New insights on heterotrophic microalgae growth on dark fermentation effluents

Several recent studies have shown the feasibility of producing both hydrogen and biolipids by coupling dark hydrogen fermentation of organic waste with lipid accumulation by microalgal heterotrophy [1]. Such combination allows the use of dark fermentation by-products (i.e. volatile fatty acids such as acetate, butyrate and lactate) as a low cost substrate to sustain microalgal heterotrophic growth. Nevertheless, little is known about the influence of mixed carbon sources on microalgal growth and some of dark fermentation by-products may act as growth inhibitor (e.g. butyrate) [2]. The objective of this study is to characterize the consumption of acetate, butyrate and lactate as single or mixed carbon sources by two lipid producing heterotrophic Chlorella species using a kinetic modeling approach. Results will give new insights on microalgal physiology for optimizing the coupling of H2 producing dark fermentation and microalgal lipid production. For this purpose, Chlorella sorokiniana (CCAP 211/8K) and Auxenochlorella protothecoides (CCAP 211/7A) were cultivated under axenic conditions and complete darkness until exhaustion of carbon sources (5 – 40 days). Synthetic media containing various concentrations of acetate, butyrate and lactate, similar to those of raw effluents, were tested. The interaction between substrate consumption and biomass growth was represented by fitting Monod and Haldane models to our data. As a result, both species were able to grow on acetate and butyrate, but not on lactate as sole carbon source. Growth on acetate followed a Monod model whereas growth on butyrate followed a Haldane model, meaning that the consumption rate of butyrate decreased at inhibitory substrate concentrations. For the first time, when the substrates were both present, a diauxic phenomenon was observed, ie. the consumption of butyrate started only after complete exhaustion of acetate. A new kinetic model taking into account acetate and butyrate consumption as well as such diauxic effect was successfully built for these strains. Growth rates on acetate were almost 10 times higher (µmax = 2 d-1) than those observed on butyrate (µmax = 0.2 d-1). This study shows the importance to focus on butyrate consumption, especially when considering mixtures of dark fermentation bacterial metabolites. Two initial parameters were identified as key factors to promote microalgal growth and butyrate exhaustion: the butyrate concentration per se and the acetate:butyrate ratio. Targeting these parameters will be the next step in an efficient process coupling dark fermentation with microalgal heterotrophy.