With the rising global energy demand and the environmental impact of fossil fuel use, biofuels have gained substantial attention as an alternative energy source (Jeswani et al., 2020). Among these, bioethanol is a liquid biofuel produced by yeasts and other microorganisms that convert carbohydrates into ethanol through fermentation. First-generation bioethanol (1G) is derived from grains, cereals, and sugarcane; however, its reliance on arable land and the resulting competition between food and fuel production have raised concerns. Second-generation bioethanol (2G) addresses these issues by utilizing lignocellulosic agricultural residues and sugar-rich industrial wastewaters as feedstock (Broda et al., 2022; Comelli, 2023).

Using lignocellulosic material as a feedstock, however, presents challenges. Lignocellulose is a complex structure comprising lignin, cellulose (hexoses: D-glucose), and hemicellulose (hexoses and pentoses: L-arabinose and D-xylose). To access these sugars, a physicochemical pretreatment with sulfuric acid and high temperatures is required, followed by enzymatic hydrolysis to release glucose from cellulose (Gil Rolón et al., 2023). Additionally, Saccharomyces cerevisiae strains commonly employed in bioethanol production lack the ability to efficiently utilize pentoses, limiting raw material conversion efficiency. Genetic modifications to enable pentose metabolism in S. cerevisiae have shown limited success, yielding low efficiencies under industrial conditions (Kuyper et al., 2005; Hahn-Hägerdal et al., 2007; Nevoigt, 2008; Lee et al., 2017; Endalur Gopinarayanan and Nair, 2019). Consequently, the study and evaluation of non-conventional yeasts with intrinsic pentose metabolism capabilities are of significant interest (Radecka et al., 2015; Geijer et al., 2022; Ndubuisi et al., 2023).

Bolzico et al (2024) evaluated xylose metabolism in non-conventional yeast strains and examined the role of oxygen in their fermentative performance, demonstrating promising results for Spathaspora passalidarum and Scheffersomyces stipitis, consistent with the findings from other studies (Kumar et al., 2009; da Cunha-Pereira et al., 2011; Cadete et al., 2012; Krahulec et al., 2012; Cadete and Rosa, 2018; Campos et al., 2022). A subsequent work by Guzmán et al (2024) further investigated S. passalidarum as a viable xylose-fermenting strain, assessing key industrial parameters such as the inoculum-to-substrate ratio, pH influence, and ethanol tolerance. This study reported a significant inhibitory effect of glucose on xylose metabolism, with pentoses only metabolized after glucose depletion, i.e., an important consideration when designing processes for mixed-sugar feedstocks. Moreover, S. passalidarum exhibited high ethanol sensitivity, with growth inhibition observed at concentrations of 20 g L⁻¹ or higher, and was also sensitive to lignocellulosic pretreatment inhibitors like furfural and 5-hydroxymethylfurfural (HMF) (Hou and Yao, 2012; Soares et al., 2020).

For effective integration of S. passalidarum into an industrial 2G ethanol process, it is essential to strategically consider this metabolic profile to maximize yields while minimizing operational time. Given the variety of mixed-sugar residues available as feedstocks for bioethanol production ( Supplementary Figure S1 ) (Friedman, 2013; Isla et al., 2013; He et al., 2014; Featherstone, 2015; Ikram et al., 2017; Comelli et al., 2018; Cruz et al., 2018; Palmonari et al., 2020; Ajala et al., 2021; Amaro Bittencourt et al., 2021; Mahmud and Anannya, 2021; Nair et al., 2022; Roukas and Kotzekidou, 2022; Zou and Chang, 2022; Gil Rolón et al., 2023) and the potential to combine sugar-rich industrial effluents with agro-industrial lignocellulosic residues (Comelli, 2023; de Oliveira Pereira et al., 2024), an expanded metabolic study of S. passalidarum across a broad carbohydrate spectrum, including hexoses, pentoses, and disaccharides, is necessary. Literature about sugar fermentation by S. passalidarum is still under development and mainly focused on glucose and xylose (Nguyen et al., 2006; Hou, 2012; Long et al., 2012; Rodrussamee et al., 2018; Ribeiro et al., 2021; Campos et al., 2022; Trichez et al., 2023; Bolzico et al., 2024; Guzmán et al., 2024; Saengphing et al., 2024), avoiding other monosaccharides and disaccharides (Cadete et al., 2009; Rodrussamee et al., 2018; Du et al., 2019; Farias and Maugeri-Filho, 2021; Pereira I de et al., 2021) ( Table 1 ). In this study, we applied an innovative methodology to evaluate S. passalidarum’s metabolism of individual carbohydrates and to understand its adaptive response to sudden “pulses” of various sugars, including glucose, galactose, mannose, fructose, maltose, sucrose, lactose, and arabinose, on xylose consumption. We also extended our research on carbon metabolism by studying arabinose, an abundant but less frequently reported pentose in lignocellulosic biomass (Jacob et al., 2023), and glycerol, a low-cost carbon source derived from industry by-products (Abad and Turon, 2012; Kaur et al., 2020). These findings contribute to a deeper understanding of S. passalidarum and provide valuable insights for selecting or combining residues as feedstock in bioethanol production processes.

Spathaspora passalidarum has been proposed as a promising non-conventional yeast for 2G ethanol production due to its ability to metabolize xylose from lignocellulosic materials and sugar-rich industrial effluents efficiently (Cadete and Rosa, 2018; Campos et al., 2022; Bolzico et al., 2024). This capacity enables greater utilization of the hemicellulosic fraction, increasing ethanol yield. However, integrating S. passalidarum into a 2G process poses challenges, notably the need for an expanded understanding of its metabolic behavior in mixed-sugar streams. For example, glucose and other carbohydrates have been shown to inhibit xylose metabolism, a phenomenon known as carbon catabolite repression (Subtil and Boles, 2012; Lane et al., 2018; Rodrussamee et al., 2018; Simpson-Lavy and Kupiec, 2019). This effect, common among yeasts and other microorganisms, leads to the prioritization of energetically favorable sugars like glucose over secondary sugars, as described by Zhao et al (Zhao et al., 2020).

Our results demonstrated a dose-dependent inhibition of xylose metabolism by glucose. A sudden pulse of glucose—or other hexoses such as galactose or mannose—was sufficient to temporarily interrupt xylose consumption, indicating that the metabolism of these sugars is prioritized over that of pentoses in this strain. The observation that a pulse of 2DG, a non−metabolizable (but phosphorylatable) glucose analog, also delayed xylose utilization, suggests that the underlying mechanism is triggered by glucose sensing or its immediate cytosolic entry, rather than full metabolic processing. A similar approach was previously reported by Ribeiro et al (Ribeiro et al., 2021); however, in their design, 2DG was present from the start of the culture, resulting in an extended lag phase followed by eventual xylose consumption. Consequently, the authors did not report a complete inhibition of pentose utilization. In contrast, the pulse−based strategy employed in the present study allowed us to capture the immediate adaptive response of the yeast to a sudden sugar shift, providing a more precise view of the transient regulatory phenomenon that occurs during ongoing xylose fermentation.

Although molecular signaling pathways in S. passalidarum remain poorly characterized, insights can be drawn from the well−studied glucose-signaling network in S. cerevisiae (Kayikci and Nielsen, 2015; Persson et al., 2022). Glucose−mediated catabolite repression primarily involves three interconnected routes: i) the HXK2/Snf1/Mig1 pathway represses the transcription of genes required for the utilization of alternative carbon sources. The glucose hexokinase HXK2 allows glucose sensing, and it is involved in Mig1 phosphorylation and in their nucleocytoplasmic distribution (Ahuatzi et al., 2007); ii) the Rgt2/Snf3 signaling cascade, which regulates the expression of HXT glucose transporters and promotes the degradation of other sugar permeases; and iii) the cAMP/PKA pathway, which stimulates fermentative growth under glucose while suppressing stress responses and respiratory metabolism. The step of glucose phosphorylation by a hexokinase could be involved in pentose repression mediated by glucose and 2DG, and the use of other non-phosphorylatable analogs like 4DG or 6DG to obtain additional information is necessary. Recently, it was reported that an overexpression of Snf3 improves signaling in the presence of xylose, suggesting that this receptor is involved in extracellular pentose sensing in S. cerevisiae (Bolzico et al., 2025).

While elucidating the specific molecular mechanism in S. passalidarum was beyond the scope of this work, our pulse−based experimental design provides a promising platform for future transcriptomic and regulatory analyses, particularly through RNA−seq, to identify the transcriptional responses and key regulators that govern hexose–pentose co−utilization in this species.

Regarding the experiments with disaccharides, it is noteworthy that interrupting a xylose culture with a maltose pulse was also able to delay pentose consumption. Subtil and Boles (2012) reported similar inhibitory effects of glucose on both xylose and arabinose in recombinant S. cerevisiae strains, with pentose metabolism resuming only after glucose depletion. They proposed a competitive inhibition mechanism where hexoses, due to their higher affinity for transporters, outcompete pentoses. Additionally, in their co-fermentation studies of xylose and maltose, they observed that maltose partially inhibited xylose consumption. This inhibition suggested that intracellular accumulation of D-glucose could also disrupt pentose metabolism. Our experiments are consistent with these findings: a maltose pulse effectively interrupted xylose metabolism, indicating that S. passalidarum prioritized maltose over xylose. Interestingly, the inhibitory effect could be partially mitigated by overexpressing the hexose transporters HXT7 and Gal2.

We could hypothesize that the accumulation and phosphorylation of glucose in the cytosol, immediately after its uptake, may represent the key step in signal transduction to inhibit pentose metabolism. Nevertheless, sucrose, which is hydrolyzed to glucose and fructose, does not trigger the same effect. This could suggest the presence of specific signaling pathways mediated by membrane receptors or transporters. Transporters, known as maltose permeases, mediate maltose transport in S. cerevisiae. Once inside the cytosol, maltose is cleaved into two glucose monomers by an enzyme with α-glucosidase activity (maltase) (Brondijk et al., 2001). In the case of sucrose, this disaccharide is mainly hydrolyzed in the periplasm into fructose and glucose through the action of an invertase (β-fructofuranosidase). Both fructose and glucose subsequently enter the cytosol via HXT-family transporters (Marques et al., 2016). Non-conventional yeasts such as Ogataea polymorpha utilize intracellular α-glucosidases for hydrolyzing sucrose, relying on specific permeases for sugar uptake. In the case of S. passalidarum, this strain showed intracellular sucrose hydrolysis and a low uptake rate, reported by Pereira et al (Pereira I de et al., 2021).

We observed that the inhibitory effect of glucose on xylose is dose-dependent, suggesting that the levels of glucose generated through disaccharide hydrolysis, as well as the rate of hydrolysis and subsequent cytosolic glucose accumulation, could influence the magnitude of the response. In S. passalidarum, sucrose exhibited a longer lag phase than maltose. This indicates that it could be more metabolically prepared for the consumption of maltose, hence its ability to delay the metabolism of xylose. Additional studies are necessary to fully elucidate these mechanisms. It is also noteworthy that the inhibitory effect of glucose can be reversed at higher xylose concentrations. Soares et al (2020) observed this phenomenon in co-fermentation trials with xylose and glucose, using lignocellulosic hydrolysates of soybean hulls with high xylose and low glucose concentrations. A xylose-glucose ratio of 3:1 reported in this work using a four-sugar medium cannot avoid glucose and maltose repression on xylose metabolism.

When designing a 2G ethanol production process from lignocellulosic material or mixed composition raw materials, the inhibitory effect of hexoses on xylose must be considered. It becomes evident that S. passalidarum prioritizes glucose as its primary carbon source, a behavior that has been reported (Guzmán et al., 2024). In lignocellulosic feedstock, pentoses are metabolized only after glucose depletion. In a mixed sugar environment—such as that potentially arising from the integration of 1G/2G processes, composed of industrial and agro-industrial effluents—fermentation times are expected to vary depending on the sugar ratios. Xylose metabolism accelerated as glucose and maltose were depleted, whereas fructose exhibited the longest lag phase and a lower consumption rate, and thus would likely represent the main contributor to the delayed fermentation when using S. passalidarum. Spathaspora passalidarum prioritized xylose over fructose and sucrose, and curiously, their consumption only started after pentose depletion, extending fermentation time and resulting in a lower ethanol yield. This point is important due to the abundance of these sugars in some industrial wastewaters that could be combined with lignocellulosic residues.

In this context, several hypotheses can be proposed to optimize fermentation times. First, employing initial or sequential mixed inocula that combine conventional Saccharomyces with non-conventional yeasts could be advantageous. Various industrial S. cerevisiae strains are highly efficient at rapidly consuming glucose, even at high concentrations, and can also contribute to the consumption of sucrose and maltose. Conversely, S. passalidarum could play a key role in the fermentation of pentoses, as well as hexoses and disaccharides.

However, despite the promise of this combination, a critical bottleneck threatens overall yield: the clear inhibition of S. passalidarum by ethanol accumulation or by the presence of inhibitors like acetate, furfural, and HMF derived from lignocellulose pretreatment. Recent studies have reported encouraging results using laboratory-adapted native strains, which exhibited improved tolerance to inhibitors and even co-fermentation capacity of glucose and xylose (González-Ramos et al., 2016; Pacheco et al., 2021; Fernandes et al., 2023; Trichez et al., 2023). Additionally, detoxification methods can be applied to remove inhibitors from sugar hydrolysates.

The generation of inhibitory compounds in lignocellulosic hydrolysates is inherently unavoidable, as the high temperature and acidic pretreatments typically employed promote the release of acetate, furfural, and HMF. The extent of inhibitor formation is also strongly influenced by the type of lignocellulosic feedstock. Consequently, a variety of detoxification strategies have been developed to mitigate their impact on fermentation performance (Parawira and Tekere, 2011). Among physical–chemical approaches, modified chitosan–chitin hybrid aerogels have demonstrated high removal efficiencies for HMF (85.1%) and furfural (99%), without compromising fermentable sugars, whereas activated carbon has shown comparable performance (Liu et al., 2024). Ion-exchange resins, particularly under alkaline conditions, selectively remove organic acids and phenolics, improving hydrolysate fermentability (Nilvebrant et al., 2001). Electrodialysis represents another effective method for the removal of acetate and phenolic compounds, although its efficiency toward furfural and HMF remains limited (Lee et al., 2013; Lee et al., 2014). Enzymatic approaches, including the use of laccases and other ligninolytic enzymes, have been shown to degrade phenolics and furan derivatives, simultaneously enhancing fermentability and reducing overall toxicity (Fernández-Sandoval et al., 2024). In addition, chemical neutralization, sulfite addition, solvent extraction, and alkaline treatments have been explored, although these methods may alter the sugar composition of the hydrolysate (Palmqvist and Hahn-hägerdal, 2000). Biological detoxification using specific microorganisms, such as Cupriavidus basilensis HMF14 and Bordetella sp. BTIITR, is particularly attractive as these strains selectively degrade furfural, HMF, and acetate without consuming sugars (Wierckx et al., 2010; Singh et al., 2019). Despite these advances, the high ethanol sensitivity of S. passalidarum remains a critical bottleneck, as the desired product itself exerts inhibitory effects. Therefore, strategies based on adaptive evolution or the development of genetically improved variants with enhanced ethanol tolerance emerge as a crucial step toward the efficient industrial exploitation of this yeast.

The molecular mechanisms governing pentose transport and consumption in non-conventional yeast strains remain less understood. Among yeasts with effective xylose metabolism, such as S. passalidarum and Sc. stipitis, xylose is taken up by both low- and high-affinity transporters. Low-affinity transporters facilitate the diffusion of xylose and glucose and are related to the S. cerevisiae HXT transporter family, with Hxt4p, Hxt5p, Hxt7p, and Gal2p mediating xylose uptake, albeit with significantly lower affinity compared to glucose (Leandro et al., 2009). In contrast, high-affinity transporters, functioning as xylose/H⁺ symporters, are crucial under low xylose conditions. While the identification and characterization of xylose transporters in non-conventional yeasts are still in early stages, some advances have been made. Examples include the low-affinity transporter Sut1–3 from Sc. stipitis CBS5774 and Candida intermedia’s GXF1 and GXS1 transporters, which facilitate diffusion and symport, respectively (Leandro et al., 2009).

Regarding arabinose, the second most abundant pentose, there are still no functional studies on arabinose transporters in yeasts. However, its uptake seems to be independent of xylose and glucose and is likely mediated by specific transporters. The first system facilitates diffusion with low affinity for arabinose, which has been characterized in Pichia (Meyerozyma) guilliermondii and Candida arabinofermentans (Fonseca et al., 2007). This kind of transport is induced by high arabinose concentrations. The authors also reported a higher-affinity arabinose transport system mediated by an arabinose/H⁺ symporter, which plays a major role under low concentrations of arabinose. Both transport systems were repressed by glucose. According to the literature, S. cerevisiae can poorly incorporate arabinose by facilitating the diffusion of hexose transporters Gal2, Hxt5, and Hxt7. Other transporters with higher affinity have been identified in Neurospora crassa (LAT-1), Myceliophthora thermophila, Penicillium chrysogenum (AraT), and Sc. stipitis (Ye et al., 2019). In the fungal pathway, L-arabinose is first converted into its corresponding polyol by the enzyme arabitol reductase (AR), and L-arabitol is subsequently oxidized to L-xylulose in a reaction mediated by L-arabinose dehydrogenase (LAD). The utilization of L-arabinose requires an additional reduction by L-xylulose reductase (LXR), converting L-xylulose into xylitol, the common intermediate for both arabinose and xylose catabolic pathways. Xylitol is then converted into D-xylulose by xylitol dehydrogenase (XDH), which is phosphorylated to xylulose-5P by a kinase (XK) (Fonseca et al., 2007). This final molecule can serve as an intermediate in the pentose phosphate pathway to produce ethanol. In filamentous fungi, L-arabinose and D-xylose reductases prefer NADPH as a cofactor, while sugar alcohol dehydrogenases strictly depend on NAD⁺. The fungal L-arabinose pathway is not redox balanced, and the cell’s capacity to regenerate NAD⁺ under low-oxygen conditions is limited, which can lead to arabitol accumulation (Fonseca et al., 2007).

Few studies on yeasts have identified some strains, such as C. arabinofermentans, C. tropicalis, C. shehatae, M. guilliermondii, Pachysolen tannophilus, Sc. stipitis, and Torulopsis sonorensis as arabinose consumers under aerobic conditions (Kurtzman and Dien, 1998; Fonseca et al., 2007; Ye et al., 2019). Most of them direct arabinose toward biomass production. However, the three strains of Candida spp. have been reported to produce traces of ethanol (Y(ps) = 0.18 g_ethanol/g_{consumed arabinose}) in media with high arabinose concentrations, as the pathway accumulates arabitol and minimal xylitol. Additionally, Sc. stipitis has been reported to produce 0.24 (g_arabitol/g_{consumed sugar}) and 0.15 (g_ethanol/g_{consumed sugar}) (Ye et al., 2019). Rhodosporidium toruloides can also accumulate D-arabitol from xylose (Jagtap and Rao, 2017). Zygosaccharomyces rouxii and Z. siamensis have shown a high capacity to transform glucose into arabitol, although they can also produce ethanol and glycerol as by-products (Saha et al., 2007; Iwata et al., 2023). Some genetically modified strains have been reported to express genes from the arabinose pathway of filamentous fungi in combination with xylose pathway genes (Ye et al., 2019). For instance, an S. cerevisiae strain expressing AR, XDH, and XK genes, along with the Sc. stipitis genes XYL1, XYL2, and XYL3 necessary for arabinose transport and metabolism, has been reported. Furthermore, this strain was subsequently modified by adding the LAD and LXR genes from Trichoderma reesei and Ambroziosyma monospora, respectively, but ethanol yields obtained remained at very low levels. Expanding the scope of research to include other non-conventional yeasts like S. passalidarum will expand our knowledge about arabinose metabolism and increase the reservoir of genes involved.

Our results showed that S. passalidarum can utilize arabinose for biomass production in a highly oxygen-dependent process but was unable to produce ethanol from this substrate. No significant ethanol production was detected, likely due to the inability to regenerate the reducing power required by the enzymes in this pathway. On the other hand, yields of 0.35 g_arabitol/g_arabinose were obtained in 70% HS reactors after 36 h of fermentation and 80% arabinose consumption. Arabitol yields between 0.43 and 0.53 g_arabitol/g_arabinose were reported for Sc. stipitis and M. guilliermondii and higher values for a few Candida species (0.6–0.8 g_arabitol/g_arabinose) (Kordowska-Wiater, 2015). Data collected until now propose that arabinose seems not able to ferment to ethanol by any wild or recombinant strain, wasting that fraction of lignocellulose.

The use of different cofactors by arabinose pathway enzymes generates a redox imbalance, especially under oxygen-limited conditions, where NAD⁺ regeneration is insufficient. As a result, the conversion of L-arabitol to L-xylulose becomes inefficient, and L-arabitol accumulates intracellularly (Fonseca et al., 2007). The accumulation of arabitol may also serve as a mechanism to regenerate NAD⁺, allowing metabolism to continue even if ethanol is not produced efficiently (Fonseca et al., 2007). Arabitol accumulation often occurs at the expense of ethanol production under certain conditions, such as osmotic stress or nitrogen limitation (Kordowska-Wiater, 2015; Yang et al., 2021). In some cases, arabitol and ethanol production can occur simultaneously, but generally, there is metabolic competition between the two pathways. The literature indicates that arabitol can act as a redox buffer, preventing oxidative damage (Sánchez-Fresneda et al., 2013), an osmoprotectant, or a carbon reserve that is not immediately used in the most demanding energy production pathways (Blakley and Spencer, 1962).

In the case of glycerol, similar results were obtained, where S. passalidarum was able to grow but not ferment using this carbon source. This topic could be interesting for inoculum production. The ability of yeast to metabolize glycerol holds significant economic value, as it enables the valorization of glycerol, an abundant and low-cost by-product of the biodiesel process (Abad and Turon, 2012). Utilizing glycerol as a carbon source can reduce the production costs of yeast biomass and enhance process sustainability. Furthermore, it supports the development of circular economy strategies by converting industrial waste into high-value bioproducts. A saturated culture of S. passalidarum obtained after 48 h of growth in glycerol was successfully used as inoculum for a subsequent fermentation in xylose, obtaining the same ethanol yield and kinetic and stoichiometric parameters compared with a 48-h inoculum produced with xylose as a carbon source (data not shown).

This study aimed to expand our knowledge on the metabolic behavior of S. passalidarum to assess its potential for 2G ethanol production. Due to its inherent ability to ferment xylose, S. passalidarum presents an interesting alternative for bioethanol production. However, understanding its performance in mixed-sugar streams is crucial for optimizing ethanol yields. Our findings show that xylose metabolism in S. passalidarum is strongly inhibited by hexoses such as glucose, mannose, and galactose, as well as by the disaccharide maltose. In contrast, S. passalidarum prioritized xylose consumption over fructose and sucrose, which were only metabolized after xylose depletion. This delayed their consumption, extending fermentation time and slightly reducing ethanol yield. Additionally, while S. passalidarum could metabolize arabinose, this conversion favored biomass generation over ethanol, indicating that this pentose would be underutilized in the feedstock.

These results provide strategic insights for designing an efficient 2G ethanol production process, enabling the effective use of sugars in raw materials and minimizing operational time. Spathaspora passalidarum shows potential as part of a combined fermentation system alongside S. cerevisiae, harnessing the complementary metabolic strengths of each yeast to achieve sequential and complete sugar utilization. However, it is necessary to focus on overcoming the high sensitivity of S. passalidarum to inhibitors and ethanol. The inhibitor removal process and laboratory-adaptive evolution are pointed out as the most promising alternatives to solve this limitation, although it is still under development. In conclusion, this work significantly enhances the metabolic understanding of S. passalidarum, encompassing fermentation dynamics across a range of sugars and sugar combinations, which have been underexplored in previous studies.