fermentation

Article

Fermentation of Dairy-Relevant Sugars by Saccharomyces,Kluyveromyces, and Brettanomyces: An Exploratory Study withImplications for the Utilization of Acid Whey, Part I

Viviana K. Rivera Flores , Timothy A. DeMarsh , Patrick A. Gibney and Samuel D. Alcaine *

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Citation: Rivera Flores, V.K.;

DeMarsh, T.A.; Gibney, P.A.;

Alcaine, S.D. Fermentation of

Dairy-Relevant Sugars by

Saccharomyces, Kluyveromyces,

and Brettanomyces: An Exploratory

Study with Implications for the

Utilization of Acid Whey, Part I.

Fermentation 2021, 7, 266.

https://doi.org/10.3390/

fermentation7040266

Academic Editors: Ana Susmozas

and Yi-Huang Hsueh

Received: 12 October 2021

Accepted: 12 November 2021

Published: 17 November 2021

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Attribution (CC BY) license (https://

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4.0/).

Department of Food Science, Cornell University, Ithaca, NY 14853, USA; vkr6@cornell.edu (V.K.R.F.);tad24@cornell.edu (T.A.D.); pag235@cornell.edu (P.A.G.)* Correspondence: alcaine@cornell.edu; Tel.: +1-607-255-9183

Abstract: Acid whey from Greek-style yogurt (YAW) is an underutilized byproduct and a challengefor the dairy industry. One alternative is the fermentation of YAW by yeasts such as Saccharomyces,Brettanomyces, and Kluyveromyces spp., to produce new styles of fermented beverages. Previousresearch in our group suggested that the sugar profiles of the dairy coproducts impacted the fer-mentation profiles produced by B. claussenii. The present work aims to describe the fermentation ofdairy sugars by S. cerevisiae, K. marxianus, and B. claussenii, under conditions comparable to those ofYAW. For this purpose, four preparations of yeast nitrogen base, each containing 40 g/L of eitherlactose (LAC), glucose (GLU), galactose (GAL), or a 1:1 mixture of glucose and galactose (GLU:GAL),all at pH 4.20, were used as fermentation media. The fermentation was performed independently byeach organism at 25 ◦C under anoxic conditions, while density, pH, cell count, ethanol, and organicacids were monitored. Non-linear modeling was used to characterize density curves, and Analysis ofVariance and Tukey’s Honest Significant Difference tests were used to compare fermentation products.K. marxianus and S. cerevisiae displayed rapid sugar consumption with consistent ethanol yields in allmedia, as opposed to B. claussenii, which showed more variable results. The latter organism exhibitedwhat appears to be a selective glucose fermentation in GLU:GAL, which will be explored in thefuture. These results provide a deeper understanding of dairy sugar utilization by relevant yeasts,allowing for future work to optimize fermentations to improve value-added beverage and ingredientproduction from YAW.

Keywords: acid whey; ethanol; non-linear modeling

1. Introduction

Over the past several years, Greek-style yogurt has gained immense popularity aroundthe world. According to Statista’s Greek yogurt report [1], this market is forecasted toreach 12.3 billion U.S. dollars worldwide by 2024. However, during Greek-style yogurtproduction, for every kilogram of product manufactured, 2 to 3 kg of yogurt acid whey(YAW) are also generated [2]. This represents a problem for dairy producers; due to thehigh biological oxygen demand of this effluent [3], its direct disposal into water streamsposes a major ecological burden [4].

Several repurposing options for this coproduct have been studied over the years,including direct land application [5]; bioconversion of its lactose into lipids for animalfeed [6]; and anaerobic digestion to produce methane for subsequent electricity genera-tion [2]. However, these applications represent relatively less-preferred options within theUnited States Environmental Protection Agency’s Food Recovery Hierarchy system [7].

More recently, attempts to address the issues caused by the production of YAW havefollowed two main paths: (i) exploring opportunities to reduce the overall volume ofthis stream; or (ii) exploring its applications in products suitable for human consump-tion [8]. With regard to the latter, various studies have investigated the development of

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probiotic beverages using this substrate as a primary ingredient [9], the use of acid wheyfor the production of probiotic biomass [10], and the variety of sensory attributes thatcan be generated through the fermentation of acid whey beverages [11], to name a few.Such approaches are justified by the natural content of lactose, minerals, and vitaminspresent in YAW, which make it an attractive source of important nutrients. Menchik,Zuber, Zuber and Moraru [3] studied the composition of YAW and found its lactose con-tent to be approximately 35 g/L; its pH to range between 4.2 and 4.5; its most abundantminerals to be potassium (157–169 mg/100 g), calcium (120–128 mg/100 g), and phos-phorus (66.5–69.2 mg/100 g); and its vitamin profile to include pantothenic acid, niacin,riboflavin, and thiamine, at the following levels: 0.364, 0.115, 0.090, and 0.080 mg/100 g,respectively. These intrinsic characteristics make YAW a suitable candidate to be used as asubstrate for novel beverages, in which its sugar, acid, mineral, and vitamin content can befully exploited.

Recent trends in the fermented beverage market, namely beverages with functionalcharacteristics or low or no alcohol [12,13], have fueled an interest in the search for non-traditional microbes that can contribute to the production of such drinks. For instance,Lawton, et al. [14] reviewed the biotechnological potential of Brettanomyces claussenii toproduce novel fermented beverages from lactose-containing substrates. Despite tradition-ally being known as a spoilage organism, multiple applications for this yeast have recentlybeen established in the beer industry [15]; this fact, combined with its ability to fermentlactose, makes it a good candidate to produce YAW-based beverages. Similarly, Karim,et al. [16] discussed the diverse range of applications of Kluyveromyces marxianus in foodand biotechnology, including its potential as a probiotic, its role in the production of volatilecompounds, and its demonstrated ability to produce bioethanol from dairy effluents likecheese whey and whey permeate, all of which make it another ideal organism to be usedto produce fermented beverages from YAW. On the other hand, in the current context ofindustrially fermented beverages, Saccharomyces cerevisiae is the predominant yeast dueto its excellent fermentation efficiency and its notably high resistance to ethanol. Eventhough the wild-type S. cerevisiae genome does not allow for the utilization of lactose as acarbon source [17], this yeast’s fermentative process can be combined with a mechanismof lactose hydrolysis to produce alcoholic beverages from YAW. Additionally, its role as ayeast model organism provides insights into the most basic aspects of yeast metabolismand makes it relevant in the exploration of fermentative processes.

Our preliminary experiments performed in YAW demonstrated changes in fermen-tation performance upon the addition of lactase [18]. Specifically, despite the observedability of B. claussenii to metabolize both of the monomers that comprise lactose (glucoseand galactose) separately, when YAW inoculated with B. claussenii was supplemented withlactase, analysis of the sugar profile of the finished fermentate revealed that the yeast hadconsumed the glucose but left a great majority of the galactose untouched. Those resultsmade clear the need for the acquisition of more fundamental knowledge regarding thefermentative behaviors of yeasts employable in the development of value-added productsderived from acid whey.

The current work aims to investigate the metabolic processes of such yeasts, namelyS. cerevisiae, K. marxianus, and B. claussenii, in a simple medium, in order to observe theseyeasts’ utilization of dairy-relevant sugars outside of the context of YAW, the chemicalcomposition of which may complicate such observation due to the presence of constituentsthat obscure the view of the yeasts’ basic metabolic processes. Such YAW constituentsinclude chemicals with buffering capacity, such as whey proteins and minerals [19], thosewith chelating activity, such as beta-lactoglobulin [20], and those with antifungal properties,such as lactoferrin [21].

While the present article describes observations made regarding fermentation underanoxic conditions, Part II of this study will present the results obtained in oxic environ-ments. Here, we aimed to explore the fermentation of dairy-relevant sugars by S. cerevisiae,K. marxianus, and B. claussenii, focusing on the potential for ethanol production, all within

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the context of a medium adjusted to the initial sugar concentration and pH conditions char-acteristic of YAW. These results provide a deeper understanding of dairy sugar utilizationby relevant yeasts, allowing for future work to optimize fermentations in order to improveand expand upon value-added beverage production from YAW, as we continue to work torealize the potential of alcoholic fermentation as a means for the reintroduction of YAWinto the food supply chain.

2. Materials and Methods2.1. Materials

The following reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA):chloramphenicol, yeast nitrogen base with amino acids, D-lactose monohydrate, D-glucose,D-galactose, and lactic acid [90%]. A K. marxianus culture was obtained from the CornellUniversity Food Safety Laboratory (isolate: FSL B9-0008). S. cerevisiae (IOC BE FRUITSTM)and B. claussenii (OYL-201) cultures were obtained from Lallemand Oenology (Edward-stown, South Australia) and Omega Yeast Labs (Chicago, IL, USA), respectively.

2.2. Preparation of Inocula

Cryopreserved cultures of K. marxianus and B. claussenii were taken from their storageat −80 ◦C and were subcultured on Potato Dextrose Agar (Hardy Diagnostics, Santa Maria,CA, USA) supplemented with 25 mg chloramphenicol per liter medium. Incubation tookplace at 30 ◦C, for a duration of 2 days for K. marxianus and 6 days for B. claussenii. Next,a single colony from each culture was inoculated into 5 mL of a sterile 12% wt/v suspensionof commercial Dry Malt Extract (Briess Malt and Ingredients Company, Chilton, WI, USA),which was then incubated for 48 h at 30 ◦C, with a constant agitation of 200 rpm. After thisstep, each culture was propagated in a flask containing 50 mL of Dry Malt Extract broth,which was incubated at the same temperature and level of agitation, until each speciesreached a cell concentration level greater than 3.2 × 108 cfu/mL (after approximately2 days). Each culture’s cell concentration and viability were monitored daily. Determi-nations of viability followed the method of Painting and Kirsop [22] with modifications:a 1:10 dilution of the culture was prepared using 1X phosphate buffered saline solution;this suspension was then diluted 1:2 with methylene blue [0.1% v/v] (Ward’s NaturalScience, Rochester, NY, USA); the diluted culture was then applied to a hemocytometerand visualized under a light microscope. New cultures were propagated for each replicate.An S. cerevisiae inoculum was prepared according to the manufacturer’s recommendations.To achieve the desired inoculation level, an appropriate mass of active dried yeast wasweighed, and was then rehydrated in sterile ultrapure water (Milli-Q Advantage A10system, MilliporeSigma, Burlington, MA, USA) for 30 min prior to the inoculation (asdescribed below).

2.3. Fermentation Media

Yeast nitrogen base with amino acids (YNB) was used as the common base for 4 dis-tinct media, each supplemented with a different fermentable carbon source. First, the de-hydrated YNB medium was reconstituted to a 1X solution, and then 40 g of either lac-tose (LAC), glucose (GLU), galactose (GAL), or a 1:1 mixture of glucose and galactose(GLU:GAL) was added per liter medium. Each treatment therefore had an overall concen-tration of fermentable sugars simulating that observed in YAW; whereas the LAC treatmentsimulated the sugar profile of YAW, the GLU:GAL treatment represented the sugar profileof YAW immediately following the full enzymatic hydrolysis of its lactose, and the GLUand GAL treatments were to serve as relatively less complex references against which tocompare the other two treatments. A solution of 22.5% lactic acid (v/v) was then addedto these preparations to adjust the pH of each to 4.2000 ± 0.0050, as measured with aniCinacTM analyzer (AMS Alliance, Rome, Italy); this resulted in a final concentration ofapproximately 0.1 g lactic acid per liter for all treatments. Finally, these preparations weresterilized using 0.45 µm polyethersulfone vacuum-membrane filters (VWR International,

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Radnor, PA, USA), and were stored at 4 ◦C until later use. New media were prepared foreach replicate.

2.4. Inoculation and Fermentation

The media were all divided into 500 mL aliquots, and with respect to each type of sugarprofile, each aliquot was inoculated with either S. cerevisiae, K. marxianus, or B. clausseniito achieve an initial concentration of approximately 4 × 106 cfu/mL. For this purpose,K. marxianus and B. claussenii starters were centrifuged at 3220× g for 2 min, supernatantswere discarded, and remaining pellets were resuspended in sterile Milli-Q water. Re-hydrated S. cerevisiae culture was used for inoculation without further treatment. Eachfermentation was carried out in a 1L Erlenmeyer flask, fitted with a stopper and an airlock(The Vintage Store, Delta, BC, Canada); airlocks were each filled with 15 mL of sterile Milli-Q water to prevent both oxygen infiltration and over-pressurization with carbon dioxide.Incubation took place at 25 ◦C for 7 days for S. cerevisiae and K. marxianus, and 18 daysfor B. claussenii; the fermentates were gently agitated (125 rpm) to maintain cultures insuspension, while approximating an anoxic environment. Three biological replicates werecarried out for each fermentation; that is, replicates were run on different days, each timeusing fresh fermentation media and newly grown cultures, as previously stated.

2.5. Data Collection2.5.1. Density, pH, and Microbial Enumeration

Samples for each type of analysis were collected every 24 h and measured in duplicate.To obtain density data, 7 mL of sample from each treatment was collected. These sampleswere degassed using an ultrasonic bath (VWR, Radnor, PA, USA) for 20 min, and wereanalyzed with a DMATM 35 density meter (Anton Paar, Graz, Austria), at 20 ◦C. For pHanalyses, a 4-mL sample of each treatment was taken and measured via the iCinac TM

analyzer. Microbial enumerations were obtained by sampling 0.5 mL from each treatment,doing serial dilutions with 1X phosphate buffered saline solution, and spread-plating onPotato Dextrose Agar supplemented with chloramphenicol at the concentration statedpreviously. Resulting plates were incubated at 30 ◦C for a duration of 24 h for K. marxianus,48 h for S. cerevisiae, and 144 h for B. claussenii. After the incubation period, counts weredetermined using a Color Q-Count model 530 (Advanced Instruments Inc., Norwood,MA, USA).

2.5.2. Analyses of Ethanol, Organic Acids, and Sugars

A sample of 20 mL was collected at the beginning and the end of each fermentationfor determination of concentrations of ethanol, lactic acid, acetic acid, and sugars (lactose,glucose, and galactose). These samples were sent to the Cornell Craft Beverage AnalyticalLaboratory (Cornell Agritech, Geneva, NY, USA), where they were analyzed using aProminence HPLC system (Shimadzu, Kyoto, Japan). This system was equipped with a300 × 7.8 mm Rezex™ ROA-Organic Acid H+ Column (Phenomenex, Torrance, CA, USA);a Photodiode Array Detector, model SPD-M20A (Shimadzu, Kyoto, Japan); and a RefractiveIndex Detector, model RID-10A (Shimadzu, Kyoto, Japan). A mobile solution of 0.005 NH2SO4 was used, and 20 µL of sample was injected at a constant rate of 0.5 mL/min.

Results are reported in percentage by volume (% v/v) for ethanol, and in g/L fororganic acids and sugars. Additionally, ethanol results were compared to the maximumachievable ethanol yields for each treatment, which were calculated using the follow-ing equation:

Maximum Ethanol Yield (% v/v) =[Sugar]0 × Theoretical Yield

7.892(1)

In which [Sugar]0 represents the initial sugar concentration in each treatment in g/L;the theoretical yield is considered to be 0.511 g ethanol/g glucose or galactose, and 0.535 gethanol/g lactose; and the denominator represents a factor of the density of ethanol at 20 ◦C.

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2.6. Statistical Analysis2.6.1. General Analysis

Exploratory statistical analyses were performed using JMP software (SAS Institute,Cary, NC, USA), and significance level was set to 0.05. ANOVA and Tukey’s HonestSignificant Difference Test were used to compare differences among means of severaltreatments at the same timepoint, and the “Matched Pairs” function was used for non-independent datapoints in longitudinal data analysis. In all graphs and tables, eachdatapoint shows the mean, and the variability range represents the standard deviationamong replicates. Cell concentration results were log-transformed and analyzed using alinear scale.

2.6.2. Non-Linear Modeling

Non-linear fitting was performed using the Specialized Modeling—Fit Curve functionin JMP. This analysis was performed for each individual species, using average density asthe response, time as the regressor, and fermentable carbon source as the group. All curveswere fitted using logistic models of 3 and 4 parameters, as well as exponential models of2 and 3 parameters. The best fit for each species was selected using the Model Comparisontool in the Fit Curve platform, based on the corrected Akaike Information Criterion andthe significance of all estimates which, according to the Wald test, had p-values of <0.0001.The models that were selected were Logistic 4P for B. claussenii, and Exponential 3Pfor both K. marxianus and S. cerevisiae. Refer to Table S1 for details about the models’predictive equations.

Once a model was selected for each species, Analysis of Means (ANOM) was usedto compare the growth rate parameters among treatments for the same species. The label“significant difference” was applied when an estimated parameter fell outside of the upperor lower decision limit of the analysis when using a significance level of 0.05; this processwas facilitated by the Compare Parameter Estimates function in the Fit Curve platform.

3. Results and Discussion3.1. Fermentation Characterization3.1.1. Density

In the present study, density was used as a proxy for the concentration of fermentablesugars in the medium in order to provide data supplementary to those of actual sugarconcentrations, which were obtained for the beginning and end of each fermentation, as de-scribed before. While continuous density decrease would indicate ongoing consumptionof sugars and potential generation of volatile compounds and ethanol, a density valuethat remained unvaried over the course of several days would indicate completion offermentation. For that reason, with regard to each individual treatment, paired t-testswere performed to compare each possible pair of days covering the duration of the fer-mentation. On the final day of a 3-consecutive-day period for which none of the possiblepairs of days displayed a significant difference (p > 0.05), the fermentation in question wasconsidered concluded. Figure 1 presents the density profiles of B. claussenii, K. marxianus,and S. cerevisiae in YNB supplemented with different carbon sources.

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Figure 1. Density profiles for anaerobic fermentations of sugars commonly found in acid whey, incubated at 25 °C. Error bars represent the standard deviation of 3 biological replicates. Supplemented sugars at 40 g/L: lactose (LAC), 1:1 glu-cose/galactose mixture (GLU:GAL), glucose (GLU), and galactose (GAL). For each species, the box below the graph pre-sents the duration of fermentation (in days) in each substrate. To define the final day of each fermentation, paired t-tests were performed. On the final day of a 3-consecutive-day period for which none of the possible pairs of days displayed a significant difference (p > 0.05), the fermentation in question was considered concluded.

Our paired t-test showed that in the presence of lactose (LAC), B. claussenii completed the fermentation in 16 days. The ability of this species to utilize lactose in dairy by-prod-ucts has been studied by other authors [23,24]. For instance, Marcus, DeMarsh and Alcaine [24] explored the fermentation profile of B. claussenii in a 10% total solid whey permeate solution under anoxic conditions. Their results showed that there was a density decrease of 0.016 g/mL after 34 days at 30 °C; however, we observed the same reduction in 16 days, indicating a higher density drop rate. The discrepancy of these results may be attributable to the differences in the fermentation media (e.g., pH and composition) as well as to the differences in experimental conditions. The mechanisms by which B. claussenii can metab-olize lactose are still unclear. It has been demonstrated that both β-galactosidases and β-glucosidases possess lactase activity [25]. While B. claussenii has previously been deter-mined to produce a β-glucosidase, which has since been characterized [26], the production of β-galactosidase by B. claussenii has not yet been thoroughly investigated, so questions remain regarding this metabolism and any associated lactose transport mechanism.

When B. claussenii was in the presence of glucose and galactose together (GLU:GAL), different results were observed. Initially, the density dropped at a noticeably higher rate than that of B. claussenii in LAC, but it ultimately bottomed out by day 5. As presented in Figure 1, this density drop was approximately half of that observed in the previously men-tioned treatment, hypothetically due to the incomplete consumption of sugars in the fer-mentation medium (refer to Section 3.2 for further discussion on sugar composition). To the best of our knowledge, no study has explored sugar utilization by B. claussenii in a mixture of these two monosaccharides under these conditions. Moktaduzzaman, et al. [27] studied the effects that addition of glucose to a galactose medium had for B. bruxellensis under oxic conditions, but that species did not exhibit a fermentative metabolism for ga-lactose.

Separate treatments involving either glucose or galactose aimed to unveil any poten-tial difference in the speed at which these monosaccharides are metabolized by B. claussenii under anoxic conditions. Our results showed that this species can utilize glucose much quicker than galactose, decreasing the density of the medium containing the former in 7 days, as opposed to 18 days for that of the latter (Figure 1). A more formal approach

Figure 1. Density profiles for anaerobic fermentations of sugars commonly found in acid whey, incubated at 25 ◦C.Error bars represent the standard deviation of 3 biological replicates. Supplemented sugars at 40 g/L: lactose (LAC),1:1 glucose/galactose mixture (GLU:GAL), glucose (GLU), and galactose (GAL). For each species, the box below the graphpresents the duration of fermentation (in days) in each substrate. To define the final day of each fermentation, paired t-testswere performed. On the final day of a 3-consecutive-day period for which none of the possible pairs of days displayed asignificant difference (p > 0.05), the fermentation in question was considered concluded.

Our paired t-test showed that in the presence of lactose (LAC), B. claussenii completedthe fermentation in 16 days. The ability of this species to utilize lactose in dairy by-productshas been studied by other authors [23,24]. For instance, Marcus, DeMarsh and Alcaine [24]explored the fermentation profile of B. claussenii in a 10% total solid whey permeate solu-tion under anoxic conditions. Their results showed that there was a density decrease of0.016 g/mL after 34 days at 30 ◦C; however, we observed the same reduction in 16 days,indicating a higher density drop rate. The discrepancy of these results may be attributableto the differences in the fermentation media (e.g., pH and composition) as well as to thedifferences in experimental conditions. The mechanisms by which B. claussenii can me-tabolize lactose are still unclear. It has been demonstrated that both β-galactosidases andβ-glucosidases possess lactase activity [25]. While B. claussenii has previously been deter-mined to produce a β-glucosidase, which has since been characterized [26], the productionof β-galactosidase by B. claussenii has not yet been thoroughly investigated, so questionsremain regarding this metabolism and any associated lactose transport mechanism.

When B. claussenii was in the presence of glucose and galactose together (GLU:GAL),different results were observed. Initially, the density dropped at a noticeably higher rate thanthat of B. claussenii in LAC, but it ultimately bottomed out by day 5. As presented in Figure 1,this density drop was approximately half of that observed in the previously mentionedtreatment, hypothetically due to the incomplete consumption of sugars in the fermentationmedium (refer to Section 3.2 for further discussion on sugar composition). To the best of ourknowledge, no study has explored sugar utilization by B. claussenii in a mixture of these twomonosaccharides under these conditions. Moktaduzzaman, et al. [27] studied the effectsthat addition of glucose to a galactose medium had for B. bruxellensis under oxic conditions,but that species did not exhibit a fermentative metabolism for galactose.

Separate treatments involving either glucose or galactose aimed to unveil any potentialdifference in the speed at which these monosaccharides are metabolized by B. clausseniiunder anoxic conditions. Our results showed that this species can utilize glucose muchquicker than galactose, decreasing the density of the medium containing the former in7 days, as opposed to 18 days for that of the latter (Figure 1). A more formal approachfor the comparison of these curves assisted in the determination of the significance of

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this difference. See Section 3.1.1.1 for a detailed explanation of this approach. One of thereasons for this behavior may relate to the transport systems responsible for the uptakeof glucose and galactose by this species. While no empirical information is currentlyavailable in the literature regarding the nature of sugar transport in B. claussenii, genesencoding high-affinity glucose transporters were identified in B. bruxellensis and predictedfor B. claussenii [28]. Of the other species we investigated, S. cerevisiae similarly exhibitsfaster growth on glucose than galactose, and galactose is primarily transported through agalactose permease encoded by the GAL2 gene [29,30].

Figure 1 presents the density decrease curves for K. marxianus; compared to those ofB. claussenii, these curves show faster rates of consumption of the sugars studied. Hence,the appropriate duration of fermentation for this species was determined to be 7 days.In LAC, K. marxianus was able to complete the fermentation by day 4; in GLU:GAL, by day6. Although no previous study has explored the ability of K. marxianus to ferment lactosespecifically in YAW, its fermentation of lactose has been studied extensively in the contextof other dairy byproducts [24,31–33]. The authors who conducted those investigationsobserved the great fermentation potential of this species for this sugar, and our resultsare in accordance with those observations. In fact, K. marxianus performed similarly in allsubstrates investigated, as it completed the fermentations in 4 days in both GLU and GAL.

As seen with K. marxianus, S. cerevisiae exhibited a rapid metabolism in these substrates.It completely reduced the density of GLU:GAL in 6 days, GLU in 3 days, and GAL in 4 days.On the other hand, LAC maintained its initial density, as expected. Given S. cerevisiae’swell-documented inability to take up lactose, this treatment served as a negative controlagainst any external contamination. The LAC treatment notwithstanding, these resultssuggest that S. cerevisiae could maintain its characteristically efficient, rapid metabolism indairy effluents, even at this pH. Moreover, the authors of the present study have previouslyobserved the efficacy of S. cerevisiae in fermenting YAW, following lactose hydrolysis [18].

3.1.1.1. Non-Linear Density Modeling

To model the density curves of all treatments for each species, we used JMP’s special-ized modeling platform. Non-linear modeling has been used to characterize fermentationsin the past; one such example is that of Speers, et al. [34], who fit sigmoid-shaped logisticfunctions to changes in degrees Plato over time during the fermentations of 7 commercialbeer brands.

Based on the results observed in the current study, the changes in density over timefor the substrates fermented by B. claussenii were best modeled by sigmoidal profiles;consequently, the model that best fitted this density decrease was a logistic equation with4 parameters (Table S1). This logistic model describes the characteristics of the fermentationprocess using the following parameters: lower asymptote (a), which is an estimation of thelowest achievable density at the end of the fermentation; upper asymptote (b), an estimationof the initial density value; growth rate (c), an estimation of the steepness of the curve or thespeed at which the density decrease happened; and inflection point (d), an estimation of thetime point at which the density decrease reached its maximum rate before it started slowingdown. Refer to Tables S1 and S2 for details regarding the model equations for all carbonsources. This S-shaped model, in which density decrease fails to commence immediatelyfollowing inoculation, suggests that this species undergoes a longer period of adaptation inthe medium before it starts the process of cell replication, and later, of fermentation. Such aphenomenon was observed with all carbon sources fermented by B. claussenii (Figure S1).

Using the same type of model to describe density decrease in substrates fermentedby the same species allows for the comparison of estimated parameters across carbonsources, especially the growth rates, which could unveil any significant difference in therates of consumption of different sugars by this species. A comparison of growth rateestimates was conducted using ANOM (Figure S2), which identified the carbon sourcefor which density reduction was significantly higher or lower than the overall reductionaverage of all sugars as pertains to one species. The upper and lower decision limits of

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this analysis served as the cutoff values to indicate significant difference. Based on thisexamination, fermentation of lactose by B. claussenii proved significantly slower comparedto the respective fermentations of the rest of the carbon sources, while the fermentationsof both glucose and the combination of glucose and galactose proved significantly faster.It is important to note that even though the hybrid carbon source composed of glucose andgalactose exhibited the growth rate with the highest absolute value, this does not constitutethe best fermentation performance among substrates, since its higher “a” value estimate(lower asymptote) indicates that the fermentation stopped short of consuming all availablefermentable sugars (Table S2).

On the other hand, results obtained from K. marxianus showed an immediate densitydecrease, which was best fitted by an exponential model with 3 parameters. This modelis described by three components: asymptote (a), an estimation of the lowest achievabledensity value; scale (b), an estimation of the difference between the highest and lowestdensity values in the process; and growth rate (c), an estimation of the speed at which thedensity decreased. Refer to Table S1 for details regarding the model equation.

The ANOM test revealed that this species ferments lactose significantly faster than itdoes other carbon sources, as observed by its estimate value, which exceeded the lowerdecision limit (Figure S2). It is worth mentioning that even though this difference isstatistically significant, overall, K. marxianus exhibited a rapid fermentation rate with allcarbon sources, a fact that may be more useful on a practical level.

The metabolism of S. cerevisiae in the substrates in which density decrease was ob-served (GLU:GAL, GLU, and GAL) was also better estimated by an exponential modelwith 3 parameters, albeit with a higher root mean square error than that associated withK. marxianus (Table S1). Our results as determined by the ANOM test did not show anysignificant differences between the growth rates of any of the carbon sources that werefermented by this species (Figure S2), showing good fermentation rates for all.

3.1.2. pH

Another relevant characteristic during fermentation is pH, as it provides informationabout changes in the medium which may influence the fermentative performance of yeasts.Figure 2 presents the changes in pH that were observed in each medium supplementedwith a different carbon source. We observed as much as a 1.9-unit reduction in pH in themedia, with pH values dropping very rapidly during the first days of fermentation anddecreasing more slowly as the fermentations ceased. This general pH reduction profileis typically seen during fermentations due to the logarithmic nature of the pH scale [35].Likewise, preliminary fermentations of YAW showed this pattern of pH reduction, yet nodecrease greater than 0.25 pH units was observed [11]. Such behavior may be the resultof the characteristic mineral composition (K, Ca and P) of YAW [3], which improves itsbuffering capacity, as seen in other dairy products by Salaün, Mietton and Gaucheron [19].

As shown in Figure 2, there was a rapid decrease in pH when B. claussenii metabolizedeither GLU or GLU:GAL, as compared to the other 2 treatments. Early in the fermentations(days 2 and 3), a significant difference was observed in pH values when comparing thegroup comprising GLU and GLU:GAL with that comprising GAL and LAC; this differencesubsequently lost its significance as LAC and GAL were more rapidly metabolized later inthe fermentations. These results are in concordance with the significantly lower densitygrowth rates (i.e., steeper curves) observed in GLU and GLU:GAL, as seen in Figure 1 andFigure S2, respectively.

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Figure 2. pH profiles for anaerobic fermentations of sugars commonly found in acid whey, incubated at 25 °C. Error bars represent the standard deviation of 3 biological replicates. Supplemented sugars at 40 g/L: lactose (LAC), 1:1 glucose/ga-lactose mixture (GLU:GAL), glucose (GLU), and galactose (GAL).

As shown in Figure 2, there was a rapid decrease in pH when B. claussenii metabo-lized either GLU or GLU:GAL, as compared to the other 2 treatments. Early in the fermen-tations (days 2 and 3), a significant difference was observed in pH values when comparing the group comprising GLU and GLU:GAL with that comprising GAL and LAC; this dif-ference subsequently lost its significance as LAC and GAL were more rapidly metabolized later in the fermentations. These results are in concordance with the significantly lower density growth rates (i.e., steeper curves) observed in GLU and GLU:GAL, as seen in Fig-ures S2 and 1, respectively.

Fermentations carried out by K. marxianus (Figure 2) showed similar pH decrease patterns for all treatments except for GAL, wherein pH values were significantly different from day 1 until the end of the fermentation (day 7).

Fermentates produced by S. cerevisiae achieved final pH levels that were comparable to the ones seen with other yeast species, except for that of LAC, for which an increased and highly variable pH was observed (Figure 2). There was an increase of almost 1 pH unit at the end of the 7-day period, potentially due to the uptake of lactic acid in its disso-ciated form by this species. Cássio, et al. [36] observed that S. cerevisiae IGC4072 trans-ported lactate inside the cell by means of a proton-lactate symport, which was associated with an alkalinization of the medium. Moreover, the final pH value observed in this treat-ment was comparable to the one seen prior to the addition of lactic acid during the prep-aration of this medium.

3.1.3. Microbial Concentration Figure 3 presents the results of cell counts of all species in different carbon sources.

The graphs on the top represent the counts for LAC and GLU:GAL, while the graphs on the bottom represent those for GLU and GAL. The dotted lines show the targeted cell concentration at the time of inoculation. As compared to this initial level, almost all cases resulted in an increase of biomass at some point in the fermentation, and no apparent decrease in this microbial concentration was observed thereafter, even following the com-plete depletion of each sugar. Prolonged stationary phase in yeast is not surprising; Wer-ner-Washburne, et al. [37] stated that during their stationary phase of growth, S. cerevisiae cells can maintain almost 100% viability for up to 3 months without any added nutrients or carbon source, though this is highly strain- and environment-dependent.

Figure 2. pH profiles for anaerobic fermentations of sugars commonly found in acid whey, incubated at 25 ◦C. Error bars rep-resent the standard deviation of 3 biological replicates. Supplemented sugars at 40 g/L: lactose (LAC), 1:1 glucose/galactosemixture (GLU:GAL), glucose (GLU), and galactose (GAL).

Fermentations carried out by K. marxianus (Figure 2) showed similar pH decreasepatterns for all treatments except for GAL, wherein pH values were significantly differentfrom day 1 until the end of the fermentation (day 7).

Fermentates produced by S. cerevisiae achieved final pH levels that were comparableto the ones seen with other yeast species, except for that of LAC, for which an increasedand highly variable pH was observed (Figure 2). There was an increase of almost 1 pH unitat the end of the 7-day period, potentially due to the uptake of lactic acid in its dissociatedform by this species. Cássio, et al. [36] observed that S. cerevisiae IGC4072 transportedlactate inside the cell by means of a proton-lactate symport, which was associated with analkalinization of the medium. Moreover, the final pH value observed in this treatment wascomparable to the one seen prior to the addition of lactic acid during the preparation ofthis medium.

3.1.3. Microbial Concentration

Figure 3 presents the results of cell counts of all species in different carbon sources.The graphs on the top represent the counts for LAC and GLU:GAL, while the graphs on thebottom represent those for GLU and GAL. The dotted lines show the targeted cell concentra-tion at the time of inoculation. As compared to this initial level, almost all cases resulted inan increase of biomass at some point in the fermentation, and no apparent decrease in thismicrobial concentration was observed thereafter, even following the complete depletionof each sugar. Prolonged stationary phase in yeast is not surprising; Werner-Washburne,et al. [37] stated that during their stationary phase of growth, S. cerevisiae cells can maintainalmost 100% viability for up to 3 months without any added nutrients or carbon source,though this is highly strain- and environment-dependent.

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Figure 3. Yeast cell concentration profiles for anaerobic fermentations of sugars commonly found in acid whey, incubated at 25 °C. Top: lactose (LAC); 1:1 glucose/galactose mixture (GLU:GAL). Bottom: glucose (GLU); galactose (GAL). Dotted lines represent targeted cell concentration at time of inoculation. Error bars represent the standard deviation of 3 biological replicates.

It is important to note that B. claussenii exhibited a slow increase in its cell concentra-tion following inoculation, reaching exponential phase somewhere between day 1 and day 2. This longer lag period correlates to the one observed in density curves and is one possible reason that the density profile of this species was best described with a logistic model rather than an exponential model, which was found to be more appropriate for the density profiles of K. marxianus and S. cerevisiae (Section 3.1.1.1).

Growth curves for substrates fermented by K. marxianus show an increase of more than 1 log cfu/mL during the first 24 h of fermentation, indicating a short lag phase in these substrates that was not captured by our sampling frequency. Various researchers have investigated the kinetics of K. marxianus growth in cheese whey under acidic pH conditions [38,39]; their studies revealed that this species can exhibit a very short lag phase (2–4 h) in this substrate, reaching stationary phase within 16 h. It is also interesting to note that microbial counts of K. marxianus in the LAC and GLU:GAL treatments exhibited sim-ilar trends, a behavior that was not observed in any other group.

As observed in Figure 3, counts corresponding to S. cerevisiae started at a lower level than the desired inoculation and had higher variability, which may be explained by the use of active dried yeast that were rehydrated prior to inoculation, as opposed to liquid cultures for the other species. However, a relevant finding was observed in LAC, in which S. cerevisiae counts neither increased nor decreased throughout the fermentation period (matched pair comparisons revealed no significant differences between these numbers). The stable cell concentration of this species could be the result of an observed ability of S. cerevisiae to survive for extended periods with no carbon addition [37] as mentioned be-fore, or could be due to the use of lactic acid as an alternative energy source in this me-dium.

3.2. Sugar Concentrations Table 1 presents the sugar concentrations at the beginning and at the end of each

fermentation for all substrates. For the purposes of this study, the B. claussenii samples were considered to be finished fermenting as of 18 days, and samples fermented by K.

Figure 3. Yeast cell concentration profiles for anaerobic fermentations of sugars commonly found in acid whey, incu-bated at 25 ◦C. Top: lactose (LAC); 1:1 glucose/galactose mixture (GLU:GAL). Bottom: glucose (GLU); galactose (GAL).Dotted lines represent targeted cell concentration at time of inoculation. Error bars represent the standard deviation of3 biological replicates.

It is important to note that B. claussenii exhibited a slow increase in its cell concentrationfollowing inoculation, reaching exponential phase somewhere between day 1 and day 2.This longer lag period correlates to the one observed in density curves and is one possiblereason that the density profile of this species was best described with a logistic modelrather than an exponential model, which was found to be more appropriate for the densityprofiles of K. marxianus and S. cerevisiae (Section 3.1.1.1).

Growth curves for substrates fermented by K. marxianus show an increase of morethan 1 log cfu/mL during the first 24 h of fermentation, indicating a short lag phase inthese substrates that was not captured by our sampling frequency. Various researchershave investigated the kinetics of K. marxianus growth in cheese whey under acidic pHconditions [38,39]; their studies revealed that this species can exhibit a very short lag phase(2–4 h) in this substrate, reaching stationary phase within 16 h. It is also interesting tonote that microbial counts of K. marxianus in the LAC and GLU:GAL treatments exhibitedsimilar trends, a behavior that was not observed in any other group.

As observed in Figure 3, counts corresponding to S. cerevisiae started at a lower levelthan the desired inoculation and had higher variability, which may be explained by theuse of active dried yeast that were rehydrated prior to inoculation, as opposed to liquidcultures for the other species. However, a relevant finding was observed in LAC, in whichS. cerevisiae counts neither increased nor decreased throughout the fermentation period(matched pair comparisons revealed no significant differences between these numbers).The stable cell concentration of this species could be the result of an observed ability ofS. cerevisiae to survive for extended periods with no carbon addition [37] as mentioned be-fore, or could be due to the use of lactic acid as an alternative energy source in this medium.

3.2. Sugar Concentrations

Table 1 presents the sugar concentrations at the beginning and at the end of eachfermentation for all substrates. For the purposes of this study, the B. claussenii samples wereconsidered to be finished fermenting as of 18 days, and samples fermented by K. marxianusand S. cerevisiae were considered to be finished after 7 days; the data representing thesugar concentrations of the finished fermentates as presented here are in conformity withthis timeline.

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Table 1. Concentrations of sugars at the beginning and at the end of the fermentation of sugars commonly found in acidwhey, incubated at 25 ◦C. Displayed data represent the mean and standard deviation of 3 biological replicates.

Supplemented Medium

Lactose * Glucose + Galactose Glucose Galactose

Species Timepoint Lactose (g/L) Glucose (g/L) Galactose (g/L) Glucose (g/L) Galactose (g/L)

B. clausseniiDay 0 32.86 ± 1.95 19.07 ± 0.15 18.84 ± 0.49 37.12 ± 0.51 35.03 ± 0.34

Day 18 ND ND 15.28 ± 0.56 ND 0.01 ± 0.02

K. marxianusDay 0 33.17 ± 1.55 18.95 ± 0.12 18.39 ± 0.29 38 ± 1.05 34.95 ± 0.49Day 7 ND ND ND ND ND

S. cerevisiaeDay 0 32.87 ± 2.05 18.64 ± 0.17 18.45 ± 0.32 37.42 ± 0.12 35.58 ± 0.63Day 7 34.16 ± 0.61 ND ND ND ND

* Glucose and galactose that could potentially result from this treatment were not detected in any sample; ND: Non-detectable. Limits ofdetection, in g/L: lactose 0.003, glucose 0.004, and galactose 0.005.

Post-fermentation sugar concentrations showed that not all B. claussenii fermentatesunderwent a complete consumption of sugars. GLU:GAL retained a substantial amount ofgalactose residue, this suggests that under the conditions provided, B. claussenii fermentedonly glucose, leaving almost all galactose intact. These results correlate very well withwhat was observed in density curves, where this treatment achieved only half the densityreduction seen with the rest of the treatments (Figure 1). The factors driving this selectiveconsumption of sugars by B. claussenii when in the presence of glucose and galactose—notin lactose—and the associated mechanisms of this behavior are still unclear. One plausiblescenario involves the repression of genes involved in galactose metabolism in this speciesimmediately following inoculation, which could be caused by the initial glucose concentra-tion in the medium; a similar phenomenon was previously observed with D. bruxellensisCBS 2499 [27] and S. cerevisiae [29,30]. Later in the fermentation, by the time B. claussenii hasfermented all of the available glucose in the medium to ethanol or acetic acid, it is possiblethat a state of intracellular redox imbalance has been established, such that despite anysubsequent expression of gene products involved in galactose metabolism, a significant lagoccurs before the cells can finally utilize the available galactose. While the full investigationof such a lag phase is outside of the scope of the current research, cell count samples takenfor this treatment on day 18 contained viable CFUs at concentrations not significantlydifferent from those immediately following the initial inoculation, indicating that by theend of sampling, the B. claussenii culture was viable but abstaining from metabolism offermentable carbon sources. These findings propose an interesting direction for futureresearch to help understand the carbon metabolism and metabolic regulation exhibitedspecifically by B. claussenii. This knowledge may be relevant in industrial processes thataim for a selective fermentation for the development of novel beverages generated fromYAW, in which context, with the utilization of the proper lactase, glucose could be rapidlyconverted to ethanol or other metabolites, while the galactose that remains could pro-vide supplemental properties (e.g., contributions to flavor, texture, and functionality, etc.).As expected, results from the fermentations performed by K. marxianus and S. cerevisiaedemonstrated full consumption of sugars in all treatments, except for that of S. cerevisiaein LAC.

Luo, Demarsh, deRiancho, Stelick and Alcaine [11] studied YAW fermentations andreported full consumption of lactose by B. claussenii and K. marxianus under anoxic con-ditions after 21 days and 20 days, respectively. Additionally, their results in S. cerevisiaefermentates, treated with a lactase for the purposes of lactose hydrolysis prior to the fer-mentation, showed complete consumption of glucose and galactose within 8 days [11].Their observations suggest that YAW has intrinsic compositional factors, unaccounted forin the design of the current research, that affect the speed of fermentation in some species,as seen with K. marxianus.

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3.3. Production of Ethanol and Organic Acids

As stated previously, samples for analyses of ethanol and organic acids were col-lected at the beginning and at the end of each fermentation; the results are presented inTables 2 and 3.

Table 2. Concentration of ethanol [% v/v] at the end of the fermentation of sugars commonlyfound in acid whey, incubated at 25 ◦C. Displayed data represent the mean and standard deviationof 3 biological replicates. Within each row, different superscripts indicate instances in which thefermentation of the four different media by the same species resulted in fermentates with significantlydifferent (p < 0.05) ethanol concentrations.

Species TimepointSupplemented Medium

Lactose Glucose +Galactose Glucose Galactose

B. claussenii Day 18 1.78 ± 0.07 a 0.77 ± 0.10 b 1.86 ± 0.09 a 1.66 ± 0.10 a

K. marxianus Day 7 2.05 ± 0.03 a,b 1.99 ± 0.01 b 1.99 ± 0.03 b 2.11 ± 0.04 a

S. cerevisiae Day 7 ND 2.00 ± 0.09 a 2.02 ± 0.06 a 1.94 ± 0.03 a

ND: Non-detectable. Limit of detection: 0.009% v/v.

Table 3. Concentrations of organic acids (lactic acid and acetic acid) at the beginning and at the end of the fermentationof sugars commonly found in acid whey, incubated at 25 ◦C. Displayed data represent the mean and standard deviationof 3 biological replicates. Within each row, different superscripts indicate instances in which the fermentation of the fourdifferent media by the same species resulted in fermentates with significantly different (p < 0.05) organic acid concentrations.

Species TimepointLactose Glucose + Galactose Glucose Galactose

Lactic Acid (g/L)

B. clausseniiDay 0 0.101 ± 0.002 0.102 ± 0.008 0.104 ± 0.004 0.104 ± 0.003

Day 18 ND ND ND ND

K. marxianusDay 0 0.1 ± 0.004 0.102 ± 0.006 0.116 ± 0.016 0.102 ± 0.007Day 7 ND 0.048 ± 0.041 ND ND

S. cerevisiaeDay 0 0.101 ± 0.002 0.104 ± 0.003 0.104 ± 0.002 0.105 ± 0.001Day 7 ND 0.099 ± 0.011 a,b 0.132 ± 0.018 a 0.096 ± 0.014 b

Acetic Acid (g/L)

B. claussenii Day 0 ND ND 0.013 ± 0.023 NDDay 18 2.728 ± 0.425 a 2.305 ± 0.77 a 1.66 ± 0.339 a 3.096 ± 0.696 a

K. marxianus Day 0 ND ND 0.014 ± 0.024 NDDay 7 0.184 ± 0.033 a 0.326 ± 0.082 a 0.311 ± 0.042 a 0.299 ± 0.08 a

S. cerevisiae Day 0 ND ND ND NDDay 7 ND 0.513 ± 0.382 a 0.348 ± 0.139 a 0.547 ± 0.309 a

ND: Non-detectable. Limit of detection: 0.091 g/L (lactic acid) and 0.001 g/L (acetic acid).

3.3.1. Ethanol

B. claussenii produced an equivalent amount of ethanol in each fermentation medium,with the exception of GLU:GAL, in which galactose consumption was minimally observed(Table 1). Fermentation of this substrate yielded less than 0.8% v/v of ethanol, comparedto an average of 1.8% v/v for other fermentable carbon sources. Such results translate toan efficiency rate of 31% for GLU:GAL, and an average rate of 77% for the other sugars,as compared to the maximum achievable ethanol yields presented in Table S3. Brettanomycesspp. are known to be effective acetic acid producers [40] thus, the lower ethanol yieldsseen in GLU:GAL could be the result of a combination of influences, including the totalconcentration of sugars consumed, and the conversion of a portion of those sugars to aceticacid, rather than ethanol (see Section 3.3.2).

K. marxianus achieved an ethanol concentration of around 2% v/v in all substrates.These results are in accordance with the ones observed by Luo, Demarsh, deRiancho,Stelick and Alcaine [11]; in their study, YAW with an initial lactose concentration of

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32.9 g/L yielded 2.33% v/v ethanol upon being fermented anaerobically by K. marxianus.The values observed in the present study represent an average fermentation efficiency of87%, as compared to the maximum achievable ethanol yields; the fraction of consumedsugar that is unaccounted for was potentially diverted to the formation of biomass or thegeneration of other metabolites.

S. cerevisiae exhibited similar levels of ethanol production for GLU, GAL, and GLU:GAL(around 2% v/v, and 84% efficiency). As has been demonstrated in innumerable previousstudies, this species is well known to dependably produce ethanol in a variety of matrices.

3.3.2. Organic Acids

Table 3 presents the concentrations of lactic acid and acetic acid at the beginning andend of each fermentation. With B. claussenii, complete depletion of initial lactic acid wasobserved, suggesting utilization of this compound as part of its metabolism. Productionof acetic acid was not significantly different between carbon sources, in spite of the lowerlevel of sugar consumption observed in GLU:GAL for this species.

With K. marxianus, lactic acid consumption was also detected. Even though we found aresidual amount of lactic acid in GLU:GAL, we considered it to be negligible. The transportof lactate anions was observed by Fonseca, et al. [41] in this species, which seems to occurby means of a monocarboxylate uniporter. Following fermentation, the concentration ofacetic acid produced by K. marxianus in each substrate was lower than that produced byB. claussenii in the same substrate; this, combined with the opposite scenario for ethanol,suggests an intrinsic inclination of K. marxianus’ metabolism toward ethanol formation,at the expense of the production of acetic acid.

Of all the carbon sources, S. cerevisiae only depleted lactic acid when in the presenceof lactose, in contrast to the metabolisms of the other species. Assimilable sugars in themedium could act as repressors of lactate’s transport in this yeast; such repression hasalready been demonstrated to occur in the presence of glucose, as reported by Cássio, Leãoand van Uden [36]. Regarding acetic acid, S. cerevisiae exhibited minimal synthesis in alltreatments except in LAC, in which no acetic acid was produced. Overall, our resultsindicate that, in this treatment, S. cerevisiae remained viable due to its ability to metabolizelactic acid, showing no indications of sugar consumption, replication, nor production ofethanol nor organic acids.

4. Conclusions

This work aimed to describe the processes of fermentation of dairy-relevant sugarsby S. cerevisiae, K. marxianus, and B. claussenii, along with each’s relative potential toproduce ethanol in media adjusted to the initial sugar concentration and pH conditionscharacteristic of YAW. Our results indicated that these species showed distinct sugarutilization rates and ethanol yields in the various substrates. Although K. marxianus andS. cerevisiae displayed rapid sugar consumption with consistent ethanol yield efficiencies inall media, B. claussenii showed variable rates of sugar utilization and variable ethanol yields.Additionally, our results revealed a difference in the sugar uptake of B. claussenii in LACas compared to that in GLU:GAL, in which this organism displayed a strong propensitytoward fermentation of glucose to the almost complete exclusion of galactose. Theseobservations point to the possibility of developing versatile biomanufacturing processesthat could target glucose for ethanol production, while leaving available residual galactosethat could confer resulting products with additional properties, or that could be usedas an input for potentially cost-effective galactose powder production. They also elicitquestions regarding the sugar metabolism of B. claussenii and offer openings to expandthe existing body of literature related to this species. Overall, these results provide adeeper understanding of dairy sugar utilization by relevant yeasts, allowing for futurework to optimize fermentations in order to improve valued-added beverage and ingredientproduction from YAW. Further studies should investigate the profiles of these fermentations

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under oxic conditions for potential organic acid synthesis, as well as the optimal operationalparameters that maximize ethanol production from YAW and other dairy byproducts.

Supplementary Materials: The following are available online at https://doi.org/10.7298/v23f-9x12,Table S1: Prediction models for density curves of anaerobic fermentations by different yeasts, innon-selective medium supplemented with sugars commonly found in acid whey, Table S2: Estimatedparameters of fitted models for density curves of anaerobic fermentations by different yeasts, Fig-ure S1: Model plots for density curves of anaerobic fermentations by different yeasts, in non-selectivemedium supplemented with sugars commonly found in acid whey, Figure S2: Analysis of Mean(ANOM) of growth rate parameters obtained per carbon source for each yeast species, Table S3:Estimated maximum achievable ethanol yields in each treatment (in% v/v), as calculated usingEquation (1).

Author Contributions: Conceptualization, S.D.A. and V.K.R.F.; methodology, V.K.R.F., T.A.D. andS.D.A.; software, V.K.R.F.; validation, V.K.R.F. and T.A.D.; formal analysis, V.K.R.F. and T.A.D.;investigation, V.K.R.F. and T.A.D.; resources, S.D.A.; data curation, V.K.R.F. and T.A.D.; writing—original draft preparation, V.K.R.F. and T.A.D.; writing—review and editing, V.K.R.F., T.A.D., S.D.A.and P.A.G.; visualization, V.K.R.F. and T.A.D.; supervision, S.D.A. and P.A.G.; project administration,S.D.A.; funding acquisition, S.D.A. All authors have read and agreed to the published version ofthe manuscript.

Funding: This research was funded by the New York State Dairy Promotion Advisory Board,appointed by the New York State Department of Agriculture and Markets (Albany, NY, USA).

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: The data presented in this study are available on request from thecorresponding author.

Acknowledgments: We would like to thank the New York State Dairy Promotion Advisory Board,appointed by the New York State Department of Agriculture and Markets (Albany, NY, USA),for funding this study. We also acknowledge the Cornell Craft Beverage Analytical Laboratory(Geneva, NY, USA), particularly Pamela Raes, for her valuable assistance executing the HPLCanalyses that allowed for the quantification of fermentation metabolites.

Conflicts of Interest: The principal investigator of this research, Sam Alcaine, is the cofounder of thestartup company Norwhey Brewing, which aims to transform yogurt whey into low-alcohol seltzers.

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