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.     

浙江雁荡山山脉叶栖酵母菌资源与物种多样性

对北雁荡山、中雁荡山、南雁荡山、西雁荡山和东雁荡山等广阔区域内的叶栖酵母菌资源进行考察,旨在弄清雁荡山山脉叶栖酵母菌资源和多样性。采用PDA平板对雁荡山地区的叶栖酵母菌资源进行分离和纯化,根据酵母菌培养物26S rDNA D1/D2序列和系统发育关系明确酵母菌的分类地位,分析该地区叶栖酵母菌群落的多样性、组成和分布情况。结果发现,分离自雁荡山山脉的1118株叶栖酵母菌可以被鉴定为56个已知类群和19个疑似新种,其中38种酵母菌在该地区比较常见。常见的酵母菌类群中,包括Derxomyces属5种,Sporobolomyces属3种,Bannoa、Bulleribasidium、Rhodosporidiobolus、Symmetrospora、Taphrina、Tilletiopsi和Udeniomyces属各2种,Bullera、Coniochaeta、Coniosporium、Cryptococcus、Elsinoe、Erythrobasidium、Fellozyma、Golubevia、Kockovaella、Kondoa、Leucosporidium、Moesziomyces、Oberwinklerozyma、Phyllozyma、Ruinenia和Saitozyma属各1种。根据常见酵母菌类群的相对频率,在该地区分离到的叶栖酵母菌中Derxomyces mrakii具有明显分布优势,并探讨了Derxomyces mrakii株间26S rDNA D1/D2序列的稳定性和变异幅度。根据雁荡山地区常见叶栖酵母菌组成和相似程度,可以推测地理上相近地区的酵母菌物种分布具有更大相似性。基于雁荡山山脉常见叶栖酵母菌种类和数量而绘制出的物种累积曲线,证明雁荡山地区绝大多数的叶栖酵母菌种类已经被分离到,研究结果可以真实反映该地区叶栖酵母菌资源和物种多样性的存在状况。     

Yeast Microflora Isolated From Brazilian Cassava Roots: Taxonomical Classification Based on Molecular Identification

A rapid molecular identification technique was applied on microbial microflora isolated from Brazilian cassava roots given a yeast profile presented in the samples analyzed. A total of 24 strain isolated from cassava were initially grouped and identified in five groups using restriction-fragment length polymorphism (RFLPs) of 5.8S-ITS rDNA region. Sequencing analysis of the domains D1 and D2 of the 26S rRNA gene or 5.8S rRNA-ITS region were used to identify different groups of yeasts. Representative colonies of yeasts of each group were isolated and identified as Debaromyces hansenii, Kodamaea ohmeri, Candida glabrata, C. haemulonii, and Pichia gullhermondii. It is hoped that these results will contribute toward selecting yeast from this microflora capable to degrade cassava starch in the near future.     

<Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>, <Emphasis Type="Italic">Cryptococcus gattii</Emphasis>: Serotypes in Venezuela

Mycoses due to yeasts belonging to other genera than Candida have become common in the last years especially in immuno-compromised patients. Species of the anamorphic basidiomycetous yeast genus Trichosporon are such opportunistic human pathogenic yeasts which cause several diseases. In this study, Trichosporon faecale is reported in Germany for the first time. The isolate was taken from a human foot, where it was associated with a tinea pedis. The fungal isolate was identified by investigating the morphology, physiology by a commercial API 32 C-set and molecular data of SSU and LSU rDNA as well as the ITS region.     

26S rDNA单链构象多态性分析在临床酵母菌菌种鉴定中的应用

摘要： 【目的】探讨酵母菌临床分离株26S rDNA D1/D2区序列种内相似性和种间差异性的快速检测方法,为临床酵母菌菌种鉴定方法的改进奠定基础。调查北京地区临床酵母菌的种群多样性,为国内酵母菌感染的流行病学研究提供新的基础数据。【方法】用5种常见临床酵母菌种的模式和权威菌株作为标准参考菌株,从北京四家综合性医院收集临床酵母菌260余株,PCR扩增其26S rDNA D1/D2区,对扩增产物进行单链构象多态性（Single-Strand Conformation Polymorphism,SSCP）分析和序列测定分析。【结果】常见病原酵母菌26S rDNA D1/D2区的SSCP图谱具有明显的种间差异性和种内相似性,可以通过该方法对菌株进行初步的菌种鉴定。D1/D2-SSCP和序列分析相结合,对260余株临床酵母菌进行了菌种鉴定,共鉴定有10个属20个种,优势属为念珠菌属(Candida),优势种及其所占比例分别是：C. albicans (57.7%), C. parapsilosis (10.0%), C. tropicalis (9.2%), C. glabrata (6.7%)和C. krusei (5.8%),并发现过去从未或很少报道致病的酵母菌种,愈来愈多地出现在临床分离菌株中。【结论】 26S rDNA D1/D2区的SSCP图谱分析为临床酵母菌株的快速鉴定提供了新的方法;北京地区酵母菌临床分离株呈种群多样性分布,C. albicans虽然仍占优势,但其它念珠菌种的比例已达42%。     

Heteroduplex mobility assay of the 26S rDNA D1/D2 region for differentiation of clinically relevant Candida species

The Heteroduplex Mobility Assay (HMA) method using the PCR amplified D1/D2 region of the 26S rDNA was tested for the differentiation of clinically relevant Candida species. Strains belonging to the same species are not expected to form heteroduplexes in this assay when their PCR products are mixed. D1/D2 HMA experiments between all Candida type strains tested showed heteroduplex formation, including Candida albicans and Candida dubliniensis. There was no heteroduplex formation when most clinical and non-type strains were tested against the type strain of their presumptive species, except when C. albicans WVE and C.␣dubliniensis TAI were analysed. Additional HMA experiments, phenotypic characterisation, and D1/D2 sequencing identified these isolates as Candida tropicalis and Candida parapsilosis, respectively. HMA provides a rapid and relatively simple molecular tool for the differentiation of potentially pathogenic Candida species.     

Occurrence and Diversity of Yeasts in the Mid-Atlantic Ridge Hydrothermal Fields Near the Azores Archipelago

The yeast community associated with deep-sea hydrothermal systems of the Mid-Atlantic Rift was surveyed for the first time. This study relied on a culture-based approach using two different growth media: a conventional culture medium for yeasts supplemented with sea salts (MYPss) and the same medium additionally supplemented with sulfur (MYPssS). For the evaluation of species diversity, a molecular approach involving minisatellite-primed polymerase chain reaction (MSP-PCR) strain typing and sequence analysis of the D1/D2 domains of the 26S rDNA was followed. In the seven water samples that were studied, the number of colony-forming units per liter (cfu/L) ranged from 0 to 5940. The nonpigmented yeasts were much more abundant than the pink-pigmented ones. This disproportion was not observed in studies of other marine systems and may be due to the unique conditions of hydrothermal vents, characterized by a rich animal and microbial diversity and therefore by the availability of organic compounds utilizable by yeasts. Higher counts of nonpigmented yeast were obtained using MYPss, whereas for pink yeasts, higher counts were obtained using MYPssS. Moreover, among pink yeasts, some of the MSP-PCR classes obtained were composed of isolates obtained only on MYPssS, which might be an indication that these isolates are adapted to the ecosystems of the hydrothermal vents. Twelve phylotypes belonged to the Ascomycota and seven phylotypes belonged to the Basidiomycota. The nonpigmented yeasts were identified as Candida atlantica, C. atmosphaerica, C. lodderae, C. parapsilosis, Exophiala dermatitidis, Pichia guilliermondii, and Trichosporon dermatis, whereas the pigmented yeasts were identified as Rhodosporidium diobovatum, R. sphaerocarpum, R. toruloides, and Rhodotorula mucilaginosa. Some of the yeasts that were found belong to phylogenetic groups that include species reported from other marine environments, and eight phylotypes represent undescribed species. The new phylotypes found at Mid-Atlantic Ridge hydrothermal fields represent 33% of the total number of yeast taxa that were found.     

Candida species in tchapalo and bangui, two traditional alcoholic beverages from Côte d'Ivoire

The increase of infections due to non-Candida albicans species made it very necessary to conduct adequate characterization to be able to identify the species of Candida isolated from traditional fermented foods. In this study, based on their hue on Candida Chromogenic Agar medium, a total of 136 yeast strains were isolated from tchapalo and bangui. Molecular identification based on PCR-RFLP of internal transcribed spacers of rDNA (ITS) and sequencing of the ITS and the D1/D2 regions allowed us to assign these isolates to seven species: Candida tropicalis, Candida inconspicua, Candida rugosa, Saccharomyces cerevisiae, Kluyveromyces marxianus, Hanseniaspora guilliermondii, Trichosporon asahii. With the respect to each beverage, six species were found among with four species are regarded as opportunistic pathogens. From these, C. tropicalis, C. inconspicua and K. marxianus were the most commonly encountered. The enzyme activities of the potential pathogens assessed using API ZYM system showed that almost strains had esterase, esterase lipase, valine and cystine arylamidase, alpha chymotrypsin, alkaline phosphatase and naphthol phosphohydrolase activities. The activity of α-glucosidase was found only in C. tropicalis and C. inconspicua strains isolated from tchapalo while β-glucosidase activity was found in all strains from tchapalo and only in C. inconspicua isolated from bangui.     

Three new species of anamorphic yeasts phenotypically and phylogenetically related toCandida mesenterica: The description ofCandida fungicola sp. nov.,Candida sagamina sp. nov., andCandida fukazawae sp. nov. isolated from fruit bodies of mushrooms

Four strains of anamorphic yeasts isolated from fruit bodies of mushrooms collected in Japan were found to represent three new species of the genusCandida. These species resembleCandida mesenterica in characteristics commonly employed in the classification of yeasts. On the basis of DNA-DNA reassociation, however, they were clearly distinguished fromC. mesenterica and from one another. Three new species,Candida fungicola, Candida sagamina, andCandida fukazawae, are proposed for these yeasts. The analysis of SSU rDNA sequences suggested that these three species were closely related to each other and toC. mesenterica andC. suecica.     

Yeasts associated with pollinating bees and flower nectar

A study of the yeast flora of 328 honey stomachs from 7 different pollinating bee species, and 342 flower nectar samples of 9 different flower species yielded 766 yeast isolates composed of 16 genera and 47 species. Most of the yeast species from both the sources belonged to the genusCandida, while the most frequently isolated yeasts wereDekkera intermedia from honey stomach andCandida blankii from flower nectar. Among the honey bees,Xylocopa sp., and among flowers,Citrus medica, yielded the highest number of yeast species. Nineteen species of yeasts belonging to 9 genera were common to both the sources.     

Development of a Simple and Rapid Method Based on Polymerase Chain Reaction–Based Restriction Fragment Length Polymorphism Analysis to Differentiate Helicobacter, Campylobacter, and Arcobacter Species

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of amplified DNA fragment of the 16S and 23S rRNA genes was performed on 35 Helicobacter, 24 Campylobacter, and 15 Arcobacter strains. PCR amplification generated a 1004-bp fragment of 16S rDNA and a 2.6-Kbp fragment of 23S rDNA from each strain. The amplicons were digested with DdeI and HpaII, respectively. For both assays, distinctive profiles were obtained for each genus. 23S rDNA PCR-RFLP analysis with HpaII enzyme identified Campylobacter and Helicobacter strains at the species level. Analysis of 16S rRNA gene with DdeI enzyme was not useful for the specific identification of Campylobacter and Arcobacter, although it discriminated among Helicobacter species. The PCR-RFLP technique allowed for the discrimination among these three related genus with only one restriction enzyme; therefore it can be a simple, rapid, and useful method for routine identification.     

Molecular characterization of carotenogenic yeasts from aquatic environments in Patagonia, Argentina

Fifteen aquatic environments (lakes, lagoons and rivers) of glacial origin in the northern Andean Patagonia (Argentina) were surveyed for the occurrence of red yeasts. Subsurface water samples were filtered and used for colony counting and yeast isolation. A preliminary quantitative analysis indicated that total yeast counts ranged between 0 and 250 cells l⁻¹. A polyphasic approach including physiological and molecular methods was used for the identification of 64 carotenogenic yeast strains. The molecular characterisation of the isolates was based on the mini/microsatellite-primed PCR technique (MSP-PCR) employing the (GTG)₅ and the M13 primers. Comparison of representative fingerprints of each group with those of the type strains of pigmented yeasts allowed the expeditious identification of 87.5% isolates. The sequence analysis of the D1/D2 domains of the 26S rDNA was employed to confirm identifications and in the characterization of the unidentified MSP-PCR groups. Teleomorphic yeast species were detected by performing sexual compatibility assays. The isolates corresponded to 6 genera and 15 yeast species, including four new yeast species of the genera Cryptococcus (1), Rhodotorula (1) and Sporobolomyces (2). Rhodotorula mucilaginosa was found in the majority of the samples and represented ca. 50% of the total number of isolates. However, this yeast was not detected in aquatic environments with very low anthropic influence. Other frequent yeast isolates were teleomorphic yeast species of Rhodosporidium babjevae, R. kratochvilovae and Sporidiobolus salmonicolor. This study represents the first report on red yeast occurrence and biodiversity in northwestern Patagonia. This revised version was published online in June 2006 with corrections to the Cover Date.     

Yeast microbiota associated with spontaneous sourdough fermentations in the production of traditional wheat sourdough breads of the Abruzzo region (Italy)

The aims of this study were to describe the yeast community of 20 sourdoughs collected from central Italy and to characterize the sourdoughs based on chemical properties. A polyphasic approach consisting of traditional culture-based tests (spore-forming and physiological tests) and molecular techniques (PCR-RFLP, RAPD-PCR, PCR-DGGE) and chemical analysis (total acidity, acids, and sugar contents), was utilized to describe the yeast population and to investigate the chemical composition of the doughs. PCR-RFLP analysis identified 85% of the isolates as Saccharomyces cerevisiae, with the other dominant species being Candida milleri (11%), Candida krusei (2.5%), and Torulaspora delbrueckii (1%). RAPD-PCR analysis, performed with primers M13 and LA1, highlighted intraspecific polymorphism among the S. cerevisiae strains. The diversity of the sourdoughs from the Abruzzo region is reflected in the chemical composition, yeast species, and strain polymorphism. Our approach using a combination of phenotypic and genotypic methods identified the yeast species in the 20 sourdough samples and provided a complete overview of the yeast populations found in sourdoughs from the Abruzzo region.     

Massive isolation of anamorphous ascomycete yeasts <Emphasis Type="Italic">Candida oleophila</Emphasis> from plant phyllosphere

Many years of research has confirmed a wide distribution of anamorphous ascomycete yeasts in the phyllosphere of diverse plants of Moscow and the Moscow oblast. Based on the standard morphological and physiological criteria, on the results of restriction analysis of the 5.8S-ITS rDNA region, and on the sequencing of the D1D2 region of 26S rDNA, these yeasts were identified as Candida oleophila Montrocher. Previous isolation of this species has been rare, possibly due to its incorrect identification. This species, together with phytobiotic basidiomycete yeasts, was shown to be dominant in the yeast epiphytic communities on the surface parts of plants. The relative abundance of C. oleophila is highest on plant fruits and increases significantly by the end of the vegetation period. Wide occurrence of this yeast species on fruits and in the phyllosphere may be related to its ability to compete with rapidly growing phytopathogenic fungi.     

Occurrence of yeasts in psittacines droppings from captive birds in Italy

Three-hundred twenty five droppings from parrots raised in the premises of 4 breeders and in several private households were cultured for yeasts. One-hundred sixty droppings (49.2%) resulted positive. From these specimens 212 isolates belonging to 27 different species were obtained. Mainly Candida species such as C. albicans, C. catenulata, C. curvata, C. famata, C. glabrata, C. guilliermondi, C. holmii, C. intermedia, C. krusei, C. lambica, C. lusitaniae, C. membranaefaciens, C. parapsilosis, C. pelliculosa, C. sake and C. valida were isolated. Debaryomyces marama, D. polymorphus, Geotrichum sp., Pichia etchelsii, P. ohmeri, Rhodotorula glutinis, R. rubra, Rhodotorula sp., Saccharomyces cerevisiae, S. kluyiveri and Zygosaccharomyces sp. were also obtained. Dark colonies on Staib medium were never observed. The psittacine birds apparently serve as carriers for several Candida species or their perfect states and to a lesser extent for other opportunistic yeasts such as Rhodotorula, Trichosporon and Saccharomyces spp., which are considered part of the transient microbiota of the gastrointestinal tract. The most striking finding was the absence of Cryptococcus spp. among the isolates. The present survey confirms the role of pet birds in carrying potential zoonotic yeasts. This revised version was published online in June 2006 with corrections to the Cover Date.     

Identification of medically important yeasts by sequence analysis of the internal transcribed spacer and D1/D2 region of the large ribosomal subunit

BackgroundThe prevalence of opportunistic yeast infections has increased in recent decades as the result of an increasing immunocompromised patient population.AimsTo evaluate ribosomal RNA (rRNA) gene sequence to identify medically important yeast species, to investigate the performance of both the rRNA gene internal transcribed spacer (ITS) and D1/D2 region in identifying clinically relevant yeasts, and to compare these results with those of a standard phenotypic method.MethodsBoth regions from 50 yeast strains, comprising 45 clinical isolates and 5 reference strains, were amplified using PCR and then sequenced. The sequences were compared to reference data available from the GenBank database of the National Center for Biotechnology Information using the BLASTn tool.ResultsUsing ID32C, 88% (44/50) of all strains were identified accurately at the species level, although 6% were misidentified; two Candida eremophila isolates were identified as Candida glabrata and Candida tropicalis, and one Saprochaete clavata isolate was identified as Saprochaete capitata. Two of the four isolates identified by phenotypic methods as Trichosporon asahii were defined so by analyzing the ITS region, but the remaining two were not distinguishable from closely related species. Based on the D1/D2 region, these four isolates had 100% sequence identity with T. asahii, Trichosporon japonicum, and Trichosporon asteroides. The isolate identified as Trichosporon inkin using ID32C could not be distinguished from Trichosporon ovoides by analyzing the ITS and D1/D2 regions.ConclusionsIdentifying medically important yeasts by sequencing the ITS and D1/D2 region is a rapid and reliable alternative to conventional identification methods. For a diagnostic algorithm, we suggest a two-step procedure integrating conventional methods (e.g. microscopic morphology on corn meal agar with Tween® 80 and API ID32C®) and sequence analysis of the ITS and D1/D2 region.     

<Emphasis Type="Italic">Candida patagonica</Emphasis> sp. nov., a new species of yeast from cellar surfaces

A novel anamorphic yeast species belonging to the genus Candida has been isolated from cellar surfaces in North Patagonia. Morphological and physiological observation and phylogenetic analysis were performed. Pseudomycelium was plentifully produced. No sexual reproduction was observed. From sequence analysis of the 26S rDNA D1/D2 region, Candida bituminiphila and Zygoascus hellenicus were the closest species with 40 and 79 bp substitutions, respectively. C. bituminiphila differed physiologically from the novel species in its ability to assimilate sucrose and erythritol, in not fermenting any sugars, in growing without some vitamin compounds, and in growing at 40°C. All these data support the hypothesis that the new yeast, named Candida patagonica, is a novel species related to C. bituminiphila. The type strain is UNCOMA 159.5 (= CECT 12029 = CBS 10443).     

Identification of species in the genus Pichia by restriction of the internal transcribed spacers (ITS1 and ITS2) and the 5.8S ribosomal DNA gene

In this study, the variability within the ribosomal DNA region spanning the internal transcribed spacers ITS1 and ITS2 and the 5.8S gene (5.8S-ITS rDNA) was used to differentiate species in the genus Pichia. The 5.8S-ITS rDNA region was PCR-amplified and the PCR product digested with the enzymes CfoI, HinfI, and HaeIII. The variability in the size of the amplified product and in the restriction patterns enabled differentiation between species in the genus Pichia, and between Pichia species and yeast species from other genera in the Yeast-id database (). Moreover, the restriction fragment length polymorphism (RFLP) patterns of the 5.8S-ITS enabled misidentified strains to be detected and revealed genetic heterogeneity between strains within the Pichia membranifaciens and Pichia nakazawae species. Ultimately, the RFLP patterns of the 5.8S-ITS rDNA failed to differentiate between some Pichia and Candida species that could be distinguished on the basis of the sequence of the 5.8S-ITS rRNA region or the sequence of the D1/D2 domain of the 26S rDNA gene.     

Sequencer-Based Capillary Gel Electrophoresis (SCGE) Targeting the rDNA Internal Transcribed Spacer (ITS) Regions for Accurate Identification of Clinically Important Yeast Species

Accurate species identification of Candida, Cryptococcus, Trichosporon and other yeast pathogens is important for clinical management. In the present study, we developed and evaluated a yeast species identification scheme by determining the rDNA internal transcribed spacer (ITS) region length types (LTs) using a sequencer-based capillary gel electrophoresis (SCGE) approach. A total of 156 yeast isolates encompassing 32 species were first used to establish a reference SCGE ITS LT database. Evaluation of the ITS LT database was then performed on (i) a separate set of (n = 97) clinical isolates by SCGE, and (ii) 41 isolates of 41 additional yeast species from GenBank by in silico analysis. Of 156 isolates used to build the reference database, 41 ITS LTs were identified, which correctly identified 29 of the 32 (90.6%) species, with the exception of Trichosporon asahii, Trichosporon japonicum and Trichosporon asteroides. In addition, eight of the 32 species revealed different electropherograms and were subtyped into 2–3 different ITS LTs each. Of the 97 test isolates used to evaluate the ITS LT scheme, 96 (99.0%) were correctly identified to species level, with the remaining isolate having a novel ITS LT. Of the additional 41 isolates for in silico analysis, none was misidentified by the ITS LT database except for Trichosporon mucoides whose ITS LT profile was identical to that of Trichosporon dermatis. In conclusion, yeast identification by the present SCGE ITS LT assay is a fast, reproducible and accurate alternative for the identification of clinically important yeasts with the exception of Trichosporon species.     

Safety and regulation of yeasts used for biocontrol or biopreservation in the food or feed chain

Yeasts have been important components of spontaneous fermentations in food and beverage processing for millennia. More recently, the potential of utilising antagonistic yeasts, e.g. Pichia anomala and Candida spp., for post-harvest biological control of spoilage fungi during storage of plant-derived produce (‘biopreservation’) has been clearly demonstrated. Although some yeast species are among the safest microorganisms known, several have been reported in opportunistic infections in humans, including P. anomala and bakers’ yeast, Saccharomyces cerevisiae. More research is needed about the dominant pathogenicity and virulence factors in opportunistic yeasts, and whether increased utilisation of biopreservative yeasts in general could contribute to an increased prevalence of yeast infections. The regulatory situation for yeasts used in post-harvest biocontrol is complex and the few products that have reached the market are mainly registered as biological pesticides. The qualified presumption of safety (QPS) approach to safety assessments of microorganisms intentionally added to food or feed, recently launched by the European Food Safety Authority, can lead to more efficient evaluations of new products containing microbial species with a sufficient body of knowledge or long-term experience on their safety. P. anomala is one of several yeast species that have been given QPS status, although the status is restricted to use of this yeast for enzyme and metabolite production purposes. With regard to authorisation of new biopreservative yeasts, we recommend that the possibility to regulate microorganisms for food biopreservation as food additives be considered.