The non-conventional, oleaginous yeast Cutaneotrichosporon oleaginosus is flagged as an industrial cell factory for generation of oleochemicals and biofuels due to its substrate flexibility and high triglyceride yields. In this study, we employed a computational Response Surface Methodology to guide and streamline the experimental media optimization matrix with 12 nitrogen and 10 carbon sources in order to provide for high biomass and lipid accumulation toward an industrially relevant fermentation process. The resulting data provide new insights into C. oleaginosus physiology under variable nutritional states. Accordingly, the lipid content % (lipid weight/yeast dry weight) is controlled by a defined interplay between carbon and nitrogen. In our experimental setup, the highest biomass (18.4 ± 2.20 g/L) and lipid yield (9 ± 0.34 g/L; 49.74 ± 5.16% g lipid weight/g yeast dry cell weight) were obtained with lactose and yeast extract as carbon and nitrogen sources at an elemental weight ratio of 120:1, respectively. Interestingly, with ammonium salts as a N-source, the intracellularly accumulated triglycerides increasingly contain saturated fatty acids, which provides a new route to generate tailored fatty acid profiles for specific oleochemicals or food applications. Our data indicate that a metabolic ceiling for lipid accumulation in C. oleaginosus is obtained with the correct carbon and nitrogen source mixture.

Due to enhanced energy content and reduced hygroscopicity compared with ethanol, n-butanol is flagged as the next generation biofuel and platform chemical. In addition to conventional cellular systems, n-butanol bioproduction by enzyme cascades is gaining momentum due to simplified process control. In contrast to other bio-based alcohols like ethanol and isobutanol, cell-free n-butanol biosynthesis from the central metabolic intermediate pyruvate involves cofactors [NAD(P)H, CoA] and acetyl-CoA-dependent intermediates, which complicates redox and energy balancing of the reaction system. We have devised a biochemical process for cell-free n-butanol production that only involves three enzyme activities, thereby eliminating the need for acetyl-CoA. Instead, the process utilizes only NADH as the sole redox mediator. Central to this new process is the amino acid catalyzed enamine–aldol condensation, which transforms acetaldehyde directly into crotonaldehyde. Subsequently, crotonaldehyde is reduced to n-butanol applying a 2-enoate reductase and an alcohol dehydrogenase, respectively. In essence, we achieved conversion of the platform intermediate pyruvate to n-butanol utilizing a biocatalytic cascade comprising only three enzyme activities and NADH as reducing equivalent. With reference to previously reported cell-free n-butanol reaction cascades, we have eliminated five enzyme activities and the requirement of CoA as cofactor. Our proof-of-concept demonstrates that n-butanol was synthesized at neutral pH and 50°C. This integrated reaction concept allowed GC detection of all reaction intermediates and n-butanol production of 148 mg L−1 (2 mM), which compares well with other cell-free n-butanol production processes.

Harvest and dewatering poses a significant economical burden for industrial algae biomass production. To mitigate these effects, energy efficient techniques for these process steps have to be developed. Flocculation of the microalgae Scenedesmus obtusiusculus in salt based medium was induced by pH-shift and alternatively by addition of two biological flocculants, chitosan, and the commercial tannin CFL-PT. This is the first time that CFL-PT is used as an algae flocculant particularly focusing on harvesting of halophilic strains. The method was characterized and subsequently optimized. In comparison to biological flocculants, induction by pH shift is far cheaper, but due to buffering effects of the brackish cultivation medium infeasible amounts of base are required to raise the pH-value. tannin appears to be superior compared to chitosan not only in the absence of algae organic matter (AOM), but tannin-based harvest is also more robust regarding culture pH in presence of AOM. A higher flocculant-demand for modified tannin compared to chitosan is offset by the lower price. Given the employed strain and cultivation conditions, cultivation time had no pronounced effect on flocculation efficiencies (FE) while algae zeta-potential and bacterial communities also remained stable.