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Insect-fungi associations are common, with effects on insect hosts ranging from detrimental to beneficial. Fungi can provide multiple benefits to insects, including nutritional benefits, detoxification of food resources, and protection from detrimental microbes. In contrast, fungi can also harm insect hosts, acting as pathogens or food-spoiling saprotrophs. Bees (clade Anthophila) commonly associate with fungi, which can be found within their gastrointestinal tracts, in stored provisions within their nests, and in the flowers that they forage on. While certain pathogenic fungi (Ascosphaera, Aspergillus, Nosema) have been well-studied for the negative impacts they have on bee hosts, the majority of the fungi associated with bees are non-pathogenic, with unknown impacts on bee health. My dissertation focuses on understanding these associations between bees and their symbiotic fungi. Chapter 1 provides a review of existing literature on bee-fungi interactions, highlighting the fungal genera most commonly associated with bees and their nests. Notably, the yeast genera Starmerella and Zygosaccharomyces are commonly found in association with a wide variety of bee species, particularly with social bees, and exhibit adaptations to the bee nest environment. Chapters 2 and 3 utilize bumble bee systems to test the impacts of these common yeast symbionts on bee health. In Chapter 2, the impacts of fungicide exposure and yeast supplementation on the health of two bumble bee species (Bombus impatiens, B. vosnesenskii) are assessed. Impacts of treatments varied by bee species, with yeasts assisting B. vosnesenskii workers in recovering from the negative impacts of fungicide exposure. For B. impatiens, yeasts were beneficial to bee survival and reproduction regardless of fungicide exposure. In Chapter 3, the mechanisms behind the positive impacts of yeasts are tested using B. impatiens. Bees were fed treatment solutions varying in the presence of living yeast cells and yeast-modified media, and bee performance was measured. Impacts of yeast treatments were variable, and depended on the source colony bees were derived from, suggesting that benefits of yeast are context dependent. Overall, this research shows that many bee species are consistently associated with fungi which can benefit bee health and reproduction. However, the effects of these yeasts are inconsistent, with context-dependent benefits. Discovering the contexts in which yeasts are beneficial to bees, and the mechanisms underlying these benefits, is necessary to fully understand the roles that these microbes play in bee health.

The objective of this study was to isolate and identify a yeast strain from the kombucha beverage and evaluate in-vitro its potential as a novel starter in beverage fermentation. Starmerella davenportii Do18 was characterized for its cholesterol reduction; growth at different conditions such as temperatures (25, 30, 37 and 42 °C), low pH (1.2, 1.5, 2.0 3.0, and 7.0), bile salts (0%, 0.25%, 0.5%, 1% and 2%) high-sucrose stress (2%, 10%, 20%, 40% and 60%); and in-vitro survival in gastric and intestinal environments. Results showed that the yeast strain has a cholesterol-lowering capacity of 45% ± 2%, grew at temperature of 37 °C and is resistant to pH 1.5, 2% bile and 40% sucrose solution, could survive in simulated gastric and intestinal environments. The physicochemical characteristics of the fermented beverages were also evaluated, which indicated that the yeast has pH reduction capacity and can produce organic acids and volatile compound such as 2-phenylethanol. Furthermore, the fermented beverage also has high total phenolics and flavonoids content and showed great antioxidant and antimicrobial activities. Therefore, the findings of this research provide strong evidence that S. davenportii Do18 has good fermentation properties, can be a potential starter in food and beverage fermentation.

A novel yeast species of Starmerella vitis f.a. sp. nov. is proposed to accommodate five strains isolated from flowers, grapes and an insect in the Azores, Canada, Hungary, Palau and Taiwan. As the strains were genetically distinct, we used parsimony network analysis based on ITS-D1/D2 sequences to delineate the species in a statistically objective manner. According to sequence comparisons and phylogenetic analysis, the novel species is most closely related to Starmerella lactis-condensi. The two species cannot be distinguished by conventional physiological tests. The type strain of Starmerella vitis f.a., sp. nov. is CBS 16418T; Mycobank number MB 835251.

The conversion of grape juice to wine starts with complex yeast communities consisting of strains that have colonised the harvested grape and/or reside in the winery environment. As the conditions in the fermenting juice gradually become inhibitory for most species, they are rapidly overgrown by the more adaptable Saccharomyces strains, which then complete the fermentation. However, there are environmental factors that even Saccharomyces cannot cope with. We show that when the sugar content is extremely high, osmotolerant yeasts, usually considered as &ldquo, spoilage yeasts&ldquo, ferment the must. The examination of the yeast biota of 22 botrytised Tokaj Essence wines of sugar concentrations ranging from 365 to 752 g∙L&minus, 1 identified the osmotolerant Zygosaccharomyces rouxii, Candida (Starmerella) lactis-condensi and Candida zemplinina (Starmerella bacillaris) as the dominating species. Ten additional species, mostly known as osmotolerant spoilage yeasts or biofilm-producing yeasts, were detected as minor components of the populations. The high phenotypical and molecular (karyotype, mtDNA restriction fragment length polymorphism (RFLP) and microsatellite-primed PCR (MSP-PCR)) diversity of the conspecific strains indicated that diverse clones of the species coexisted in the wines. Genetic segregation of certain clones and interactions (antagonism and crossfeeding) of the species also appeared to shape the fermenting yeast biota.

Non-Saccharomyces yeasts are prevalent at the onset of grape must fermentations and can have a significant influence on the final wine product. In contrast to Saccharomyces cerevisiae, the biosynthetic pathways leading to aroma compound formation in these non-conventional yeasts, in particular those that are derived from amino acid metabolism, remains largely unexplored. Within a synthetic must environment, we investigated the amino acid utilization of four species (Hanseniaspora uvarum, Hanseniaspora osmophila, Zygosaccharomyces rouxii, Starmerella bacillaris) and S. cerevisiae. We report on the differential uptake preferences for amino acids with H. uvarum displaying the most rapid uptake of most amino acids. To investigate the fate of amino acids and their direct contribution to aroma synthesis in H. uvarum, H. osmophila and Z. rouxii, musts were supplemented with single amino acids. Aroma profiling undertaken after three days showed the synthesis of specific aroma compounds by the respective yeast was dependent on the specific amino acid supplementation. H. osmophila showed similarities to S. cerevisiae in both amino acid uptake and the synthesis of aroma compounds depending on the nitrogen sources. This study shows how the uptake of specific amino acids contributes to the synthesis of aroma compounds in wine fermentations using different non-Saccharomyces yeasts.

International audience; Nature is harnessed since ancient times to fulfill human needs, and yeast culture has been mastered for bakery, brewery, or the preparation of beverages. In this context, the two recently discovered yeast species Starmerella reginensis and Starmerella kourouensis, belonging to a genus related to fermentative activities in the literature, were explored via untargeted metabolomics approaches. Ultrahigh-performance liquid chromatography hyphenated with tandem mass spectrometry and a deep investigation of molecular networks and spectral data allowed the annotation of, respectively, 439 and 513 metabolites for S. reginensis and S. kourouensis, with approximatively 30% compound annotations and 40% chemical class annotations for both yeast strains. These analyses and Fourier transform ion cyclotron resonance mass spectrometry accurate metabolic profiles unveiled a rich content of alkaloids, lipids, amino acids, and terpenoids for S. reginensis. S. kourouensis presents a similar profile with more sulfated compounds. In short, these results enrich the current knowledge about Starmerella yeast secondary metabolites and reveal their significant structural diversity of small molecules.

The evolution of alcoholic fermentation in the non-conventional Wickerhamiella/Starmerella (W/S) yeast clade is characterized by the loss of native pyruvate decarboxylase (PDC1) and alcohol dehydrogenase (ADH) genes. In some species, the reacquisition of this via was achieved through horizontal gene transfer (HGT) of ADH and co-option of a native decarboxylase, Aro10. This work aimed to build on the previous knowledge regarding alcoholic fermentation in the W/S clade, by combining in silico data obtained from newly sequenced genomes, with phenotypic assays, aiming to assess alcoholic fermentation abilities and characterization of Adh enzymes, in a subset of species. Three independent HGT events were previously reported to have introduced different bacterial ADH1 in distinct subgroups of the W/S clade. Subgroups A, B and C harbour ADH1a, ADH1b and ADH1c, respectively. Subgroup ADH0 does not carry an ADH1. In this work, data supporting these three HGT events was obtained and two new HGT events of bacterial ADH6 were detected. Most ADH6 genes were acquired in the ancestor of the subgroups reported for ADH1, while one was found in a ADH0 species (Wickerhamiella slavikovae) that seemingly lacks other alcoholic fermentation genes (ADH1, PDC1 and ARO10). As for the remaining species, while ARO10 is present, PDC1 is absent. Ethanol production was generally observed in subgroup A, while its assimilation was verified in subgroups B and C, suggesting that Adh proteins are functional. It was confirmed that Adh1a from Starmerella bombicola is involved in the interconversion of acetaldehyde and ethanol, using NAD(H) and NADP(H) as cofactors, which contrasts with the specificity of Adh proteins from yeasts towards NAD(H). To further understand the evolution of the alcoholic fermentation in the W/S clade, it is essential to combine comparative genomics with the characterization of these enzymes to evaluate their role on the central carbon metabolism. A evolução da fermentação alcoólica no clado de leveduras não convencionais Wickerhamiella/Starmerella (W/S) é caracterizada pela perda dos genes nativos da piruvato descarboxilase (PDC1) e das álcool desidrogenases (ADH). Em algumas espécies, a reaquisição desta via foi conseguida através de transferências horizontais de genes (HGT) ADH e pela cooptação de uma descarboxilase nativa, Aro10. Este trabalho teve como objetivo partir do conhecimento prévio relativo à fermentação alcoólica no clado W/S, combinando dados in silico de novos genomas sequenciados, com ensaios fenotípicos, de forma a avaliar as capacidades fermentativas e caracterizar as enzimas Adh, num conjunto de espécies. Três eventos HGT independentes foram previamente identificados como tendo introduzido diferentes ADH1 bacterianos nos distintos subgrupos do clado W/S. Os subgrupos A, B e C possuem ADH1a, ADH1b e ADH1c, respetivamente. O subgrupo ADH0 não possui ADH1. Neste trabalho, dados que suportam estes três eventos HGT foram obtidos e dois novos eventos HGT de ADH6 bacterianos foram detetados. A maioria dos genes ADH6 foram adquiridos nos mesmos ancestrais dos subgrupos reportados para ADH1, enquanto um foi encontrado numa espécie ADH0 (Wickerhamiella slavikovae) que aparentemente não possui outros genes da fermentação alcoólica (ADH1, PDC1 e ARO10). Em relação às espécies restantes, enquanto ARO10 está presente, PDC1 está ausente. A produção de etanol foi geralmente observada no subgrupo A, enquanto a sua assimilação foi verificada nos subgrupos B e C, sugerindo que as proteínas Adh são funcionais. Foi confirmado o papel da Adh1a de Starmerella bombicola na interconversão de acetaldeído e etanol, usando NAD(H) e NADP(H) como cofatores, o que contrasta com a especificidade das proteínas Adh de leveduras relativamente a NAD(H). Para compreender melhor a evolução da fermentação alcoólica no clado W/S, é essencial combinar genómica comparativa com a caracterização destas enzimas, de forma a avaliar o seu papel no metabolismo central de carbono.

Sophorolipids are biobased compounds produced by the genera Starmerella and Pseudohyphozyma that gain exponential interest from academic and industrial stakeholders due to their mild and environmental friendly characteristics. Currently, industrially relevant sophorolipid volumetric productivities are reached up to 3.7 g center dot L-1 center dot h(-1) and sophorolipids are used in the personal care and cleaning industry at small scale. Moreover, applications in crop protection, food, biohydrometallurgy and medical fields are being extensively researched. The research and development of sophorolipids is at a crucial stage. Therefore, this work presents an overview of the state-of-the-art on sophorolipid research and their applications, while providing a critical assessment of scientific techniques and standardisation in reporting. In this review, the genuine sophorolipid producing organisms and the natural role of sophorolipids are discussed. Subsequently, an evaluation is made of innovations in production processes and the relevance of in-situ product recovery for process performance is discussed. Furthermore, a critical assessment of application research and its future perspectives are portrayed with a focus on the self-assembly of sophorolipid molecules. Following, genetic engineering strategies that affect the sophorolipid physiochemical properties are summarised. Finally, the impact of sophorolipids on the bioeconomy are uncovered, along with relevant future perspectives.

Kombucha fermentation is initiated by transferring a solid-phase cellulosic pellicle into sweetened tea and allowing the microbes that it contains to initiate the fermentation. This pellicle, commonly referred to as a symbiotic culture of bacteria and yeast (SCOBY), floats to the surface of the fermenting tea and represents an interphase environment, where embedded microbes gain access to oxygen as well as nutrients in the tea. To date, various yeast and bacteria have been reported to exist within the SCOBY, with little consensus as to which species are essential and which are incidental to Kombucha production. In this study, we used high-throughput sequencing approaches to evaluate spatial homogeneity within a single commercial SCOBY and taxonomic diversity across a large number (n = 103) of SCOBY used by Kombucha brewers, predominantly in North America. Our results show that the most prevalent and abundant SCOBY taxa were the yeast genus Brettanomyces and the bacterial genus Komagataeibacter, through careful sampling of upper and lower SCOBY layers. This sampling procedure is critical to avoid over-representation of lactic acid bacteria. K-means clustering was used on metabarcoding data of all 103 SCOBY, delineating four SCOBY archetypes based upon differences in their microbial community structures. Fungal genera Zygosaccharomyces, Lachancea and Starmerella were identified as the major compensatory taxa for SCOBY with lower relative abundance of Brettanomyces. Interestingly, while Lactobacillacae was the major compensatory taxa where Komagataeibacter abundance was lower, phylogenic heat-tree analysis infers a possible antagonistic relationship between Starmerella and the acetic acid bacterium. Our results provide the basis for further investigation of how SCOBY archetype affects Kombucha fermentation, and fundamental studies of microbial community assembly in an interphase environment.

Yeast species have been spontaneously participating in food production for millennia, but the scope of applications was greatly expanded since their key role in beer and wine fermentations was clearly acknowledged. The workhorse for industry and scientific research has always been Saccharomyces cerevisiae. It occupies the largest share of the dynamic yeast market, that could further increase thanks to the better exploitation of other yeast species. Food-related ‘non-conventional’ yeasts (NCY) represent a treasure trove for bioprospecting, with their huge untapped potential related to a great diversity of metabolic capabilities linked to niche adaptations. They are at the crossroad of bioprocesses and biorefineries, characterized by low biosafety risk and produce food and additives, being also able to contribute to production of building blocks and energy recovered from the generated waste and by-products. Considering that the usual pattern for bioprocess development focuses on single strains or species, in this review we suggest that bioprospecting at the genus level could be very promising. Candida, Starmerella, Kluyveromyces and Lachancea were briefly reviewed as case studies, showing that a taxonomy- and genome-based rationale could open multiple possibilities to unlock the biotechnological potential of NCY bioresources.

Microbial diversity present on grapes in wineries, and throughout fermentation has been associated with important metabolites for final wine quality. Although microbiome-metabolome associations have been well characterized and could be used as indicators of wine quality, the impact of regionality on the microbiome and metabolome is not well known. Additionally, studies between microbiome and metabolome have been conducted on single species grape such as Vitis vinifera instead of other species and interspecific hybrids. Although the Pennsylvania wine industry is relatively young compared to California, the industry has been experiencing rapid growth over the past decade and is expected to continue to grow in the future. Pennsylvania’s climate of cold winters and high levels of rainfall throughout the growing season favors cultivation of interspecific hybrid grapes such as Vitis ssp. Chambourcin, one of the most commonly grown hybrid varieties in the state. Chambourcin is a prime candidate for studying the impact of regionality on microbiome-metabolome interactions as interspecific hybrid varieties could shape the future of winemaking. Here, we identify for the first time the regional distribution of microbial communities and their interactions with volatile metabolome during fermentation (0–20 days) by integrating high throughput Illumina sequencing (16S and ITS) and headspace-solid phase microextraction-gas chromatography-mass spectrometry. Analyzing 88 samples from nine wineries in the Central and East Pennsylvania regions, we observed high microbial diversity during early stages of fermentation (1–4 days) where non-Saccharomyces yeasts such as Starmerella and Aureobasidium and non-Oenococcus bacteria, Sphingomonas, likely contribute to microbial terroir to the resulting wines. Furthermore, key differentiators between two regions in Pennsylvania, as identified by LEfSe analysis, include the fungal genera Cladosporium and Kazachstania and the bacterial genera Lactococcus and Microbacterium. Moreover, 29 volatile fermentation metabolites were discriminated significantly (variable importance in projection > 1) between the two regions as shown by Partial Least Squares-Discriminant Analysis. Finally, Spearman’s correlation identified regional differences of microbial-metabolite associations throughout fermentation that could be used for targeted microbiome manipulation to improve wine quality and preserve regionality. In summary, these results demonstrate the microbial signatures during fermentation and differential microorganisms and metabolites further support impact of regionality on Chambourcin wines in Pennsylvania.

International audience; The aim of the present study was to assess the cultivable microbiota of "mothers" of Vino cotto collected from production of different years 1890, 1895, 1920, 1975, 2008. A total of 73 yeasts and 81 bacteria were isolated. Starmerella lactis-condensi, Starmerella bacillaris, Hanseniaspora uvarum, Saccharomyces cerevisiae, Hanseniaspora guillermondi and Metschnikowia pulcherrima were identified. Bacteria isolates belonged to lactic acid bacteria (Lactiplantibacillus plantarum and Pediococcus pentosaceus) and acetic acid bacteria (Gluconobacter oxydans). Remarkable biodiversity was observed for Starm. bacillaris, as well as L. plantarum and G. oxydans. Organic acids and volatile compounds were also determined. Malic and succinic acids were the main ones with values ranging from 8.49 g/L to 11.76 g/L and from 4.15 g/L to 7.73 g/L respectively, while citric acid was present at low concentrations (

The pollen stores of bumble bees host diverse microbiota that influence overall colony fitness. Yet, the taxonomic identity of these symbiotic microbes is relatively unknown. In this descriptive study, we characterized the microbial community of pollen provisions within captive-bred bumble bee hives obtained from two commercial suppliers located in North America. Findings from 16S rRNA and ITS gene-based analyses revealed that pollen provisions from the captive-bred hives shared several microbial taxa that have been previously detected among wild populations. While diverse microbes across phyla Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Ascomycota were detected in all commercial hives, significant differences were detected at finer-scale taxonomic resolution based on the supplier source. The causative agent of chalkbrood disease in honey bees, Ascosphaera apis, was detected in all hives obtained from one supplier source, although none of the hives showed symptoms of infection. The shared core microbiota across both commercial supplier sources consisted of two ubiquitous bee-associated groups, Lactobacillus and Wickerhamiella/Starmerella clade yeasts that potentially contribute to the beneficial function of the microbiome of bumble bee pollen provisions.

Among typical Portuguese sausages, the cacholeira blood sausage undoubtedly represents one of the most popular preparations. To the authors' knowledge, a lack of information on both the microbiota and the volatile organic compounds (VOCs) of this blood-containing sausage emerges from the available scientific literature. This study represents the first characterization of physico-chemical, microbiological and volatile traits of Portuguese cacholeira blood sausage. To this end, ready-to-eat cacholeira blood sausages were collected from two production batches manufactured in summer (batch 1) and autumn (batch 2). Viable counts showed active microbial communities mainly composed by lactic acid bacteria, coagulase negative cocci, enterococci and eumycetes. The metataxonomic approach showed a simple bacterial composition, which was dominated by Lactobacillus sakei in both the analyzed batches (1 and 2) considered. Carnobacterium, Enterococcus, Kluyvera, Lactococcus and Serratia were found as minor genera. The mycobiota varied according to the production season. Batch 1 was dominated by Starmerella apicola, Debaryomyces hansenii and Candida tropicalis, whereas batch 2 was dominated by D. hansenii. Moreover, Aspergillus spp., Kurtzmaniella zeylanoides, Saccharomyces cerevisiae, Kurtzmaniella santamariae, Brettanomyces bruxellensis and Pichia kluyveri were detected in both the batches as minority species. Seventy-two volatile compounds were identified, including esters, phenols, terpenoids, acids, alcohols, ketones, aldehydes, lactones, furans, sulphur and nitrogen compounds. Significant differences were seen in the amount of some compounds, as a feasible consequence of differences in the raw materials, artisan production and seasonality.

International audience; Analysis of yeasts isolated from various biotopes in French Guiana led to the identification of two strains isolated from flowers and designated CLIB 1634(T) and CLIB 1707(T). Comparison of the D1/D2 domain of the large subunit (LSU D1/D2) rRNA gene sequences of CLIB 1634(T) and CLIB 1707(T) to those in the GenBank database revealed that these strains belong to the Starmerella Glade. Strain CLIB 1634(T) was shown to diverge from the closely related Starmerella apicola type strain CBS 2868 T with a sequence divergence of 1.34 and 1.30 %, in the LSU D1/D2 rRNA gene and internal transcribed spacer (ITS) sequences respectively. Strain CLIB 1634(T) and Candida apicola CBS 2868(T) diverged by 3.81 and 14.96 % at the level of the protein-coding gene partial sequences EF-1 alpha and RPB2, respectively. CLIB 1707(T) was found to have sequence divergence of 3.88 and 9.16 % in the LSU D1/D2 rRNA gene and ITS, respectively, from that of the most closely related species Starmerella ratchasimensis type strain CBS 10611(T). The species Starmerella reginensis f.a., sp. nov. and Starmerella kourouensis f.a., sp. nov. are proposed to accommodate strains CLIB 1634(T) (=CBS 15247(T)) and CLIB 1707(T) (=CBS 15257(T)), respectively.

Six novel yeast species, Starmerella camargoi f.a., sp. nov., Starmerella ilheusensis f.a., sp. nov., Starmerella litoralis f.a., Starmerella opuntiae f.a., sp. nov., sp. nov., Starmerella roubikii f.a., sp. nov. and Starmerella vitae f.a, sp. nov. are proposed to accommodate 19 isolates recovered from ephemeral flowers or bees in Brazil, Costa Rica and Belize. Sequence analysis of the ITS-5.8S region (when available) and the D1/D2 domains of the large subunit of the rRNA gene showed that the six novel yeasts are phylogenetically related to several species of the Starmerella clade. The type strains are Starmerella camargoi f.a., sp. nov. UFMG-CM-Y595T (=CBS 14130T; Mycobank number MB 822640), Starmerella ilheusensis f.a., sp. nov. UFMG-CM-Y596T (=CBS CBS14131T; MB 822641), Starmerella litoralis f.a., sp. nov. UFMG-CM-Y603T (=CBS14104T; MB 822642), Starmerella opuntiae f.a., sp. nov. UFMG-CM-Y286T (=CBS 13466T; MB 822643), Starmerella roubikii f.a., sp. nov. UWOPS 01-191.1 (=CBS 15148; MB 822645) and Starmerella vitae f.a., sp. nov. UWOPS 00-107.2 (=CBS 15147T; MB 822646). In addition, 25 species currently assigned to the genus Candida are reassigned formally to the genus Starmerella.

Glycolipid microbial biosurfactants, such as sophorolipids (SLs), generate high industrial interest as 100% biobased alternatives for traditional surfactants. A well-known success story is the efficient SL producer Starmerella bombicola, which reaches titers well above 200g/L. Recent engineering attempts have enabled the production of completely new types of molecules by S. bombicola, e.g. the bolaform SLs. Scale-up of bolaform SL production was performed at 150L scale. The purified product was evaluated in detergent applications, as classic SLs are mostly applied in eco-friendly detergents. In this paper, we show that they can be used as green and non-irritant surfactants in for example (automatic) dishwashing applications. However, due to the presence of an ester function in the biosurfactant molecule a limited chemical stability at higher pH values (>6.5) was noticed, (therefore called 'non-symmetrical' (nsBola)) which, is a major drawback that will most likely inhibit market introduction. An integrated bioprocess design (IBPD) strategy was thus applied to resolve this issue. The strategy was to replace the fed fatty acids with fatty alcohols, to generate so-called "symmetrical bolaform (sBola) sophorosides (SSs)," containing two instead of one glycosidic bond. Next to a change in feeding strategy, the blocking of the fatty alcohols from metabolizing/oxidizing through the suggested omega-oxidation pathway was necessary. For the latter, two putative fatty alcohol oxidase genes (fao1 and fao2) were identified in the S. bombicola genome and deleted in the bolaform SL producing strain (Delta at Delta sble). Shake flask experiments for these new strains (Delta at Delta sble Delta fao1 and Delta at Delta sble Delta fao2) were performed to evaluate if the fed fatty alcohols were directly implemented into the SL biosynthesis pathway. Indeed, sBola sophorosides (SSs) production up to 20 g/L was observed for the Delta at Delta sble Delta fao1 strain. Unexpectedly, the Delta at Delta sble Delta fao2 strain only produced minor amounts of sBola sophorosides (SSs), and mainly nsBola SLs (alike the parental Delta at Delta sble strain). The sBola sophorosides (SSs) were purified and their symmetrical structure was confirmed by NMR. They were found to be significantly more stable at higher pH, opening up the application potential of the biosurfactant by enhancing its stability properties.

Nutrition is vital for health and immune function in honey bees (Apis mellifera). The effect of diets enriched with bee-associated yeasts and essential oils of Mexican oregano (Lippia graveolens) was tested on survival, food intake, accumulated fat body tissue, and gene expression of vitellogenin (Vg), prophenoloxidase (proPO) and glucose oxidase (GOx) in newly emerged worker bees. The enriched diets were provided to bees under the premise that supplementation with yeasts or essential oils can enhance health variables and the expression of genes related to immune function in worker bees. Based on a standard pollen substitute, used as a control diet, enriched diets were formulated, five with added bee-associated yeasts (Starmerella bombicola, Starmerella etchellsii, Starmerella bombicola 2, Zygosaccharomyces mellis, and the brewers’ yeast Saccharomyces cerevisiae) and three with added essential oils from L. graveolens (carvacrol, thymol, and sesquiterpenes). Groups of bees were fed one of the diets for 9 or 12 days. Survival probability was similar in the yeast and essential oils treatments in relation to the control, but median survival was lower in the carvacrol and sesquiterpenes treatments. Food intake was higher in all the yeast treatments than in the control. Fat body percentage in individual bees was slightly lower in all treatments than in the control, with significant decreases in the thymol and carvacrol treatments. Expression of the genes Vg, proPO, and GOx was minimally affected by the yeast treatments but was adversely affected by the carvacrol and thymol treatments.

Among many alternatives, biodiesel is considered as one of the most promising biofuels for phasing out fossil-derived fuels and mitigating their adverse ecological impacts. However, conventional alkali- or acid-catalyzed transesterification of plant oils triggers food security, economic, and even environmental (e.g., land-use change and greenhouse gas emissions) challenges. In contrast, advanced biodiesel (i.e., second-generation biodiesel) could be synthesized from oleaginous microorganisms such as fungal and yeast. The most oleaginous fungal genera are Alternaria, Aspergillus, Chaetomium, Colletotrichum, Cunninghamella, Epicoccum, Mortierella, Mucor, Thamnidium, and Zygorhynchus. On the other hand, Yarrowia, Candida, Rhodotorula, Rhodosporidium, Aureobasidium, Cryptococcus, Metschnikowia, Kodamaea, Trichosporon, Sporidiobolus, Sporobolomyces, Occultifur, Starmerella, Pichia, and Lipomyces are the most common oleaginous yeast genera. Typically, the main lipid classes produced by oleaginous fungi and yeasts are polyunsaturated fatty acids (PUFAs, such as arachidonic acid, c-linolenic acid, dihomo-γ-linolenic acid, docosahexaenoic acid, and eicosapentanoic acid) and triglycerides (TGs) and sterol esters (SEs), respectively. Alternatively, oleaginous yeast or fungal-origin lipases could be exploited in biodiesel production via enzymatic transesterification approach. It is worth mentioning that the most widely used lipases in biodiesel industry are those extracted from yeasts (e.g., Candida antarctica and Candida rugosa) and filamentous fungi (e.g., Aspergillus niger, Penicillium expansum, Rhizomucor miehei, Rhizopus oryzae, and Thermomyces lanuginosus). In this chapter, the current status for the application of different single-cell oil (SCO)-producing filamentous fungi and yeast species is scrutinized in the first part. Same section also deals with some technological strategies for reducing SCO production cost with a particular attention on the abilities of these microorganisms for assimilating different organic wastes. In the second part of the chapter, yeast and fungal lipases as biocatalyst for biodiesel production are discussed. Overall, despite some improvements, further research in this field with respect to identification of novel oleaginous microorganisms (with high lipase and/or lipid content) as well as development of more efficient technologies (microbial fermentation, oil extraction, and biodiesel production) must be conducted to address some techno-economic constraints of SCO-based biodiesel industry.

The Wickerhamiella and Starmerella genera form a clade (W/S clade) that branches close to Yarrowia lipolytica in the Saccharomycotina species tree. It comprises approximately 90 recognized species and 50 putative new species not formally described yet. The large majority of the members of the W/S clade are ecologically associated with flowers and floricolous insects. Many species exhibit unusual metabolic traits, like fructophily and the production of sophorolipids, which are glycolipids that can be used as environmentally friendly biosurfactants. Genomic data have not only firmly established the W/S clade but have also revealed a tumultuous evolution of metabolism marked by losses and gains of important metabolic pathways, among which alcoholic fermentation. Possibly the most surprising finding brought to light by comparative genomics concerned the large number of genes acquired by some species of the W/S clade from bacteria through horizontal gene transfer, many of which were shown to be functional in their new setting. This was facilitated by the genetic tractability of one species in the clade, Starmerella bombicola, which is used for the industrial production of sophorolipids. We suggest that high-density coverage of genome sequencing in this clade, combined with the possibility to conduct molecular genetics experiments in at least one species, has the potential to set the stage for yet more exciting discoveries concerning the evolution of yeast metabolism.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. The yeasts belonging to the Wickerhamiella and Starmerella genera (W/S clade) share a distinctive evolutionary history marked by loss and subsequent reinstatement of alcoholic fermentation mediated by horizontal gene transfer events. Species in this clade also share unusual features of metabolism, namely the preference for fructose over glucose as carbon source, a rare trait known as fructophily. Here we show that fructose may be the preferred sugar in W/S-clade species because, unlike glucose, it can be converted directly to mannitol in a reaction with impact on redox balance. According to our results, mannitol is excreted to the growth medium in appreciable amounts along with other fermentation products such as glycerol and ethanol but unlike the latter metabolites mannitol production increases with temperature. We used comparative genomics to find genes involved in mannitol metabolism and established the mannitol biosynthesis pathway in W/S-clade species Starmerella bombicola using molecular genetics tools. Surprisingly, mannitol production seems to be so important that St. bombicola (and other W/S-clade species) deploys a novel pathway to mediate the conversion of glucose to fructose, thereby allowing cells to produce mannitol even when glucose is the sole carbon source. Using targeted mutations and 13C-labeled glucose followed by NMR analysis of end-products, we showed that the novel mannitol biosynthesis pathway involves fructose-6-phosphate as an intermediate, implying a key role for a yet unknown fructose-6-P phosphatase. We hypothesize that mannitol production contributed to mitigate the negative effects on redox balance of the ancient loss of alcoholic fermentation in the W/S clade. Presently, mannitol also seems to play a role in stress protection. info:eu-repo/semantics/publishedVersion

Secondary peat swamp forest (PSF) arise by degradation of primary PSF as a result of fire and human activities. Yeasts diversity of Kuan Kreng (KK) and Rayong Botanical Garden (RBG) PSF, which are two secondary PSF in southern and in eastern Thailand, respectively, were investigated. Yeasts were isolated from soil and peat soil by the dilution plate and enrichment techniques. From six samples collected from KK PSF, 35 strains were obtained, and they were identified based on the sequence analysis of the D1/D2 region of the large subunit (LSU) rRNA gene 13 species in 12 genera, and one potential new species of the genus Galactomyces were detected. Thirty-two strains were obtained from six samples collected from RBG PSF and 26 strains were identified as 13 known yeast species in 11 genera, whereas six strains were found to represent two potential new species of the genera Papiliotrema and Moesziomyces. Among yeast strains isolated from KK PSF, the number of strains in the phylum Ascomycota and Basidiomycota were equal, whereas there were slightly fewer strains in Ascomycota than in Basidiomycota among the strains obtained from RBG PSF. The yeast strains were evaluated for their antagonistic activities against fungal pathogens which cause rice diseases (Fusarium moniliforme, Helminthosporium oryzae, Rhizoctonia solani, Curvularia lunata and Pyricularia grisea) and postharvest disease of fruits (Phytophthora palmivora, Lasiodiplodia theobromae and Colletotrichum gloeosporioides). Twelve strains of seven species were found to be antagonistic yeast strains. Starmerella kuoi DMKU-SPS13-6, Hanseniaspora lindneri DMKU ESS10-9 and Piskurozyma taiwanensis DMKU-SPS12-2 capable to inhibit R. solani by 70.1-76.2%, Wickerhamomyces anomalus DMKU SPS6-1 and three Rhodotorula taiwanensis strains (DMKU SPS8-1, DMKU ESS9-3, DMKU SPS9-2) inhibited C. lunata by 69.8-71.9%, Hanseniaspora lindneri DMKU ESS10-9 and Scheffersomyces spartinae DMKU SPS9-3 inhibited P. grisea by 81.9-84.4% and four Papiliotrema laurentii strains (DMKU-SPS15-1, DMKU-ESS11-2, DMKU-ESS8-2, DMKU-ESS6-4) inhibited P. palmivora by 53.2-59.5%.

Nectar is frequently inhabited by a limited number of microorganisms. Nonetheless, these species can quickly attain relatively high cell densities. This is quite surprising because of the limited availability of nutrients and unbalanced Carbon/Nitrogen ratios. Because nectar yeasts commonly aggregate around pollen and pollen grains are particularly rich in proteins, it has been suggested that the presence of pollen in nectar contributes to enhanced growth of yeasts in nectar, but compelling experimental evidence is still lacking. In this study, we conducted in vitro growth experiments to investigate whether the addition of pollen to sugar water increased growth of yeasts that naturally occur in nectar and honey provisions: Metschnikowia reukaufii, Starmerella orientalis and Torulaspora delbueckii. Our results indicate that yeasts benefit from the addition of pollen to a sugar-dominated medium, but the effects depended on type of pollen used. Overall, these results demonstrate that pollen plays an important role in the population dynamics of nectar-inhabiting yeasts and supports the idea that the chemical composition and the concentration of dehisced pollen may be more important factors determining the population growth of nectar yeasts than the chemistry of the nectar itself. ispartof: FEMS MICROBIOLOGY LETTERS vol:366 issue:15 ispartof: location:England status: published

Toddy is a traditional mild-alcoholic drink of India, which is produced from fresh palm saps by natural fermentation. We studied the successional changes in bacterial and fungal communities during the natural fermentation (0 h-96 h) of toddy. During fermentation, alcohol content of the fermenting saps increased significantly from 0.6 %±0.15 to 5.6 %±0.02, pH decreased from 6.33 %±0.02-3.93 ± 0.01, volatile and titratable acidity acidity (g/100 mL) increased from 0.17 ± 0.02 (0 h) to 0.48 ± 0.02 (96 h) and 1.30 ± 0.005 (0 h) to 2.47 ± 0.005 (96 h), respectively. Total sugar content and ˚BRIX also decreased during the fermentation. Firmicutes (78.25 %) was the most abundant phylum followed by Proteobacteria (21.57 %). Leuconostoc was the most abundant genus in the early stages of fermentation. However, Lactobacillus and Gluconoacetobacter were found abundant with increase in pH during the later phases of fermentation (72 h-96 h). Ascomycota (99.02 %) was the most abundant fungal phylum. Hanseniaspora was the abundant yeast in the initial stages of fermentation, whereas the population of Saccharomyces increased significantly after 24 h of fermentation. Torulaspora, Lachancea and Starmerella showed their heterogeneous distribution throughout the fermentation. Computational analysis of metagenomes based on KEGG and MetaCyc databases showed different predictive functional profiles such as folate biosynthesis, glutathione metabolism, terpenoids biosynthesis and biosynthesis of amino acids with significant differences between the fresh palm saps and fermenting saps during toddy fermentation.

The physicochemical characteristics and yeasts diversity in honey samples from 17 species of stingless bees of the genera Nannotrigona, Melipona, Plebeia, Scraptotrigona, and Tetragonisca cultivated in Southern Brazil were determined. The sugar content, moisture, water activity, pH, reducing sugars/total sugar ratio, and total yeast population varied significantly among the honey from the different bee species. The highest yeast population was found in the Plebeia's honey samples and correlated with their high water-activity. Sixteen yeast species were identified based on the nuclear large subunit (26S) ribosomal RNA partial sequences. The genera Starmerella and Zygosaccharomyces were found predominant, with a high prevalence of Starmerella sp., S. etchellsii, and S. apicola. Some yeast species were only identified in honey samples from specific bee species indicating a close relationship between the yeasts and the insects. For the first time, Wickerhamomyces sydowiorum in honey is being reported. In general, the yeast species isolated from stingless bee honey samples demonstrated high osmotolerance and low sugar assimilation.

Pollen stored by bees undergoes a fermentation marked by the presence of lactic acid bacteria and yeasts. It results in bee bread. Past studies have singled out Starmerella (Candida) magnoliae as the most common yeast species in honey bee-stored bee bread. Starmerella species are ecological specialists with potential biotechnological value. The rarity of recent studies on yeasts in honey bees prompted us to generate new information on yeast diversity during the conversion of bee-collected pollen to bee bread. Bees and stored pollen from two apiaries in Belgium were sampled, a yeast isolation protocol was developed, yeast isolates were grouped according to their macro- and micromorphology, and representative isolates were identified using DNA sequences. Most of the 252 identified isolates belonged to the genera Starmerella, Metschnikowia, and Zygosaccharomyces. The high abundance of yeasts in fresh bee bread decreased rapidly with the storage duration. Starmerella species dominated fresh bee bread, while mostly Zygosaccharomyces members were isolated from aged bee bread. Starmerella (Candida) apis, a rarely isolated species, was the most frequent and abundant species in fresh bee bread. Yeasts from the bee&rsquo, s honey stomach and from pollen pellets obtained from bees hind legs were dominated by Metschnikowia species. The distinctive communities from pollen pellets over fresh bee bread to aged bee bread indicate a non-random distribution of these yeasts.

In Brazilian savannas, stingless bees are associated with yeast communities composed mainly of Starmerella species. This study aimed to establish the distribution of yeasts associated with the bee species Frieseomelitta varia, Scaptotrigona aff. postica, Scaptotrigona polysticta, Tetragonisca angustula angustula, Melipona compressipes manaosensis and Melipona scutellaris in northern savannas and the importance of yeasts in the resource partitioning of bees. We collected 380 bees from six species, which were individually placed in Petri plates containing rich medium for 15 min. Yeasts were then isolated, resulting in the identification of 54 species, 48 of which were ascomycetous, while six were basidiomycetous. Torulaspora delbrueckii was the most frequently isolated yeast species in this study, followed by Pichia membranifaciens, Starmerella apicola, Pichia kluyveri, Starmerella meliponinorum and Starmerella bombicola. The total number of Starmerella species identified showed that these species are prevalent in association with these stingless bees. These communities differ from stingless bee-associated yeasts found in the southern savannas of Brazil. Although stingless bees are generalist foragers, there are some yeasts showing high prevalence in only one bee species, resulting in differences in niche breadth that may point to a role of yeasts in the resource partitioning of bees in Brazilian savannas.