One-carbon compounds (C1) such as methanol are an attractive feedstock for biomanufacturing. To make these feedstocks accessible to popular industrial strains, recent efforts have been directed towards establishing C1-assimilation pathways in otherwise heterotrophic strains. In this study, we want to enable methanol-utilization in the yeast S. cerevisiae by expressing the synthetic formolase (FLS) pathway. The FLS pathway consists of three steps which link methanol to the central carbon metabolism of the cell: methanol oxidation, formaldehyde condensation to the three-carbon compound dihydroxyacetone (DHA), and DHA phosphorylation resulting in the glycolytic intermediate DHAP. The condensation reaction is catalyzed by the artificial FLS enzyme, but although the enzyme has been successfully applied in enzymatic cascades for in vitro C1-fixation, its functional expression in S. cerevisiae has not been demonstrated. Our approach is to construct an auxotrophic strain which relies on FLS activity for growth and can serve as a platform to screen FLS variants in vivo. Moreover, such a strain presents a suitable starting point for adaptive laboratory evolution aimed at improving methanol utilization in S. cerevisiae.

Ethanol is produced at industrial scale from non-agricultural feedstocks by gas fermentation, while research on other low-emission processes for ethanol production is accelerating. In view of its degree of reduction, water solubility and relatively low toxicity, ethanol is an interesting candidate to replace sugars in aerobic, zero-emission processes for yeast-based production of whole-cell protein and low-molecular-weight compounds. Currently, little information is available on specific growth rates, biomass yields and biomass composition of yeast species during growth on synthetic medium with ethanol as sole carbon source. In this study, strains of 52 Saccharomycotina yeasts were screened for their growth characteristics on ethanol. After first screening in microtiter plates, 21 fast-growing strains that were further analysed in aerobic shake-flask cultures showed specific growth rates of 0.12-0.46 h−1. Five fast-growing strains were further studied in aerobic, ethanol-limited chemostats (dilution rate 0.10 h−1). Strains of the industrial yeasts Saccharomyces cerevisiae and Kluyveromyces lactis, whose genomes lack genes for a proton-coupled Complex-I NADH dehydrogenase, both showed biomass yields of 0.6 g biomass (g ethanol)−1. Of three yeasts whose genome does contain Complex-I genes, Phaffomyces thermotolerans, showed the same biomass yield as S. cerevisiae, while Ogataea parapolymorpha and Cyberlindnera jadinii showed biomass yields of 0.67 ± 0.01 and 0.73 ± 0.00 g g−1, respectively. The biomass yield of C. jadinii, which also showed the highest protein content of the 5 yeasts tested in chemostats, corresponded to 88% of the theoretical biomass yield in a scenario where growth is limited by assimilation rather than by energy metabolism.

Emerging low-emission production technologies make ethanol an interesting substrate for yeast biotechnology, but information on growth rates and biomass yields of yeasts on ethanol is scarce. Strains of 52 Saccharomycotina yeasts were screened for growth on ethanol. The 21 fastest strains, among which representatives of the Phaffomycetales order were overrepresented, showed specific growth rates in ethanol-grown shake-flask cultures between 0.12 and 0.46 h⁻¹. Seven strains were studied in aerobic, ethanol-limited chemostats (dilution rate 0.10 h⁻¹). Saccharomyces cerevisiae and Kluyveromyces lactis, whose genomes do not encode Complex-I-type NADH dehydrogenases, showed biomass yields of 0.59 and 0.56 gbiomass g_ethanol⁻¹, respectively. Different biomass yields were observed among species whose genomes do harbour Complex-I-encoding genes: Phaffomyces thermotolerans (0.58 g g⁻¹), Pichia ethanolica (0.59 g g⁻¹), Saturnispora dispora (0.66 g g⁻¹), Ogataea parapolymorpha (0.67 g g⁻¹), and Cyberlindnera jadinii (0.73 g g⁻¹). Cyberlindnera jadinii biomass showed the highest protein content (59 ± 2%) of these yeasts. Its biomass yield corresponded to 88% of the theoretical maximum that is reached when growth is limited by assimilation rather than by energy availability. This study suggests that energy coupling of mitochondrial respiration and its regulation will become key factors for selecting and improving yeast strains for ethanol-based processes.

Background
Elimination of greenhouse gas emissions in industrial biotechnology requires replacement of carbohydrates by alternative carbon substrates, produced from CO2 and waste streams. Ethanol is already industrially produced from agricultural residues and waste gas and is miscible with water, self-sterilizing and energy-dense. The yeast C. jadinii can grow on ethanol and has a history in the production of single-cell protein (SCP) for feed and food applications. To address a knowledge gap in quantitative physiology of C. jadinii during growth on ethanol, this study investigates growth kinetics, growth energetics, nutritional requirements, and biomass composition of C. jadinii strains in batch, chemostat and fed-batch cultures.

Results
In aerobic, ethanol-limited chemostat cultures, C. jadinii CBS 621 exhibited a maximum biomass yield on ethanol (Y max X/S) of 0.83 gbiomass (gethanol)−1 and an estimated maintenance requirement for ATP (mATP) of 2.7 mmolATP (gbiomass)−1 h−1. Even at specific growth rates below 0.05 h−1, a stable protein content of approximately 0.54 gprotein (gbiomass)−1 was observed. At low specific growth rates, up to 17% of the proteome consisted of alcohol dehydrogenase proteins, followed by aldehyde dehydrogenases and acetyl-CoA synthetase. Of 13 C. jadinii strains evaluated, 11 displayed fast growth on ethanol (μmax > 0.4 h−1) in mineral medium without vitamins, and CBS 621 was found to be a thiamine auxotroph. The prototrophic strain C. jadinii CBS 5947 was grown on an inorganic salts medium in fed-batch cultures (10-L scale) fed with pure ethanol. Biomass concentrations in these cultures increased up to 100 gbiomass (kgbroth)−1, with a biomass yield of 0.65 gbiomass (gethanol)−1. Model-based simulation, based on quantitative parameters determined in chemostat cultures, adequately predicted biomass production. A different protein content of chemostat- and fed-batch-grown biomass (54 and 42%, respectively) may reflect the more dynamic conditions in fed-batch cultures.

Conclusions
Analysis of ethanol-grown batch, chemostat and fed-batch cultures provided a quantitative physiology baseline for fundamental and applied research on C. jadinii. Its high maximum growth rate, high energetic efficiency of ethanol dissimilation, simple nutritional requirements and high protein content, make C. jadinii a highly interesting platform for production of SCP and other products from ethanol.

Hydroxylamine is a key intermediate in several biological reactions of the global nitrogen cycle. However, the role of hydroxylamine in anammox is still not fully understood. In this work, the impact of hydroxylamine (also in combination with other substrates) on the metabolism of a planktonic enrichment culture of the anammox species Ca. Kuenenia stuttgartiensis was studied. Anammox bacteria were observed to produce ammonium both from hydroxylamine and hydrazine, and hydroxylamine was consumed simultaneously with nitrite. Hydrazine accumulation - signature for the presence of anammox bacteria - strongly depended on the available substrates, being higher with ammonium and lower with nitrite. Furthermore, the results presented here indicate that hydrazine accumulation is not the result of the inhibition of hydrazine dehydrogenase, as commonly assumed, but the product of hydroxylamine disproportionation. All kinetic parameters for the identified reactions were estimated by mathematical modelling. Moreover, the simultaneous consumption and growth on ammonium, nitrite and hydroxylamine of anammox bacteria was demonstrated, this was accompanied by a reduction in the nitrate production. Ultimately, this study advances the fundamental understanding of the metabolic versatility of anammox bacteria, and highlights the potential role played by metabolic intermediates (i.e. hydroxylamine, hydrazine) in shaping natural and engineered microbial communities.