Beyond Saccharomyces: Exploring the Bioethanol Potential of Wickerhamomyces anomalus and Diutina rugosa in Xylose and Glucose Co-Fermentation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The experimental article "Beyond Saccharomyces: Exploring the Bioethanol Potential of Wickerhamomyces anomalus and Diutina rugosa in Xylose and Glucose Co-Fermentation" is devoted to assessing the productivity of the studied strains under conditions similar to lignocellulosic ones. The authors studied kinetics, sugar consumption, and ethanol production, paying particular attention to catabolite repression and the inhibitory effect of ethanol on xylose utilization. The advantage of the work is the use of literature from 2023-2025 in the introduction, which undoubtedly confirms the relevance of the work. In terms of its subject matter, the manuscript is suitable for the publication "Fermentation". However, before publication, the authors need to eliminate a number of comments, which are presented below.

1. Point 2.4. It is very interesting, based on what the authors chose exactly these ratios of glucose and xylose. Did the authors rely on the composition of real hydrolysates? If so, which ones?
2. Point 2.6. For clarity, it is recommended to provide calculation formulas.
3. Table 1. The table shows "Best" and "Worst". I would like to see clarifications on what these conditions are.
4. Point 4. Discussion. Lines 323-325. I would like to see real examples with which the authors compare the obtained results.

5. The highest ethanol yield is observed at glucose concentrations of 20 g / l and acidity of 20 g / l. For other glucose and xylose ratios, the ethanol yield is lower. What is the probability that the use of the Diutina rugose strain on real lignocellulose hydrolysates with other glucose and xylose ratios will be effective? I would like the authors to give recommendations or make predictions on the use of the strain on real hydrolysates. What can be the concentration of sugars for an effective process? What types of raw materials and pre-treatment of this raw material can be used to achieve such an ethanol yield?

Author Response

Comment 1 (Section 2.4): “It is very interesting, based on what the authors chose exactly these ratios of glucose and xylose. Did the authors rely on the composition of real hydrolysates? If so, which ones?”

Response 1: Thank you for this important comment. We selected the glucose/xylose ratios (i.e., 20 g/L glucose + 5 g/L xylose; 40 g/L glucose + 5 g/L xylose; 20 g/L glucose + 10 g/L xylose; 20 g/L glucose + 20 g/L xylose; 5 g/L glucose + 40 g/L xylose; and 20 g/L glucose + 40 g/L xylose) based on published data on lignocellulosic hydrolysates. For example, studies on hydrolysates derived from sugarcane bagasse, corn stover, and wheat straw report glucose concentrations ranging from approximately 15 to 40 g/L and xylose levels from 5 to 40 g/L [e.g., Woźniak et al., 2025 (https://doi.org/10.3390/su17010287); Subudhi et al., 2024 (https://doi.org/10.1016/j.jtice.2024.105616)]. By testing these different ratios, our experimental design captures the variability encountered in real industrial scenarios, allowing us to evaluate the yeast performance under conditions that closely mimic practical hydrolysates. Manuscript modification: This explanation was inserted in Section 2.4 on Page 3, Lines 135 to 137 (new text highlighted in red).

 

Comment 2 (Section 2.6 – Calculation Formulas): “For clarity, it is recommended to provide calculation formulas.”

Response 2: We appreciate the suggestion. To improve clarity, we have added the formulas for calculating key kinetic and yield parameters in Section 2.6 on Page 4, Lines 171–196 (new text highlighted in red).

 

Comment 3 (Table 1 – “Best” and “Worst” Clarification): “The table shows 'Best' and 'Worst'. I would like to see clarifications on what these conditions are.”

Response 3: Thank you for your comment. To clarify, we have added a footnote to Table 1 that explains “Best” refers to the highest value obtained among the experimental conditions (A–F), and “Worst” refers to the lowest value. For example, the highest ethanol yield (0.45 g/g) was recorded under Condition D (20 g/L glucose + 20 g/L xylose), while the lowest yield (0.22 g/g) was observed under Condition E (5 g/L glucose + 40 g/L xylose). Manuscript Modification:
A footnote with this clarification was added to Table 1 on Page 11, Lines 322-325 (new text highlighted in red).

 

Comment 4 (Discussion, Lines 323–325 – Real-World Comparisons): “I would like to see real examples with which the authors compare the obtained results.”

Response 4: We thank the reviewer for this suggestion. In the Discussion, we have now included direct comparisons with literature data. The revised text reads as follows:

“For instance, engineered S. cerevisiae strains have been reported to achieve ethanol yields ranging from 0.42 to 0.48 g/g under mixed-sugar fermentations [53,54], which is comparable to our maximum yield of 0.45 g/g observed with D. rugosa under Condition D. Similarly, non-conventional yeasts such as S. stipitis typically achieve yields around 0.40 g/g under similar conditions [55], underscoring that D. rugosa can perform at levels competitive with established yeast systems.”

Manuscript Modification: This comparative discussion was inserted in the Discussion section on Page 13, Lines 414–419 (new text highlighted in red).

 

Comment 5 (Applicability on Real Hydrolysates and Recommendations): “The highest ethanol yield is observed at glucose concentrations of 20 g/L and xylose concentrations of 20 g/L. For other glucose and xylose ratios, the ethanol yield is lower. What is the probability that the use of the Diutina rugosa strain on real lignocellulose hydrolysates with other glucose and xylose ratios will be effective? I would like the authors to give recommendations or make predictions on the use of the strain on real hydrolysates. What can be the concentration of sugars for an effective process? What types of raw materials and pre-treatment of this raw material can be used to achieve such an ethanol yield?”

Response 5: Thank you for raising this application-oriented question. Based on our experimental findings, the optimal performance of D. rugosa was observed at a balanced sugar composition of 20 g/L glucose and 20 g/L xylose. However, real lignocellulosic hydrolysates generally contain a total sugar concentration between 40 and 60 g/L, with variable glucose:xylose ratios depending on the feedstock and pretreatment method. Studies have shown that feedstocks such as sugarcane bagasse, corn stover, and wheat straw, when subjected to appropriate pretreatments (e.g., dilute-acid or enzymatic hydrolysis), can produce hydrolysates within this range. We predict that D. rugosa will remain effective provided the overall sugar concentration is maintained, even if the individual ratios vary. We therefore recommend that industrial processes focus on pretreatment methods that yield balanced sugar profiles to minimize catabolite repression and maximize ethanol productivity. Pilot-scale studies are necessary to confirm these predictions under real operating conditions. Manuscript Modification: A new paragraph discussing the applicability of D. rugosa on real lignocellulosic hydrolysates was added in the Discussion section on Page 14, Lines 430-441 (new text highlighted in red).

Reviewer 2 Report

Comments and Suggestions for Authors

Beyond Saccharomyces: Exploring the Bioethanol Potential of Wickerhamomyces anomalus and Diutina rugosa in Xylose and Glucose Co-Fermentation

The authors in this study evaluated two non-Saccharomyces yeasts, Wickerhamomyces anomalus UEMG-LF-Y2 and Diutina rugosa UEMG-LF-Y4, in mixed-sugar conditions. It demonstrated their ability to co-ferment glucose and xylose in a laboratory setting.

 

I think the authors ought to highlight a bit more about co-fermentation and co-culture, focusing on how important they are in the interactions between microbes and fermentation processes. The paper could give a lot of useful information by going into more detail about how these approaches can improve pathways, encourage synergistic effects between different microorganisms, and make the fermentation process run more efficiently overall. Discussing with regard to particular scenarios and how these methods could be used in different fields, like biotechnology, food production, or wastewater treatment, would also enhance the subject matter.  

 

 

• Is there any observable synergy between the two used yeasts, Wickerhamomyces anomalus and Diutina rugosa? Two strains in a co-culture demonstrate both synergistic and competitive interactions. This intricate interaction is a crucial element of microbial ecology and carries substantial implications.

 

• As shown in Figure 2, both strains exhibited higher specific growth rates (μ, h-1) in glucose than in xylose at all tested concentrations. rugosa Y4 consistently outperformed W. anomalus Y2, reaching maximal μ values of 0.347 h-1 (glucose) and 0.241 h-1 (xylose), whereas W. anomalus attained 0.299 h-1 and 0.164 h-1, respectively. Growth rates in both yeasts tended to plateau at substrate concentrations of 20 g/L or higher--- Was substrate concentration the sole factor affecting the specific growth rate? Furthermore, did the nature of the substrate have any influence?

 

• What are the efficiencies of ethanol production and glucose production in this study?

 

• Did the authors investigate the individual effects of glucose and xylose tested by Wickerhamomyces anomalus and Diutina rugosa to assess the efficacy of these yeast strains?

 

 

• anomalus Y2 exhibited lower xylose utilization efficiency compared to D. rugosa Y4, with a residual xylose concentration detected in all conditions where xylose was present. In contrast, D. rugosa Y4 demonstrated a more efficient co-metabolism of glucose and xylose, particularly under Conditions E and F, where over 80% of the available xylose was consumed within 48 h---- Is this data and experimental design genuinely rational? It seems rather noticeable that both yeast strains can exhibit varying potentials when exposed to different substrates such as glucose and xylose.

 

• Building on these metabolic distinctions, a notable finding was the differential response to 2-deoxyglucose (2-DG), which induces catabolite repression. The inhibitory effect was particularly evident in anomalus, suggesting a higher sensitivity to glucose repression mechanisms, while D. rugosa maintained residual growth (Figure 1), indicating a comparatively lower susceptibility--- Considering figure 4, if Wickerhamomyces anomalus indicated a greater sensitivity to glucose repression mechanisms, how is it that the second highest ethanol production was recorded under high glucose levels?

 

• In this study, both strains exhibited relatively low biomass yields (Table 1), suggesting a metabolic preference for ethanol production rather than extensive proliferation--- Could the authors expand upon this discussion, highlighting its significance in an industrial context?

 

• This study illustrates the capacity of anomalus UEMG-LF-Y2 and D. rugosa UEMG-LF-Y4 to co-ferment glucose and xylose under laboratory conditions, yet their performance under industrial-scale stresses—such as high ethanol concentrations and inhibitory compounds—remains to be determined---- What specific inhibitors should be considered for future evaluations to improve productivity?

 

 

Author Response

Comment 1: “I think the authors ought to highlight a bit more about co-fermentation and co-culture, focusing on how important they are in the interactions between microbes and fermentation processes. The paper could give a lot of useful information by going into more detail about how these approaches can improve pathways, encourage synergistic effects between different microorganisms, and make the fermentation process run more efficiently overall. Discussing with regard to particular scenarios and how these methods could be used in different fields, like biotechnology, food production, or wastewater treatment, would also enhance the subject matter.”

Response 1: Thank you for your insightful comment. We agree that a broader discussion on co-fermentation and co-culture strategies could enhance the paper. In the revised manuscript, we have added a new paragraph in the Discussion (inserted on Page 12, Lines 356–366) that elaborates on the significance of these approaches. In this paragraph, we discuss:

• How co-culture strategies can lead to improved substrate utilization, enhanced metabolic pathways, and synergistic interactions between different microorganisms.
• The potential benefits of co-fermentation in various fields, including biotechnology, food production, and wastewater treatment.
• That while our current study evaluates the strains individually, future work will explore the synergistic and competitive interactions in co-cultures.
  New text (in red) includes:

“In addition to the individual fermentation capabilities demonstrated by W. anomalus and D. rugosa, co-fermentation and co-culture strategies have been shown to significantly enhance overall process efficiency by promoting synergistic interactions among diverse microbial populations. These interactions can lead to improved substrate conversion, more balanced metabolic fluxes, and mitigation of inhibitory effects during fermentation. For example, in various biotechnological applications and wastewater treatment processes, mixed microbial cultures have achieved higher yields and increased robustness compared to monocultures [35,36]. Although our present study evaluated the strains individually, future work will investigate co-culture systems to explore the potential synergistic effects between these yeasts, which may further optimize mixed-sugar fermentations for industrial bioethanol production”

 

Comment 2: “Is there any observable synergy between the two used yeasts, Wickerhamomyces anomalus and Diutina rugosa? Two strains in a co-culture demonstrate both synergistic and competitive interactions. This intricate interaction is a crucial element of microbial ecology and carries substantial implications.”

Response 2: We appreciate the reviewer’s interest in the potential synergy between the two strains. In our current study, the experiments were conducted with each strain in monoculture to clearly characterize their individual fermentation profiles. However, we acknowledge that co-culturing these yeasts could lead to both synergistic and competitive interactions that might further enhance fermentation performance. We have now included a sentence in the Discussion (added on Page 12, Lines 363–366) stating:

“Although our present study evaluated the strains individually, future work will investigate co-culture systems to explore the potential synergistic effects between these yeasts, which may further optimize mixed-sugar fermentations for industrial bioethanol production.”

 

Comment 3: “As shown in Figure 2, both strains exhibited higher specific growth rates (μ, h⁻¹) in glucose than in xylose at all tested concentrations. ... Was substrate concentration the sole factor affecting the specific growth rate? Furthermore, did the nature of the substrate have any influence?”

Response 3: Thank you for this question. We have expanded the discussion regarding the factors affecting the specific growth rate. In the revised manuscript (inserted on Page 7, Lines 246–252), we now clarify that while substrate concentration is a key determinant, the nature of the substrate also plays a significant role. We note that:

- Glucose, being a preferred carbon source, is metabolized more efficiently than xylose.

- Other factors such as substrate transport efficiency and the inherent metabolic pathways influence the observed growth rates.

New text (in red) reads:

“It is important to note that substrate concentration is not the sole factor influencing the specific growth rate. The intrinsic nature of the substrate also plays a critical role. Glucose, being a preferred carbon source, is metabolized more efficiently due to more direct entry into central metabolic pathways. In contrast, xylose requires additional metabolic conversion steps, which can limit its utilization rate. Consequently, the observed differences in specific growth rates are attributed both to the concentration of the substrates and to their inherent metabolic properties.”

 

Comment 4: “What are the efficiencies of ethanol production and glucose production in this study? Did the authors investigate the individual effects of glucose and xylose tested by Wickerhamomyces anomalus and Diutina rugosa to assess the efficacy of these yeast strains?”

Response 4: Thank you for your valuable comment. Our study reports detailed efficiency metrics, including ethanol yield (g EtOH per g sugar) and sugar consumption rates (Qs), which are presented in Table 1 and described in Section 3.3. Additionally, individual fermentations using either glucose or xylose as the sole carbon source were conducted to determine the inherent substrate-specific performance of each strain. These individual assays provided a solid basis for evaluating the efficiency of mixed-sugar fermentations and clearly demonstrated the differential substrate utilization profiles of Wickerhamomyces anomalus and Diutina rugosa (see also Figure 2). We believe that the combined data adequately reflect the performance and efficacy of these yeast strains under both single and mixed-sugar conditions.

New text (in red) reads (inserted on Page 4, Lines 141–142):

“Individual fermentations using either glucose or xylose were conducted to determine the inherent substrate-specific performance of each strain.”

 

Comment 5: “anomalus Y2 exhibited lower xylose utilization efficiency compared to D. rugosa Y4, with a residual xylose concentration detected in all conditions where xylose was present. In contrast, D. rugosa Y4 demonstrated a more efficient co-metabolism of glucose and xylose, particularly under Conditions E and F, where over 80% of the available xylose was consumed within 48 h---- Is this data and experimental design genuinely rational? It seems rather noticeable that both yeast strains can exhibit varying potentials when exposed to different substrates such as glucose and xylose.”

Response 5: We appreciate the reviewer’s concern regarding the rationality of the experimental design. The differences in substrate utilization between the two strains are indeed genuine and reflect their inherent metabolic capabilities. Our design—testing various glucose/xylose ratios—was intentionally chosen to mimic the variability in sugar compositions observed in industrial lignocellulosic hydrolysates. We have now added a brief explanation (see Page 14, Lines 430–441) clarifying that the observed differences confirm that each yeast strain possesses a distinct capacity for sugar uptake and metabolism, which supports the rationale behind our experimental approach.

New text (in red) may state:

“Although our optimal ethanol yield of 0.45 g EtOH/g sugar was observed under Condition D (20 g/L glucose + 20 g/L xylose), real lignocellulosic hydrolysates typically exhibit a broader range of sugar concentrations, often with a total sugar content between 40 and 60 g/L. Studies have shown that feedstocks such as sugarcane bagasse, corn stover, and wheat straw - when subjected to appropriate pretreatment methods (e.g., dilute-acid or enzymatic hydrolysis) - yield hydrolysates with such sugar compositions [62,63]. Based on our findings, we predict that D. rugosa will remain effective as long as the overall sugar concentration is maintained within this range, even if the individual glucose:xylose ratio deviates from 1:1. We recommend that industrial applications focus on optimizing pretreatment strategies to maximize both glucose and xylose release, thereby reducing the inhibitory effects of catabolite repression and enhancing ethanol productivity. Future pilot-scale studies are warranted to validate these predictions under industrial conditions.”

 

Comment 6: “Building on these metabolic distinctions, a notable finding was the differential response to 2-deoxyglucose (2-DG), which induces catabolite repression. The inhibitory effect was particularly evident in anomalus, suggesting a higher sensitivity to glucose repression mechanisms, while D. rugosa maintained residual growth (Figure 1), indicating a comparatively lower susceptibility--- Considering figure 4, if Wickerhamomyces anomalus indicated a greater sensitivity to glucose repression mechanisms, how is it that the second highest ethanol production was recorded under high glucose levels?”

Response 6: Thank you for raising this intriguing point. Although W. anomalus exhibits higher sensitivity to 2-DG (and thus to glucose repression), its metabolic network may be channelling carbon flux preferentially toward ethanol production under high glucose conditions. In other words, even if xylose metabolism is repressed, the abundant glucose can still support significant ethanol production. We have added a sentence to the Discussion (inserted on Page 13, Lines 385–387) to address this apparent contradiction, explaining that differences in metabolic flux distribution can result in high ethanol production despite the observed sensitivity to catabolite repression.

New text (in red) may read:

“It is plausible that W. anomalus, despite its sensitivity to glucose repression, allocates a larger fraction of the available carbon toward ethanol production under high glucose conditions, which explains the relatively high ethanol titers observed.”

 

Comment 7: “In this study, both strains exhibited relatively low biomass yields (Table 1), suggesting a metabolic preference for ethanol production rather than extensive proliferation--- Could the authors expand upon this discussion, highlighting its significance in an industrial context?”

Response 7: We agree that the low biomass yields observed have important industrial implications. In the revised Discussion (inserted on Page 14, Lines 475–478), we have expanded our discussion to emphasize that lower biomass formation can be advantageous in industrial fermentations, as it indicates that a higher proportion of the substrate is directed towards ethanol production. We further discuss that, in an industrial context, optimizing the balance between biomass formation and product yield is crucial, and a metabolic preference for ethanol is desirable when the primary goal is maximizing biofuel production.

New text (in red) may state:

“From an industrial perspective, lower biomass yields can be beneficial as they imply that more substrate carbon is converted into ethanol rather than being used for cell growth. This metabolic allocation is particularly advantageous when the objective is to maximize ethanol production in large-scale fermentations.”

 

Comment 8: “This study illustrates the capacity of anomalus UEMG-LF-Y2 and D. rugosa UEMG-LF-Y4 to co-ferment glucose and xylose under laboratory conditions, yet their performance under industrial-scale stresses—such as high ethanol concentrations and inhibitory compounds—remains to be determined---- What specific inhibitors should be considered for future evaluations to improve productivity?”

Response 8: Thank you for this important question. We have now expanded our discussion regarding the potential industrial challenges and the need for further studies. In the revised Discussion (inserted on Page 15, Lines 487–492), we mention that future evaluations should consider inhibitors commonly found in lignocellulosic hydrolysates, such as furfural, hydroxymethylfurfural (HMF), acetic acid, and various phenolic compounds. We note that these inhibitors can negatively impact yeast metabolism and that assessing the tolerance of W. anomalus and D. rugosa to these compounds will be essential for industrial applications.

New text (in red) may read:

“Future studies should evaluate the performance of these strains in the presence of inhibitors commonly found in lignocellulosic hydrolysates, such as furfural, hydroxymethylfurfural (HMF), acetic acid, and phenolic compounds, which are typically generated during pretreatment processes. Assessing and enhancing the tolerance of W. anomalus and D. rugosa to these inhibitors is critical for improving ethanol productivity and scaling up the fermentation processes to industrial levels.”

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have provided a commendable description and response to the comments. No further comments from my side. The manuscript can be accepted for publication and further processing.

Author Response

Reply to Academic Editor

Comment 1 (Section 2.4): “However the authors should add a paragraph about the safety of Wickerhamomyces anomalus and Diutina rugosa (i.e. if they are GRAS and/or edible strains) as they are compared to baker's yeast for ethanol production”

Response 1: We sincerely thank the Academic Editor for the thoughtful suggestion regarding the importance of addressing the safety aspects of W. anomalus and D. rugosa. As requested, we have now incorporated the following paragraph into the Discussion section, on page 12, lines 356 to 369 of the revised manuscript:

“From an industrial and regulatory perspective, the safety of yeast strains is a critical consideration when evaluating their potential for bioethanol production. W. anomalus is widely recognized for its application in food preservation, biomass energy generation, and aquaculture feed, and is generally considered safe due to its long-standing use in food-related fermentations [35,36]. Notably, the European Food Safety Authority (EFSA) classifies W. anomalus as a Biosafety Level 1 organism, indicating a low risk to human health, and several killer strains of this species have been applied as biocontrol agents against molds and bacteria in the agro-food sector [37]. In contrast, D. rugosa is not formally classified as Generally Recognized as Safe (GRAS), but it has long been used in industrial biotechnology, particularly in the sustainable production of lipases from agro-industrial residues. Despite the absence of a GRAS designation, D. rugosa has not been associated with pathogenicity under controlled fermentation conditions [38]. These considerations reinforce the suitability of both strains for ethanol production under non-food industrial settings.”

 

Kind regards,
Caio Roberto Soares Bragança (on behalf of all authors)