Introduction

In recent decades, biofuels have drawn considerable attention due to fossil fuel depletion and adverse climate effects of fossil fuel burning (Martins, et al., 2019; Kung, 2019; Kumar and Singh, 2020). Microalgae are introduced as a unique biomass feedstock for biofuels since they have several advantages including fast growth rates, resistance to extreme environmental conditions, feasible and eco-friendly large-scale production, and simple life cycle. Microalgae-derived biofuels production can be coupled with flue gas CO2 mitigation, wastewater treatment, and high-value chemical compounds extraction. However, their production is not yet financially competitive with fossil fuels and conventional biofuels . Microalgal storage compounds which are the prime biofuel precursors, starch, and lipids, are used as the substrates for bioethanol and biodiesel production, respectively. Since these storage compounds’ accumulation is not linearly delineated to the growth rate, one of the technical challenges in making biofuels cost-effective is to increase starch and lipids productivity. The most frequently reported approaches are nutrient starvation/limitation including nitrogen , phosphorus , and sulfur , salt stress (Takagi and Yoshida, 2006; Pancha et al., 2015) and light intensity stress . Nitrogen starvation is reported as the most convenient technique to enhance energy storage components . Most studies to date have been focused on two-stage production. In the first stage, microalgae are grown in the non-limiting growth medium. In the second stage, microalgae are triggered by one of the above-mentioned nutrient limitations and/or stress approaches to induce storage polymer production . However, under stress, nutrient or light limitation conditions, along with the accumulation of storage compounds, the system is exposed to extended unfavorable environmental conditions, which lead to high expenses of metabolic energy and decreasing productivity . To overcome this problem, a single-stage continuous system (chemostat) with a nutrient-limited medium can be explored. The main advantage of using chemostat is that the liquid dilution rate controls the growth rate of the biomass with a defined limiting substrate. When steady-state conditions are reached at specific dilution rates, biomass productivity, medium concentrations, and intracellular biochemical composition remain constant . This permits the optimization of the dilution rate to a determined value for maximal storage compound productivity in microalgae .
Another major obstacle in the profitable production of biofuels and scale-up is to maintain the microalgae cultivation systems monoculture, due to high-priced sterilization of inlet streams and reactor system. On the other hand, the use of environmental water bodies as inoculum has this opportunity to introduce new suitable microalgae species for biodiesel production which are offered by nature’s microbial diversity. The natural selection and competition is introduced by Environmental Biotechnology which is targeted at enrichment and maintaining a characteristic or function as an alternative to a specific species in a system in order to engineer the ecosystem rather than the organisms . Furthermore, several studies claimed that biomass yield improved in mixed-species growth systems relative to algal monocultures grown under the same resource supply conditions. Previous related studies have shown that the strategy of chemostat selection could be successfully applied to obtain a stable enrichment of a polyhydroxyalkanoates (PHA) storing microbial community .
The main objectives of this study were to investigate the impacts of different nitrogen concentrations in feeding medium on mixed microalgae communities in a chemostat reactor. Especially the starch and lipids productivities were monitored as they have the potential for the production of biofuels. Furthermore, the nitrogen loading rates (NLR) were designed to direct the culture to be under light- or nitrogen-limited conditions that elucidate the microalgae tendency to accumulate lipids or starch as a dominant intracellular energy reservoir compound. For all NLRs, the CO2 supply was unlimited.