The recovery and sustainable exploitation of industrial waste is a theme that cuts across all production chains, and there are international policies that introduce guidelines to solve this problem. An example is represented by goal 12 of the United Nations 2030 Agenda, where “Ensure sustainable consumption and production patterns” is identified among the priorities (Tsalis et al., 2020). Cheese whey (CW), the liquid by-product resulting from the curdling process in cheese-making (Osorio-González et al., 2022), is considered one of the most important pollutants of the dairy industry due to the presence of lactose and hardly-biodegradable proteins. Furthermore, several studies estimated a growing production of CW in the next decades (Lappa et al., 2019; Figueroa Pires et al., 2021). This industrial waste represents approximately 90% of the milk volume (Figueroa Pires et al., 2021) and retains 55% of the milk nutrients (Pendón et al., 2021). Specifically, most of the lactose in milk remains in the CW, contributing to 90% of the total organic load; with the remaining part represented by fats and proteins (Prazeres et al., 2012; Figueroa Pires et al., 2021). CW is characterized by high Biochemical Oxygen Demand (BOD) and Chemical Oxygen Demand (COD) values, equal to 27–60 kg m⁻³ and 50–102 kg m⁻³, respectively (Prazeres et al., 2012; Rocha and Guerra, 2020). The growing interest in the use of renewable feedstock to produce biochemicals, biomaterials, and biofuels also involves agro-industrial and food waste, such as CW. Significant financial resources are needed for CW disposal in order to lower the polluting load and prevent environmental issues like excessive oxygen consumption, soil eutrophication and depletion, and groundwater contamination (Caballero et al., 2021; Dinkci, 2021). Because of the high nutritional content, roughly 50% of whey is utilized for food (Asunis et al., 2020; Zotta et al., 2020; Barba, 2021) and animal nutrition sectors (El-Tanboly et al., 2017; Palmieri et al., 2017; Zou and Chang, 2022; Arshad et al., 2023).

A biorefinery approach for the valorization of this dairy by-product is the extraction of valuable proteins by membrane filtration methods and the recovery of a lactose-rich effluent called cheese whey permeate (CWP) (Nobre Macedo et al., 2020; Zandona et al., 2021). Nonetheless, a key challenge is that many wild-type microorganisms are unable to metabolize lactose directly. Over the past two decades, various methods have been developed to enable Saccharomyces cerevisiae to consume lactose, including enzymatic conversion of lactose into glucose and galactose using free or immobilized enzymes, as well as genetic modifications to enhance lactose metabolism (Zou and Chang, 2022). However, most strains showed unwanted characteristics, such as genetic instability or inability to produce ethanol with high concentration and/or yield (Rubio-Texeira, 2006; Zou et al., 2013).

Since the purchase of the commercial β-galactosidase enzyme at the industrial process scale is not compatible with a cost-effective process, non-conventional yeast strains that naturally exhibit β-galactosidase activity, such as Kluyveromyces marxianus, have been recently studied (Rocha and Guerra, 2020). K. marxianus exhibits superior traits, such as the ability to grow on a wide range of substrates with high specific growth rates, tolerance to high temperatures (up to 52 °C) (Fonseca et al., 2008), as well as the capacity to produce ethanol with high yield and productivity and valuable bioproducts, such as proteins and cell biomass (Carvalho et al., 2021). Together with the GRAS (Generally Recognized As Safe) classification, these features make this yeast an appealing microorganism for application in CW valorisation (Martynova et al., 2016; Rouanet et al., 2020).

Up to now, the majority of the biotechnological processes reported in the scientific literature, aiming to convert lactose present in CW, is based on the use of β-galactosidase for the preliminary hydrolysis of lactose to give its two monosaccharides, namely glucose and galactose (Awasthi et al., 2022). Lactose hydrolysis has various advantages, including nutritional and technical benefits; indeed, due to the increasing lactose-intolerant population, the commercial interest in β-galactosidase manufacturing is tied to large-scale food and medicinal uses (Fonseca et al., 2008; Rollini et al., 2008). As well documented in the literature, K. marxianus can be effectively used for the production of β-galactosidase from CW (Bansal et al., 2008; Yadav et al., 2014; Bilal et al., 2022; Singh and Sambyal, 2023). Moreover, only a few Kluyveromyces yeast species have significant potential for the efficient production of bioethanol from CWP (Díez-Antolínez et al., 2018). Particularly, K. marxianus exhibits advantageous physiological traits, such as the ability to thrive in harsh industrial environments and effectively utilize lactose-rich substrates, that make it a promising whole-cell biocatalyst for simultaneous production of bioethanol and β-galactosidase starting from industrial side-streams and/or waste in the perspective of modern circular bioeconomy (Beniwal et al., 2017; Karim et al., 2020).

However, from an industrial point of view, the selection of a proper yeast strain with appropriate physiological features is crucial to ensure an efficient lactose utilization from CWP and a competitive and selective production of ethanol and β-galactosidase with respect to other yeast species (Fonseca et al., 2013; Morrissey et al., 2015).

In this context, the present study aims to provide a comprehensive physiological characterization of three K. marxianus strains and assess their potential for the sustainable and profitable valorization of CWP, mainly through bioethanol production. In particular, K. marxianus DSM 5422, K. marxianus DSM 7239, and K. marxianus DSM 5572 were selected due to their ability to efficiently ferment lactose (Ozmihci and Kargi, 2007a,b,c,d; Lane et al., 2011; Rocha et al., 2011; Ortiz-Merino et al., 2018; Leandro et al., 2019; de Albuquerque et al., 2021). The proposed physiological characterization was mainly focused on the study of the β-galactosidase enzyme activity for lactose hydrolysis and its correlation to the fermentation of CWP to ethanol. For this purpose, preliminary microplate growth tests on various carbon sources and measurements of β-galactosidase enzyme activity were carried out to increase the knowledge of K. marxianus physiology and increase its industrial biotechnology applications (Fonseca et al., 2008). Subsequently, flask fermentation tests were carried out to evaluate the performance of these three strategic yeast strains in ethanol production from the waste-derived CWP and define the best microorganism for the proposed third-generation biorefinery model.

Considering the high specific growth rate of K. marxianus DSM 5422 in the presence of a wide range of lactose concentrations (20–200 g/L), it can be considered a promising candidate for industrial applications in modern biorefinery schemes. Moreover, in the preliminary screening, the temperature of 37 °C represented the most favorable condition for the growth of the selected yeast strain. The optimal lactose concentration for the maximization of μ_max was 130 g/L for both K. marxianus DSM 5422 and DSM 7239. Lactose concentration in CWP is usually comprised in the range 140–180 g/L (Díez-Antolínez et al., 2016). For this reason, the preliminary results obtained for the three strains in MTP tests encouraged further scale-up to the flask to validate these findings. Similar results on the physiology of K. marxianus were reported by Saini et al. (2017), who found that an increase in sugar concentration in the fermentation medium up to 150 g/L of lactose led to a corresponding rise in colony-forming units of these yeast strains. However, higher concentrations generated significant osmotic stress for cells, which caused a decrease in cell vitality and the quantified colony-forming units (Saini et al., 2017).

Regarding the β-galactosidase activity test, statistically significant differences were not observed in the activity between the exponential growth phase and the stationary phase of the selected yeasts. These findings were consistent with previous studies (Bacci Junior et al., 1996). On the contrary, significant differences in enzymatic activity were observed between the yeast strains. In particular, K. marxianus DSM 7239 showed a maximum β-galactosidase activity of 27.8 ± 0.9 U mg⁻¹ after 16 h of incubation.

As reported in the study of Rollini et al. (2008), in most K. marxianus strains, β-galactosidase is primarily located inside the cell (as intracellular enzyme). Preliminary tests conducted on the supernatant of non-permeabilized cells confirmed the intracellular localization for all the strains studied in the present work. Moreover, tests conducted on untreated cells, as a control, indicated that permeabilization of cells increased the β-galactosidase activity, which is mainly located inside the cells. Not significant differences were detected between the yeast strains under the adopted test conditions. These findings are in accordance with other similar studies (Bacci Junior et al., 1996; Rollini et al., 2008).

Regarding the kinetic study of β-galactosidase activity through the monitoring of the ONPG hydrolysis to give ONP, the K_M values of the three strains and their enzymes' affinity for the substrate were similar at 30 °C and slightly different at 37 °C. Differently, significant differences were observed in V_max values, both at 30 and 37 °C. The significantly higher V_max for the DSM 7239 strain could indicate that the catalytic activity of its β-galactosidase is intrinsically higher in the adopted experimental conditions. Moreover, this result could also be related to the overexpression of β-galactosidase by this strain, namely, a higher amount of enzyme produced per unit of biomass, compared to the other strains. At the same time, the absence of any significant differences in the enzymatic activity of β-galactosidase between the exponential and stationary growth phases excluded a diverse level of expression of the enzyme in different cell-cycle stages. These results aligned with the higher β-galactosidase activity (V₀/mg) observed at increasing ONPG concentrations at both temperatures considered.

The K_M values obtained for K. marxianus DSM 5422, DSM 7239, and DSM 5572 in the present investigation (23–50 mM) were higher than those reported in the literature for other strains, such as K. marxianus ATCC 16045 (2.3 mM) and K. marxianus CCT 7082 (3.3 mM) (Cavalcante Braga et al., 2013; Selvarajan and Mohanasrinivasan, 2015). This difference may be due to the use of a crude enzyme preparation from permeabilized cells in this study, as opposed to other studies where the test was conducted using purified enzymes (Osorio-González et al., 2022). Furthermore, the permeabilization process can increase the usefulness of CW for yeast that does not consume lactose. For example, permeabilized K. marxianus cells were used in co-culture with S. cerevisiae in hydrolysed CW (Yadav et al., 2014) to produce mixed-culture biomass. Another example of its application was the use of permeabilized K. marxianus cells for lactose hydrolysis in CW. This was then fermented by Aureobasidium pullulans in a mixed culture to produce polymalic acid. This expands the applications of K. marxianus to co-culture systems for other fermentative processes involving non-lactose-consuming microorganisms (Xia et al., 2021).

The enzyme kinetics study revealed substantial inter-strain variability in β-galactosidase performance, despite the similarity in substrate affinity (K_M). Across both tested temperatures (30 °C and 37 °C), the three strains exhibited comparable K_M values, suggesting that the affinity of their β-galactosidases for the synthetic substrate ONPG is not the major determinant of their physiological differences. However, clear divergence emerged in their maximum catalytic rate (V_max) under high substrate availability.

Among the strains, DSM 7239 consistently displayed the highest V_max values, especially at elevated ONPG concentrations. These kinetic features, correlate well with the high specific activities measured in permeabilized cell extracts (27.8 ± 0.9 U mg⁻¹), reinforcing the notion that DSM 7239 is the most efficient lactose-hydrolyser.

By contrast, DSM 5422 and DSM 5572 showed lower V_max values, reflecting a reduced enzymatic activity compared with DSM 7239. However, these strains exhibited similar kinetic profiles to each other, with no significant differences in hydrolytic activity across the range of ONPG concentrations tested. This suggests that their enzymatic machinery for lactose hydrolysis is more balanced, leading to steadier lactose assimilation during fermentation.

Regarding flask fermentation on SSM-50 g/L lactose, μ_max values were higher at flask scale than in MTP tests. Nevertheless, the micro-cultivation experimental set-up proved useful for predicting the physiological behavior of the investigated strains. Indeed, at both the MTP and flask scales, a temperature of 37 °C produced a better specific growth rate than 30 °C. Metabolic activity in yeast produces heat, causing the temperature of the bioreactor to rise during fermentation. Using thermotolerant yeast strains could therefore play a significant role in high-temperature fermentation, as it reduces the risk of contamination and cooling costs at an industrial scale (Saini et al., 2017). Another attractive feature is that K. marxianus showed a higher specific growth rate at 42 °C, which gives it an advantage over bacteria in a non-sterile industrial process (Pendón et al., 2021). Most bacterial and fungal contaminants of industrial fermentations thrive below 40 °C; thus, maintaining the process at ≥42 °C provides a selective environment favoring thermotolerant yeasts such as K. marxianus (Pendón et al., 2021). This reduces the need for costly sterilization steps, which represent a significant burden in large-scale operations (Tesfaw, 2023). Lower contamination pressure translates into greater process robustness, reduced downtime, and lower operating costs in non-sterile biorefineries (Saini et al., 2017). Another benefit relates to energy balance. Fermentation processes generate metabolic heat, and cooling is a substantial cost factor in large bioreactors. By using strains capable of performing efficiently at 42–45 °C, the cooling demand is reduced (Pattanakittivorakul et al., 2024). This aligns with the goal of lowering the carbon footprint of bioprocessing. However, running at higher temperatures has implications for yeast physiology and medium composition. K. marxianus is known to tolerate up to 52 °C (Fonseca et al., 2008), but performance differences between strains are significant, as shown in this study. Above 42 °C, certain strains exhibit reduced growth or impaired lactose uptake, which may limit ethanol productivity if not carefully matched with process design (Kosaka et al., 2022). Additionally, prolonged exposure to elevated temperatures can increase cellular stress, potentially leading to reduced viability in repeated-batch or continuous fermentations (Fu et al., 2019). This may necessitate strain adaptation, evolutionary engineering, or process strategies such as cell recycling to maintain stable performance. Medium composition also becomes critical at high temperature (Flores-Cosío et al., 2024). In complex nutrient-poor substrates, such as CWP, nitrogen limitation may interact synergistically with thermal stress, slowing down lactose metabolism. Supplementation with low-cost nitrogen sources (e.g., urea or corn steep liquor) may therefore be required in large-scale applications to sustain fermentation rates under non-sterile, thermophilic conditions. Finally, downstream processing considerations should not be overlooked. Higher fermentation temperatures can facilitate ethanol recovery by distillation, as the feed enters distillation columns closer to operating conditions, thereby reducing energy inputs (Kumakiri et al., 2021). However, foaming and viscosity issues may be exacerbated under thermophilic conditions and must be managed through process engineering.

For all these reasons, 42 °C was selected as the optimal temperature for this process and was investigated in shake flask experiments using SSM-lactose-rich and CWP-based media.

To validate the fermentation results obtained in the preliminary MTP tests in the presence of a higher lactose concentration (130 g/L), similar to that in the CWP, a flask-scale investigation of the physiological performance of the three strains was performed at 42 °C. DSM 7239 displayed the highest β-galactosidase specific activity, as well as superior catalytic efficiency at elevated ONPG concentrations compared to DSM 5422 and DSM 5572. However, despite this enhanced hydrolytic capacity, DSM 7239 consumed lactose more slowly, accumulated less biomass, and produced significantly less ethanol in both semi-synthetic media and CWP. This apparent contradiction highlights that efficient lactose hydrolysis does not necessarily translate into efficient fermentation. Several factors may contribute to this outcome. First, although DSM 7239 is proficient in breaking down lactose into glucose and galactose, its ability to channel these monosaccharides into central carbon metabolism and alcoholic fermentation pathways may be impaired at high substrate concentrations and elevated temperatures (Fu et al., 2019; Kosaka et al., 2022). Indeed, the strain showed slower lactose utilization kinetics at 42 °C compared to DSM 5422 and DSM 5572, suggesting that heat stress may negatively affect downstream metabolic steps such as galactose catabolism or glycolytic flux regulation, even if hydrolysis itself is not limiting (Beniwal et al., 2017). In addition, excessive galactose release could contribute to a metabolic bottleneck, since galactose assimilation in yeasts is typically slower than glucose and is tightly regulated (Beniwal et al., 2017). If galactose metabolism is inefficient, this could lead to suboptimal carbon flux into ethanol production, despite efficient lactose cleavage (Lyutova and Naumova, 2023). Third, the nutrient composition of CWP may exacerbate this imbalance. With its low nitrogen and micronutrient content, CWP imposes additional stress on yeast metabolism. DSM 7239, which already shows sensitivity to elevated temperature and osmotic pressure, may be more vulnerable to these combined stresses, further reducing its fermentative performance. In contrast, DSM 5422 and DSM 5572, while showing lower β-galactosidase activity, appear to balance lactose hydrolysis with efficient assimilation of glucose and galactose, achieving near-theoretical ethanol yields even in nutrient-limited whey permeate. Taken together, these findings suggest that DSM 7239 is an effective lactose utilizer rather than an efficient ethanol producer. Its high enzymatic activity could make it an attractive candidate for biotechnological applications where lactose hydrolysis itself is the desired outcome (e.g., production of lactose-free dairy products or bioconversion processes requiring glucose and galactose release). However, for ethanol production from CWP, DSM 5422 and DSM 5572 represent superior candidates due to their balanced metabolism, thermotolerance, and robustness in nutrient-limited conditions.

Díez-Antolínez et al. (2016) conducted flask fermentation tests in synthetic media supplemented with 130 g/L lactose at 35 °C for 48 h, using strains DSM 5422 and DSM 7239. They reported ethanol production of 52.9 and 48.8 g/L for these strains, respectively, corresponding to yields of 0.41 and 0.36 g/g. In the present study, a higher ethanol yield was observed for DSM 5422 (0.48 ± 0.03 g/g with production of around 60 g/L of ethanol) in just 24 h. In contrast, DSM 7239 exhibited lower ethanol production (41.61 g/L) than that reported by Dìez-Antolìnez et al., but a higher yield (0.49 ± 0.03 g/g). As the compositions of the media used in this study and in the study by Dìez-Antolìnez et al. were similar, it can be concluded that the impaired growth and slower lactose consumption rate observed in DSM 7239 were due to its sensitivity to higher temperatures (42 °C instead of 35 °C).

This physiological behavior, observed in the fermentation of CWP by all strains with respect to SSM, could be explained by the low concentration of amino acids (an organic nitrogen source) in CWP. While a high C/N ratio generally supports fermentation (Sezmis et al., 2018; Pendón et al., 2021), the presence of sufficient nitrogen in the medium is crucial for efficient alcoholic fermentation, and deficiency can negatively impact overall fermentation performance (Prazeres et al., 2012).

As reported in Section 2.3, the CWP provided by the Italian company Distilleria Bartin Srl contained only 1 g/L of total nitrogen and around 140 g/L of lactose. This corresponds to a very high C/N ratio in the presence of a relatively low concentration of the nitrogen source.

Furthermore, although higher temperatures offer several advantages, such as a higher lactose consumption rate, as observed in the previous experiment with SSM and 130 g/L of lactose, the combined effects of high-temperature stress and deficiency in nitrogen and other micronutrients likely impaired lactose consumption in the test. These conditions may have enhanced the inhibitory effect of other factors, such as osmotic stress (Tesfaw, 2023).

Industrial fermentation introduces a variety of stressors that can impact the performance and viability of microbial strains (Pattanakittivorakul et al., 2024; Rocha et al., 2011; Saini et al., 2017). In CWP fermentation processes, these stressors include high temperatures, osmotic stress, organic acids, and ethanol accumulation (Rocha et al., 2011; Tesfaw, 2023). Thus, determining the “safe” range of temperature, substrate, product and inhibitor concentrations is essential in this context (Dreżek et al., 2023). While K. marxianus is known for its thermotolerance (Fonseca et al., 2008), high temperatures can still pose challenges by destabilizing cellular structures and inhibiting metabolic processes: proteins and membranes are particularly susceptible to thermal damage, which can lead to reduced growth rates and lower ethanol yields (Fu et al., 2019; Montini et al., 2022). Since low lactose content results in low ethanol titer, direct whey to bioethanol fermentation is not cost-effective (Tesfaw, 2023). Consequently, starting the fermentation with higher lactose concentrations is one of the key strategies to achieve a higher ethanol concentration (Saini et al., 2017). However, a high amount of sugar causes osmotic stress that results in reduced growth and cell survival, which prevents most yeast strains from growing and fermenting high-gravity substrate (Díez-Antolínez et al., 2016; Saini et al., 2017). CWP also contains salts, mainly NaCl, KCl, and calcium phosphates (Prazeres et al., 2012). Salts, together with the high lactose concentration, contribute to osmotic stress affecting membrane integrity, impairing nutrient transport, and increasing the energy demand for maintaining cellular homeostasis. Wongso reported that ethanol fermentation by K. marxianus is not affected by NaCl concentrations up to 35 g/L, suggesting a degree of salt tolerance that varies by strain and environmental conditions (Wongso, 1993). The by-product of acid coagulation is acid whey. Either organic acids like citric or acetic are added during its processing. In particular, acid whey is a by-product of making Greek yogurt and acid-coagulated cheeses, such as ricotta and cottage cheese (Rocha-Mendoza et al., 2021). These weak acids can inhibit cell growth and fermentation performance (Martynova et al., 2016). Ethanol, the desired product of CWP fermentation, can act as an inhibitor at high concentrations by disrupting membrane fluidity and impairing metabolic processes (Saini et al., 2017). Ethanol stress can also exacerbate the effects of other stressors, such as osmotic pressure and temperature (Costa et al., 2014; Saini et al., 2017).

Moreover, regarding the toxic effect of high concentrations of ethanol on K. marxianus, in the present study, strains DSM 5422 and DSM 5572 showed cells vitality up to 61–63 g/L on SSM liquid medium supplemented with 130 g/L of lactose after 24 h, demonstrating a high resistance to high ethanol concentration. Our results are consistent with what was observed by Costa et al. (2014), who studied the K. marxianus strains ATCC 8554 and CCT 4086 able to withstand ethanol concentrations as high as 60 g/L, while the K. marxianus UFV-3 strain was only able to grow at concentrations as high as 40 g/L. The ability of K. marxianus to tolerate ethanol is a critical trait for its application in industrial bioethanol production. Several studies have shown that the typical ethanol tolerance of K. marxianus strains is around 60 g/L (Costa et al., 2014; Diniz et al., 2017). Dreżek et al. (2023) investigated the effect of exogenous ethanol on the growth of K. marxianus WUT240. Ethanol was added when cultures reached an OD₆₀₀ of 0.8–1.0, at final concentrations of 12.5–100 g/L, alongside a control without ethanol. Growth, monitored via OD₆₀₀ measurements, showed a 47% reduction compared to the control at ethanol concentrations of 62.5 g/L, while concentrations of 75 and 100 g/L almost completely inhibited growth (77% and 88%, respectively). However, through adaptive evolution or metabolic engineering, this tolerance could be significantly enhanced (Fu et al., 2019). For instance, da Silveira et al. reported that initial ethanol tolerance in K. marxianus strains could be improved from 60 to 100 g/L following an adaptive laboratory evolution (ALE) strategy. This improvement was attributed to metabolic changes, including increased accumulation of ergosterol and unsaturated fatty acids (UFAs), which stabilized the cell membrane, both in control and stress conditions (da Silveira et al., 2020). Furthermore, Pal and Vij (2022) found that the ALE approach increased the ethanol tolerance of K. marxianus MTCC1389 strain from 80 to 120 g/L, leading to a 42.9% improvement in ethanol production (110.5 g/L vs. 78.3 g/L). Similarly, Mo et al. (2019) demonstrated that evolved K. marxianus strains subjected to ethanol stress over 100 days developed enhanced tolerance through transcriptional regulation of multiple metabolic pathways, such as sterol biosynthesis and membrane fortification, suggesting that in K. marxianus, ethanol tolerance improvement perhaps relied primarily on transcriptional regulation rather than DNA mutation.

Based on the results of the present study, K. marxianus DSM 5422 was found to be the most suitable microorganism for converting CWP into bioethanol, thanks to its high biomass production, lactose conversion and ethanol yield. Using K. marxianus DSM 5422 in a modern biorefinery scheme to valorize CWP is a strategic move, as it eliminates the need to purchase commercial β-galactosidase enzymes at an industrial scale, thanks to its natural enzymatic activity. This makes the process more cost-effective and scalable. Furthermore, its ability to grow on a wide range of substrates and lactose concentrations (50–200 g/L), with high specific growth rates at temperatures of up to 42 °C, ensures high ethanol yields (very close to the maximum theoretical value), making this GRAS yeast strain a strategic microorganism for CWP and CW valorization in the context of the circular bioeconomy.

In conclusion, these results are important, especially from the perspective of the process scalability and economic feasibility of the proposed biorefinery scheme, as reported in our recent study (Colacicco et al., 2024) focused on the process scale-up simulation and techno-economic assessment of ethanol fermentation from cheese whey using K. marxianus. The process was simulated for a facility in Apulia, Italy, treating 539 m³/day of CW. The model integrated three steps: membrane filtration to recover proteins and concentrate lactose, fermentation to ethanol, and anaerobic digestion of residues for energy and soil conditioner. Three scenarios with different yields and lactose concentrations were assessed. Techno-economic analysis showed ethanol production costs ranging from 1.43 to 2.57 e/kg, with the best performance achieved at higher substrate concentrations and improved yeast efficiency. Sensitivity analysis revealed strong dependence on CW cost or credit, highlighting the importance of policy frameworks. Overall, CW ethanol production is feasible, especially under a gate-fee system, and could serve as a sustainable plug-in solution for dairy-based biorefineries.

This study investigated three promising commercial strains of the yeast species Kluyveromyces marxianus (DSM 5422, KDSM 7239, and DSM 5572) as attractive candidates for the sustainable production of bioethanol from CWP at high lactose concentration and high temperatures. Preliminary physiological characterization of the K. marxianus strains was performed using MTP tests on synthetic media containing various lactose concentrations (50–200 g/L) at 30 and 37 °C. The natural activity of the β-galactosidase enzyme in the different strains was studied alongside a kinetic study in an MTP assay to correlate fermentation performance with β-galactosidase expression. Subsequently, a flask-scale study was conducted in an SSM containing 130 g/L lactose at temperatures of 30, 37, and 42 °C to evaluate the impact of temperature on yeast growth, lactose conversion, and ethanol production. Optimal conditions for lactose utilization and ethanol production were identified as 130 g/L lactose at 42 °C for all yeasts. However, K. marxianus DSM 5422 exhibited superior performance in both synthetic media and real CWP liquid medium by achieving high lactose conversion (approximately 65% of the initial concentration, equivalent to around 95 g/L), substantial biomass (approximately 6 g/L) and significant ethanol production (approximately 43 g/L), resulting in an ethanol yield of 0.50 ± 0.13 g/g. Notably, this value is very close to the maximum theoretical limit. These findings emphasized the importance of selecting appropriate fermentation conditions to maximize yield while maintaining process stability. By expanding knowledge of K. marxianus physiology and optimizing its performance in CWP fermentation, this research has contributed to the development of economically and environmentally sustainable bioethanol production processes, while addressing the valorization of a major agro-industrial waste product from the dairy sector.