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The ability of Zygosaccharomyces bailii to grow at low pH and in the presence of considerable amounts of weak organic acids, at lethal condition for Saccharomyces cerevisiae , increased the interest in the biotechnological potential of the yeast. To understand the mechanism of tolerance and growth effect of weak acids on Z. bailii , we evaluated the physiological and macromolecular changes of the yeast exposed to sub lethal concentrations of lactic acid. Lactic acid represents one of the important commodity chemical which can be produced by microbial fermentation. We assessed physiological effect of lactic acid by bioreactor fermentation using synthetic media at low pH in the presence of lactic acid. Samples collected from bioreactors were stained with propidium iodide (PI) which revealed that, despite lactic acid negatively influence the growth rate, the number of PI positive cells is similar to that of the control. Moreover, we have performed Fourier Transform Infra-Red (FTIR) microspectroscopy analysis on intact cells of the same samples. This technique has been never applied before to study Z. bailii under this condition. The analyses revealed lactic acid induced macromolecular changes in the overall cellular protein secondary structures, and alterations of cell wall and membrane physico-chemical properties.

The success of the biorefinery concept will require efficient, robust and versatile cell factories. Currently, the major part of industrial microorganisms are used because of historical grounds, rather than being selected for a specific application. Additionally, demands for increased productivity, wider substrate range utilization, and production of nonconventional compounds lead to a great interest in further improving the currently used industrial workhorses (hosts) and the selection or development of strains with novel properties. The model yeast Saccharomyces cerevisiae is the main microorganism used for first generation ethanol production. When moving from first to second generation of production, one of the major obstacles for a viable development is the toxic effect of compounds released during the pre-treatment of lignocellulosic biomasses, which are the more sustainable feedstock utilized. In the first part of this work, two different approaches to improve S. cerevisiae tolerance to compounds deriving from biomass pre-treatment are described. Firstly, the effects of overexpressing genes encoding the transcription factor (YAP1) and the mitochondrial NADH-cytochrome b5 reductase (MCR1) was evaluated in an industrial xylose-consuming S. cerevisiae strain. During batch fermentation on undiluted and undetoxified spruce hydrolysate overexpression of either genes resulted in faster hexose catabolism. The second approach revealed that acetic acid tolerance of S. cerevisiae can be increased by engineering it to endogenously produce L-ascorbic acid (L-AA). In the second part of the work, since the currently used industrial yeasts represent only the tip of the proverbial iceberg of the genetic diversity present in nature, different non-saccharomyces yeasts were investigated for their potential industrial applications: Kluyveromyces marxianus (CBS 712), the oleaginous yeasts Rhodosporidium toruloides (DSM 4444), Lipomyces starkeyi (DSM 70295) and Cryptococcus curvatus (DSM 70022), Zygosacchromyces bailii and finally, Candida lignohabitans. Overall, the work performed resulted in the development of industrial S. cerevisiae strains with improved traits that can match the requirements of lignocellulosic hydrolysate fermentation. The work also contributed to a better understanding of the metabolism and physiology of different non-saccharomyces yeasts with a great industrial potential.

Background Microbial lipids can represent a valuable alternative feedstock for biodiesel production in the context of a viable bio-based economy. This production can be driven by cultivating some oleaginous microorganisms on crude-glycerol, a 10 % (w/w) by-product produced during the transesterification process from oils into biodiesel. Despite attractive, the perspective is still economically unsustainable, mainly because impurities in crude glycerol can negatively affect microbial performances. In this view, the selection of the best cell factory, together with the development of a robust and effective production process are primary requirements. Results The present work compared crude versus pure glycerol as carbon sources for lipid production by three different oleaginous yeasts: Rhodosporidium toruloides (DSM 4444), Lipomyces starkeyi (DSM 70295) and Cryptococcus curvatus (DSM 70022). An efficient yet simple feeding strategy for avoiding the lag phase caused by growth on crude glycerol was developed, leading to high biomass and lipid production for all the tested yeasts. Flow-cytometry and fourier transform infrared (FTIR) microspectroscopy, supported by principal component analysis (PCA), were used as non-invasive and quick techniques to monitor, compare and analyze the lipid production over time. Gas chromatography (GC) analysis completed the quali-quantitative description. Under these operative conditions, the highest lipid content (up to 60.9 % wt/wt) was measured in R. toruloides, while L. starkeyi showed the fastest glycerol consumption rate (1.05 g L−1 h−1). Being productivity the most industrially relevant feature to be pursued, under the presented optimized conditions R. toruloides showed the best lipid productivity (0.13 and 0.15 g L−1 h−1 on pure and crude glycerol, respectively). Conclusions Here we demonstrated that the development of an efficient feeding strategy is sufficient in preventing the inhibitory effect of crude glycerol, and robust enough to ensure high lipid accumulation by three different oleaginous yeasts. Single cell and in situ analyses allowed depicting and comparing the transition between growth and lipid accumulation occurring differently for the three different yeasts. These data provide novel information that can be exploited for screening the best cell factory, moving towards a sustainable microbial biodiesel production. Electronic supplementary material The online version of this article (doi:10.1186/s12934-016-0467-x) contains supplementary material, which is available to authorized users.

Background: The yeast Kluyveromyces marxianus features specific traits that render it attractive for industrial applications. These include production of ethanol which, together with thermotolerance and the ability to grow with a high specific growth rate on a wide range of substrates, could make it an alternative to Saccharomyces cerevisiae as an ethanol producer. However, its ability to co-ferment C5 and C6 sugars under oxygen-limited conditions is far from being fully characterized.Results: In the present study, K. marxianus CBS712 strain was cultivated in defined medium with glucose and xylose as carbon source. Ethanol fermentation and sugar consumption of CBS712 were investigated under different oxygen supplies (1.75%, 11.00% and 20.95% of O2) and different temperatures (30°C and 41°C). By decreasing oxygen supply, independently from the temperature, both biomass production as well as sugar utilization rate were progressively reduced. In all the tested conditions xylose consumption followed glucose exhaustion. Therefore, xylose metabolism was mainly affected by oxygen depletion. Loss in cell viability cannot explain the decrease in sugar consumption rates, as demonstrated by single cell analyses, while cofactor imbalance is commonly considered as the main cause of impairment of the xylose reductase (KmXR) - xylitol dehydrogenase (KmXDH) pathway. Remarkably, when these enzyme activities were assayed in vitro, a significant decrease was observed together with oxygen depletion, not ascribed to reduced transcription of the corresponding genes.Conclusions: In the present study both oxygen supply and temperature were shown to be key parameters affecting the fermentation capability of sugars in the K. marxianus CBS712 strain. In particular, a direct correlation was observed between the decreased efficiency to consume xylose with the reduced specific activity of the two main enzymes (KmXR and KmXDH) involved in its catabolism. These data suggest that, in addition to the impairment of the oxidoreductive pathway being determined by the cofactor imbalance, post-transcriptional and/or post-translational regulation of the pathway enzymes contributes to the efficiency of xylose catabolism in micro-aerobic conditions. Overall, the presented work provides novel information on the fermentation capability of the CBS712 strain that is currently considered as the reference strain of the genus K. marxianus. © 2014 Signori et al.; licensee BioMed Central Ltd.

Biotechnological processes are of increasing significance for industrial production of fine and bulk chemicals, including biofuels. Unfortunately, under operative conditions microorganisms meet multiple stresses, such as non-optimal pH, temperature, oxygenation and osmotic stress. Moreover, they have to face inhibitory compounds released during the pretreatment of lignocellulosic biomasses, which constitute the preferential substrate for second-generation processes. Inhibitors include furan derivatives, phenolic compounds and weak organic acids, among which acetic acid is one of the most abundant and detrimental for cells. They impair cellular metabolism and growth, reducing the productivity of the process: therefore, the development of robust cell factories with improved production rates and resistance is of crucial importance. Here we show that a yeast strain engineered to endogenously produce vitamin C exhibits an increased tolerance compared to the parental strain when exposed to acetic acid at moderately toxic concentrations, measured as viability on plates. Starting from this evidence, we investigated more deeply: (a) the nature and levels of reactive oxygen species (ROS); (b) the activation of enzymes that act directly as detoxifiers of reactive oxygen species, such as superoxide dismutase (SOD) and catalase, in parental and engineered strains during acetic acid stress. The data indicate that the engineered strain can better recover from stress by limiting ROS accumulation, independently from SOD activation. The engineered yeast can be proposed as a model for further investigating direct and indirect mechanism(s) by which an antioxidant can rescue cells from organic acid damage; moreover, these studies will possibly provide additional targets for further strain improvements. © 2013 John Wiley & Sons, Ltd.

Development of robust yeast strains that can efficiently ferment lignocellulose-based feedstocks is one of the requirements for achieving economically feasible bioethanol production processes. With this goal, several genes have been identified as promising candidates to confer improved tolerance to S. cerevisiae. In most of the cases, however, the evaluation of the genetic modification was performed only in laboratory strains, that is, in strains that are known to be quite sensitive to various types of stresses. In the present study, we evaluated the effects of overexpressing genes encoding the transcription factor (YAP1) and the mitochondrial NADH-cytochrome b5 reductase (MCR1), either alone or in combination, in an already robust and xylose-consuming industrial strain of S. cerevisiae and evaluated the effect during the fermentation of undiluted and undetoxified spruce hydrolysate. Overexpression of either gene resulted in faster hexose catabolism, but no cumulative effect was observed with the simultaneous overexpression. The improved phenotype of MCR1 overexpression appeared to be related, at least in part, to a faster furaldehyde reduction capacity, indicating that this reductase may have a wider substrate range than previously reported. Unexpectedly a decreased xylose fermentation rate was also observed in YAP1 overexpressing strains and possible reasons behind this phenotype are discussed.