The production of fuel ethanol from sugarcane-based raw materials in Brazil is a successful example of a large-scale bioprocess that delivers an advanced biofuel at competitive prices and low ...environmental impact. Two to three fed-batch fermentations per day, with acid treatment of the yeast cream between consecutive cycles, during 6–8 months of uninterrupted production in a nonaseptic environment are some of the features that make the Brazilian process quite peculiar. Along the past decades, some wild
Saccharomyces cerevisiae
strains were isolated, identified, characterized, and eventually, reintroduced into the process, enabling us to build up knowledge on these organisms. This information, combined with physiological studies in the laboratory and, more recently, genome sequencing data, has allowed us to start clarifying why and how these strains behave differently from the better known laboratory, wine, beer, and baker's strains. All these issues are covered in this minireview, which also presents a brief discussion on future directions in the field and on the perspectives of introducing genetically modified strains in this industrial process.
Abstract Bioethanol is a sustainable energy alternative and can contribute to global greenhouse-gas emission reductions by over 60%. Its industrial production faces various bottlenecks, including ...sub-optimal efficiency resulting from bacteria. Broad-spectrum removal of these contaminants results in negligible gains, suggesting that the process is shaped by ecological interactions within the microbial community. Here, we survey the microbiome across all process steps at two biorefineries, over three timepoints in a production season. Leveraging shotgun metagenomics and cultivation-based approaches, we identify beneficial bacteria and find improved outcome when yeast-to-bacteria ratios increase during fermentation. We provide a microbial gene catalogue which reveals bacteria-specific pathways associated with performance. We also show that Limosilactobacillus fermentum overgrowth lowers production, with one strain reducing yield by ~5% in laboratory fermentations, potentially due to its metabolite profile. Temperature is found to be a major driver for strain-level dynamics. Improved microbial management strategies could unlock environmental and economic gains in this US $ 60 billion industry enabling its wider adoption.
Fully defined laboratory media have 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. We 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 physiology of Saccharomyces cerevisiae in different actual molasses-based media. We demonstrate the flexibility of the medium by investigating the effect of nitrogen availability on the ethanol yield during fermentation. Here we present in detail the development of a fully defined synthetic molasses medium and the physiology of yeast strains in this medium compared to industrial molasses. This tailor-made medium was able to satisfactorily reproduce the physiology of S. cerevisiae in industrial molasses. Thus, 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.
Simultaneous intracellular depolymerization of xylo-oligosaccharides (XOS) and acetate fermentation by engineered Saccharomyces cerevisiae offers significant potential for more cost-effective ...second-generation (2G) ethanol production. In the present work, the previously engineered S. cerevisiae strain, SR8A6S3, expressing enzymes for xylose assimilation along with an optimized route for acetate reduction, was used as the host for expressing two β-xylosidases, GH43-2 and GH43-7, and a xylodextrin transporter, CDT-2, from Neurospora crassa, yielding the engineered SR8A6S3-CDT-2-GH34-2/7 strain. Both β-xylosidases and the transporter were introduced by replacing two endogenous genes, GRE3 and SOR1, that encode aldose reductase and sorbitol (xylitol) dehydrogenase, respectively, and catalyse steps in xylitol production. The engineered strain, SR8A6S3-CDT-2-GH34-2/7 (sor1Δ gre3Δ), produced ethanol through simultaneous XOS, xylose, and acetate co-utilization. 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. Xylan, a common polysaccharide in lignocellulosic residues, enables recombinant strains to outcompete contaminants in fermentation tanks, as XOS transport and breakdown occur intracellularly. Furthermore, acetic acid is a ubiquitous toxic component in lignocellulosic hydrolysates, deriving from hemicellulose and lignin breakdown. Therefore, 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.
Biotechnological Applications of Biomass provides a comprehensive overview of the current state of the art of biomass utilization in agriculture and pharmaceuticals. The information contained herein ...is useful to researchers and other readers interested in biomass utilization and production of bioproducts.
The engineering of xylo-oligosaccharide-consuming
strains is a promising approach for more effective utilization of lignocellulosic biomass and the development of economic industrial fermentation ...processes. Extending the sugar consumption range without catabolite repression by including the metabolism of oligomers instead of only monomers would significantly improve second-generation ethanol production This review focuses on different aspects of the action mechanisms of xylan-degrading enzymes from bacteria and fungi, and their insertion in
strains to obtain microbial cell factories able of consume these complex sugars and convert them to ethanol. Emphasis is given to different strategies for ethanol production from both extracellular and intracellular xylo-oligosaccharide utilization by
strains. The suitability of
for ethanol production combined with its genetic tractability indicates that it can play an important role in xylan bioconversion through the heterologous expression of xylanases from other microorganisms.
Recently, in Brazil, corn ethanol industries are being installed and the integration with sugar/energy-cane has been proposed, using bagasse for cogeneration and the juice to dilute the corn. ...However, this integration may have some limitations, such as the quality of the cane juice and potential contamination by microorganisms brought with the cane from the field. In this article, we first tested the effects of mixing energy cane juice with corn on fermentative parameters. We also assessed the effects of Lactobacilli. contamination on organic acids produced during the fermentation and fermentation parameters and proposed the use of ionizing radiation to replace antibiotics as a disinfection control method. Our results showed that mixing energy cane juice with corn does not have any negative effect on fermentation parameters, including ethanol production. The contamination with Lactobacilli. considerably increased the production of acetic, lactic, and succinic acid, reducing the pH and ethanol content from 89.2 g L−1 in the sterilized treatment to 72.9 g L−1 in the contaminated treatment. Therefore, for the integration between corn and cane to be applied on an industrial scale, it is essential to have effective disinfection before fermentation. Ionizing radiation (20 kGy) virtually disinfected the wort, showing itself to be a promising technology; however, an economic viability study for adopting it in the industry should be carried out.
Lactic acid is the monomeric unit of polylactide (PLA), a bioplastic widely used in the packaging, automotive, food, and pharmaceutical industries. Previously, the yeast
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
and points to ways for improving production of lactic acid using
as a biocatalyst.
This study aimed to develop non-GMO freezing-resistant hybrid yeast for frozen French bread dough. The hybrids were generated by direct mating, using Saccharomyces cerevisiae strains from the ethanol ...industry (PE-2) and a baker's yeast (FLE). Four viable hybrids were generated and named F5P1, F5P33, F5P40, and F5P42. According to the 96-well microplate analyses, the growth curves of the hybrids after freezing and subsequent thawing presented similar behavior to those without freezing. In a performance test after the freezing step, F5P1 and F5P40 achieved a higher development index, with values of (147.1 ± 2.5) % and (102.1 ± 5.6) %, respectively. The peak reached by F5P1, after 50 min, was similar (p > 0.05) to that of FLE, which was at 110 min. The difference between the time required for F5P1 to achieve the growth peak with and without freezing was 10 min, whereas for FLE, this difference was three times higher, which highlights the improving freezing resistance of the hybrid. Therefore, the hybrid F5P1 showed a leavening capacity similar to FLE (p > 0.05) and improved freezing resistance, which indicates its potential for application to frozen French bread dough.
•Direct mating is a simple and efficient methodology to generate non-GMO hybrids.•Aqueous suspension of wheat flour is suitable to estimate yeast leavening capacity.•Freezing resistance and leavening capacity can be inherited from parental strains.•Hybridization allows to achieve desirable traits using industrial strains.
A synthetic medium to simulate sugarcane molasses Lino, Felipe Senne de Oliveira; Basso, Thiago Olitta; Sommer, Morten Otto Alexander
Biotechnology for biofuels,
08/2018, Letnik:
11, Številka:
1
Journal Article
Odprti dostop
Developing novel microbial cell factories requires careful testing of candidates under industrially relevant conditions. However, this frequently occurs late during the strain development process. ...The availability of laboratory media that simulate industrial-like conditions might improve cell factory development, as they allow for strain construction and testing in the laboratory under more relevant conditions. While sugarcane molasses is one of the most important substrates for the production of biofuels and other bioprocess-based commodities, there are no defined media that faithfully simulate it. In this study, we tested the performance of a new synthetic medium simulating sugarcane molasses.
Laboratory scale simulations of the Brazilian ethanol production process, using both sugarcane molasses and our synthetic molasses (SM), demonstrated good reproducibility of the fermentation performance, using yeast strains, PE-2 and Ethanol Red™. After 4 cycles of fermentation, the final ethanol yield (g
g
) values for the SM ranged from 0.43 ± 0.01 to 0.44 ± 0.01 and from 0.40 ± 0.01 to 0.46 ± 0.01 for the molasses-based fermentations. The other fermentation parameters (i.e., biomass production, yeast viability, and glycerol and acetic acid yield) were also within similar value ranges for all the fermentations. Sequential pairwise competition experiments, comparing industrial and laboratory yeast strains, demonstrated the impact of the media on strain fitness. After two sequential cocultivations, the relative abundance of the laboratory yeast strain was 5-fold lower in the SM compared to the yeast extract-peptone-dextrose medium, highlighting the importance of the media composition on strain fitness.
Simulating industrial conditions at laboratory scale is a key part of the efficient development of novel microbial cell factories. In this study, we have developed a synthetic medium that simulated industrial sugarcane molasses media. We found good agreement between the synthetic medium and the industrial media in terms of the physiological parameters of the industrial-like fermentations.
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