Effect of the use of commercial Saccharomyces strains in a newly established winery in Ronda (Málaga, Spain)

An ecological study of the yeasts present in a spontaneous and an inoculated fermentation in red wine was carried out in 2005 vintage in a winery located in the Denomination of Origin "Sierras de Málaga" (Málaga, southern of Spain). The winery operated by the first time with the 2003 vintage and since then, has used commercial yeast inocula to start alcoholic fermentation. Yeast isolates were identified by PCR-RFLP analysis of the 5.8S-ITS region from the ribosomal DNA and by mitochondrial DNA RFLP analysis. Except for non-Saccharomyces yeasts found in the fresh must before fermentation, all the isolates were found to be commercial Saccharomyces cerevisiae strains employed by the winery during the successive vintages; thus, no indigenous Saccharomyces yeasts were isolated during fermentation. The same four restriction patterns were found in non inoculated and inoculated vats, although with different frequencies. The use of commercial yeast starter in a new established winery seems to have prevented the development of a resident indigenous Saccharomyces flora.     

Identification and characterization of yeasts in sugarcane silages

Aims: To enumerate the micro‐organisms and to identify the yeast species present during the ensilage of different sugarcane (Saccharum spp.) cultivars. Method: Samples of sugarcane silage were collected at 10, 20, 30 and 40 days from the start of fermentation. Population levels of lactic acid bacteria (LAB), mesophilic facultative anaerobic (MFA) bacteria, filamentous fungi and yeasts were determined. Nine species of yeasts were classified according to traditional methods and confirmed using molecular techniques. Conclusions: LAB dominated the ensiling process of sugarcane, although yeasts were present at relatively high population levels throughout the whole fermentation period. The detected species of yeasts varied according to sugarcane cultivar and time of fermentation. Torulaspora delbrueckii was the predominant yeast, followed by Pichia anomala and Saccharomyces cerevisiae. Significance and Impact of the Study: Knowledge of the population of micro‐organisms in general, and of yeasts in particular, present during the fermentation of sugarcane is of fundamental importance in the development of more effective ensiling processes.     

Yeast assessment during alcoholic fermentation inoculated with a natural “pied de cuve” or a commercial yeast strain

The present study has been carried out in an organic winery established in 2003 in the Denomination of Origin “Sierras de Málaga” (Southern Spain) region during the 2007 vintage. The aim of this work was to ascertain the yeast microflora present in the winery and during the vinifications and to obtain a collection of autochthonous S. cerevisiae strains from this area. Yeast populations from three vats containing fermenting musts from different grape varieties were analysed. Two of them were inoculated with a natural “pied de cuve” while the third one was sown with a rehydrated commercial yeast strain. A total of 382 yeasts were isolated and identified, initially by restriction analysis of ribosomal DNA and further by sequencing of this region. Non-Saccharomyces yeasts were found in all three musts but they practically disappeared as the fermentations progressed. Analysis of mitochondrial DNA RFLP revealed 13 different restriction patterns of Saccharomyces cerevisiae strains, five of them similar to those of commercial strains used in the winery. Commercial strains were found even in vats inoculated with a “pied de cuve” generated by spontaneous fermentation of a must sample. The analysis of samples recovered from different winery surfaces and equipments demonstrated that non-Saccharomyces and both commercial and autochthonous Saccharomyces strains were part of the resident microflora in the winery. Biodiversity of autochthonous S. cerevisiae in fermentation vats was low but two of them were able to compete with the commercial ones and they were isolated even at the end of the fermentation.     

Yeast heterogeneity during spontaneous fermentation of black Conservolea olives in different brine solutions

Aims: To assess the yeast community structure and dynamics during Greek‐style processing of natural black Conservolea olives in different brine solutions. Methods and Results: Black olives were subjected to spontaneous fermentation in 6% (w/v) NaCl brine solution or brine supplemented with (i) 0·5% (w/v) glucose, (ii) 0·2% (v/v) lactic acid and (iii) both glucose and lactic acid. Yeast species diversity was evaluated at the early (2 days), middle (17 days) and final (35 days) stages of fermentation by restriction fragment length polymorphism and sequence analyses of the 5·8S internal transcribed spacer and the D1/D2 ribosomal DNA (rDNA) regions of isolates. Analysis revealed a relatively broad range of biodiversity composed of 10 genera and 17 species. In all treatments, yeasts were the main micro‐organisms involved in fermentation together with lactic acid bacteria that coexisted throughout the processes. Metschnikowia pulcherrima was the dominant yeast species at the onset of fermentation, followed by Debaryomyces hansenii and Aureobasidium pullulans. Species heterogeneity changed as fermentations proceeded and Pichia membranifaciens along with Pichia anomala evolved as the main yeasts of olive elaboration, prevailing at 17 and 35 days of the process. Molecular techniques allowed for the identification of five yeast species, namely A. pullulans, Candida sp., Candida silvae, Cystofilobasidium capitatum and M. pulcherrima, which have not been reported previously in black olive fermentation. Conclusions: By using molecular techniques, a rich yeast community was identified from Conservolea black olive fermentations. Metschnikowia pulcherrima was reported for the first time to dominate in different brines at the onset of fermentation, whereas Pichia anomala and P. membranifaciens evolved during the course. The addition of glucose and/or lactic acid perturbed yeast succession and dominance during fermentation. Significance and Impact of the Study: Yeasts have an important role in black olive fermentation and contribute to the development of the organoleptic characteristics of the final product. At the same time, certain species can cause significant spoilage. The present study adds to a better knowledge of yeast communities residing in olive fermentations towards a well‐controlled process with minimization of product’s losses.     

Identification of yeasts within Saccharomyces sensu stricto complex by PCR-fingerprinting

Biological relatedness makes species characterization of the industrially important Saccharomyces sensu stricto complex difficult. In this paper we present a set of PCR-fingerprinting markers based in Single Primer Amplification Reactions (SPAR) that, together with PCR-ribotyping and single gene RFLP analysis, can effectively identify individual species and fully characterize the hybrid nature of industrial isolates. With those markers, all six yeast species of the sensu stricto complex could be discriminated and we also identified errors in the previous taxonomic characterization of certain wine yeasts. The unique patterns generated by the SPAR markers could be useful in monitoring yeast populations during industrial fermentation processes and can be used to detect the appearance of yeast hybrids in these environments.     

Physiological and genetic stability of hybrids of industrial wine yeasts Saccharomyces sensu stricto complex

Aims: The aim of this study was to examine the physiological and genetic stability of hybrids of industrial wine yeasts Saccharomyces sensu stricto complex subjected to acidic stress during fermentation. Methods and Results: Laboratory‐constructed yeast hybrids, one intraspecific Saccharomyces cerevisiae × S. cerevisiae and three interspecific S. cerevisiae ×Saccharomyces bayanus, were subcultured in aerobic or anaerobic conditions in media with or without l ‐malic acid. Changes in the biochemical profiles, karyotypes and mitochondrial DNA profiles of the segregates were assessed after 50–190 generations. All yeast segregates showed a tendency to increase the range of the tested compounds utilized as sole carbon sources. Interspecific hybrids were alloaneuploid and their genomes tended to undergo extensive rearrangement especially during fermentation. The karyotypes of segregates lost up to four and appearance up to five bands were recorded. The changes in their mtDNA patterns were even broader reaching 12 missing and six additional bands. These hybrids acquired the ability to sporulate and significantly changed their biochemical profiles. The alloaneuploid intraspecific S. cerevisiae hybrid was characterized by high genetic stability despite the phenotypic changes. l ‐malic acid was not found to affect the extent of genomic changes of the hybrids, which suggests that their demalication ability is combined with resistance to acidic stress. Conclusions: The results reveal the plasticity and extent of changes of chromosomal and mitochondrial DNA of interspecific hybrids of industrial wine yeast especially under anaerobiosis. They imply that karyotyping and restriction analysis of mitochondrial DNA make it possible to quickly assess the genetic stability of genetically modified industrial wine yeasts but may not be applied as the only method for their identification and discrimination. Significance and Impact of the Study: Laboratory‐constructed interspecific hybrids of industrial strains may provide a model for studying the adaptive evolution of wine yeasts under fermentative stress.     

Enrichment of a continuous culture of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> with the yeast <Emphasis Type="Italic">Issatchenkia orientalis</Emphasis> in the production of ethanol at increasing temperatures

A fermentation system was continuously fed with sugar-cane syrup and operated with recycling of Saccharomyces cerevisiae cells at temperatures varying from 30 to 47°C. The aim of the present work was to obtain and study the colonies of isolates showing elongated cells of yeasts which were sporadically observed at the end of this continuous process. Based on a sequence of assays involving methods of classical taxonomy and RAPD-PCR, two groups of isolates showing characteristics of non-Saccharomyces yeasts were identified in the yeast population where S. cerevisiae was the dominant yeast. The largest group of non-Saccharomyces yeasts, resulting from a slow proliferation over the 2 months, reached a final level of 29.6% at the end of the process. RAPD-PCR profiles obtained for the isolates of this dominant non-Saccharomyces yeast indicated that they were isolates of Issatchenkia orientalis. Pichia membranifaciens was the only species of non-Saccharomyces yeast detected together with I. orientalis but at a very low frequency. The optimum temperature for ethanol formation shown by the isolate 195B of I. orientalis was 42°C. This strain also showed a faster ethanol formation and biomass accumulation than the thermotolerant strain of S. cerevisiae used as the starter of this fermentation process. Some isolates of I. orientalis were also able to grow better at 40°C than at 30°C on plates containing glycerol as carbon source. Yeasts able to grow and produce ethanol at high temperatures can extend the fermentation process beyond the temperature limits tolerated by S. cerevisiae.     

Phenotypic and genotypic diversity of wine yeasts used for acidic musts

The aim of this study was to examine the physiological and genetic stability of the industrial wine yeasts Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum under acidic stress during fermentation. The yeasts were sub-cultured in aerobic or fermentative conditions in media with or without l-malic acid. Changes in the biochemical profiles, karyotypes, and mitochondrial DNA profiles were assessed after minimum 50 generations. All yeast segregates showed a tendency to increase the range of compounds used as sole carbon sources. The wild strains and their segregates were aneuploidal or diploidal. One of the four strains of S. cerevisiae did not reveal any changes in the electrophoretic profiles of chromosomal and mitochondrial DNA, irrespective of culture conditions. The extent of genomic changes in the other yeasts was strain-dependent. In the karyotypes of the segregates, the loss of up to 2 and the appearance up to 3 bands was noted. The changes in their mtDNA patterns were much broader, reaching 5 missing and 10 additional bands. The only exception was S. bayanus var. uvarum Y.00779, characterized by significantly greater genome plasticity only under fermentative stress. Changes in karyotypes and mtDNA profiles prove that fermentative stress is the main driving force of the adaptive evolution of the yeasts. l-malic acid does not influence the extent of genomic changes and the resistance of wine yeasts exhibiting increased demalication activity to acidic stress is rather related to their ability to decompose this acid. The phenotypic changes in segregates, which were found even in yeasts that did not reveal deviations in their DNA profiles, show that phenotypic characterization may be misleading in wine yeast identification. Because of yeast gross genomic diversity, karyotyping even though it does not seem to be a good discriminative tool, can be useful in determining the stability of wine yeasts. Restriction analysis of mitochondrial DNA appears to be a more sensitive method allowing for an early detection of genotypic changes in yeasts. Thus, if both of these methods are applied, it is possible to conduct the quick routine assessment of wine yeast stability in pure culture collections depositing industrial strains.     

Dynamics and characterization of yeasts during natural fermentation of Italian sausages

A multiphasic approach was used to investigate the yeast ecology in Italian fermented sausages. Culture-dependent and -independent methods were applied to identify the yeast species during the maturation process and to characterize the numerically dominant species. Plating analysis and subsequent molecular identification of the isolates highlighted the dominance of Debaryomyces hansenii, but at least other three yeast species -Candida zeylanoides, Pichia triangularis and Metschnikowia pulcherrima - contributed to the fermentation as well. Direct denaturing gradient gel electrophoresis analysis confirmed that D. hansenii was the main yeast species present and its activity was also demonstrated. No other yeasts species were detected on the direct denaturing gradient gel electrophoresis gels, whereas DNA of Penicillium farinosum, Penicillium viridicatum and Mucor racemosus were present. Molecular characterization by RAPD-PCR analysis of the D. hansenii isolates demonstrated a shift in its population from the beginning to the end of the maturation of the sausages. Strains present during the early stages of the fermentation were grouped in clusters that differed from those isolated in the final phases of the maturation, underlining the genetic differences between these two populations of D. hansenii. However, all the isolates were able to grow in the presence of 3.5% sodium chloride and at 10 degrees C, evidence that these parameters did not select the species present at the end of the maturation period.     

Multivariate analysis to discriminate yeast strains with technological applications in table olive processing

This survey uses a multivariate classification analysis to discriminate yeast strains with interesting biochemical activities for the processing of table olives among a collection of 32 isolates belonging to 16 different yeast species. Lipase, esterase and β-glucosidase activities (desirable characteristics) were quantitatively evaluated in both extracellular and cellular fractions for all isolates in different types of culture media. The study of the quantitative data by cluster and principal component analyses led to the identification of several Wickerhamomyces anomalus, Candida boidinii and Candida diddensiae isolates with promising characteristics (the best global activity levels), clearly differentiated from the rest of the yeasts. The results obtained in this work open up new alternatives to this methodology for the study, classification and selection of the most suitable yeasts to be used as starters, alone or in combination with lactic acid bacteria, during table olive processing.     

Changes in the intracellular concentrations of the adenosine phosphates and nicotinamide adenine dinucleotides ofSaccharomyces cerevisiae during batch fermentation

Significant changes in the intracellular concentrations of adenosine phosphates and nicotinamide adenine dinucleotides were observed during fermentation of grape must by three different strains ofSaccharomyces cerevisiae: S. cerevisiae var.cerevisiae, a typical fermentative yeast strain and two flor-veil-forming strains,S. cerevisiae var.bayanus andS. cerevisiae var.capensis. The intracellular concentration of ATP was always higher inS. cerevisiae var.cerevisiae than in the flor-veil-forming strains. NAD⁺ and NADP⁺ concentrations decreased at faster rates in the flor-veil-forming yeasts than in the other yeast but NADH concentration was the same in all yeasts for the first 10 days of fermentation. NADPH concentration was always lower inS. cerevisiae var.cerevisiae than in the other yeasts and this yeast also showed higher rates of growth and fermentation during the early stages of the fermentation and the presence of non-viable cells at the end of fermentation. In contrast, the flor-veil-forming strains maintained growth and fermentation capabilities for a relatively long time and viable cells were present throughout the entire fermentation process (31 days).The authors are with the Department of Microbiology, Faculty of Sciences, University of Cordoba, Avda. San Alberto Magno s/n, 14004-Córdoba, Spain     

Ecology of yeasts in polluted water

The general size and composition of the extant yeast populations in 13 polluted freshwater habitats were surveyed. Subsequently the yeast populations in three of the 13 locations were quantitatively determined and compared. The three locations had (A) low pollution levels, (B) heavy industrial waste pollution, and (C) heavy domestic waste pollution.The yeast population at location A was dominated byRhodotorula andCryptococcus isolates. At station BRhodotorula andCandida were predominant.Candida isolates were in the majority at location C andRhodotorula strains were second in frequency, but were much lower in proportion of the population than at the other two habitats.These polluted waters in general had large yeast populations, ranging as high as 27,000 yeasts per 100 ml, and averaging approximately 3000 yeasts per 100 ml.The presence of human wastes was especially associated with large increases in the proportion ofCandida yeasts in the environment. The genusRhodotorula was consistently present at all locations, but the genusCryptococcus was a major component of the yeast population only in non-polluted or lightly polluted fresh water.We appreciate the assistance of the Calumet Area Surveillance Program, Federal Water Pollution Control Administration, Chicago, in the collection of water samples. This investigation was supported by U.S. Public Health Service Research Grant No. AI 04642 from the National Institute of Allergy and Infectious Diseases.     

Genetic diversity in commercial wineries: effects of the farming system and vinification management on wine yeasts

Aims: Analysis of the diversity and distribution of wine yeasts isolated from organically and conventionally grown grapes, and during the subsequent fermentation with or without starter cultures in six different commercial wineries. Methods and Results: PCR‐RFLP screening of isolates revealed the involvement of ten different species. Saccharomyces cerevisiae, scarcely isolated from grapes, was the dominant species during the latter phases of fermentation, identifying 108 different genotypes by means of SSR analysis. Species and strains’ diversity and presence were strongly influenced by the farming system used to grow the grapes and the system of vinification. Conclusions: Organic farming management was more beneficial in terms of diversity and abundance than the conventional one. Induced fermentation generated a great replacement of native yeasts. Although winery‐resident yeasts resulted to be predominant in the process, some noncommercial strains originally in the vineyard were found in final stages of the fermentation, confirming that autochthonous strains of S. cerevisiae are capable to conduct the fermentation process up to its end. Significance and Impact of the Study: The study of natural yeast communities from commercial vineyards and wineries is an important step towards the preservation of native genetic resources. Our results have special relevance because it is the first time that the real situation of the yeast ecology of alcoholic fermentation in commercial wineries belonging to the relevant wine‐producing Appellation of Origin ‘Vinos de Madrid’ is shown.     

Molecular identification of wine yeasts at species or strain level: a case study with strains from two vine-growing areas of Greece

The composition of wine yeast populations, present during spontaneous fermentation of musts from two wine-producing areas of Greece (Amyndeon and Santorini) and followed for two consecutive years, were studied using a range of molecular techniques. Internal Transcribed Spacer (ITS) ribotyping was convincingly applied for yeast species identification, proving its usefulness as a reliable tool for the rapid characterization of species composition in yeast population studies. Restriction Fragment Length Polymorphism (RFLP) of mitochondrial DNA (mtDNA) was shown to be a convenient criterion for the detection of intraspecies genetic diversity of both Saccharomyces and non-Saccharomyces isolate populations. Similarly, polymorphism of amplified delta interspersed element sequences provided an additional criterion for S. cerevisiae strain differentiation. Comparative analysis of S. cerevisiae genetic diversity, using mtDNA restriction patterns and delta-amplification profiles, showed a similar discriminative power of the two techniques. However, by combining these approaches it was possible to distinguish/characterize strains of the same species and draw useful conclusions about yeast diversity during alcoholic fermentation. The most significant findings in population dynamics of yeasts in the spontaneous fermentations were (i) almost complete absence of non-S.cerevisiae species from fermentations of must originating from the island Santorini, (ii) a well recorded strain polymorphism in populations of non-Saccharomyces species originating from Amyndeon and (iii) an unexpected polymorphism concerning S. cerevisiae populations, much greater than ever reported before in similar studies with wine yeasts of other geographical regions.     

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.     

Yeast populations associated with the artisanal cheese produced in the region of Serra da Canastra, Brazil

The aim of this work was to describe the yeast populations present during the manufacturing of Minas cheese of the region of Serra da Canastra, Minas Gerais state, Brazil. Canastra cheese is produced from raw cow’s milk at the farmhouse level using artisanal procedures and natural whey cultures as starters. Samples from 10 farms were studied, and they included: raw milk, natural starter, cheese curd before salting and cheese after 5 days of ripening. The most frequent yeasts in whey, curd and cheese were Debaryomyces hansenii, Kluyveromyces lactis, Kodamaea ohmeri and Torulaspora delbrueckii. Many yeast isolates were able to produce proteases, lipases and β-galactosidades. Production of these enzymes by yeasts in the cheese would contribute to the development of the characteristic flavor and smell during the ripening process.     

Indigenous and inoculated yeast fermentation of gabiroba (<Emphasis Type="Italic">Campomanesia pubescens</Emphasis>) pulp for fruit wine production

The objectives of this study were to evaluate the potential of gabiroba Campomanesia pubescens (DC) O. Berg in the production of a beverage fermented using selected and wild yeasts from indigenous fermentation, analyze the volatile compounds profile present during the process of fermentation, and evaluate the sensory quality of the final beverage produced. Throughout the process of fermentation, when Saccharomyces cerevisiae UFLA CA 1162 was inoculated, there were stable viable populations around 9 log cells ml⁻¹. During indigenous fermentation, yeast population increased from 3.7 log CFU ml⁻¹ to 8.1 log CFU ml⁻¹ after 14 days. The diversity and dynamics of the yeast population during indigenous fermentation observed by PFGE analysis showed five different karyotyping profiles in the first days of fermentation. After the seventh day, there was a higher frequency of a similar S. cerevisiae profile. The yeast non-Saccharomyces were identified by sequencing of the ITS region as Candida quercitrusa and Issatchenkia terricola. Inoculated fermentations yielded a higher amount of alcohol than indigenous ones, indicating the efficiency of selected strains. There was also a greater concentration of higher alcohols, which are usually responsible for the flavor found in alcoholic beverages. Based on the characteristics of the pulp and acceptance in the sensory analysis, gabiroba fruits showed good potential for use in the production of fermented beverage.     

Experimental approach for target selection of wild wine yeasts from spontaneous fermentation of “Inzolia” grapes

The aim of this research was the study of indigenous yeasts isolated from spontaneous fermentation of Inzolia grapes, one of the most widespread native white grapes in Sicily (Italy). The use of selective medium for the isolation and the screening for sulphur dioxide tolerance were useful for the first selection among 640 isolates. The yeasts characterized by high SO₂ tolerance were identified at species level by restriction analysis of ITS region; although the majority of isolates were identified as S. cerevisiae, some non-Saccharomyces yeasts were found. Forty-seven selected yeasts, both S. cerevisiae and non-Saccharomyces yeasts, were characterized for genetic and technological diversity. The genetic polymorphism was evaluated by RAPD-PCR analysis, whereas the technological diversity was analyzed by determining the main secondary compounds in the experimental wines obtained by inoculating these yeasts. Both the molecular and metabolic profiles of selected yeasts were able to clearly discriminate S. cerevisiae from non-Saccharomyces yeasts. This research was useful for the constitution of a collection of selected indigenous yeast strains, including S. cerevisiae and non-Saccharomyces species possessing interesting enological traits. This collection represents a source of wild yeasts, among of which it is possible to select indigenous starters able to maintain the specific organoleptic characteristics of Inzolia wine.     

Yeast Diversity and Persistence in Botrytis-Affected Wine Fermentations

Culture-dependent and -independent methods were used to examine the yeast diversity present in botrytis-affected (“botrytized”) wine fermentations carried out at high (~30°C) and ambient (~20°C) temperatures. Fermentations at both temperatures possessed similar populations of Saccharomyces, Hanseniaspora, Pichia, Metschnikowia, Kluyveromyces, and Candida species. However, higher populations of non-Saccharomyces yeasts persisted in ambient-temperature fermentations, with Candida and, to a lesser extent, Kluyveromyces species remaining long after the fermentation was dominated by Saccharomyces. In general, denaturing gradient gel electrophoresis profiles of yeast ribosomal DNA or rRNA amplified from the fermentation samples correlated well with the plating data. The direct molecular methods also revealed a Hanseniaspora osmophila population not identified in the plating analysis. rRNA analysis also indicated a large population (>10⁶ cells per ml) of a nonculturable Candida strain in the high-temperature fermentation. Monoculture analysis of the Candida isolate indicated an extreme fructophilic phenotype and correlated with an increased glucose/fructose ratio in fermentations containing higher populations of Candida. Analysis of wine fermentation microbial ecology by using both culture-dependent and -independent methods reveals the complexity of yeast interactions enriched during spontaneous fermentations.     

Microbiota of the <Emphasis Type="Italic">Planalto de Bolona</Emphasis>: an artisanal cheese produced in uncommon environmental conditions in the Cape Verde Islands

The present study aimed to evaluate the dominant microbial community naturally present in the Planalto de Bolona cheese, produced in the Cape Verde Islands. Samples of milk, curd and cheese from two different producers were examined through culture-dependent and independent-methods. Traditional plating and genetic identification of lactic acid bacteria (LAB) and yeast isolates were carried out. Moreover, DNA and RNA extracted directly from samples were subjected to Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE). Concerning the LAB population, a total of 278 isolates were identified: Lactococcus lactis subsp. lactis and Enterococcus faecium represented the most isolated species. Regarding yeasts, the analysis of isolates throughout the manufacturing period showed a consistent presence of the genus Candida. Divergences in species detection between culture-dependent and culture-independent methods were observed, as well as between DNA and RNA analysis. PCR-DGGE underlined high heterogeneity among bacterial species while yeast microbiota was dominated by Aureobasidium pullulans at DNA level. The obtained results represent a first approach in the understanding of the Planalto de Bolona cheese microbial ecology and consequently may constitute a first step towards the full comprehension of the microbiota of this artisanal cheese produced in unusual environmental conditions in the Cape Verde Islands.