Convergent expansions of keystone gene families drive metabolic innovation in a major eukaryotic clade.

Many remarkable innovations have repeatedly occurred across vast evolutionary distances. When convergent traits emerge on the tree of life, they are sometimes driven by the same underlying gene families, while other times many different gene families are involved. Conversely, a gene family may be repeatedly recruited for a single trait or many different traits. To understand the general rules governing convergence at both genomic and phenotypic levels, we systematically tested associations between 56 binary metabolic traits and gene count in 14,710 gene families from 993 species of Saccharomycotina yeasts. Using a recently developed phylogenetic approach that reduces spurious correlations, we discovered that gene family expansion and contraction was significantly linked to trait gain and loss in 45/56 (80%) of traits. While 601/746 (81%) of significant gene families were associated with only one trait, we also identified several 'keystone' gene families that were significantly associated with up to 13/56 (23%) of all traits. These results indicate that metabolic innovations in yeasts are governed by a narrow set of major genetic elements and mechanisms.

Diversity of endophytic bacteria with antimicrobial potential isolated from marine macroalgae from Yacila and Cangrejos beaches, Piura-Peru.

Endophytic bacteria found in marine macroalgae have been studied for their potential antimicrobial activity, consequently, they could serve as a valuable source of bioactive compounds to control pathogenic bacteria, yeasts, and fungi. Algae endophytic bacteria were isolated from Caulerpa sp., Ulva sp., Ahnfeltiopsis sp., and Chondracantus chamissoi from Yacila and Cangrejo Beaches (Piura, Peru). Antimicrobial assays against pathogenic bacteria were evaluated using cross-culture, over-plate, and volatile organic compound tests. Afterward, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of selected crude extracts were determined, also ITS molecular analysis, antifungal activity, and PCR of iturin, fengycin, and surfactin genes were performed for bacteria strains exhibiting better activity. Forty-six algae endophytic bacteria were isolated from algae. Ten strains inhibited gram-positive pathogenic bacteria (Enterococcus faecalis, Staphylococcus epidermidis, S. aureus, and Listeria monocytogenes), and 12 inhibited gram-negative bacteria (Escherichia coli and Salmonella enteric sv typhimurium). Bacteria with better activity belong to Bacillus sp., Kluyvera ascorbata, Pantoea agglomerans, Leclercia adecarboxylata, and Enterobacter sp., which only four showed antifungal activities against Candida albicans, C. tropicalis, Colletotrichium sp., Fusarium sp., Fusarium oxysporum, and Alternaria sp. Furthermore, K. ascorbata YAFE21 and Bacillus sp. YCFE4 exhibited iturin and fengycin genes. The results indicate that the algae endophytic bacteria found in this study, particularly K. ascorbata YAFE21, Bacillus sp. YCFR6, L. adecarboxylata CUFE2, Bacillus sp. YUFE8, Enterobacter sp. YAFL1, and P. agglomerans YAFL6, could be investigated as potential producers of antimicrobial compounds due to their broad activity against various microorganisms.

Functional potential of a new plant-based fermented beverage: Benefits through non-conventional probiotic yeasts and antioxidant properties.

Functional foods represent one of the fastest-growing, newer food category, and plant sources with functional properties are increasingly used as analogues of fermented milk-based derivatives. In this study, blended wort-rooibos beverages fermented with probiotic yeasts are proposed for the first time. Benefits of functional, non-conventional Lachancea thermotolerans (Lt101), Kazachstania unispora (Kum3-B3), Meyerozyma guilliermondii (Mg112), Meyerozyma caribbica (Mc58) and Debaryomyces hansenii (Dh36) yeast strains and the content of bioactive metabolites were evaluated. Viability tests on the probiotic yeasts confirmed previous results obtained in other matrices. The functional footprint of probiotic yeasts Lt101, Mg112 and Dh36 was confirmed by a balanced nutritional profile of the final drinks, also supported by aromatic and sensory analyses. In vitro estimated glycaemic index ranged between 77 % and 87 % without any influence on glycaemic response. Strains Dh36, Mc58, Kum3-B3 and Mg112 showed high antioxidant capacity and high total phenolic content, supporting the health promoting effect of the beverages.

Potential probiotic and functional properties of Brettanomyces strains isolated from kombucha tea.

Kombucha, a beverage traditionally obtained through the fermentation of tea, is believed to have beneficial health properties. Therefore, characterizing the microorganisms responsible for this fermentation is essential to demonstrate its potential health benefits and to identify candidates for new probiotics. In this study, four probiotic yeast strains isolated from kombucha tea were identified, by the PCR-RFLP analysis of the ribosomal ITS region and the sequence of the D1/D2 domain of the 26S rDNA, as Brettanomyces bruxellensis (UVI55 and UVI56) and B. anomalus (UVI57 and UVI58). Properties relevant to probiotics were also studied in these strains. All of them showed excellent survival in simulated gastric (99%-100%) and duodenal (95%-100%) juices. The ability to self-aggregate (38%-100%), adhesion to xylene (15%-50%) and, above all, adhesion to Caco-2 cells (4%-21%), revealed its potential capacity to adhere to the intestinal epithelium. In addition, the tested strains showed excellent antioxidant capacity (82%-94%), antimicrobial activity against different pathogens (Escherichia coli, Staphylococcus aureus, Salmonella enterica, Listeria monocytogenes, and Bacillus cereus), as well as remarkable cytotoxic activity against colon, melanoma and ovarian tumor cell lines. Finally, using Caenorhabditis elegans as a model, strain UVI56 exhibited ability to both extend the lifespan of the nematode and protect it against infection by S. enterica. These results support the probiotic and functional properties of the analyzed strains. In conclusion, the study revealed that kombucha tea could be a source of potential probiotics that contribute to its health-promoting properties and that the characterized Brettanomyces strains could be exploited directly as probiotics or for the development of new functional foods.

The impact of simultaneous inoculation with Torulaspora delbrueckii and Hanseniaspora uvarum combined with Saccharomyces cerevisiae on chemical and sensory quality of Sauvignon blanc wines.

Non-Saccharomyces yeasts have great potential in improving wine quality, showing personality characteristics, and highlighting the terroir of wine. In this study, we evaluated the impact of simultaneous inoculation with the non-Saccharomyces yeasts Torulaspora delbrueckii or (and) Hanseniaspora uvarum in combination with Saccharomyces cerevisiae (EC1118 or VL3) on the aromatic compounds and sensory quality of Sauvignon blanc wines. The growth of yeast groups in the alcoholic fermentation process was tracked using fluorescence in situ hybridization. The presence of non-Saccharomyces yeast notably impacted the distribution of S. cerevisiae and was related to the species of yeast. The co-fermentation of H. uvarum and S. cerevisiae improved the content of total esters, especially acetate esters. Simultaneous inoculation of T. delbrueckii or (and) H. uvarum significantly increased the content of total terpenes, especially linalool. Similar results were found for some higher alcohols and organic acids. Sensory evaluation showed that the wines mixed fermentation with H. uvarum had significantly tropical fruit aroma characteristics. Citrus and mineral notes, typical aroma characteristics of Sauvignon blanc wine, were enhanced by mixed fermentation strategies with T. delbrueckii or (and) H. uvarum and different S. cerevisiae. Hence, co-fermentation by T. delbrueckii or H. uvarum combined with S. cerevisiae could significantly improve the sensory quality of Sauvignon blanc wine.

Codon usage bias in yeasts and its correlation with gene expression, growth temperature, and protein structure.

Codon usage bias (CUB) has been described in viruses, prokaryotes, and eukaryotes and has been linked to several cellular and environmental factors, such as the organism's growth temperature, gene expression levels, and regulation of protein synthesis and folding. Most of the studies in this area have been conducted in bacteria and higher eukaryotes, in some cases with different results. In this study, a comparative analysis of CUB in yeasts isolated from cold and template environments was performed in order to evaluate the correlation of CUB with yeast optimal temperature of growth (OTG), gene expression levels, cellular function, and structure of encoded proteins. Among the main findings, highly expressed ORFs tend to have a more similar CUB within and between yeasts, and a direct correlation between codons ending in C and expression level was generally found. A low correspondence between CUB and OTG was observed, with an inverse correlation for some codons ending in C. The clustering of yeasts based on their CUB partially aligns with their OTG, being more consistent for yeasts with lower OTG. In most yeasts, the abundance of preferred codons was generally lower at the 5' end of ORFs, higher in segments encoding beta strand, lower in segments encoding extracellular and transmembrane regions, and higher in "translation" and "energy metabolism" pathways, especially in highly expressed ORFs. Based on our findings, it is suggested that the abundance and distribution of preferred and non-preferred codons along mRNAs contribute to proper protein folding and functionality by regulating protein synthesis rates, becoming a more important factor under conditions that require faster protein synthesis in yeasts.

Effect of Hanseniaspora vineae and Saccharomyces cerevisiae co-fermentations on aroma compound production in beer.

In recent years, the boom of the craft beer industry refocused the biotech interest from ethanol production to diversification of beer aroma profiles. This study analyses the fermentative phenotype of a collection of non-conventional yeasts and examines their role in creating new flavours, particularly through co-fermentation with industrial Saccharomyces cerevisiae. High-throughput solid and liquid media fitness screening compared the ability of eight Saccharomyces and four non-Saccharomyces yeast strains to grow in wort. We determined the volatile profile of these yeast strains and found that Hanseniaspora vineae displayed a particularly high production of the desirable aroma compounds ethyl acetate and 2-phenethyl acetate. Given that H. vineae on its own can't ferment maltose and maltotriose, we carried out mixed wort co-fermentations with a S. cerevisiae brewing strain at different ratios. The two yeast strains were able to co-exist throughout the experiment, regardless of their initial inoculum, and the increase in the production of the esters observed in the H. vineae monoculture was maintained, alongside with a high ethanol production. Moreover, different inoculum ratios yielded different aroma profiles: the 50/50 S. cerevisiae/H. vineae ratio produced a more balanced profile, while the 10/90 ratio generated stronger floral aromas. Our findings show the potential of using different yeasts and different inoculum combinations to tailor the final aroma, thus offering new possibilities for a broader range of beer flavours and styles.

Oligomeric Structure of Yeast and Other Invertases Governs Specificity.

Invertases, or ß-fructofuranosidases, are metabolic enzymes widely distributed among plants and microorganisms that hydrolyze sucrose and release fructose from various substrates. Invertase was one of the earliest discovered enzymes, first investigated in the mid-nineteenth century, becoming a classical model used in the primary biochemical studies on protein synthesis, activity, and the secretion of glycoproteins. However, it was not until 20 years ago that a member of this family of enzymes was structurally characterized, showing a bimodular arrangement with a ß-propeller catalytic domain, and a ß-sandwich domain with unknown function. Since then, many studies on related plant and fungal enzymes have revealed them as basically monomeric. By contrast, all yeast enzymes in this family that have been characterized so far have shown sophisticated oligomeric structures mediated by the non-catalytic domain, which is also involved in substrate binding, and how this assembly determines the particular specificity of each enzyme. In this chapter, we will review the available structures of yeast invertases to elucidate the mechanism regulating oligomer formation and compare them with other reported dimeric invertases in which the oligomeric assembly has no apparent functional implications. In addition, recent work on a new family of invertases with absolute specificity for the α-(1,2)-bond of sucrose found in cyanobacteria and plant invertases is highlighted.

The Effect of Sucrose and Yeast Extract on Total Phenolic, Flavonoid, and Anthocyanin of Lactic-Acid-Fermented Mangosteen Fruit Peel (Garcinia mangostana L.).

Objectives: This study aimed to determine the most suitable concentration of sucrose and yeast extract (SYE) and its impact on the levels of total phenol, flavonoid, and anthocyanin (TPFA) for lactic acid fermentation in mangosteen fruit peel. Materials and Methods: In this study, the primary components were mangosteen fruit peel, SYE, and lactic acid bacteria starter. The experimental design was conducted using the Factorial Design method. The colorimetric method was used to determine the total phenol (Folin-Ciocalteu reagent) and total flavonoid (AlCl3 reagent). In addition, the differential pH method was used to determine the total anthocyanins using KCl and the CH3COONa reagent. Results: The addition of SYE during the fermentation of mangosteen fruit peel significantly increased the concentrations of TPFA compared with the control (p value of 0.0001). The high sucrose concentration and low yeast extract produced the highest TPFA levels in mangosteen rind fermentation. Conclusion: The use of SYE affects the levels of TPFA in lactic acid-fermented mangosteen fruit peel, with the most suitable concentrations obtained using sucrose (45 g/L) and yeast extract (2.5 g/L).

Culturable yeast community associated with grape must and honey bees sampled from apiaries located in the vineyards.

AIM: In this study, we investigated culturable yeast community, present in grape must sampled from vineyards with apiaries on the borders, and in honey bees collected in these apiaries. METHODS AND RESULTS: To this aim, yeasts isolated from spontaneously fermented grapes randomly collected in two vineyards (P1 and P2) with apiaries on the borders (A1 and A2) were compared to those isolated from spontaneously fermented grapes collected from a vineyard without apiary (P4). At the same time, yeast community was analyzed on bees collected in each apiary placed in the vineyards, in comparison to yeasts isolated from an apiary (A3) located far from the vineyards. The analysis was performed for two consecutive years (2021 and 2022). The isolated yeasts were identified by restriction analysis of amplified ITS region, followed by sequencing of ITS fragment.Our research showed that the presence of apiaries seems to increase yeast counts of grape must, in particular of Saccharomyces cerevisiae; furthermore, the permanence of apiaries in the vineyards allowed the recovering of these yeasts also from bees. CONCLUSIONS: Our findings seem to corroborate the role of bees as vectors and reservoirs of oenologically relevant yeasts, such as a source of non-conventional yeasts with potential biotechnological applications.

The 'Erlenmeter': a low-cost, open-source turbidimeter for no-sampling phenotyping of microorganism growth.

This work presents a low-cost, open-source turbidimeter, the 'Erlenmeter', designed to monitor the growth of microorganisms in batch cultures. It is easy to build, based exclusively on inexpensive off-the-shelf electronic components and 3D-printed parts. The Erlenmeter allows measuring the optical density of cultures on standard Erlenmeyer flasks without the need to open the flasks to collect aliquots, ensuring speed, minimal use of consumables, and elimination of the risk of contamination. These features make it particularly well-suited not just for routine research assays but also for experimental teaching. Here we illustrate the use of the Erlenmeter turbidimeter to record the growth of the microalga Phaeodactylum tricornutum, of the bacterium Escherichia coli, and of the yeast Saccharomyces cerevisiae, model organisms that are widely used in research and teaching. The Erlenmeter allows a detailed characterization of the growth curves of all organisms, confirming its usefulness for studying microbial populations dynamics both for research purposes and in classroom settings.

First Data on the Investigation of Gut Yeasts in Hermit Beetle (Osmoderma barnabita Motschulsky, 1845) Larvae in Lithuania.

In this study, yeasts from the gut of O. barnabita larvae were isolated and molecularly identified. It is worth noting that this research provides the first analysis of the gut yeast community in O. barnabita larvae in Lithuania, which is a significant contribution to the field. Two hermit-like L3-praepupa instars were collected from a decaying oak log in Lithuania. The isolation, morphology, biochemistry, and physiology of the yeast isolates were characterized using standards commonly employed in yeast taxonomy studies. The isolates were identified by sequencing the large subunit (26S) rDNA (D1/D2 domain of the LSU). All gut compartments were colonized by the yeast. A total of 45 yeast strains were obtained from the gut of both O. barnabita larvae, with 23 strains originating from Larva 1, 16 strains from Larva 2, and 6 strains from the galleries. According to our identification results of the 45 yeast strains, most of the species were related to Ascomycota, with most of them belonging to the Saccharomycetales order. Yeasts of the genera Candida, Debaryomyces, Meyerozyma, Priceomyces, Schwanniomyces, Spencermartinsiella, Trichomonascus, and Blastobotrys were present in gut of O. barnabita larvae. Species of the Trichosporonales order represented the Basidiomycota phylum.

Identification of Food Spoilage Fungi Using MALDI-TOF MS: Spectral Database Development and Application to Species Complex.

Fungi, including filamentous fungi and yeasts, are major contributors to global food losses and waste due to their ability to colonize a very large diversity of food raw materials and processed foods throughout the food chain. In addition, numerous fungal species are mycotoxin producers and can also be responsible for opportunistic infections. In recent years, MALDI-TOF MS has emerged as a valuable, rapid and reliable asset for fungal identification in order to ensure food safety and quality. In this context, this study aimed at expanding the VITEK® MS database with food-relevant fungal species and evaluate its performance, with a specific emphasis on species differentiation within species complexes. To this end, a total of 380 yeast and mold strains belonging to 51 genera and 133 species were added into the spectral database including species from five species complexes corresponding to Colletotrichum acutatum, Colletotrichum gloeosporioides, Fusarium dimerum, Mucor circinelloides complexes and Aspergillus series nigri. Database performances were evaluated by cross-validation and external validation using 78 fungal isolates with 96.55% and 90.48% correct identification, respectively. This study also showed the capacity of MALDI-TOF MS to differentiate closely related species within species complexes and further demonstrated the potential of this technique for the routine identification of fungi in an industrial context.

Genotyping and Phenotyping of Indigenous Saccharomyces cerevisiae from a New Zealand Organic Winery and Commercial Sources Using Inter-Delta and MALDI-TOF MS Typing.

We used inter-delta typing (IDT) and MALDI-TOF profiling to characterize the genetic and phenotypic diversity of 45 commercially available winemaking Saccharomyces cerevisiae strains and 60 isolates from an organic winemaker from Waipara, New Zealand, as a stratified approach for predicting the commercial potential of indigenous isolates. A total of 35 IDTs were identified from the commercial strains, with another 17 novel types defined among the Waipara isolates. IDT 3 was a common type among strains associated with champagne production, and the only type in commercial strains also observed in indigenous isolates. MALDI-TOF MS also demonstrated its potential in S. cerevisiae typing, particularly when the high-mass region (m/z 2000-20,000) was used, with most indigenous strains from each of two fermentation systems distinguished. Furthermore, the comparison between commercial strains and indigenous isolates assigned to IDT 3 revealed a correlation between the low-mass data (m/z 500-4000) analysis and the recommended use of commercial winemaking strains. Both IDT and MALDI-TOF analyses offer useful insights into the genotypic and phenotypic diversity of S. cerevisiae, with MALDI-TOF offering potential advantages for the prediction of applications for novel, locally isolated strains that may be valuable for product development and diversification.

Occurrence and Persistence of Saccharomyces cerevisiae Population in Spontaneous Fermentation and the Relation with "Winery Effect".

The yeast Saccharomyces cerevisiae ensures successful fermentation in winemaking, although the persistent use of commercial strains lead to the loss of aroma complexity of wines. Hence, the research of indigenous S. cerevisiae with proper oenological features and well adapted to specific wine-growing areas become of great interest for winemakers. Here, 206 pure cultures of S. cerevisiae were isolated from two wineries during a two-year sampling campaign and bio-typed through interdelta sequences analyses with the aim to evaluate the occurrence and persistence of the S. cerevisiae wild population linked to each winery. Both wineries belong to the same Verdicchio DOC wine area (Castelli di Jesi), and never used commercial yeasts during fermentation. Results showed 19 different biotypes with a specific population of S. cerevisiae in each winery, without cross-contamination with each other and with commercial starter strains. Moreover, inside each winery a persistence of some dominant biotypes was observed over time (three biotypes in winery 1; 95% of isolates in the two years and one biotype in winery 2; 20% of isolates in the two years), indicating a sort of "winery-effect". The evaluation of S. cerevisiae populations for the oenological characters by microfermentations showed a proper and well distinct aromatic imprinting on the resulted wines supporting the concept of "winery effect".

Apple Blossom Agricultural Residues as a Sustainable Source of Bioactive Peptides through Microbial Fermentation Bioprocessing.

This study explored the impact of starter-assisted fermentation on apple blossoms to enhance their potential as a source of antioxidant and antifungal molecules. Fructobacillus fructosus PL22 and Wickerhamomyces anomalus GY1 were chosen as starters owing to their origin and promising ability to modify plant secondary metabolites. An initial assessment through microbiological and physicochemical analyses showed superior outcomes for starter-assisted fermentation compared to the spontaneous process. Enzymatic hydrolysis of proteins, primarily controlled by starters, orchestrated the generation of new low-molecular-weight peptides. W. anomalus GY1 also induced modifications in the phenolic profile, generating a diverse array of bioactive metabolites. These metabolic changes, particularly the release of potentially bioactive peptides, were associated with significant antioxidant activity and marked antifungal efficacy against three common mold species. Our results shed light on the potential of microbial starters to valorize agricultural wastes and convert them into a valuable resource for industry.

Quantification methods of Candida albicans are independent irrespective of fungal morphology.

The ability of Candida albicans to switch its morphology from yeast to filaments, known as polymorphism, may bias the methods used in microbial quantification. Here, we compared the quantification methods [cell/mL, colony forming units (CFU)/mL, and the number of nuclei estimated by viability polymerase chain reaction (vPCR)] of three strains of C. albicans (one reference strain and two clinical isolates) grown as yeast, filaments, and biofilms. Metabolic activity (XTT assay) was also used for biofilms. Comparisons between the methods were evaluated by agreement analyses [Intraclass and Concordance Correlation Coefficients (ICC and CCC, respectively) and Bland-Altman Plot] and Pearson Correlation (α = 0.05). Principal Component Analysis (PCA) was employed to visualize the similarities and differences between the methods. Results demonstrated a lack of agreement between all methods irrespective of fungal morphology/growth, even when a strong correlation was observed. Bland-Altman plot also demonstrated proportional bias between all methods for all morphologies/growth, except between CFU/mL X vPCR for yeasts and biofilms. For all morphologies, the correlation between the methods were strong, but without linear relationship between them, except for yeast where vPCR showed weak correlation with cells/mL and CFU/mL. XTT moderately correlated with CFU/mL and vPCR and weakly correlated with cells/mL. For all morphologies/growth, PCA showed that CFU/mL was similar to cells/mL and vPCR was distinct from them, but for biofilms vPCR became more similar to CFU/mL and cells/mL while XTT was the most distinct method. As conclusions, our investigation demonstrated that CFU/mL underestimated cells/mL, while vPCR overestimated both cells/mL and CFU/mL, and that the methods had poor agreement and lack of linear relationship, irrespective of C. albicans morphology/growth.1.

Chromosome-level genome assembly of an auxotrophic strain of the pathogenic yeast Candida parapsilosis.

We report the genome sequence of the pathogenic yeast Candida parapsilosis strain SR23 (CBS 7157) used in a number of experimental studies. The nuclear genome assembly consists of eight chromosome-sized contigs with a total size of 13.04 Mbp (N50 2.09 Mbp) and a G+C content of 38.7%.

Identification and antifungal susceptibility profile of uncommon yeast species at Fattouma Bourguiba University Hospital in Tunisia.

Despite the severe impact of uncommon yeast fungal infections and the pressing need for more research on the topic, there are still few studies available on the identification, epidemiology, and susceptibility profile of those pathogens. The aims of the current study were to define the profile of uncommon yeast species at Fattouma Bourguiba University Hospital using phenotypic, molecular, and proteomic methods and to study their antifungal susceptibility profile. Pre-identified uncommon yeast species were collected from 2018 to 2021. These isolates were further identified using phenotypic methods (ID32C® system and Vitek2® YST), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and sequencing. The antifungal susceptibility profile was studied using the reference CLSI broth microdilution method. In total, 30 strains were collected during the study period. Referring to the sequencing, the most isolated uncommon species were Saprochaete capitata, Candida lusitaniae, Candida kefyr, Candida inconspicua, and Candida guilliermondii. A total of 90% of isolates were correctly identified by MALDI-TOF MS compared to 76.7% and 63.3% by ID32® C and VITEK® 2 YST, respectively. The isolated species showed variable responses to antifungals. Candida guilliermondii showed increased azole minimum inhibitory concentrations. Misidentification of uncommon yeast species was common using commercial phenotypic methods. The high percentage of concordance of MALDI-TOF results with sequencing highlights its high performance and usefulness as a routine diagnosis tool.

There is still little information on the epidemiology of uncommon emergent yeasts, although their implication in severe diseases and mainly invasive infections. Thus, the importance of an accurate identification and antifungal susceptibility testing for a better monitoring of related infections.

Non-sterile cultivation of Yarrowia lipolytica in fed-batch mode for the production of lipids and biomass.

A reduction in the cost of production and energy requirement is necessary for developing sustainable commercial bioprocesses. Bypassing sterilization, which is an energy and cost-intensive part of bioprocesses could be a way to achieve this. In this study, nonsterile cultivation of Yarrowia lipolytica was done on a synthetic medium containing acetic acid as the sole carbon source using two different strategies in the fed-batch mode. The contamination percentages throughout the process were measured using flow cytometry and complemented using brightfield microscopy. Maximum biomass and lipid yields of 0.57 (g biomass/g substrate) and 0.17 (g lipids/g substrate), respectively, and maximum biomass and lipid productivities of 0.085 and 0.023 g/L/h, respectively, were obtained in different fed-batch strategies. Feeding at the point of stationary phase resulted in better biomass yield and productivity with less than 2% contamination till 48 h. Feeding to maintain a minimum acetic level resulted in better lipid yield and productivity with less than 2% contamination during the complete process. The results of this study demonstrate the potential for cultivating Y. lipolytica in nonsterile conditions and monitoring the contamination throughout the process using flow cytometry.