acid as an example) can be a recommended feedstock for C. curvatus’ culture. A pH-stat culture maintained at pH 7.0 by feeding acetic acid produced 68.8 g/L dry cell weight and 37.0 g/L lipid in a 72 h culture process. HPE from synthetic wastewater (with sucrose as carbon source) was tested as an
Conclusion
VFA was shown to be a favorable feedstock in C. curvatus culture for lipid production, and C. curvatus’ culture with HPE from synthetic wastewater demonstrated that no inhibitor to C. curvatus growth was produced in the hydrogen production process. Although FW-HPE contained inhibitor(s), it successfully supported C. curvatus growth in continuous culture. However, the lipid content of produced biomass was low due to the high nitrogen concentration in FW-HPE. Nitrogen-deficient raw materials are
Acknowledgments
The Washington State Department of Ecology provided funding for this project.
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