The influence of pH, temperature and carbon source (glucose and maltose) on growth rate and ethanol yield of Dekkera bruxellensis was investigated using a full-factorial design. Growth rate and ethanol yield were lower on maltose than on glucose. In controlled oxygen-limited batch cultivations, the ethanol yield of the different combinations varied from 0.42 to 0.45 g (g glucose)(-1) and growth rates varied from 0.037 to 0.050 h(-1). The effect of temperature on growth rate and ethanol yield was negligible. It was not possible to model neither growth rate nor ethanol yield from the full-factorial design, as only marginal differences were observed in the conditions tested. When comparing three D. bruxellensis strains and two industrial isolates of Saccharomyces cerevisiae, S. cerevisiae grew five times faster, but the ethanol yields were 0-13% lower. The glycerol yields of S. cerevisiae strains were up to six-fold higher compared to D. bruxellensis, and the biomass yields reached only 72-84% of D. bruxellensis. Our results demonstrate that D. bruxellensis is robust to large changes in pH and temperature and may have a more energy-efficient metabolism under oxygen limitation than S. cerevisiae.

The ethanol production process of a Swedish alcohol production plant was dominated by Dekkera bruxellensis and Lactobacillus vini, with a high number of lactic acid bacteria. The product quality, process productivity, and stability were high; thus, D. bruxellensis and L. vini can be regarded as commercial ethanol production organisms.We analyzed the population dynamics of yeasts and lactic acid bacteria (LAB) in a Swedish ethanol production plant. The production process runs as a continuous fermentation with recirculation of the yeasts. The substrate for fermentation is formed from wheat starch. The material is first liquefied with ␣-glucoamylase at 90°C and then further degraded by ␣-glucosidase at 60°C to release fermentable sugars. By this procedure, about 96% of the starch is degraded to fermentable sugar. The glucose concentration in the fermentor is always below 0.1 g/liter because of the substrate-limited continuouscultivation method (B. Johansson, personal communication). The process is started by mixing 1 ton of baker's yeast (Jästbolaget, Sollentuna, Sweden; cell viability, Ͼ90%) with the substrate in the fermentor (fermentor size, about 100 m 3 ). According to observations of the staff, it usually takes up to 3 weeks until stable fermentation is obtained; thereafter, the process can run stably for 2 or even more years. During the first 3 weeks, the process is prone to infections and stuck fermentation. After stabilization, the staff noticed a change in the cell shape of the production yeast, which was regarded by them either as a physiological adaptation to the harsh conditions in the fermentor (high cell density, sugar and oxygen limitation, a pH of about 3.5, and a temperature of 35°C) or as a selected genetic variant of the inoculated baker's yeast. Our intention was to analyze this genetic variant but also to investigate the role of potentially contaminating LAB.Samples were taken in January, March, and July 2006 from the fermentor, which had been running stably since July 2005. Appropriate dilutions of the fermentation broth were spread onto plates selective for either yeasts (malt extract agar containing 20 g/liter malt extract and 0.1 g/liter chloramphenicol) or LAB (de Man-Rogosa-Sharp medium containing 0.1 g/liter Delvocid). Surprisingly, during the first two samplings the number of LAB was very high, constituting about 70% of the total cell number (yeast plus LAB). At the last sampling, this relation had changed and the LAB were less than 10% of the total cell number (Table 1). However, these changes did not influence the ethanol concentration in the fermentor (B. Johansson, personal communication). The quantifications are based on CFU determinations on the selective media described above. It was not possible to perform microscopic counts because of the high number of particles in the industrial medium that were difficult to distinguish from microbial cells. From every sample, 20 yeast and 20 bacterial colonies were randomly chosen for PCR fingerprint analysis. For a comparison, we also anal...

This study investigated lipid production from the hemicellulosic fraction of birch wood by the oleaginous yeast Lipomyces starkeyi. Birch wood chips were thermochemically pretreated by hot water extraction, and the liquid phase, containing 45.1 g/l xylose as the major sugar, 13.1 g/l acetic acid and 4.7 g/l furfural, was used for cultivations of L. starkeyi CBS1807. The hydrolysate strongly inhibited yeast growth; the strain could only grow in medium containing 30% hydrolysate at pH 6. At pH 5, growth stopped already upon the addition of about 10% hydrolysate. In fed-batch cultures fed with hydrolysate or a model xylose-acetic acid mixture, co-consumption of xylose and acetic acid was observed, which resulted in a pH increase. This phenomenon was utilized to establish a pH-stat fed-batch cultivation in which, after an initial feeding, hydrolysate or model mixture was connected to the pH-regulation system of the bioreactor. Under these conditions we obtained growth and lipid production in cultures grown on either xylose or glucose during the batch phase. In cultivations fed with model mixture, a maximum lipid content of 60.5% of the cell dry weight (CDW) was obtained; however, not all xylose was consumed. When feeding hydrolysate, growth was promoted and carbon sources were completely consumed, resulting in higher CDW with maximum lipid content of 51.3%. In both cultures the lipid concentration was 8 g/l and a lipid yield of 0.1 g/g carbon source was obtained. Lipid composition was similar in all cultivations, with C18:1 and C16:0 being the most abundant fatty acids.

Background Rhodotorula toruloides is a promising platform organism for production of lipids from lignocellulosic substrates. Little is known about the metabolic aspects of lipid production from the lignocellolosic sugar xylose by oleaginous yeasts in general and R. toruloides in particular. This study presents the first proteome analysis of the metabolism of R. toruloides during conversion of xylose to lipids. Results Rhodotorula toruloides cultivated on either glucose or xylose was subjected to comparative analysis of its growth dynamics, lipid composition, fatty acid profiles and proteome. The maximum growth and sugar uptake rate of glucose-grown R. toruloides cells were almost twice that of xylose-grown cells. Cultivation on xylose medium resulted in a lower final biomass yield although final cellular lipid content was similar between glucose- and xylose-grown cells. Analysis of lipid classes revealed the presence of monoacylglycerol in the early exponential growth phase as well as a high proportion of free fatty acids. Carbon source-specific changes in lipid profiles were only observed at early exponential growth phase, where C18 fatty acids were more saturated in xylose-grown cells. Proteins involved in sugar transport, initial steps of xylose assimilation and NADPH regeneration were among the proteins whose levels increased the most in xylose-grown cells across all time points. The levels of enzymes involved in the mevalonate pathway, phospholipid biosynthesis and amino acids biosynthesis differed in response to carbon source. In addition, xylose-grown cells contained higher levels of enzymes involved in peroxisomal beta-oxidation and oxidative stress response compared to cells cultivated on glucose. Conclusions The results obtained in the present study suggest that sugar import is the limiting step during xylose conversion by R. toruloides into lipids. NADPH appeared to be regenerated primarily through pentose phosphate pathway although it may also involve malic enzyme as well as alcohol and aldehyde dehydrogenases. Increases in enzyme levels of both fatty acid biosynthesis and beta-oxidation in xylose-grown cells was predicted to result in a futile cycle. The results presented here are valuable for the development of lipid production processes employing R. toruloides on xylose-containing substrates. Electronic supplementary material The online version of this article (10.1186/s13068-019-1478-8) contains supplementary material, which is available to authorized users.