Identification of Dekkera bruxellensis as a major contaminant yeast in continuous fuel ethanol fermentation

AIMS: To identify and characterize the main contaminant yeast species detected in fuel-ethanol production plants in Northeast region of Brazil by using molecular methods. METHODS AND RESULTS: Total DNA from yeast colonies isolated from the fermentation must of industrial alcohol plants was submitted to PCR fingerprinting, D1/D2 28S rDNA sequencing and species-specific PCR analysis. The most frequent non-Saccharomyces cerevisiae isolates were identified as belonging to the species Dekkera bruxellensis, and several genetic strains could be discriminated among the isolates. The yeast population dynamics was followed on a daily basis during a whole crop harvesting period in a particular industry, showing the potential of D. bruxellensis to grow faster than S. cerevisiae in industrial conditions, causing recurrent and severe contamination episodes. CONCLUSIONS: The results showed that D. bruxellensis is one of the most important contaminant yeasts in distilleries producing fuel-ethanol from crude sugar cane juice, specially in continuous fermentation systems. SIGNIFICANCE AND IMPACT OF THE STUDY: Severe contamination of the industrial fermentation process by Dekkera yeasts has a negative impact on ethanol yield and productivity. Therefore, early detection of D. bruxellensis in industrial musts may avoid operational problems in alcohol-producing plants.     

Polyhexamethyl biguanide can eliminate contaminant yeasts from fuel-ethanol fermentation process

Industrial ethanol fermentation is a non-sterile process and contaminant microorganisms can lead to a decrease in industrial productivity and significant economic loss. Nowadays, some distilleries in Northeastern Brazil deal with bacterial contamination by decreasing must pH and adding bactericides. Alternatively, contamination can be challenged by adding a pure batch of Saccharomyces cerevisiae-a time-consuming and costly process. A better strategy might involve the development of a fungicide that kills contaminant yeasts while preserving S. cerevisiae cells. Here, we show that polyhexamethyl biguanide (PHMB) inhibits and kills the most important contaminant yeasts detected in the distilleries of Northeastern Brazil without affecting the cell viability and fermentation capacity of S. cerevisiae. Moreover, some physiological data suggest that PHMB acts through interaction with the yeast membrane. These results support the development of a new strategy for controlling contaminant yeast population whilst keeping industrial yields high.     

Rapid detection of Obesumbacterium proteus from yeast and wort using polymerase chain reaction

AIMS: To design and evaluate PCR primers for the rapid detection of Obesumbacterium proteus. METHODS AND RESULTS: The 16S rDNA from a wild-type Obesumbacterium proteus biotype II isolate was sequenced and the resultant data used to produce specific primers for this organism. These primers discriminated between biotype I (nonbrewery) and biotype II isolates. In addition, the primers were able to detect Obesumbacterium proteus in wort and in yeast slurries in the presence of competitive bacteria. The primers were tested against a range of other beer spoilage bacteria for any cross-reactions. None were detected. CONCLUSIONS:Obesumbacterium proteus primers can detect this contaminant without generating cross-reactions to related species. SIGNIFICANCE AND IMPACT OF THE STUDY: The primers generated in this study can now be used for PCR detection assays that will contribute to early detection of this important process contaminant.     

New PCR-based methods for yeast identification

AIMS: To characterize reference yeast strains and identify indigenous strains isolated from wine fermentations by PCR methods. METHODS AND RESULTS: We compared several PCR techniques for yeast identification. We used oligonucleotide primers that are complementary to (i) intron splice sites, (ii) REP and (iii) ERIC elements to produce PCR fingerprints that display specific patterns between the different yeast species. These three techniques were used to characterize 41 reference yeast strains belonging to 15 different species and to identify 40 indigenous strains isolated from grape must and wine fermentations. Species-specific banding patterns were obtained with the three PCR-techniques with different degrees of intraspecific differentiation depending on the method. By comparing the PCR fingerprints of unknown isolates with those produced by reference strains, we identified yeast strains isolated from an industrial wine fermentation. CONCLUSIONS: All three PCR techniques are rapid, reliable and simple methods of yeast identification. As far as we know, this is the first time that the primers designed for amplifying repetitive elements in bacteria have been successfully used in yeast. SIGNIFICANCE AND IMPACT OF THE STUDY: Industry needs rapid, reliable and simple methods of yeast identification. The proposed PCR techniques will allow to achieve this objective.     

Monitoring of Saccharomyces and Hanseniaspora populations during alcoholic fermentation by real-time quantitative PCR

Real-time, or quantitative, PCR (QPCR) was developed for the rapid quantification of two of the most important yeast groups in alcoholic fermentation (Saccharomyces spp. and Hanseniaspora spp.). Specific primers were designed from the region spanning the internal transcribed spacer 2 (ITS2) and the 5.8S rRNA gene. To confirm the specificity of these primers, they were tested with different yeast species, acetic acid bacteria and lactic acid bacteria. The designed primers only amplified for the intended group of species and none of the PCR assays was positive for any other wine microorganisms. This technique was performed on reference yeast strains from pure cultures and validated with both artificially contaminated wines and real wine fermentation samples. To determine the effectiveness of the technique, the QPCR results were compared with those obtained by plating. The design of new primers for other important wine yeast species will enable to monitor yeast diversity during industrial wine fermentation and to detect the main spoilage yeasts in wine.     

Ecological survey of Saccharomyces cerevisiae strains from vineyards in the Vinho Verde Region of Portugal

One thousand six hundred and twenty yeast isolates were obtained from 54 spontaneous fermentations performed from grapes collected in 18 sampling sites of three vineyards (Vinho Verde Wine Region in northwest Portugal) during the 2001-2003 harvest seasons. All isolates were analyzed by mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) and a pattern profile was verified for each isolate, resulting in a total of 297 different profiles, that all belonged to the species Saccharomyces cerevisiae. The strains corresponding to seventeen profiles showed a wider temporal and geographical distribution, being characterized by a generalized pattern of sporadic presence, absence and reappearance. One strain (ACP10) showed a more regional distribution with a perennial behavior. In different fermentations ACP10 was either dominant or not, showing that the final outcome of fermentation was dependent on the specific composition of the yeast community in the must. Few of the grape samples collected before harvest initiated a spontaneous fermentation, compared to the samples collected after harvest, in a time frame of about 2 weeks. The associated strains were also much more diversified: 267 patterns among 1260 isolates compared to 30 patterns among 360 isolates in the post- and pre-harvest samples, respectively. Fermenting yeast populations have never been characterized before in this region and the present work reports the presence of commercial yeast strains used by the wineries. The present study aims at the development of strategies for the preservation of biodiversity and genetic resources as a basis for further strain development.     

Detection and Identification of Wild Yeast Contaminants of the Industrial Fuel Ethanol Fermentation Process

Monitoring for wild yeast contaminants is an essential component of the management of the industrial fuel ethanol manufacturing process. Here we describe the isolation and molecular identification of 24 yeast species present in bioethanol distilleries in northeast Brazil that use sugar cane juice or cane molasses as feeding substrate. Most of the yeast species could be identified readily from their unique amplification-specific polymerase chain reaction (PCR) fingerprint. Yeast of the species Dekkera bruxellensis, Candida tropicalis, Pichia galeiformis, as well as a species of Candida that belongs to the C. intermedia clade, were found to be involved in acute contamination episodes; the remaining 20 species were classified as adventitious. Additional physiologic data confirmed that the presence of these major contaminants cause decreased bioethanol yield. We conclude that PCR fingerprinting can be used in an industrial setting to monitor yeast population dynamics to early identify the presence of the most important contaminant yeasts.     

Molecular survey of aeroplane bacterial contamination

AIMS: To examine bacterial contamination of passenger aircraft and to identify aeroplane environments posing the greatest potential health risk. METHODS AND RESULTS: DNA was extracted from ten environmental samples collected on four different flights (three domestic, one international) from a variety of surfaces frequently touched by passengers. PCR clone libraries were made from the DNA samples using bacterial-specific 16S ribosomal DNA (rDNA) primers. A total of 271 bacterial rDNA sequences were obtained. We used BLAST analysis of the rDNA clone sequences to identify sequences in Genbank with the highest sequence similarity. The majority of the rDNA clones obtained from aeroplane environments were more than 97% identical to rDNA sequences from cultured bacterial species. Samples collected from the cabin surfaces (e.g., tray tables and arm rests) had undetectable levels of DNA and produced no PCR products. Bacterial diversity was highest on lavatory surfaces, including door handles, toilet handles, and sink faucets. Sequence data from these surfaces detected species from 58 different bacterial genera, and many of the best BLAST hits matched rDNA sequences of cultured species known to be opportunistic pathogens. The most frequently observed species came from five genera commonly associated with humans: Streptococcus, Staphylococcus, Cornybacterium, Proprionibacterium and Kocuria. CONCLUSIONS: The results show that there is a large diversity of bacterial contamination on aeroplanes, including organisms known to be opportunistic pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results indicate that aeroplanes have the potential to spread an enormous diversity of bacterial species among passengers and destinations. Aeroplane lavatories present an especially significant concern to public health.     

绍兴黄酒酒药中酵母菌的物种资源

酒药是绍兴黄酒酿造中使用的重要糖化发酵剂。2013年绍兴黄酒冬酿期间,从绍兴地区5个主要黄酒生产企业,采集古越龙山、会稽山、塔牌、沈永和、女儿红及鉴湖等6个品牌酒药样本,在数量、种类和分布规律等方面对黄酒酒药内酵母菌物种资源进行了系统研究。研究发现,会稽山酒药和沈永和酒药蕴含酵母菌数量较多而且与其他样本相比差异显著（P<0.01）,而塔牌酒药中酵母菌数量显著少于其他5个品牌（P<0.05）。遵循酵母菌分类原则,从绍兴黄酒酒药中分离鉴定出3种酵母菌,扣囊复膜孢酵母Saccharomycopsis fibuligera、酿酒酵母Saccharomyces cerevisiae和隐球酵母Cryptococcus sp.。其中,扣囊复膜孢酵母Saccharomycopsis fibuligera在6个品牌黄酒酒药内的分布占有绝对优势,该种酵母菌可能对绍兴黄酒酒药的生产性能来说至关重要。比较分析酒药中酵母菌物种资源,可以为阐明绍兴黄酒的发酵机制提供基础数据和理论基础。     

PCR-fingerprinting and RAPD approaches for tracing the source of yeast contamination in a carbonated orange juice production chain

AIMS: To investigate the sort and the origin of the contamination of a packed fruit juice. METHODS AND RESULTS: Fifty-eight yeast isolates were collected in a survey of two different visits to a carbonated orange juice factory. In each visit, samples were collected, six times, from seven points in the production chain. For each visit, no significant differences were observed among the yeast average values obtained in the control points considered. The random amplified polymorphic DNA (RAPD) with primer P24 and the PCR-fingerprinting with the microsatellites primers (GTG)5 and (GAC)5 were used, in order to discriminate the isolates, rendering 29 composite profiles; the most frequent one (24/58) was profile c, which included the yeast isolates from the final product and strains isolated before and after the pasteurization of the juice. These contaminant strains were identified as Pichia galeiformis by sequence analysis of D1/D2 26S rRNA gene. CONCLUSIONS: The results obtained point to an inefficient pasteurization of the juice related to the fouling of the heat-transfer surfaces of the plate-type exchanger. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of PCR-fingerprinting and RAPD assays showed to be very useful in tracking the route of contamination in a carbonated juice production chain.     

Molecular assessment of indigenous yeast population from traditional balsamic vinegar

AIMS: To identify and describe the indigenous yeast population involved in traditional balsamic vinegar (TBV) fermentation. METHODS AND RESULTS: Using the restriction analysis of the ribosomal region 5.8S (5.8S rRNA) and the internal transcribed spacers 1 and 2 (5.8S-ITS region) we were able to group 133 strains isolated from 17 cooked grape must samples into 10 different yeast species, included into 4 genera. Moreover, we sequenced the D1/D2 domains of the 26S rRNA and confirmed the reliability of each identification at species level. Most strains belonged to the genus Zygosaccharomyces. In particular, Zygosaccharomyces bailii was found in 41% of the samples, followed by Saccharomyces cerevisiae, Zygosaccharomyces pseudorouxii and Candida stellata. Strains belonging respectively to Zygosaccharomyces mellis, Zygosaccharomyces bisporus, Zygosaccharomyces rouxii, Hanseniaspora valbyensis, Hanseniaspora osmophila and Candida lactis-condensi species were also detected. Despite the great number of species recovered, the mtDNA restriction profiles showed low variability at strain level. Saccharomyces cerevisiae isolates with an higher degree of intraspecific variance were considered an exception. CONCLUSIONS: Many different indigenous yeast species were recovered and TBV yeasts population seems to be far more complex than what was reported in previous literature. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has allowed us to gain a better understanding of the indigenous yeast species of TBV cooked must.     

Design and evaluation of PCR primers for analysis of bacterial populations in wine by denaturing gradient gel electrophoresis

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal DNA (rDNA) is routinely used to compare levels of diversity of microbial communities and to monitor population dynamics. While using PCR-DGGE to examine the bacteria in wine fermentations, we noted that several commonly used PCR primers for amplifying bacterial 16S rDNA also coamplified yeast, fungal, or plant DNA present in samples. Unfortunately, amplification of nonbacterial DNA can result in a masking of bacterial populations in DGGE profiles. To surmount this problem, we developed two new primer sets for specific amplification of bacterial 16S rDNA in wine fermentation samples without amplification of eukaryotic DNA. One primer set, termed WLAB1 and WLAB2, amplified lactic acid bacteria, while another, termed WBAC1 and WBAC2, amplified both lactic acid bacterial and acetic acid bacterial populations found in wine. Primer specificity and efficacy were examined with DNA isolated from numerous bacterial, yeast, and fungal species commonly found in wine and must samples. Importantly, both primer sets effectively distinguished bacterial species in wine containing mixtures of yeast and bacteria.     

Design and Evaluation of PCR Primers for Analysis of Bacterial Populations in Wine by Denaturing Gradient Gel Electrophoresis

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal DNA (rDNA) is routinely used to compare levels of diversity of microbial communities and to monitor population dynamics. While using PCR-DGGE to examine the bacteria in wine fermentations, we noted that several commonly used PCR primers for amplifying bacterial 16S rDNA also coamplified yeast, fungal, or plant DNA present in samples. Unfortunately, amplification of nonbacterial DNA can result in a masking of bacterial populations in DGGE profiles. To surmount this problem, we developed two new primer sets for specific amplification of bacterial 16S rDNA in wine fermentation samples without amplification of eukaryotic DNA. One primer set, termed WLAB1 and WLAB2, amplified lactic acid bacteria, while another, termed WBAC1 and WBAC2, amplified both lactic acid bacterial and acetic acid bacterial populations found in wine. Primer specificity and efficacy were examined with DNA isolated from numerous bacterial, yeast, and fungal species commonly found in wine and must samples. Importantly, both primer sets effectively distinguished bacterial species in wine containing mixtures of yeast and bacteria.     

Quantification by real-time PCR of cellulolytic bacteria in the rumen of sheep after supplementation of a forage diet with readily fermentable carbohydrates: effect of a yeast additive

AIM: To examine the effect of concentrate and yeast additive on the number of cellulolytic bacteria in the rumen of sheep. METHODS AND RESULTS: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens were quantified using real-time PCR (targeting 16S rDNA) in parallel to cellulolytic flora enumeration with cultural techniques. Whatever the conditions tested, R. flavefaciens was slightly more abundant than F. succinogenes, with both species outnumbering R. albus. Before feeding, the shift from hay to hay plus concentrate diet had no effect on rumen pH and on the number of the three specie; while after feeding, the concentrate-supplemented diet induced a decrease (-1 log) of the number of the three species concomitant with the rumen acidification. Overall, the presence of the live yeast resulted in a significant increase (two- to fourfold) of the Ruminococci. CONCLUSION: The use of real-time PCR allowed us to show changes in the number of cellulolytic bacterial species in vivo in response to diet shift and additives that could not be as easily evidenced by classical microbial methods. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the understanding of the negative impact of readily fermentable carbohydrates on rumen cellulolysis and the beneficial effect of yeast on rumen fermentation.     

Scaling-up in industrial winemaking using low electric current as an alternative to sulfur dioxide addition

AIMS: To better understand the outcome of employing low electric current (LEC) technology as a new preservation and alternative in wine technology, and to contribute to its development. It is used in industrial-scale winemaking with commercial yeast (Saccharomyces cerevisiae) during the grape must fermentation. METHODS AND RESULTS: LEC (200 mA, time 16 days) was applied to fresh grape must as an alternative method to the usual sulfur dioxide addition used in the industrial process; two tanks, each 30,000 l, were employed for parallel fermentations. The results show that LEC decreased the survival time and increased the death rate of apiculate yeasts, whereas it did not affect the growth and survival of S. cerevisiae. A comparison was made of the main chemical and sensory parameters of the wines obtained. CONCLUSIONS: The results have demonstrated that the low-voltage treatment had a positive effect on the grape juice fermentation (yeast microflora) during the early stages of winemaking. SIGINIFICANCE AND IMPACT OF THE STUDY: These results could be of significant importance in developing, for 'biological wine', new winemaking technologies for an innovative control process of yeast fermentation.     

Saccharomyces cerevisiae wine yeast populations in a cold region in Argentinean Patagonia. A study at different fermentation scales

AIMS: To study the diversity and dynamics of indigenous Saccharomyces wine populations during Malbec spontaneous fermentation, a representative Patagonian red wine, at both industrial and laboratory scale. METHODS AND RESULTS: Two molecular techniques, including restriction fragment length polymorphism of mitochondrial (mt) DNA and polymorphism of amplified delta interspersed element sequences, were used for characterization of indigenous yeasts at strain level. The mtDNA restriction patterns showed the major discriminative power; however, by combining the two molecular approaches it was possible to distinguish a larger number of strains and, therefore, draw more representative conclusions about yeast diversity. Although a great diversity of wild Saccharomyces cerevisiae strains was observed, only nine represented more than half of the total Saccharomyces yeast biota analysed; five of these were common and took over the Malbec must fermentation in both vinifications. CONCLUSIONS: Many different indigenous S. cerevisiae strains were identified; nevertheless, the dominant strains in both industrial and laboratory vinification processes were just a few and the same. SIGNIFICANCE AND IMPACT OF THE STUDY: Small-scale fermentation appears to be a valuable tool in winemaking, one especially helpful in evaluating microbiological aspects of as well as possible interactions between inoculated selected strains and native strains.     

Proteomic analysis of a distilling strain of Saccharomyces cerevisiae during industrial grain fermentation

The fermentation performance of industrial yeast strains is influenced, among other things, by their genetic composition and the nature of the fermentable sugar, availability of nitrogen, and temperature. Therefore, to manipulate the fermentation process, it is important to understand, at a molecular level, the changes occurring in the yeast cell throughout industrial fermentation processes. With this aim in mind, using two-dimensional gel electrophoresis and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS), we have examined the proteome of distillers yeast in an industrial context. Using yeast sampled from a local grain whisky distillery, we have prepared a detailed reference map of the proteome of distillers yeast and have examined in some detail the alterations in protein levels that occur throughout fermentation. In particular, as fermentation progresses, there is a significant increase in the levels of a variety of proteins involved in protecting against stress and nitrogen limitation. These results therefore give an insight into the stresses that yeast are exposed to in industrial fermentations and reveal some of the proteins and enzymes that are either necessary or important for efficient fermentation.     

Correlation between DNA of trichothecene-producing Fusarium species and deoxynivalenol concentrations in wheat-samples

AIMS: Correlations between DNA content of trichothecene-producing Fusarium spp. and concentration of the key mycotoxin deoxynivalenol (DON) in cereal samples. METHODS AND RESULTS: A LightCycler PCR-based assay was used to quantify the DNA from trichothecene-producing Fusarium spp. in 300 wheat samples. DNA concentrations ranged from not detectable to 16.3 mg kg-1 whereas DON concentrations (GC/MS data) varied from not detectable to 34.3 mg kg-1. Data analysis revealed a coefficient of correlation r=0.9557 between DON concentrations and DNA-amounts over all samples. An interval of confidence for P=95% was calculated based on samples with DON concentrations < or = 1.5 mg kg-1 (n=234). CONCLUSIONS: Quantification of 32 samples of Fusarium-contaminated wheat was performed within 45 min. Data analysis allowed estimation of DON contamination from quantitative PCR data in the wheat samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The method described is useful for the screening of cereals in industrial quality control.     

Microbial interactions during sugar cane must fermentation for bioethanol production: does quorum sensing play a role?

Microbial interactions represent important modulatory role in the dynamics of biological processes. During bioethanol production from sugar cane must, the presence of lactic acid bacteria (LAB) and wild yeasts is inevitable as they originate from the raw material and industrial environment. Increasing the concentration of ethanol, organic acids, and other extracellular metabolites in the fermentation must are revealed as wise strategies for survival by certain microorganisms. Despite this, the co-existence of LAB and yeasts in the fermentation vat and production of compounds such as organic acids and other extracellular metabolites result in reduction in the final yield of the bioethanol production process. In addition to the competition for nutrients, reduction of cellular viability of yeast strain responsible for fermentation, flocculation, biofilm formation, and changes in cell morphology are listed as important factors for reductions in productivity. Although these consequences are scientifically well established, there is still a gap about the physiological and molecular mechanisms governing these interactions. This review aims to discuss the potential occurrence of quorum sensing mechanisms between bacteria (mainly LAB) and yeasts and to highlight how the understanding of such mechanisms can result in very relevant and useful tools to benefit the biofuels industry and other sectors of biotechnology in which bacteria and yeast may co-exist in fermentation processes.