non-Saccharomyces Wine Yeasts Research Articles

Biocontrol and Enzymatic Activity of Non-Saccharomyces Wine Yeasts: Improvements in Winemaking

Biocontrol and Enzymatic Activity of Non-Saccharomyces Wine Yeasts: Improvements in Winemaking

White grape variety Maraština as a promising source of non-Saccharomyces yeasts intended as starter cultures

Non-Saccharomyces wine yeasts are recognized for their ability to enhance the aroma complexity of wines. New trends in winemaking have highlighted the concept of microbiological terroir thus encouraging the use of indigenous non-Saccharomyces starter cultures in mixed fermentations with Saccharomyces cerevisiae to overcome the lack of authenticity and provide the wines with unique regional character and distinct aromatic profiles. In this study, 257 indigenous non-Saccharomyces isolates were obtained for the first time from 11 Maraština vineyards located within the winegrowing region of Dalmatia (Croatia). The isolates were identified at the molecular level and characterized for desirable oenological traits such as growth at different temperatures, high ethanol and SO2 tolerance, and for negative features such as high hydrogen sulphide (H2S) and acetic acid production as well as hydroxycinnamic acid decarboxylase (HCDC) activity. Among 27 identified species, those from Aureobasidium genera prevailed, followed by Metschnikowia, Hanseniaspora and Lachancea. The representatives from different species showed high SO2 (up to 250 mg/L) and ethanol (up to 12%) tolerance, low acetic acid, and medium-low H2S production. The HCDC activity was detected exclusively among Metschnikowia, Aureobasidium and Kabatiella species. Some regionally distinct characteristics within the isolates from the same species were observed. The encouraging insights into potential new starter cultures emerged after the evaluation of enzymatic activities of selected isolates. Most of them showed high leucine and valine arylamidase activity, whereas Hanseniaspora and Metschnikowia isolates were additionally characterized by high β-glucosidase and esterase activity, respectively.

To each their own: Delving into the vitaminic preferences of non-Saccharomyces wine yeasts

Considering the growing interest in non-Saccharomyces wine yeasts, and notably in the context of mixed fermentations with S. cerevisiae, understanding their nutritional behaviors is essential to ensure better management of these fermentations. The vitaminic consumption of three non-Saccharomyces yeasts (Starmerella bacillaris, Metschnikowia pulcherrima and Torulaspora delbrueckii) was investigated during their growth in wine-like conditions, providing initial evidence that they consume different vitamers. The vitamin consumption profiles during their growth highlighted releases of certain vitamers by the yeasts before re-assimilation, strongly suggesting the existence of synthesis pathways. Not only did the essential character of vitamin B1, in particular, appear to be a trait common to these yeasts, since all its vitamers are consumed, this investigation also provided evidence of the existence of species-dependent preferences for their vitaminic sources. These different behaviors were quite striking in certain vitamers, as was observed in nicotinamide: while it was consumed by T. delbrueckii, it was left untouched by S. bacillaris and produced by M. pulcherrima during growth. Furthermore, this offers grounds for further investigation into these yeasts’ requirements, and provides the first tool for managing vitamin resources during mixed fermentations with S. cerevisiae, and for preventing nutritive deficiencies from occurring.

Optimizing growth and biomass production of non-Saccharomyces wine yeast starters by overcoming sucrose consumption deficiency.

The use of non-Saccharomyces yeasts as starters in winemaking has increased exponentially in the last years. For instance, non-conventional yeasts have proven useful for the improvement of the organoleptic profile and biocontrol. Active dry yeast starter production has been optimized for Saccharomyces cerevisiae, which may entail problems for the propagation of non-Saccharomyces yeasts. This work shows that the poor growth of Hanseniaspora vineae and Metschnikowia pulcherrima in molasses is related to a deficient sucrose consumption, linked to their low invertase activity. In order to address this issue, simple modifications to the cultivation media based hydrolysis and the reduction of sucrose concentration were performed. We performed biomass propagation simulations at a bench-top and bioreactor scale. The results show that cultivation in a hexose-based media improved biomass production in both species, as it solves their low invertase activity. The reduction in sugar concentration promoted a metabolic shift to a respiratory metabolism, which allowed a higher biomass yield, but did not improve total biomass production, due to the lower sugar availability. To evaluate the technological performance of these adaptations, we performed mixed grape juice fermentations with biomass produced in such conditions of M. pulcherrima and S. cerevisiae. The analysis of wines produced revealed that the different treatments we have tested did not have any negative impact on wine quality, further proving their applicability at an industrial level for the improvement of biomass production.

Metschnikowia pulcherrima as biocontrol agent and wine aroma enhancer in combination with a native Saccharomycescerevisiae

One of the main objectives for a sustainable winemaking process is the reduction of the use of sulfur dioxide. In this regard, non-Saccharomyces wine yeasts are proposed as biocontrol agent in different steps of wine production chain. Here, a selected strain of Metschnikowia pulcherrima (DiSVA 269) and a native Saccharomyces cerevisiae low sulfite producer strain (DiSVA 708) were investigated. After preliminary laboratory trials, winemaking process at industrial level showed an effective biocontrol action (reduction of c.a. 1 Log order of wild yeasts) of M. pulcherrima inoculated at prefermentative stage in cold clarification (48 h at 10 °C) and during the subsequent fermentation process. The combination of M. pulcherrima/S. cerevisiae led a distinctive aromatic profile of wines both in laboratory and winery trials with a significant enhancement of ethyl butyrate, ethyl hexanoate, isoamyl acetate and β-phenyl ethanol. Moreover the use of the two selected strains was the best combination to enhance volatile thiols (3-mercaptohexan-1-ol and 3-mercaptoexil acetate) that well correlate with the sensory analysis (tropical fruits). The overall results indicate that the combined use of M. pulcherrima DiSVA 269 and native S. cerevisiae DiSVA 708 led a biocontrol action and an improvement of aromatic and sensorial profile of wine with low SO2 content.

Metschnikowia pulcherrima in Cold Clarification: Biocontrol Activity and Aroma Enhancement in Verdicchio Wine

Non-Saccharomyces wine yeasts are not only proposed to improve the sensory profile of wine but also for several distinctive promising features. Among them, biocontrol action at different steps of the wine production chain could be a suitable strategy to reduce the use of sulfur dioxide. In this work, the activity of a selected strain of Metschnikowia pulcherrima was evaluated as inoculum in cold clarification with the aim to reduce SO2 and improve the aromatic profile of the wine. Fermentation processes were carried out at the winery level for two consecutive vintages using a pied de cuve as the starter inoculum coming from indigenous Saccharomyces cerevisiae strains. M. pulcherrima revealed an effective bio-protectant action during the pre-fermentative stage even if the timely and appropriate starter inoculum in the two years permitted the effective control of wild yeasts during the fermentation also in the control trials. In general, the main oenological characters did not show differences if compared with an un-inoculated trial, while the inoculum of M. pucherrima in cold clarification determined an enhancement of ethyl hexanoate, isobutanol, acetaldehyde, and geraniol even if they are considered in different amounts for each year. Indeed, the analytical and sensory profiles of wines were also influenced by the vintage and variation pied the cuve population. Nonetheless, the overall results indicated that M. pulcherrima led to biocontrol action and an improvement of the aromatic and sensory profile of the wine.

Synthesis of Aroma Compounds as a Function of Different Nitrogen Sources in Fermentations Using Non-Saccharomyces Wine Yeasts.

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.

Screening of Saccharomyces and Non-Saccharomyces Wine Yeasts for Their Decarboxylase Activity of Amino Acids.

The type and quantity of precursor amino acids present in grape must that are used by wine yeasts affect the organoleptic and health properties of wine. The aim of this work was to conduct a preliminary screening among Saccharomyces and non-Saccharomyces indigenous strains, which were previously isolated from different Italian regional grape varieties. This was performed in order to evaluate their decarboxylase activity on certain important amino acids-such as arginine, proline, serine, and tyrosine-that are present in grape must. In particular, a qualitative test on 122 wine yeasts was performed on a decarboxylase medium using arginine, proline, serine, and tyrosine as precursor amino acids. Our results showed a considerable variability among the microbial species tested for this parameter. Indeed, Saccharomyces cerevisiae strains exhibited a high decarboxylase capability of the four amino acids tested; moreover, only 10% of the total (i.e., a total of 81) did not show this trait. A high recovery of decarboxylation ability for at least one amino acid was also found for Zygosaccharomyces bailii and Hanseniaspora spp. These findings can, therefore, promote the inclusion of decarboxylase activity as an additional characteristic in a wine yeast selection program in order to choose starter cultures that possess desirable technological traits; moreover, this also can contribute to the safeguarding of consumer health.

Selection of Non-Saccharomyces Wine Yeasts for the Production of Leavened Doughs

Background: Non-conventional yeasts (NCY) (i.e., non-Saccharomyces) may be used as alternative starters to promote biodiversity and quality of fermented foods and beverages (e.g., wine, beer, bakery products). Methods: A total of 32 wine-associated yeasts (Campania region, Italy) were genetically identified and screened for decarboxylase activity and leavening ability. The best selected strains were used to study the leavening kinetics in model doughs (MDs). A commercial strain of Saccharomyces cerevisiae was used as the control. The volatile organic profiles of the inoculated MDs were analyzed by solid phase microextraction/gas chromatography-mass spectrometry (SPME/GC-MS). Results: Most of strains belonged to the NCY species Hanseniaspora uvarum, Metschnikowia pulcherrima, Pichia kudriavzevii, Torulaspora delbruekii, and Zygotorulaspora florentina, while a few strains were S. cerevisiae. Most strains of H. uvarum lacked decarboxylase activity and showed a high leaving activity after 24 h of incubation that was comparable to the S. cerevisiae strains. The selected H. uvarum strains generated a different flavor profile of the doughs compared to the S. cerevisiae strains. In particular, NCY reduced the fraction of aldehydes that were potentially involved in oxidative phenomena. Conclusions: The use of NCY could be advantageous in the bakery industry, as they can provide greater diversity than S. cerevisiae-based products, and may be useful in reducing and avoiding yeast intolerance.

Evaluation of Different Molecular Markers for Genotyping Non-Saccharomyces Wine Yeast Species

Wine quality is determined by the particular yeast strains prevailing at various stages of fermentation. Therefore, the ability to make an easy, fast, and unambiguous discrimination of yeasts at the strain level is of great importance. Here, the tandem repeat-tRNA (TRtRNA) method with the 5GAC or ISSR-MB primer sets and random amplified polymorphic DNA (RAPD) analysis with (GTG)3, R5, and RF2 oligonucleotides were tested on various non-Saccharomyces wine yeast species. The TRtRNA-PCR employing ISSR-MB showed the highest capacity in discriminating Lachancea thermotolerans and Metschnikowia pulcherrima isolates. RAPD with RF2 was the most efficient method in resolving Starmerella bacillaris isolates, although it produced few polymorphic bands. RAPD with R5 showed the highest capacity to discriminate among the Issatchenkia orientalis, Hanseniaspora guilliermondii, and Pichia anomala isolates. RAPD with either R5 or RF2 exhibited the highest ability to discriminate among the Torulaspora delbrueckii isolates. RAPD with (GTG)3 was the most discriminating method for the H. uvarum isolates. Here we concluded that both TRtRNA-PCR and RAPD-PCR offer rapid means for typing non-Saccharomyces species. However, each method performs better for a given species when paired with a particular primer set. The present results can be useful in wine research for the fast fingerprinting of non-Saccharomyces yeasts.

Impact of Non-Saccharomyces Wine Yeast Strains on Improving Healthy Characteristics and the Sensory Profile of Beer in Sequential Fermentation.

The use of non-Saccharomyces yeasts in brewing is a useful tool for developing new products to meet the growing consumer demand for innovative products. Non-Saccharomyces yeasts can be used both in single and in mixed fermentations with Saccharomyces cerevisiae, as they are able to improve the sensory profile of beers, and they can be used to obtain functional beers (with a low ethanol content and melatonin production). The aim of this study was to evaluate this capacity in eight non-Saccharomyces strains isolated from Madrid agriculture. For this purpose, single fermentations were carried out with non-Saccharomyces strains and sequential fermentations with non-Saccharomyces and the commercial strain SafAle S-04. The Wickerhamomyces anomalus strain CLI 1028 was selected in pure culture for brewing beer with a low ethanol content (1.25% (v/v)) for its fruity and phenolic flavours and the absence of wort flavours. The best-evaluated strains in sequential fermentation were CLI 3 (Hanseniaspora vineae) and CLI 457 (Metschnikowia pulcherrima), due to their fruity notes as well as their superior bitterness, body, and balance. Volatile compounds and melatonin production were analysed by GC and HPLC, respectively. The beers were sensory-analysed by a trained panel. The results of the study show the potential of non-Saccharomyces strains in the production of low-alcohol beers, and as a flavour enhancement in sequential fermentation.

The effect of growth rate on the production and vitality of non-Saccharomyces wine yeast in aerobic fed-batch culture.

Non-Saccharomyces wine yeasts are of increasing importance due to their influence on the organoleptic properties of wine and thus the factors influencing the biomass production of these yeasts, as starter cultures, are of commercial value. Therefore, the effects of growth rates on the biomass yield (Yx/s) and fermentation performance of non-Saccharomyces yeasts at bench and pilot scale were examined. The fermentative performance and (Yx/s) were optimised, in aerobic fed-batch cultivations, to produce commercial wine seed cultures of Lachancea thermotolerans Y1240, Issatchenkia orientalis Y1161 and Metschnikowia pulcherrima Y1337. Saccharomyces cerevisiae (Lalvin EC1118) was used as a benchmark. A Crabtree positive response was shown by L. thermotolerans in a molasses-based industrial medium, at growth rates exceeding 0.21h-1 (µcrit), resulting in a Yx/s of 0.76g/g at 0.21h-1 (46% of µmax) in the aerobic bioreactor-grown fed-batch culture at bench scale. At pilot scale and 0.133h-1 (36% of µmax), this yeast exhibited ethanol concentrations reaching 10.61g/l, as a possible result of substrate gradients. Crabtree negative responses were observed for I. orientalis and M. pulcherrima resulting in Yx/s of 0.83g/g and 0.68g/g, respectively, below 32% of µmax. The Yx/s of M. pulcherrima, I. orientalis and L. thermotolerans was maximised at growth rates between 0.10 and 0.12h-1 and the fermentative capacity of these yeasts was maximised at these lower growth rates.

Optimised Extraction and Preliminary Characterisation of Mannoproteins from Non-Saccharomyces Wine Yeasts.

The exogenous application of yeast-derived mannoproteins presents many opportunities for the improvement of wine technological and oenological properties. Their isolation from the cell wall of Saccharomyces cerevisiae has been well studied. However, investigations into the efficiency of extraction methods from non-Saccharomyces yeasts are necessary to explore the heterogeneity in structure and composition that varies between yeast species, which may influence wine properties such as clarity and mouthfeel. In this study, nine yeast strains were screened for cell wall mannoprotein content using fluorescence microscopy techniques. Four species were subsequently exposed to a combination of mechanical and enzymatic extraction methods to optimize mannoprotein yield. Yeast cells subjected to 4 min of ultrasound treatment applied at 80% of the maximum possible amplitude with a 50% duty cycle, followed by an enzymatic treatment of 4000 U lyticase per g dry cells weight, showed the highest mannoprotein-rich yield from all species. Furthermore, preliminary evaluation of the obtained extracts revealed differences in carbohydrate/protein ratios between species and with increased enzyme incubation time. The results obtained in this study form an important step towards further characterization of extraction treatment impact and yeast species effect on the isolated mannoproteins, and their subsequent influence on wine properties.

Sterol uptake analysis in Saccharomyces and non-Saccharomyces wine yeast species.

Sterols are essential components of the yeast membrane and their synthesis requires oxygen. Yet, Saccharomyces cerevisiae has developed the ability to take up sterols from the medium under anaerobiosis. Here we investigated sterol uptake efficiency and the expression of genes related to sterol import in Saccharomyces and non-Saccharomyces wine yeast species fermenting under anaerobic conditions. The sterol uptake efficiency of 39 strains was evaluated by flow cytometry (with 25-NBD Cholesterol, a fluorescent cholesterol probe introduced in the medium) and we found an important discrepancy between Saccharomyces and non-Saccharomyces wine yeast species that we correlated to a lower final cell population and a lower fermentation rate. A high uptake of sterol was observed in the various Saccharomyces strains. Spot tests performed on 13 of these strains confirmed the differences between Saccharomyces and non-Saccharomyces strains, suggesting that the presence of the sterol uptake transporters AUS1 and PDR11 could cause these discrepancies. Indeed, we could not find any homologue to these genes in the genome of Hanseniaspora uvarum, H. guillermondii, Lachancea thermotolerans, Torulaspora delbreueckii, Metschnikowia pulcherrima, or Starmarella bacillaris species. The specialization of sterol import function for post genome-duplication species may have favored growth under anaerobiosis.

The potential of spent coffee grounds hydrolysates fermented with Torulaspora delbrueckii and Pichia kluyveri for developing an alcoholic beverage: The yeasts growth and chemical compounds modulation by yeast extracts

This study evaluated the effects of yeast extracts (YE) addition (0 % and 0.25 %, w/v) on the no-volatile and volatile compounds of spent coffee grounds (SCG) hydrolysates fermented with single-cultures of two non-Saccharomyces wine yeasts, Torulaspora delbrueckii and Pichia kluyveri. The added YE improved the growth of both T. delbrueckii and P. kluyveri, especially P. kluyveri, resulting in higher ethanol production (1.98 % vs 1.47 %, v/v) by the latter yeast. In addition, the added YE did not impact on most of the alkaloids production regardless of yeast type, while significantly decreasing the contents of chlorogenic, and caffeic acids in SCG hydrolysates fermented with P. kluyveri. Furthermore, more odor-active compounds such as acetate esters and 2-phenylethyl alcohol were produced when YE was added, and P. kluyveri generated significantly higher amounts of esters compared to that of T. delbrueckii. Moreover, YE addition showed a more noticeable effect on the fermentation performance of P. kluyveri relative to that of T. delbrueckii. These findings indicated the potential of SCG hydrolysates fermented with evaluated non-Saccharomyces yeasts and may expand the applications on utilizing SCG to develop new value-added alcoholic products.

The Genus Metschnikowia in Enology.

Over the last decade, several non-Saccharomyces species have been used as an alternative yeast for producing wines with sensorial properties that are distinctive in comparison to those produced using only Saccharomyces cerevisiae as the classical inoculum. Among the non-Saccharomyces wine yeasts, Metschnikowia is one of the most investigated genera due to its widespread occurrence and its impact in winemaking, and it has been found in grapevine phyllospheres, fruit flies, grapes, and wine fermentations as being part of the resident microbiota of wineries and wine-making equipment. The versatility that allows some Metschnikowia species to be used for winemaking relies on an ability to grow in combination with other yeast species, such as S. cerevisiae, during the first stages of wine fermentation, thereby modulating the synthesis of secondary metabolites during fermentation in order to improve the sensory profile of the wine. Metschnikowia exerts a moderate fermentation power, some interesting enzymatic activities involving aromatic and color precursors, and potential antimicrobial activity against spoilage yeasts and fungi, resulting in this yeast being considered an interesting tool for use in the improvement of wine quality. The abovementioned properties have mostly been determined from studies on Metschnikowia pulcherrima wine strains. However, M. fructicola and M. viticola have also recently been studied for winemaking purposes.

Basal catalase activity and high glutathione levels influence the performance of non-Saccharomyces active dry wine yeasts

Non-Saccharomyces wine yeasts are useful tools for producing wines with complex aromas or low ethanol content. Their use in wine would benefit from their production as active dry yeast (ADY) starters to be used as co-inocula alongside S. cerevisiae. Oxidative stress during biomass propagation and dehydration is a key factor in determining ADY performance, as it affects yeast vitality and viability. Several studies have analysed the response of S. cerevisiae to oxidative stress under dehydration conditions, but not so many deal with non-conventional yeasts. In this work, we analysed eight non-Saccharomyces wine yeasts under biomass production conditions and studied oxidative stress parameters and lipid composition. The results revealed wide variability among species in their technological performance during ADY production. Also, for Metschnikowia pulcherrima and Starmerella bacillaris, better performance correlates with high catalase activity and glutathione levels. Our data suggest that non-Saccharomyces wine yeasts with an enhanced oxidative stress response are better suited to grow under ADY production conditions.

Pilot Scale Fermentations of Sangiovese: An Overview on the Impact of Saccharomyces and Non-Saccharomyces Wine Yeasts

The production of wines with peculiar analytical and sensorial profiles, together with the microbiological control of the winemaking process, has always been one of the main objectives of the wine industry. In this perspective, the use of oenological starters containing non-Saccharomyces yeasts can represent a valid tool for achieving these objectives. Here we present the results of seven pilot scale fermentations, each of which was inoculated with a different non-Saccharomyces yeast strain and after three days with a commercial Saccharomyces cerevisiae starter. The fermentations were carried out in double on 70 L of Sangiovese grape must, the most widely planted red grape variety in Italy and particularly in Tuscany, where it is utilized for the production of more than 80% of red wines. Fermentations were monitored by assessing both the development of the microbial population and the consumption of sugars at the different sampling times. The impact of the different starters was assessed after stabilization through the evaluation of the standard analytical composition of the resulting wines, also taking into account polysaccharides and volatile compounds. Moreover, quantitative descriptive sensory analyses were carried out. Compared to the control wines obtained by inoculating the S. cerevisiae starter strain, those inoculated with non-Saccharomyces/Saccharomyces mixed starters presented a significant differentiation in the chemical-analytical composition. Moreover, sensory analysis revealed differences among wines mainly for intensity of color, astringency, and dryness mouthfeel perception.

Non-Saccharomyces Yeasts and Organic Wines Fermentation: Implications on Human Health

A relevant trend in winemaking is to reduce the use of chemical compounds in both the vineyard and winery. In organic productions, synthetic chemical fertilizers, pesticides, and genetically modified organisms must be avoided, aiming to achieve the production of a “safer wine”. Safety represents a big threat all over the world, being one of the most important goals to be achieved in both Western society and developing countries. An occurrence in wine safety results in the recovery of a broad variety of harmful compounds for human health such as amines, carbamate, and mycotoxins. The perceived increase in sensory complexity and superiority of successful uninoculated wine fermentations, as well as a thrust from consumers looking for a more “natural” or “organic” wine, produced with fewer additives, and perceived health attributes has led to more investigations into the use of non-Saccharomyces yeasts in winemaking, namely in organic wines. However, the use of copper and sulfur-based molecules as an alternative to chemical pesticides, in organic vineyards, seems to affect the composition of grape microbiota; high copper residues can be present in grape must and wine. This review aims to provide an overview of organic wine safety, when using indigenous and/or non-Saccharomyces yeasts to perform fermentation, with a special focus on some metabolites of microbial origin, namely, ochratoxin A (OTA) and other mycotoxins, biogenic amines (BAs), and ethyl carbamate (EC). These health hazards present an increased awareness of the effects on health and well-being by wine consumers, who also enjoy wines where terroir is perceived and is a characteristic of a given geographical area. In this regard, vineyard yeast biota, namely non-Saccharomyces wine-yeasts, can strongly contribute to the uniqueness of the wines derived from each specific region.

Nitrogen Preferences during Alcoholic Fermentation of Different Non-Saccharomyces Yeasts of Oenological Interest.

Non-Saccharomyces yeasts have long been considered spoilage microorganisms. Currently, oenological interest in those species is increasing, mostly due to their positive contribution to wine quality. In this work, the fermentative capacity and nitrogen consumption of several non-Saccharomyces wine yeast (Torulaspora delbrueckii, Lachancea thermotolerans, Starmerella bacillaris, Hanseniaspora uvarum, and Metschnikowia pulcherrima) were analyzed. For this purpose, synthetic must with three different nitrogen compositions was used: a mixture of amino acids and ammonium, only organic or inorganic nitrogen. The fermentation kinetics, nitrogen consumption, and yeast growth were measured over time. Our results showed that the good fermentative strains, T. delbrueckii and L. thermotolerans, had high similarities with Saccharomyces cerevisiae in terms of growth, fermentation profile, and nitrogen assimilation preferences, although L. thermotolerans presented an impaired behavior when only amino acids or ammonia were used, being strain-specific. M. pulcherrima was the non-Saccharomyces strain least affected by the nitrogen composition of the medium. The other two poor fermentative strains, H. uvarum and S. bacillaris, behaved similarly regarding amino acid uptake, which occurred earlier than that of the good fermentative species in the absence of ammonia. The results obtained in single non-Saccharomyces fermentations highlighted the importance of controlling nitrogen requirements of the wine yeasts, mainly in sequential fermentations, in order to manage a proper nitrogen supplementation, when needed.