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Lactic acid is the monomeric unit of polylactide (PLA), a bioplastic widely used in the packaging, automotive, food, and pharmaceutical industries. Previously, the yeast Komagataella phaffii was genetically modified for the production of lactate from glycerol. For this, the bovine L-lactate dehydrogenase- (LDH)-encoding gene was inserted and the gene encoding the pyruvate decarboxylase (PDC) was disrupted, resulting in the GLp strain. This showed a yield of 67% L-lactic acid and 20% arabitol as a by-product in batches with oxygen limitation. Following up on these results, the present work endeavored to perform a detailed study of the metabolism of this yeast, as well as perturbing arabitol synthesis in an attempt to increase lactic acid titers. The GLp strain was cultivated in a glycerol-limited chemostat at different dilution rates, confirming that the production of both lactic acid and arabitol is dependent on the specific growth rate (and consequently on the concentration of the limiting carbon source) as well as on the oxygen level. Moreover, disruption of the gene encoding arabitol dehydrogenase (ArDH) was carried out, resulting in an increase of 20% in lactic acid and a 50% reduction in arabitol. This study clarifies the underlying metabolic reasons for arabitol formation in K. phaffii and points to ways for improving production of lactic acid using K. phaffii as a biocatalyst.

Background The use of thermotolerant yeast strains can improve the efficiency of ethanol fermentation, allowing fermentation to occur at temperatures higher than 40 °C. This characteristic could benefit traditional bio-ethanol production and allow simultaneous saccharification and fermentation (SSF) of starch or lignocellulosic biomass. Results We identified and characterized the physiology of a new thermotolerant strain (LBGA-01) able to ferment at 40 °C, which is more resistant to stressors as sucrose, furfural and ethanol than CAT-1 industrial strain. Furthermore, this strain showed similar CAT-1 resistance to acetic acid and lactic acid, and it was also able to change the pattern of genes involved in sucrose assimilation (SUC2 and AGT1). Genes related to the production of proteins involved in secondary products of fermentation were also differentially regulated at 40 °C, with reduced expression of genes involved in the formation of glycerol (GPD2), acetate (ALD6 and ALD4), and acetyl-coenzyme A synthetase 2 (ACS2). Fermentation tests using chemostats showed that LBGA-01 had an excellent performance in ethanol production in high temperature. Conclusion The thermotolerant LBGA-01 strain modulates the production of key genes, changing metabolic pathways during high-temperature fermentation, and increasing its resistance to high concentration of ethanol, sugar, lactic acid, acetic acid, and furfural. Results indicate that this strain can be used to improve first- and second-generation ethanol production in Brazil.

Environmental pollution by heavy metals is currently a problem of great concern for human health. In this context, this study aims to contribute with the synthesis and characterization of chitosan functionalized with three different ionic liquids (n-butylammonium acetate, sec-butylammonium acetate, and tert-butylammonium acetate) followed by its application in hexavalent chromium effluent treatment. The adsorbents synthesized (ChN, ChS, and ChT) were characterized by SEM, EDS, FTIR, BET, RDD, PSD, and XRD techniques. Afterward, the influences of temperature, contact time, and pH on the Cr(VI) adsorption process were evaluated. The solution with pH 3 displayed the highest adsorption capacities (107.31, 104.60, and 107.97 mg.g-1 for ChN, ChS, and ChT, respectively). The kinetic data were better adjusted to the Weber-Morris kinetic model with an ideal time of 2 h. Furthermore, the influence of temperature was evaluated using the Freundlich and Langmuir isotherms, with maximum capacities of 142.05 (ChN), 131.58 (ChS), and 146.63 mg.g-1 (ChT). The adsorbent displayed enhanced adsorption properties in comparison with raw chitosan by an intensification of the electrostatic interaction between amino groups and hexavalent chromium. Finally, the reusability was investigated, and significant results were observed (84.33 ± 4.87%) in the adsorption process after 4 cycles.

Abstract Background The use of thermotolerant yeast strains can improve the efficiency of ethanol fermentation, allowing fermentation to occur at temperatures higher than 40 °C. This increment in temperature could benefit traditional bio-ethanol production and allow simultaneous saccharification and fermentation (SSF) of starch or lignocellulosic biomass. Results We identified and characterized the physiology of a new thermotolerant strain able to fermentate at 40 °C while producing high yields of ethanol. Our results showed that, in comparison to the industrial yeast CAT-1, our strain was more resistant to various stressors generated during the production of first- and second-generation ethanol, and it also was able to change the pattern of genes involved in sucrose assimilation (SUC2 and AGT1). The formation of secondary products of fermentation was different at 40ºC, with reduced expression of genes involved in the formation of glycerol (GPD2), acetate (ALD6 and ALD4), and acetyl-CoA (ACS2). Conclusion The LBGA-01 strain is a thermotolerant strain that modulates the production of key genes, changing metabolic pathways during high-temperature fermentation, and increasing its tolerance to the high concentration of ethanol, sugar, acetic lactic, acetic acid, furfural and HMF. This indicates that this strain can be used to improve first- and second-generation ethanol production in Brazil.

Ethanolic fermentation is frequently performed under conditions of low nitrogen. In Saccharomyces cerevisiae , nitrogen limitation induces macroautophagy, including the selective removal of mitochondria, also called mitophagy. Shiroma and co-workers (2014) showed that blocking mitophagy by deletion of the mitophagy specific gene ATG32 increased the fermentation performance during the brewing of Ginjo sake. In this study, we tested if a similar strategy could enhance alcoholic fermentation in the context of fuel ethanol production from sugarcane in Brazilian biorefineries. Conditions that mimic the industrial fermentation process indeed induce Atg32-dependent mitophagy in cells of S. cerevisiae PE-2, a strain frequently used in the industry. However, after blocking mitophagy, no significant differences in CO 2 production, final ethanol titres or cell viability were observed after five rounds of ethanol fermentation, cell recycling and acid treatment, as commonly performed in sugarcane biorefineries. To test if S. cerevisiae ’s strain background influences this outcome, cultivations were carried out in a synthetic medium with strains PE-2, Ethanol Red (industrial) and BY (laboratory), with and without a functional ATG32 gene, under oxic and oxygen restricted conditions. Despite the clear differences in sugar consumption, cell viability and ethanol titres, among the three strains, we could not observe any significant improvement in fermentation performance related to the blocking of mitophagy. We conclude with caution that results obtained with Ginjo sake yeast is an exception and cannot be extrapolated to other yeast strains and that more research is needed to ascertain the role of autophagic processes during fermentation. Importance Bioethanol is the largest (per volume) ever biobased bulk chemical produced globally. The fermentation process is very well established, and industries regularly attain nearly 85% of maximum theoretical yields. However, because of the volume of fuel produced, even a small improvement will have huge economic benefits. To this end, besides already implemented process improvements, various free energy conservation strategies have been successfully exploited at least in laboratory strains to increase ethanol yields and decrease by-product formation. Cellular housekeeping processes have been an almost unexplored territory in strain improvement. Shiroma and co-workers previously reported that blocking mitophagy by deletion of the mitophagy receptor gene ATG32 in Saccharomyces cerevisiae led to a 2.1% increase in final ethanol titres during Japanese sake fermentation. We found in two commercially used bioethanol strains (PE-2 and Ethanol Red) that ATG32 deficiency does not lead to a significant improvement in cell viability or ethanol levels during fermentation with molasses or in a synthetic complete medium. More research is required to ascertain the role of autophagic processes during fermentation conditions.

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