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.     

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.     

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.     

Analysis of yeasts derived from natural fermentation in a Tokaj winery

The diversity of yeast flora was investigated in a spontaneously fermenting sweet white wine in a Tokaj winery. The non-Saccharomyces yeasts dominating the first phase of fermentation were soon replaced by a heterogeneous Saccharomycespopulation, which then became dominated by Saccharomyces bayanus. Three Saccharomyces sensu stricto strains isolated from various phases of fermentation were tested for genetic stability, optimum growth temperature, tolerance to sulphur dioxide, copper and ethanol as well as for the ability to produce hydrogen sulphide and various secondary metabolites known to affect the organoleptic properties of wines. The analysis of the single-spore cultures derived from spores of dissected asci revealed high stability of electrophoretic karyotypes and various degrees of heterozygosity for mating-types, the fermentation of galactose and the production of metabolic by-products. The production levels of the by-products did not segregate in a 2:2 fashion, suggesting that the synthesis of these compounds is under polygenic control.     

Isolation of thermotolerant,fermentative yeasts growing at 52°C and producing ethanol at 45°C and 50°C

Five thermotolerant, alcohol-producing yeast cultures were isolated from samples obtained from India. Two were identified as ofKluyveromyces marxianus. All five grew on plate-cultures up to 52°C, with maximum growth rates in liquid culture at 40°C. All produced relatively high alcohol concentrations: 5.7 to 7.0% (w/v) at 45°C and 5.0 to 5.5% (w/v) at 50°C when growing on 14.0% (w/v) glucose. All five isolates fermented diluted molasses containing 16.0% (w/v) total sugars, producing 5.6 to 6.0% (w/v) alcohol concentrations. Supplementing the molasses with P, K, Mg and Mn resulted in a 13 to 20% increase in alcohol production at 40°C. The maximum amounts of alcohol produced on supplemented molasses were 7.5 to 8.0 and 6.5 to 7.0% (w/v) at 37°C and 40°C, respectively.     

Selective antimicrobial action of chitosan against spoilage yeasts in mixed culture fermentations

The effect of chitosan on Saccharomyces cerevisiae (the yeast that carries out alcohol fermentation), Brettanomyces bruxellensis and Brettanomyces intermedius (contaminants of alcohol fermentations), was investigated. The effect of chitosan was tested on each yeast, as well as on mixed cultivations of S. cerevisiae + B. bruxellensis and S. cerevisiae + B. intermedius. Chitosan enhanced the lag period of both strains of Brettanomyces (80 h for B. bruxellensis and 170 h for B. intermedius with 6 and 2 g/l chitosan, respectively). The growth rate of S. cerevisiae was inversely proportional to the chitosan concentration; the former was 50% when 6 g/l polysaccharide was used. Moreover, in mixed cultivations of S. cerevisiae and Brettanomyces strains, it was found that both B. bruxellensis and B. intermedius failed to grow while growth of S. cerevisiae was not affected (using 3 and 6 g/l chitosan, respectively). An interesting collateral result was that the presence of chitosan accelerated the consumption of glucose in the mixed cultivations (60 h instead of 120 h).     

刺梨自然发酵过程中非酿酒酵母多样性分析

【目的】分析刺梨果实自然发酵过程中非酿酒酵母菌群特征,为筛选优质刺梨非酿酒酵母提供参考。【方法】基于Illumina MiSeq高通量测序技术和WL营养琼脂鉴定培养基纯种分离技术,分析刺梨果实自然发酵1 d(F1)、3 d(F3)、5 d(F5)和15 d(F15)4个阶段及YPD培养基富集培养样本中非酿酒酵母种群组成和多样性。【结果】高通量测序分析结果共获得182个OTUs(operational taxonomic units,OTUs),归属于81个属107个种;物种多样性分析结果表明,刺梨果实自然发酵前期,优势非酿酒酵母为汉逊酵母(Hanseniasporasp.)和伯顿丝孢毕赤酵母(Hyphopichiaburtonii),二者在样本F1中分别占42.59%和26.85%;随着自然发酵的不断进行,二者的比例逐渐降低,在第15天(F15),Hanseniaspora sp.和H.burtonii比例降低至7.73%和0.52%。相反,Pichia sporocuriosa和未培养的酵母,随着自然发酵不断进行所占比例逐渐增大,分别由F1中的0.23%和0.33%增至F15中的37.26%和32.62%。此外,采用WL营养琼脂鉴定培养基纯种分离和鉴定技术,从刺梨上分离到Hanseniasporasp.、H.burtonii、克鲁维毕赤酵母(Pichia kluyveri)、P.sporocuriosa和异常威克汉姆酵母(Wickerhamomyces anomalus)5种类型的可培养非酿酒酵母。【结论】刺梨果实上存在着丰富的非酿酒酵母菌资源,研究刺梨自然发酵过程中非酿酒酵母多样性,为酵母资源开发和利用奠定基础。     

Preparation of hydrolysates from tobacco stalks and ethanolic fermentation by Saccharomyces cerevisiae

Chipped tobacco stalks were subjected to steam pretreatment at 205 °C for either 5 or 10 min before enzymatic hydrolysis. Glucose (15.4–17.1 g/l) and xylose (4.5–5.0 g/l) were the most abundant monosaccharides in the hydrolysates. Mannose, galactose and arabinose were also detected. The hydrolysate produced by pretreatment for 10 min contained higher levels of all sugars than the 5 min-pretreated hydrolysate. The amounts of inhibitory compounds found in the hydrolysates were relatively low and increased with increasing pretreatment time. The hydrolysates were fermented with baker's yeast. Ethanol yield, maximum volumetric productivity and specific productivity were used as criteria of fermentability of the hydrolysates. The fermentation of the hydrolysates was only slightly inhibited compared to reference solutions having a similar composition of fermentable sugars. The ethanol yield in the hydrolysates was 0.38–0.39 g/g of initial fermentable sugars, whereas it was 0.42 g/g in the reference. The biomass yield was twofold lower in the hydrolysates than in the reference. The fermentation inhibition caused by the tobacco stalk hydrolysates was less than that caused by sugarcane bagasse hydrolysates obtained under the same hydrolysis conditions.     

Yeast communities in a natural tequila fermentation

Fresh and cooked agave,Drosophila spp., processing equipment, agave molasses, agave extract, and fermenting must at a traditional tequila distillery (Herradura, Amatitan, Jalisco, México) were studied to gain insight on the origin of yeasts involved in a natural tequila fermentations. Five yeast communities were identified. (1) Fresh agave contained a diverse mycobiota dominated byClavispora lusitaniae and an endemic species,Metschnikowia agaveae. (2)Drosophila spp. from around or inside the distillery yielded typical fruit yeasts, in particularHanseniaspora spp.,Pichia kluyveri, andCandida krusei. (3)Schizosaccharomyces pombe prevailed in molasses. (4) Cooked agave and extract had a considerable diversity of species, but includedSaccharomyces cerevisiae. (5) Fermenting juice underwent a gradual reduction in yeast heterogeneity.Torulaspora delbrueckii, Kluyveromyces marxianus, andHanseniaspora spp. progressively ceded the way toS. cerevisiae, Zygosaccharomyces bailii, Candida milleri, andBrettanomyces spp. With the exception ofPichia membranaefaciens, which was shared by all communities, little overlap existed. That separation was even more manifest when species were divided into distinguishable biotypes based on morphology or physiology. It is concluded that crushing equipment and must holding tanks are the main source of significant inoculum for the fermentation process.Drosophila species appear to serve as internal vectors. Proximity to fruit trees probably contributes to maintaining a substantialDrosophila community, but the yeasts found in the distillery exhibit very little similarity to those found in adjacent vegetation. Interactions involving killer toxins had no apparent direct effects on the yeast community structure.     

Improvement on laboratory fermentation equipment to study Saccharomyces cerevisiae metabolism

Beside being an ordinary fermenter, the present equipment was conceived to sample the medium, to store the samples and to record photographs of the yeasts. Ten sensors were used to measure gas exchanges. During the growth of ScM1 (a Saccharomyces cerevisiae strain) on glucose, we could observe two different linear decreases of CO₂ production rates (18.17±0.12 mmol CO₂ h^–2 (g biomass)^–1 and 8.67±0.12 mmol CO₂ h^–2 (g biomass)^–1), together with a sudden variation of slope during the respiro-fermentative phase. Nomenclature F_in InletairFlowl h ^–1 F_out OutletgasFlowl h ^–1 _in Inletairtemperature°C_out Outletgastemperature°CP _atm AtmosphericPressuremmHgP _in InletairOverPressuremmHgP _out OutletgasOverPressuremmHgDODissolvedO ₂ mg l^–1 pO₂ PartialPressureO ₂ in Outlet gas % (v/v) pCO₂ PartialPressureCO ₂ in Outlet gas % (v/v) Int(t) Whole number of hours     

Lead biosorption by waste biomass of Corynebacterium glutamicum generated from lysine fermentation process

Biomass waste, mainly Corynebacterium glutamicum, is generated from large-scale lysine fermentation process. In this study, protonated C. glutamicum biomass was evaluated as a biosorbent for the removal of lead from synthetic wastewater. As Pb2+ were bound to the biomass, the solution pH deceased, indicating that protons in the biomass were exchanged with lead ions. The Corynebacterium biomass bound Pb2+ at up to 2.74 mmol g(-1) at pH 5, where lead does not precipitate. Compared with other biosorbents and conventional sorbents, such as natural zeolite, activated carbon and synthetic ion exchange resin, the protonated C. glutamicum biomass was considered to be a useful biomaterial for lead biosorption.     

Growth and fermentation behaviour of Brettanomyces/Dekkera yeasts under different conditions of aerobiosis

Brettanomyces/Dekkera yeasts grow in wine and their presence is often associated with spoiling activity. In this report, we investigated on the influence of different conditions of aerobiosis on growth and fermentation behaviour of these spoilage yeasts in wine. Results showed that in all conditions tested the Brettanomyces strain consumed all sugars, taking wine fermentation to completion. Strict-anaerobic conditions limited the growth of Brettanomyces. Both anaerobiosis (using a fermentation trap) and strict anaerobiosis did not negatively affect the principal by-products of fermentation whereas semi-anaerobiosis caused an increase of acetic acid, acetaldehyde and ethyl acetate that negatively affected the fermentation profile of resulting products.     

Influence of killer strains of Saccharomyces cerevisiae on wine fermentation

The effect of killer strains of Saccharomyces cerevisiae on the growth of sensitive strains during must fermentation was studied by using a new method to monitor yeast populations. The capability of killer yeast strains to eliminate sensitive strains depends on the initial proportion of killer yeasts, the susceptibility of sensitive strains, and the treatment of the must. In sterile filtered must, an initial proportion of 2-6% of killer yeasts was responsible for protracted fermentation and suppression of isogenic sensitive strains. A more variable initial proportion was needed to get the same effect with non-isogenic strains. The suspended solids that remain in the must after cold-settling decreased killer toxin effect. The addition of bentonite to the must avoided protracted fermentation and the suppression of sensitive strains; however, the addition of yeast dietary nutrients with yeast cell walls did not, although it decreased fermentation lag.     

Direct ethanol fermentation from lignocellulosic biomass by Antarctic basidiomycetous yeast Mrakia blollopis under a low temperature condition

Antarctic basidiomycetous yeast Mrakia blollopis SK-4 has unique fermentability for various sugars under a low temperature condition. Hence, this yeast was used for ethanol fermentation from glucose and also for direct ethanol fermentation (DEF) from cellulosic biomass without/with Tween 80 at 10 °C. Maximally, 48.2 g/l ethanol was formed from 12% (w/v) glucose. DEF converted filter paper, Japanese cedar and Eucalyptus to 12.2 g/l, 12.5 g/l and 7.2 g/l ethanol, respectively. In the presence of 1% (v/v) Tween 80, ethanol concentration increased by about 1.1–1.6-fold compared to that without Tween 80. This is the first report on DEF using cryophilic fungi under a low temperature condition. We consider that M. blollopis SK-4 has a good potential for ethanol fermentation in cold environments.     

Formation of biogenic amines as criteria for the selection of wine yeasts

This work deals with biogenic amine production by yeast strains isolated from grapes and wines. A total of 50 strains were tested for their capacity to produce biogenic amines in wine. In general, all the species produced very low or non-detectable amounts of histamine, whereas methylamine and agmatine were formed by all the species considered. The highest concentration of total biogenic amines was formed by Brettanomyces bruxellensis, with an average value of 15 mg/l, followed by Saccharomyces cerevisiae with an average of 12.14 mg/l. The other species formed less than 10 mg of total biogenic amines per litre. Wines fermented with the most fermentative strains of S. cerevisiae species had the highest contents of ethanolamine, from 2.3 to 16 mg/l, and of agmatine, from 3.1 to 7.5 mg/l. The strains of the other species, which exhibited a low fermentative ability, Kloeckera apiculata, B. bruxellensis and Metschnikowia pulcherrima, varied in the production of agmatine and phenylethylamine. A significant variability in the production of cadaverine was characteristic of Candida stellata strains, which varied also in ethanolamine production. Our results emphasize the importance of using selected strains of S. cerevisiae, not only for the expression of desirable technological traits, but also to avoid potentially negative effects on human health. Therefore, the characterization of strains of S. cerevisiae for the 'production of biogenic amines' becomes of applicative interest.     

Evaluation of bacterial contamination in a fed-batch alcoholic fermentation process

Alcoholic fermentation by a commercial baker's yeast in a fed-batch process with cell recycling and high-test molasses as substrate was strongly inhibited by Lactobacillus fermentum CCT 1407 after a few recycles. When total acidity (mainly lactic acid) exceeded 4.8 g/l broth it seriously interfered with yeast bud formation and viability and above 6.0 g/l it decreased alcoholic efficiency.     

Genetic identification of naturalSaccharomyces sensu stricto yeasts from Finland,Holland and Slovakia

Genetic and karyotypic studies of naturalSaccharomyces sensu stricto yeasts from Finland, Holland and Slovakia revealed three wild sibling-species:Saccharomyces cerevisiae, Saccaromyces bayanus andSaccharomyces paradoxus.     

中条山野生葡萄产香酵母的筛选及其混菌发酵对葡萄酒香气成分的影响

【背景】产香酵母可赋予葡萄酒独特的香气,因此,分离筛选优良产香酵母对酿造具有地域风味的特色葡萄酒具有重要意义。【目的】从中条山野生葡萄中筛选产香酵母,进行种群鉴定和生理生化特性研究,并将其应用于葡萄酒发酵过程,研究其对葡萄酒香气成分的影响。【方法】采用稀释涂布平板法从中条山野葡萄中分离筛选酵母菌,对其进行分子生物学鉴定。优选其中具有显著香气的产香酵母,与酿酒酵母F15进行混合发酵,采用气相色谱质谱联用(gas chromatograph-mass spectrometer,GC-MS)对香气成分进行分析,采用半定量法测定香气成分含量。【结果】共分离获得各种菌株13株,26S rRNA基因D1/D2区序列分析表明它们分布于Issatchenkia、Torulaspora、Pichia、Saccharomyces和Rhodotorula等5个不同属内。优选其中一株香气较为浓郁的酵母菌株Issatchenkia orientalis strain XS-6开展研究,结果发现该菌株最高耐受乙醇浓度为8%,最高耐受NaCl浓度为6%,最适生长温度为38℃。与酿酒酵母F15混菌发酵的葡萄酒中共检测出31种香气成分。香气物质总含量较单菌发酵增加19.8%,其中11种香气成分含量增加明显,尤其是具有玫瑰香气的苯乙醇。醇类与酯类物质含量较单菌发酵增加19.6%,并发现了香草酸乙酯(ethyl vanillate)、邻苯二甲酸二丁酯(dibutyl phthalate)等7种新的酯类物质。【结论】产香酵母XS-6对乙醇、NaCl、温度等具有良好的耐受性,而且与酿酒酵母F15混菌发酵对西拉葡萄酒香气成分具有明显的影响,可能在改善葡萄酒风味方面具有潜在的应用价值。     

Improvement of direct ethanol fermentation from woody biomasses by the Antarctic basidiomycetous yeast, Mrakia blollopis, under a low temperature condition

The Antarctic basidiomycetous yeast Mrakia blollopis SK-4 can quite uniquely ferment various sugars under low temperature conditions. When strain SK-4 fermented lignocellulosic biomass using the direct ethanol fermentation (DEF) technique, approximately 30% to 65% of the theoretical ethanol yield was obtained without and with the addition of the non-ionic surfactant Tween 80, respectively. Therefore, DEF from lignocellulosic biomass with M. blollopis SK-4 requires the addition of a non-ionic surfactant to improve fermentation efficiency. DEF with lipase converted Eucalyptus and Japanese cedar to 12.6 g/l, and 14.6 g/l ethanol, respectively. In the presence of 1% (v/v) Tween 80 and 5 U/g-dry substrate lipase, ethanol concentration increased about 1.4- to 2.4-fold compared to that without Tween 80 and lipase. We therefore consider that the combination of M. blollopis SK-4 and DEF with Tween 80 and lipase has good potential for ethanol fermentation in cold environments.     

The use of karyotyping in the systematics of yeasts

The use of electrophoretic karyotyping in systematics of yeasts is discussed. New data are provided on the karyotypes of the medically important fungiHortaea werneckii, Filobasidiella (=Cryptococcus)neoformans, andMalassezia species.Hortaea werneckii has twelve to eighteen bands of chromosomal DNA, ranging in size between 500 and 2300 kb. The karyotypes ofFilobasidiella neoformans consist of seven to fourteen bands of chromosomal DNA. The varietiesneoformans andbacillispora cannot be separated by their karyotypes, and no obvious correlation was found with serotypes, geography or habitat. All strains ofMalassezia pachydermatis studied have similar karyotypes consisting of five bands, whereas inM. furfur, four different karyotypes are prevalent. However, each of these karyotypes is stable.