Exploring industrial and natural Saccharomyces cerevisiae strains for the bio-based economy from biomass: the case of bioethanol

Saccharomyces cerevisiae is the preferred microorganism for the production of bioethanol from biomass. Industrial strain development for first-generation ethanol from sugar cane and corn mostly relies on the historical know-how from high gravity beer brewing and alcohol distilleries. However, the recent design of yeast platforms for the production of second–generation biofuels and green chemicals from lignocellulose exposes yeast to different environments and stress challenges. The industrial need for increased productivity, wider substrate range utilization, and the production of novel compounds leads to renewed interest in further extending the use of current industrial strains by exploiting the immense, and still unknown, potential of natural yeast strains. This review describes key metabolic engineering strategies tailored to develop efficient industrial and novel natural yeast strains towards bioethanol production from biomass. Furthermore, it shapes how proof-of-concept studies, often advanced in academic settings on natural yeast, can be upgraded to meet the requirements for industrial applications. Academic and industrial research should continue to cooperate on both improving existing industrial strains and developing novel phenotypes by exploring the vast biodiversity available in nature on the road to establish yeast biorefineries where a range of biomass substrates are converted into valuable compounds.     

A simple and effective set of PCR-based molecular markers for the monitoring of the Saccharomyces cerevisiae cell population during bioethanol fermentation

One of the defining features of the fermentation process used in the production of bioethanol from sugarcane feedstock is the dynamic nature of the yeast population. Minisatellite molecular markers are particularly useful for monitoring yeast communities because they produce polymorphic PCR products that typically display wide size variations. We compared the coding sequences derived from the genome of the sugarcane bioethanol strain JAY270/PE-2 to those of the reference Saccharomyces cerevisiae laboratory strain S288c, and searched for genes containing insertion or deletion polymorphisms larger than 24 bp. We then designed oligonucleotide primers flanking nine of these sites, and used them to amplify differentially sized PCR products. We analyzed the banding patterns in the most widely adopted sugarcane bioethanol strains and in several indigenous yeast contaminants, and found that our marker set had very good discriminatory power. Subsequently, these markers were used to successfully monitor the yeast cell populations in six sugarcane bioethanol distilleries. Additionally, we showed that most of the markers described here are also polymorphic among strains unrelated to bioethanol production, suggesting that they may be applied universally in S. cerevisiae. Because the relatively large polymorphisms are detectable in conventional agarose gels, our method is well suited to modestly equipped on-site laboratories at bioethanol distilleries, therefore providing both cost and time savings.     

The ability to use nitrate confers advantage to <Emphasis Type="Italic">Dekkera bruxellensis</Emphasis> over <Emphasis Type="Italic">S. cerevisiae</Emphasis> and can explain its adaptation to industrial fermentation processes

The yeast Dekkera bruxellensis has been regarded as a contamination problem in industrial ethanol production because it can replace the originally inoculated Saccharomyces cerevisiae strains. The present study deals with the influence of nitrate on the relative competitiveness of D. bruxellensis and S. cerevisiae in sugar cane ethanol fermentations. The industrial strain D. bruxellensis GDB 248 showed higher growth rates than S. cerevisiae JP1 strain in mixed ammonia/nitrate media, and nitrate assimilation genes were only slightly repressed by ammonia. These characteristics rendered D. bruxellensis cells with an ability to overcome S. cerevisiae populations in both synthetic medium and in sugar cane juice. The results were corroborated by data from industrial fermentations that showed a correlation between high nitrate concentrations and high D. bruxellensis cell counts. Moreover, the presence of nitrate increased fermentation efficiency of D. bruxellensis cells in anaerobic conditions, which may explain the maintenance of ethanol production in the presence of D. bruxellensis in industrial processes. The presence of high levels of nitrate in sugar cane juice may be due to its inefficient conversion by plant metabolism in certain soil types and could explain the periodical episodes of D. bruxellensis colonization of Brazilian ethanol plants.     

Regulation of Lactobacillus plantarum contamination on the carbohydrate and energy related metabolisms of Saccharomyces cerevisiae during bioethanol fermentation

During the industrial bioethanol fermentation, Saccharomyces cerevisiae cells are often stressed by bacterial contaminants, especially lactic acid bacteria. Generally, lactic acid bacteria contamination can inhibit S. cerevisiae cell growth through secreting lactic acid and competing with yeast cells for micronutrients and living space. However, whether are there still any other influences of lactic acid bacteria on yeast or not? In this study, Lactobacillus plantarum ATCC 8014 was co-cultivated with S. cerevisiae S288c to mimic the L. plantarum contamination in industrial bioethanol fermentation. The contaminative L. plantarum-associated expression changes of genes involved in carbohydrate and energy related metabolisms in S. cerevisiae cells were determined by quantitative real-time polymerase chain reaction to evaluate the influence of L. plantarum on carbon source utilization and energy related metabolism in yeast cells during bioethanol fermentation. Contaminative L. plantarum influenced the expression of most of genes which are responsible for encoding key enzymes involved in glucose related metabolisms in S. cerevisiae. Specific for, contaminated L. plantarum inhibited EMP pathway but promoted TCA cycle, glyoxylate cycle, HMP, glycerol synthesis pathway, and redox pathway in S. cerevisiae cells. In the presence of L. plantarum, the carbon flux in S. cerevisiae cells was redistributed from fermentation to respiratory and more reducing power was produced to deal with the excess NADH. Moreover, L. plantarum contamination might confer higher ethanol tolerance to yeast cells through promoting accumulation of glycerol. These results also highlighted our knowledge about relationship between contaminative lactic acid bacteria and S. cerevisiae during bioethanol fermentation.     

Comparison of PCR-RFLP with 21-plex PCR and rDNA Sequencing for Identification of Clinical Yeast Isolates

Non-albicans Candida species and other rare yeasts have emerged as major opportunistic pathogens in fungal infections. Identification of opportunistic yeasts in developing countries is mainly performed by phenotypic assay, which are time-consuming and prone to errors. The aim of the present study was to evaluate PCR-RFLP as a routinely used identification technique for the most clinically important Candida species in Iran and make a comparison with a novel multiplex PCR, called 21-plex PCR. One hundred and seventy-three yeast isolates from clinical sources were selected and identified with sequence analysis of the D1/D2 domains of rDNA (LSU rDNA) sequencing as the gold standard method. The results were compared with those obtained by PCR-RFLP using MspI restriction enzyme and the 21-plex PCR. PCR-RFLP correctly identified 93.4% of common pathogenic Candida species (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and P. kudriavsevii (=?C. krusei)) and was able to identify 45.5% of isolates of the uncommon yeast species compared to the D1/D2 rDNA sequencing. Compared with PCR-RFLP, all common Candida species and 72.7% of uncommon yeast species were correctly identified by the 21-plex PCR. The application of the 21-plex PCR assay as a non-sequence-based molecular method for the identification of common and rare yeasts can reduce turnaround time and costs for the identification of clinically important yeasts and can be applied in resource-limited settings.

     

Identification of bacteria and yeasts from <Emphasis Type="Italic">in vitro</Emphasis> and surface-sterilized field samples of <Emphasis Type="Italic">Ensete ventricosum</Emphasis> by rDNA analysis

Bacterial and fungal contaminants of enset (Ensete ventricosum) cultures and microbes associated with surface-sterilized field material were identified by 16S/26S rDNA sequencing. Ten bacterial species were identified in 16 isolates from in vitro cultures and seven in 10 isolates from field clones. Three yeast species and one filamentous fungus were recorded as in vitro contaminants, whereas five yeast species were isolated from the field material. The bacterium, Pseudomonas reactans (6 isolates), and the yeast, Torulaspora delbrueckii (8 isolates), were the most frequent in vitro contaminants. Most of the bacterial species isolated from in vitro enset were Gram-positive and hitherto unrecorded as in vitro contaminants. The difficulty in controlling the in vitro contaminants is due to their apparent endogenous nature and their resistance to antimicrobial drugs.     

Yeast population dynamics of industrial fuel-ethanol fermentation process assessed by PCR-fingerprinting

Yeast population used in industrial production of fuel-ethanol may vary according to the plant process condition and to the environmental stresses imposed to yeast cells. Therefore, yeast strains isolated from a particular industrial process may be adapted to such conditions and should be used as starter strain instead of less adapted commercial strains. This work reports the use of PCR-fingerprinting method based on microsatellite primer (GTG)₅ to characterize the yeast population dynamics along the fermentation period in six distilleries. The results show that indigenous fermenting strains present in the crude substrate can be more adapted to the industrial process than commercial strains. We also identified new strains that dominate the yeast population and were more present either in molasses or sugar cane fermenting distilleries. Those strains were proposed to be used as starters in those industrial processes. This is the first report on the use of molecular markers to discriminate Saccharomyces cerevisiae strains from fuel-ethanol producing process.     

Yeast population dynamics of industrial fuel-ethanol fermentation process assessed by PCR-fingerprinting

Yeast populations used in industrial production of fuel-ethanol may vary according to the plant process conditions and to the environmental stresses imposed on yeast cells. Therefore, yeast strains isolated from a particular industrial process may be adapted to such conditions and should be used as the starter strain instead of less adapted commercial strains. This work reports the use of a PCR-fingerprinting method based on microsatellite primer (GTG)₅ to characterize the yeast population dynamics during the fermentation period in six distilleries. The results show that indigenous fermenting strains present in the crude substrate can be more adapted to the industrial process than commercial strains. We also identified new strains that dominated the yeast population and were more commonly present either in molasses or sugar cane fermenting distilleries. Those strains were proposed to be used as starters in those industrial processes. This is the first report on the use of molecular markers to discriminate Saccharomyces cerevisiae strains from the fuel-ethanol producing process.     

Production of bioethanol from four species of duckweeds (Landoltia punctata,Lemna aequinoctialis,Spirodela polyrrhiza,and Wolffia arrhiza) through optimization of saccharification process and fermentation with Saccharomyces cerevisiae

Duckweeds are promising potential sources for bioethanol production due to their high starch content and fast growth rate. We assessed the potential for four species, Landoltia punctata, Lemna aequinoctialis, Spirodela polyrrhiza, and Wolffia arrhiza, for bioethanol production. We also optimized a possible production procedure, which must include saccharification to convert starch to soluble sugars that can serve as a substrate for fermentation. Duckweeds were cultivated on 10% Hoagland solution for 12 days, harvested, dried, homogenized, and dissolved in solutions that were tested as substrates for bioethanol production by the yeast Saccharomyces cerevisiae. First, we optimized the saccharification process, including the ideal ratio of the enzyme used to convert starch into simple sugars. The greatest starch-to-sugar conversion was obtained when the α-amylase and amyloglucosidase was 2:1 (v/v) and with a 24 h incubation period at 50 °C. After saccharification, the solutions were incubated with the yeast, S. cerevisiae. The fermentation process was carried out for 48 h with 10% (v/v) yeast inoculum. The ethanol content was maximal approximately 24 h after the start of incubation, and the sugars and protein were minimal, with little change over the next 24 h. The final ethanol concentration obtained were 0.19, 0.17, 0.19, and 0.16 g ethanol/g dry biomass for L. punctata, L. aequinoctialis, S. polyrrhiza, and W. arrhiza respectively. We suggest that these four species of duckweed have the potential to serve sources of bioethanol and hope that the procedure we have optimized proves useful in the endeavour.     

Yeast selection for fuel ethanol production in Brazil

Brazil is one of the largest ethanol biofuel producers and exporters in the world and its production has increased steadily during the last three decades. The increasing efficiency of Brazilian ethanol plants has been evident due to the many technological contributions. As far as yeast is concerned, few publications are available regarding the industrial fermentation processes in Brazil. The present paper reports on a yeast selection program performed during the last 12 years aimed at selecting Saccharomyces cerevisiae strains suitable for fermentation of sugar cane substrates (cane juice and molasses) with cell recycle, as it is conducted in Brazilian bioethanol plants. As a result, some evidence is presented showing the positive impact of selected yeast strains in increasing ethanol yield and reducing production costs, due to their higher fermentation performance (high ethanol yield, reduced glycerol and foam formation, maintenance of high viability during recycling and very high implantation capability into industrial fermenters). Results also suggest that the great yeast biodiversity found in distillery environments could be an important source of strains. This is because during yeast cell recycling, selective pressure (an adaptive evolution) is imposed on cells, leading to strains with higher tolerance to the stressful conditions of the industrial fermentation.     

A comparison of clinical and food Saccharomyces cerevisiae isolates on the basis of potential virulence factors

Saccharomyces cerevisiae is the most widely used yeast in industrial/commercial food and beverage production and is even consumed as a nutritional supplement. Various cases of fungemia caused by this yeast species in severely debilitated traumatized or immune-deficient patients have been reported in recent years, suggesting that this species could be an opportunistic pathogen in such patients. To determine whether the industrial S. cerevisiae strains can be included in this virulent group of strains, we carried out a comparative study between clinical and industrial yeasts based on the various phenotypic traits associated with pathogenicity in two other yeast species (Candida albicans and Cryptococcus neoformans). The majority of the clinical isolates were found to secrete higher levels of protease and phospholipase, grow better at 42°C and show strong pseudohyphal growth relative to industrial yeasts. However three industrial yeast strains, one commercial wine strain, baker’s yeast and one commercial strain of S. cerevisiae (var. boulardii), were exceptions and based on their physiological traits these yeasts would appear to be related to clinical strains.     

Genotypic and phenotypic characterization of industrial autochthonous Saccharomyces cerevisiae for the selection of well-adapted bioethanol-producing strains

In northwestern Argentina, sugarcane-derived industrial fermentation is being extensively used for bioethanol production, where highly adaptive native strains compete with the baker's yeast Saccharomyces cerevisiae traditionally used as starter culture. Yeast populations of 10 distilleries from Tucumán (Argentina) were genotypic and phenotypic characterized to select well-adapted bioethanol-producing autochthonous strains to be used as starter cultures for the industrial production of bioethanol fuel. From the 192 isolates, 69.8% were identified as S. cerevisiae, 25.5% as non-Saccharomyces, and 4.7% as Saccharomyces sp. wild yeasts. The majority of S. cerevisiae isolates (68.5%) were non-flocculating yeasts, while the flocculating strains were all obtained from the only continuous fermentation process included in the study. Simple Sequence Repeat analysis revealed a high genetic diversity among S. cerevisiae genotypes, where all of them were very different from the original baker's strain used as starter. Among these, 38 strains multi-tolerant to stress by ethanol (8%), temperature (42.5 °C) and pH (2.0) were obtained. No major differences were found among these strains in terms of ethanol production and residual sugars in batch fermentation experiments with cell recycling. However, only 10 autochthonous strains maintained their viability (more than 80%) throughout five consecutive cycles of sugarcane-based fermentations. In summary, 10 autochthonous isolates were found to be superior to baker's yeast used as starter culture (S. cerevisiae Calsa) in terms of optimal technological, physiological and ecological properties. The knowledge generated on the indigenous yeast populations in industrial fermentation processes of bioethanol-producing distilleries allowed the selection of well-adapted bioethanol-producing strains.     

Co-utilization of L-arabinose and D-xylose by laboratory and industrial Saccharomyces cerevisiae strains

Background  

Fermentation of lignocellulosic biomass is an attractive alternative for the production of bioethanol. Traditionally, the yeast Saccharomyces cerevisiae is used in industrial ethanol fermentations. However, S. cerevisiae is naturally not able to ferment the pentose sugars D-xylose and L-arabinose, which are present in high amounts in lignocellulosic raw materials.     

Identification of pathogenic yeast species by polymerase chain reaction amplification of the RPS0 gene intron fragment

Aims: This work focuses on the development of a method for the identification of pathogenic yeast. With this aim, we target the nucleotide sequence of the RPS0 gene of pathogenic yeast species with specific PCR primers. PCR analysis was performed with both the genomic DNA, whole cells of clinical isolates of Candida species and clinical samples. Methods and Results: A single pairs of primers, deduced from the nucleotide sequence of the RPS0 gene from pathogenic yeast, were used in PCR analysis performed with both the genomic DNA and whole cells of clinical isolates of Candida species and clinical samples. The primers designed are highly specific for their respective species and produce amplicons of the expected sizes and fail to amplify any DNA fragment from the other species tested. The set of primers was tested successfully for the identification of yeast from colonies, blood cultures and clinical samples. These results indicate that genes containing intron sequences may be useful for designing species‐specific primers for the identification of fungal strains by PCR. The sensitivity of the method with genomic DNA was evaluated with decreasing DNA concentrations (200 ng to 1 pg) and different cell amounts (10⁷–10⁵ cells). Conclusion: The results obtained show that the amplification of RPS0 sequences may be suitable for the identification of pathogenic and other yeast species. Significance and Impact of the Study: Identification of Candida species using molecular approaches with high discriminatory power is important in determining adequate measures for the interruption of transmission of this yeast. The approach described in this work is based on standard technology, and it is specific, sensitive and does not involve complex and expensive equipment. Furthermore, the method developed in this work not only can be used in eight yeast species, but also provides the basis to design primers for other fungi species of clinical, industrial or environmental interest.     

Enzymatic saccharification of sugar cane bagasse by continuous xylanase and cellulase production from cellulomonas flavigena PR‐22

Cellulase (CMCase) and xylanase enzyme production and saccharification of sugar cane bagasse were coupled into two stages and named enzyme production and sugar cane bagasse saccharification. The performance of Cellulomonas flavigena (Cf) PR‐22 cultured in a bubble column reactor (BCR) was compared to that in a stirred tank reactor (STR). Cells cultured in the BCR presented higher yields and productivity of both CMCase and xylanase activities than those grown in the STR configuration. A continuous culture with Cf PR‐22 was run in the BCR using 1% alkali‐pretreated sugar cane bagasse and mineral media, at dilution rates ranging from 0.04 to 0.22 1/h. The highest enzymatic productivity values were found at 0.08 1/h with 1846.4 ± 126.4 and 101.6 ± 5.6 U/L·h for xylanase and CMCase, respectively. Effluent from the BCR in steady state was transferred to an enzymatic reactor operated in fed‐batch mode with an initial load of 75 g of pretreated sugar cane bagasse; saccharification was then performed in an STR at 55°C and 300 rpm for 90 h. The constant addition of fresh enzyme as well as the increase in time of contact with the substrate increased the total soluble sugar concentration 83% compared to the value obtained in a batch enzymatic reactor. This advantageous strategy may be used for industrial enzyme pretreatment and saccharification of lignocellulosic wastes to be used in bioethanol and chemicals production from lignocellulose. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:321–326, 2016     

Response of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> to Cadmium and Nickel Stress: The Use of the Sugar Cane Vinasse as a Potential Mitigator

Most of the metals released from industrial activity, among them are cadmium (Cd) and nickel (Ni), inhibit the productivity of cultures and affect microbial metabolism. In this context, the aim of this work was to investigate the capacity of sugar cane vinasse to mitigate the adverse effects of Cd and Ni on cell growth, viability, budding rate and trehalose content of Saccharomyces cerevisiae, likely because of adsorption and chelating action. For this purpose, the yeast was grown batch-wise in YED medium supplemented with selected amounts of vinasse and Cd or Ni. The negative effects of Cd and Ni on S. cerevisiae growth and the mitigating one of sugar cane vinasse were quantified by an exponential model. Without vinasse, the addition of increasing levels of Cd and Ni reduced the specific growth rate, whereas in its presence no reduction was observed. Consistently with the well-proved toxicity of both metals, cell viability and budding rate progressively decreased with increasing their concentration, but in the presence of vinasse the situation was remarkably improved. The trehalose content of S. cerevisiae cells followed the same qualitative behavior as cell viability, even though the negative effect of both metals on this parameter was stronger. These results demonstrate the ability of sugar cane vinasse to mitigate the toxic effects of Cd and Ni.     

The consequences of Lactobacillus vini and Dekkera bruxellensis as contaminants of the sugarcane-based ethanol fermentation

This work describes the effects of the presence of the yeast Dekkera bruxellensis and the bacterium Lactobacillus vini on the industrial production of ethanol from sugarcane fermentation. Both contaminants were quantified in industrial samples, and their presence was correlated to a decrease in ethanol concentration and accumulation of sugar. Then, laboratory mixed-cell fermentations were carried out to evaluate the effects of these presumed contaminants on the viability of Saccharomyces cerevisiae and the overall ethanol yield. The results showed that high residual sugar seemed the most significant factor arising from the presence of D. bruxellensis in the industrial process when compared to pure S. cerevisiae cultures. Moreover, when L. vini was added to S. cerevisiae cultures it did not appear to affect the yeast cells by any kind of antagonistic effect under stable fermentations. In addition, when L. vini was added to D. bruxellensis cultures, it showed signs of being able to stimulate the fermentative activity of the yeast cells in a way that led to an increase in the ethanol yield.