Use of fermentative metabolites for heterotrophic microalgae growth: Yields and kinetics
Introduction
Production of microalgae has gained a lot of interest in the past decades due to their ability to synthesize biomolecules having potential industrial applications such as energy generation. The use of microalgae to produce biofuels offers the main advantage of improving biomass and lipid yields and land use when compared to oil crops. Georgianna and Mayfield (2012) assessed that microalgae could produce the same amount of oil than palm oil using six times less area. Due to higher growth rates, expressed in days instead of months or years, and lipid contents up to 80% of dry biomass, microalgae are very promising candidates to produce massively and constantly biofuels (Wu et al., 2012).
Growing microalgae in darkness and on organic compounds presents several advantages compared to autotrophic cultivation systems: (1) a higher biomass density can be achieved, leading to reduce the costs of microalgae harvesting compared with low density systems operated in autotrophy (Doucha and Lívanský, 2011). (2) Higher growth rates are observed in heterotrophy, reducing the time of cultivation (Kim et al., 2013). (3) Higher lipid yields can be achieved in heterotrophic cultures, improving economic competitiveness of microalgae biofuels (Wan et al., 2012).
Industrial production of heterotrophic microalgae is hampered by the high economic and environmental costs of glucose, commonly used as main carbon source. Therefore, glycerol, acetate or wastewaters containing glucose, such as whey permeate, are considered as the most promising alternatives of low cost carbon substrates (Espinosa-Gonzalez et al., 2014). Glycerol is a by-product of biodiesel and can be used to sustain heterotrophic algal growth and reduces the overall process cost (Heredia-Arroyo et al., 2010). Acetate is a by-product of anaerobic digestion and often accumulates in dark fermentation processes. Interestingly, microalgae can easily convert acetate into acetyl-CoA which is the main precursor for lipid synthesis (Ramanan et al., 2013). Auxenochlorella protothecoides was successfully grown on glycerol and acetate, with maximal biomass concentrations of 3.97 and 3.62 g L−1 and maximal lipids contents of 20.33 and 52.38% of dry biomass, respectively (Heredia-Arroyo et al., 2010). These results were very similar to the values reported with glucose, i.e. 4.25 g L−1 of biomass and a lipid content of 25.25% (Heredia-Arroyo et al., 2010). The use of other organic substrates, such as sucrose, lactose and ethanol may not support substantial heterotrophic growth of microalgae (Perez-Garcia et al., 2011b).
Coupling dark fermentation, producing hydrogen and volatile fatty acids (VFA), such as acetate, butyrate and lactate, with microalgal mixotrophic bioprocesses has been recently investigated with the purpose of lowering the costs of the overall process by finding new sources of substrates. Hu et al. (2013) showed the feasibility of growing mixotrophically a newly isolated Chlorella sp. on acidogenic swine effluents containing a mixture of acetate, propionate and butyrate. The effluent had to be diluted 8 folds in order to promote the microalgae growth and it was assumed that growth inhibition was caused by high concentrations of VFAs. Consistently, Liu et al. (2012) pointed out an inhibitory effect on mixotrophic growth of Chlorella vulgaris when butyrate concentration was higher than 0.1 g L−1. In contrast, Wen et al. (2013) found that butyrate was degraded before acetate but after valerate and ethanol by Chlorella protothecoides in heterotrophic conditions on anaerobically digested sugarcane wastewaters. Venkata Mohan and Prathima Devi (2012) used a mixed culture of microalgae, containing species of Scenedesmus and Chlorella, in order to convert dark fermentation effluents into microalgal biomass (1.42 g L−1) and microalgal lipids (26.4% of dry weight), under mixotrophic conditions. They reported that acetate was a preferred substrate compared to butyrate and propionate. Ren et al. (2013) used sterilized dark fermentation effluents, composed of at least 95% of acetate and ethanol, to sustain heterotrophic growth of Scenedesmus sp. In this study, acetate was completely removed but not ethanol. The subsequent biomass production and lipids content reached 1.98 g L−1 and 40.9% of dry weight, respectively. Liu et al. (2013) pointed out that butyrate removal was higher under heterotrophic than mixotrophic conditions due to the competition between organic and inorganic carbon uptake. Considering all these studies, the heterotrophic growth of microalgae on a mixture of organic substrates is still difficult to estimate mainly because of the lack of a clear behavior when a mixture of substrates is used.
A. protothecoides and Chlorella sorokiniana are two well-known lipid-producing microalgae, their lipids content can be as high as 57 and 61.5% of dry weight, respectively (Ramanna et al., 2014, Wang et al., 2013). The aim of this study was to characterize the growth of A. protothecoides and C. sorokiniana, under strict heterotrophic conditions and in presence of three organic acids, i.e. acetate, butyrate and lactate, mainly generated in dark fermentation processes. A. protothecoides and C. sorokiniana kinetic parameters were assessed using a global kinetic model fitting biomass growth and organic carbon removal.
Section snippets
Microalgae strains and culture conditions
C. sorokiniana (CCAP 211/8K) and A. protothecoides (CCAP 211/7A) were obtained from the CCAP culture collection (United Kingdom). A modified BG11 medium (UTEX, http://www.utex.org/) was used to pre-cultivate the inoculum. Sodium bicarbonate (10 mM), chlorure ammonium (5 mM) and dipotassium phosphate (0.31 mM) were used as inorganic carbon (C), nitrogen (N) and phosphorus (P) sources, respectively. Since A. protothecoides is auxotrophic for thiamine (vitamin B1) (Huss et al., 1999), the medium was
High cell growth using acetate
Both C. sorokiniana and A. protothecoides growth was efficient and rapid for the four different concentrations of acetate (0.1–1 gC L−1) (Fig. 1A and C). Acetate was completely exhausted in less than 1.5 days and less than 2 days, for C. sorokiniana and A. protothecoides, respectively (Fig. 1B and D). The end of the biomass growth occurred when acetate was completely exhausted. These results showed that acetate did not inhibit microalgae growth even for concentrations as high as 1 gC L−1. This might
Conclusion
Microalgal heterotrophy should provide a mean to successfully produce biofuels if low cost carbon sources are used. The growth of microalgae on synthetic dark fermentation effluent, composed mainly of acetate, butyrate and lactate, was evaluated and modeled. Defining the optimal butyrate concentration and acetate:butyrate ratio should promote microalgae growth. Acclimation of microalgae (during four weeks) to the inhibitory butyrate may substantially improve biomass growth. The use of raw
Acknowledgements
This study was funded by the National Institute of Agronomic Research (INRA) and the University of Montpellier II, France.
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