Biology subjects

Basso, T. O.

Publications and source records attributed to Basso, T. O..

6 recordsLinked to original sources

bioRxiv2023

Metabolic engineering of Saccharomyces cerevisiae for second-generation ethanol production from xylo-oligosaccharides and acetate

AO_SCPLOWBSTRACTC_SCPLOWSimultaneous intracellular depolymerization of xylo-oligosaccharides (XOS) and acetate fermentation by engineered Saccharomyces cerevisiae offers an advance towards more cost-effective second-generation (2G) ethanol production. As xylan is one of the most abundant polysaccharides present in lignocellulosic residues, the transport and breakdown of XOS in an intracellular environment might bring a competitive advantage for recombinant strains in competition with contaminating microbes, which are always present in fermentation tanks; furthermore, acetic acid is a ubiquitous toxic component in lignocellulosic hydrolysates, deriving from hemicellulose and lignin breakdown. In the present work, the previously engineered S. cerevisiae strain, SR8A6S3, expressing NADPH-linked xylose reductase (XR), NAD+-linked xylitol dehydrogenase (XDH) (for xylose assimilation), as well as NADH-linked acetylating acetaldehyde dehydrogenase (AADH) and acetyl-CoA synthetase (ACS) (for an NADH-dependent acetate reduction pathway), was used as the host for expressing of two {beta}-xylosidases, GH43-2 and GH43-7, and a xylodextrin transporter, CDT-2, from Neurospora crassa, yielding the engineered strain SR8A6S3-CDT2-GH432/7. Both {beta}-xylosidases and the transporter were introduced by replacing two endogenous genes, GRE3 and SOR1, that encode aldose reductase and sorbitol (xylitol) dehydrogenase, respectively, which catalyse steps in xylitol production. Xylitol accumulation during xylose fermentation is a problem for 2G ethanol production since it reduces final ethanol yield. The engineered strain, SR8A6S3-CDT2-GH432/7, produced ethanol through simultaneous co-utilization of XOS, xylose, and acetate. The mutant strain produced 60% more ethanol and 12% less xylitol than the control strain when a hemicellulosic hydrolysate was used as a mono- and oligosaccharide source. Similarly, the ethanol yield was 84% higher for the engineered strain using hydrolysed xylan compared with the parental strain. The consumption of XOS, xylose, and acetate expands the capabilities of S. cerevisiae for utilization of all of the carbohydrate in lignocellulose, potentially increasing the efficiency of 2G biofuel production. HighlightsO_LIIntegration of XOS pathway in an acetate-xylose-consuming S. cerevisiae strain; C_LIO_LIIntracellular fermentation of XOS, acetate and xylose improved ethanol production; C_LIO_LIDeletion of both sor1{Delta} and gre3{Delta} reduced xylitol production. C_LI

bioengineering↗

bioRxiv2023

A Fully Defined Synthetic Medium Mimicking Sugar Cane Molasses

BackgroundYeast research in the context of food/beverage production and industrial biotechnology faces a dilemma: to use real industrial media or to use fully defined laboratory media? While the former option might lead to experiments closer to industrial conditions, the latter has the advantage of allowing for reproducibility and comparability of results among different laboratories, as well as being suitable for the investigation of how different individual components affect microbial or process performance. It is undoubtable that the development of a synthetic must a few decades ago led to important advances in wine yeast research. ResultsWe developed a fully defined medium that mimics sugarcane molasses, a frequently used medium in different industrial processes where yeast is cultivated. The medium, named 2SMol, builds upon a previously published semi-defined formulation and is conveniently prepared from some stock solutions: C-source, organic N, inorganic N, organic acids, trace elements, vitamins, Mg+K, and Ca. We validated the 2SMol recipe in a scaled-down sugarcane biorefinery model, comparing the performance of different yeast strains in different real molasses-based media. We also showcase the flexibility of the medium by investigating the effect of nitrogen availability on the ethanol yield during fermentation. ConclusionsHere we present in detail the development of a fully defined synthetic molasses medium, and we hope the 2SMol formulation will be valuable to researchers both in academia and industry to obtain new insights and developments in industrial yeast biotechnology.

bioengineering↗

bioRxiv2022

Physiology and mathematical modeling of immobilized Saccharomyces spp. in beer fermentation

There is an ever-increasing demand for reduction of unit operations and a growing interest in the physiology of yeasts used in beer fermentation. In this context, cell immobilization is an interesting alternative, since it reduces steps to separate biomass from fermented broth. Yet, physiological alterations in yeast metabolism caused by immobilization are still to be fully described. Thus, the main objective of this work was to evaluate the physiology of three brewers S. cerevisiae yeast strains (SY025, SY067 and SY001) immobilized on a porous cellulose-based support. Batch fermentations in malt extract 12 {degrees}P were carried out for all strains both in free and immobilized forms in order to compare kinetic parameters obtained from distinct process conditions. Mathematical modeling was performed following two viewpoints: modeling of fermentation kinetics by parameter estimation from experimental data and application of a reaction-diffusion model for estimation of substrate concentration gradient inside the immobilization support. Moreover, fermentations with different initial substrate and biomass concentrations were carried out using strain SY025, aiming to evaluate their influence over flavor compounds, using statistical models. Compared to free cells, immobilized yeasts showed both higher glycerol yield (SY025, 40%; SY067, 53%; SY001, 19%) and biomass yield in the system (SY025, 67%; SY067, 78%; SY001, 56%). On the other hand, free cells presented higher ethanol yields when compared to immobilized ones (SY025, 9%; SY067, 9%; and SY001, 13%). According to the model developed, a substrate gradient inside the support was predicted, but with low mass transfer limitations. KEY POINTSO_LIYeast immobilization not always hinder biomass growth, here it was stimulated. C_LIO_LIA classic kinetic model describes accurately immobilized yeast fermentations. C_LIO_LIPhysiology changes occur in immobilization even with low mass transfer limitations. C_LI

bioengineering↗

bioRxiv2021

Blocking mitophagy does not improve fuel ethanol production in Saccharomyces cerevisiae

Ethanol 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 differences in CO2 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. cerevisiaes 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 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. ImportanceBioethanol 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 2.12% 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 an 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.

microbiology↗

bioRxiv2021

Strain dynamics of specific contaminant bacteria modulate the performance of ethanol biorefineries

Bioethanol is a viable alternative for fossil fuels, and its use has lowered CO2 emissions by over 500 million tonnes in Brazil alone by replacing more than 40% of the national gasoline consumption. However, contaminant bacteria reduce yields during fermentation. Our understanding of these contaminants is limited to targeted studies, and the interplay of the microbial community and its impact on fermentation efficiency remains poorly understood. Comprehensive surveying and longitudinal analysis using shotgun metagenomics of two major biorefineries over a production season revealed similar patterns in microbial community structure and dynamics throughout the entire fermentation system. Strain resolution metagenomics identified specific Lactobacillus fermentum strains as strongly associated with poor industrial performance and laboratory-scale fermentations revealed yield reductions of up to 4.63{+/-}1.35% depending on the specific contaminating strains. Selective removal of these strains could reduce emissions from the bioethanol industry by more than 2x106 tonnes per year. Using the large-scale Brazilian ethanol fermentations as a model system for studying microbiome-phenotype relationships this study further demonstrates how high-resolution metagenomics can identify culprits of large scale industrial biomanufacturing.

microbiology↗

bioRxiv2021

Homo-and heterofermentative lactobacilli are differently affected by lignocellulosic inhibitory compounds

Second generation (2G) ethanol is produced through the use of lignocellulosic biomass. However, the pretreatment processes generates a variety of molecules (furan derivatives, phenolic compounds and organic acids) that act as inhibitors of microbial metabolism, and thus reduce the efficiency of the fermentation step in this process. In this context, the present study aimed to investigate the effect of furan derivatives on the physiology of lactic acid bacteria (LAB) strains that are potential contaminants of ethanol production. Homofermentative and heterofermentative strains of laboratory LAB and isolated from first generation ethanol fermentation were used. LAB strains were challenge to grow in the presence of furfural and hydroxymethyylfurfural (HMF). We found that the effect of HMF and furfural on the growth rate of LAB is dependent of the metabolic type, and growth kinetics in the presence of these compounds is enhanced for heterofermentative LAB, whereas is inhibitory to homofermentative LAB. Sugar consumption and product formation were also enhanced in the presence of furaldehydes in heterofermentative LAB, that displayed an effective detoxification kinetics when compared to the homofermentative LAB. This knowledge is important because LAB can be explored both within the scope of bio-detoxification, being applied before the fermentation. Key points- Heterofermentative LAB presented the ability to decrease the concentrations of furfural and HMF - LAB can be used in the bio-detoxification to remove the inhibitors before fermentations - The presence of furan derivatives had a growth stimulus observed in heterofermentative LAB

microbiology↗