Bioprocess Integration of Candida ethanolica and Chlorella vulgaris for Sustainable Treatment of Organic Effluents in the Honey Industry

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study proposes an effective solution based on two sequential bioprocesses using real effluent from an Argentine honey-exporting facility. The synergistic combination of complementary microorganisms and different effluents enhances microalgal growth and improves the overall sustainability of an integrated wastewater management approach for honey production. It is interesting. However, a comparison of the proposed method with conventional methods in terms of cost and efficiency would strengthen the study.

1. To facilitate comparison, Table 1 and Table 2 can be merged into a single table.
2. The number of keywords can be reduced.
3. Graphical Abstract was not clear.
4. In Figure 5, the y-axis unit should use a larger scale.

Author Response

This study proposes an effective solution based on two sequential bioprocesses using real effluent from an Argentine honey-exporting facility. The synergistic combination of complementary microorganisms and different effluents enhances microalgal growth and improves the overall sustainability of an integrated wastewater management approach for honey production. It is interesting. However, a comparison of the proposed method with conventional methods in terms of cost and efficiency would strengthen the study.

1. To facilitate comparison, Table 1 and Table 2 can be merged into a single table.

We appreciate the suggestion. However, merging the tables would require formatting them into a single-page layout, which we believe would reduce readability and make the information harder to interpret. We have consulted the Editor for further guidance on this matter.

Thank you for the suggestion; the keywords have been revised accordingly.

1. Graphical Abstract was not clear.

Thank you for the observation; the graphical abstract has been improved for better clarity and resolution.

1. In Figure 5, the y-axis unit should use a larger scale.

The correction has been implemented.

Reviewer 2 Report

Comments and Suggestions for Authors

The primary objective is to develop and evaluate a sequential biological treatment for industrial effluents from the honey industry by integrating two bioprocesses: an initial stage using Candida ethanolica for sugar removal, followed by a second stage involving Chlorella vulgaris for nutrient removal and COD reduction.

1) Technical and scientific errors and speculative conclusions

- Page 9, lines 343–347: The assertion that C. vulgaris’s flexibility "allows the microalgae to produce O₂, fix CO₂, and assimilate various sources of organic carbon" is plausible but should be supported with specific culture condition data to confirm whether autotrophic or mixotrophic metabolism predominated.

- Page 11, lines 370–372: The resilience to variable sunlight and scaling-up potential is overgeneralised without pilot-scale or long-term validation.

- Page 11, lines 377–379: The claim of carbon footprint mitigation is valid in principle but speculative in this context unless quantified by a life cycle assessment (LCA).

2) Introduction

- The lack of prior studies on honey effluent treatment is asserted but should be more rigorously supported with a systematic literature review or comparative data.

- No mention is made of mixotrophy or nutrient-limited cultivation of Chlorella, which is directly relevant to the study’s novelty.

- Novelty is underemphasised. Suggestion: “This is the first integrated approach reported for treating honey industry effluents using a native yeast-microalgae system with in situ effluent recycling and dual waste valorisation potential.”

- Have other yeast strains been compared in preliminary tests, or why was Candida ethanolica H3 selected over others?

- It needs improvement. See my suggested references below.

3) Methods

- Was COD increase after yeast treatment due to cell lysis, metabolite release, or oxygen limitation? Was DOC or BOD monitored?

- Clarify whether the sequential system is designed for batch or continuous operation.

4) Results and Discussion

- Was the light spectrum controlled or characterised? This affects photosynthetic activity significantly.

- Can the authors elaborate on the economic feasibility and energy input of their system compared to other decentralised solutions?

- Include statistical significance indicators in Figures 3–5.

- The absence of lipid/productivity data from C. vulgaris limits the valorisation claim.

- Use of RTW as a nutrient source is interesting—discuss potential risks (e.g., faecal contamination).

- Provide a process flow diagram with retention times to improve clarity for real-world applications.

5) Conclusions

- Are there plans for scaling up this system? Any preliminary techno-economic or LCA analysis?

6) Suggested references

- "Strain selection and adaptation of a fungal‑yeast‑microalgae consortium for sustainable bioethanol production and wastewater treatment from livestock wastewater": https://doi.org/10.1186/s12934-024-02537-4

It can be used in the Introduction (where you contextualize microbial consortia involving yeasts and microalgae for effluent treatment). Also in the Methods and Discussion (when comparing C. ethanolica + C. vulgaris with similar integrated systems).

- “The Role of the Microalgae–Bacteria Consortium in Biomass Formation and Its Application in Wastewater Treatment Systems”: https://doi.org/10.3390/app14146083

It can be used after discussing the synergy between microalgae and heterotrophic microorganisms such as yeasts, and the advantages of microbial interactions for bioremediation and biomass valorisation. Also in the Results and Discussion, when reporting on the nutrient removal efficiency, COD reduction, and growth performance of Chlorella vulgaris under different light and inoculum densities. Finally, to To support the sustainability and circular economy potential of the proposed integrated yeast–microalgae bioprocess, especially regarding by-product recovery.

- “Co-Fermentation of Chlorella vulgaris with Oleaginous Yeast in Starch Processing Effluent as a Carbon-Reducing Strategy for Wastewater Treatment and Biofuel Feedstock Production”: https://doi.org/10.3390/fermentation9050476

It can be used in the Results and Discussion, particularly when interpreting the co-culture benefits in COD reduction, carbon conversion efficiency, and biomass valorization. This paper reports concurrent cultivation of C. vulgaris with oleaginous yeast (Rhodotorula spp.) in real industrial effluent, achieving high TOC removal (~88–96%) and impressive lipid yields (~1.8 g/L), clearly illustrating the biomass valorisation potential of yeast–microalgae systems.

- “Effect of microalgae and bacteria inoculation on the startup of bioreactors for paper pulp wastewater and biofuel production”: https://doi.org/10.1016/j.jenvman.2024.121305

It can be used in the Introduction: the manuscript discusses the synergy between microalgae and heterotrophic microorganisms (like yeasts) in co-culture systems. It can can support and expand this concept with their findings on photogranules and the symbiotic interaction between microalgae and bacteria for treating industrial wastewater. Also, in the Results and Discussion: the manuscript reports that higher light and cell density increased C. vulgaris biomass and nutrient removal. Therefore, it provides a detailed analysis of nutrient uptake (N and P), pigment production, and lipid yield in mixed cultures. When the manuscript discusses the valorisation of biomass and metabolite production, this paper could be highly useful, as it quantifies lipid and pigment yields from microalgal biomass, highlighting biofuel and bioproduct potential.

Comments on the Quality of English Language

Requires moderate English revision for clarity and flow.

Author Response

The primary objective is to develop and evaluate a sequential biological treatment for industrial effluents from the honey industry by integrating two bioprocesses: an initial stage using Candida ethanolica for sugar removal, followed by a second stage involving Chlorella vulgaris for nutrient removal and COD reduction.

1) Technical and scientific errors and speculative conclusions

- Page 9, lines 343–347: The assertion that C. vulgaris’s flexibility "allows the microalgae to produce O₂, fix CO₂, and assimilate various sources of organic carbon" is plausible but should be supported with specific culture condition data to confirm whether autotrophic or mixotrophic metabolism predominated.

We appreciate the comment. Organic matter in the wastewater could be converted through CO₂ fixation, as reflected in the observed increase in biomass (Figure 5). The paragraph has been revised to clarify this concept.

 

- Page 11, lines 370–372: The resilience to variable sunlight and scaling-up potential is overgeneralised without pilot-scale or long-term validation.

We appreciate the comment; the paragraph was modified.

- Page 11, lines 377–379: The claim of carbon footprint mitigation is valid in principle but speculative in this context unless quantified by a life cycle assessment (LCA).

We appreciate the comment; the paragraph was modified.

 

2) Introduction

- The lack of prior studies on honey effluent treatment is asserted but should be more rigorously supported with a systematic literature review or comparative data.

We appreciate the comment; the paragraph was modified.

- No mention is made of mixotrophy or nutrient-limited cultivation of Chlorella, which is directly relevant to the study’s novelty.

We appreciate the comment; the paragraph was modified.

- Novelty is underemphasised. Suggestion: “This is the first integrated approach reported for treating honey industry effluents using a native yeast-microalgae system with in situ effluent recycling and dual waste valorisation potential.”

We appreciate the comment; the paragraph was modified.

- Have other yeast strains been compared in preliminary tests, or why was Candida ethanolica H3 selected over others?

Thank you for the comment. The selection of Candida ethanolica H3 is described in detail in the cited reference (8- Sánchez Novoa, J.G.; Domínguez, F.G.; Pajot, H.; de Cabo, L.I.; Navarro Llorens, J.M.; Marconi, P.L. Isolation and assessment of highly sucrose-tolerant yeast strains for honey processing factory’s effluent treatment, AMB Express. 14, 2024, 125, https://doi.org/10.1186/s13568-024-01771-8), and this information has been integrated into the Introduction and Materials & Methods sections.

 

- It needs improvement. See my suggested references below.

3) Methods

- Was COD increase after yeast treatment due to cell lysis, metabolite release, or oxygen limitation? Was DOC or BOD monitored?

Thank you for the observation. DOC and BOD were not monitored in this study. However, cell lysis was unlikely, as biomass increased during treatment. The COD increase (measured as detailed in the Materials & Methods section, at the beginning and at the end of the treatments, and observed in Tables 1 and 2) may be attributed to oxygen limitation, metabolite accumulation, and suspended biomass. The bioreactor used was non-aerated and lacked any oxygen supplementation.

- Clarify whether the sequential system is designed for batch or continuous operation.

We appreciate the comment; the paragraph was modified (line 175).

 

4) Results and Discussion

- Was the light spectrum controlled or characterised? This affects photosynthetic activity significantly.

Thank you for the suggestion. Light intensity was measured using a LI-190R Quantum Sensor, which quantifies photosynthetically active radiation (PAR).

- Can the authors elaborate on the economic feasibility and energy input of their system compared to other decentralised solutions?

Thank you for the suggestion. A detailed economic or energy analysis is beyond the scope of this study but could be addressed in future work.

- Include statistical significance indicators in Figures 3–5.

The figures have been updated to include statistical significance indicators.

- The absence of lipid/productivity data from C. vulgaris limits the valorisation claim.

We appreciate the comment. However, lipid and productivity analyses were not within the scope of this study.

- Use of RTW as a nutrient source is interesting—discuss potential risks (e.g., faecal contamination).

Thank you for the suggestion. A discussion on potential risks associated with RTW, such as faecal contamination, has been added to the Results and Discussion section.

- Provide a process flow diagram with retention times to improve clarity for real-world applications.

Figure 1 has been updated to include a process flow diagram with retention times for improved clarity.

5) Conclusions

- Are there plans for scaling up this system? Any preliminary techno-economic or LCA analysis?

Thank you for the comment. A techno-economic or life cycle assessment (LCA) analysis was not conducted in this study, as it falls beyond its current scope. However, these aspects will be considered in future research.

6) Suggested references

- "Strain selection and adaptation of a fungal‑yeast‑microalgae consortium for sustainable bioethanol production and wastewater treatment from livestock wastewater": https://doi.org/10.1186/s12934-024-02537-4

It can be used in the Introduction (where you contextualize microbial consortia involving yeasts and microalgae for effluent treatment). Also in the Methods and Discussion (when comparing C. ethanolica + C. vulgaris with similar integrated systems).

- “The Role of the Microalgae–Bacteria Consortium in Biomass Formation and Its Application in Wastewater Treatment Systems”: https://doi.org/10.3390/app14146083

It can be used after discussing the synergy between microalgae and heterotrophic microorganisms such as yeasts, and the advantages of microbial interactions for bioremediation and biomass valorisation. Also in the Results and Discussion, when reporting on the nutrient removal efficiency, COD reduction, and growth performance of Chlorella vulgaris under different light and inoculum densities. Finally, to To support the sustainability and circular economy potential of the proposed integrated yeast–microalgae bioprocess, especially regarding by-product recovery.

- “Co-Fermentation of Chlorella vulgaris with Oleaginous Yeast in Starch Processing Effluent as a Carbon-Reducing Strategy for Wastewater Treatment and Biofuel Feedstock Production”: https://doi.org/10.3390/fermentation9050476

It can be used in the Results and Discussion, particularly when interpreting the co-culture benefits in COD reduction, carbon conversion efficiency, and biomass valorization. This paper reports concurrent cultivation of C. vulgaris with oleaginous yeast (Rhodotorula spp.) in real industrial effluent, achieving high TOC removal (~88–96%) and impressive lipid yields (~1.8 g/L), clearly illustrating the biomass valorisation potential of yeast–microalgae systems.

- “Effect of microalgae and bacteria inoculation on the startup of bioreactors for paper pulp wastewater and biofuel production”: https://doi.org/10.1016/j.jenvman.2024.121305

It can be used in the Introduction: the manuscript discusses the synergy between microalgae and heterotrophic microorganisms (like yeasts) in co-culture systems. It can can support and expand this concept with their findings on photogranules and the symbiotic interaction between microalgae and bacteria for treating industrial wastewater. Also, in the Results and Discussion: the manuscript reports that higher light and cell density increased C. vulgaris biomass and nutrient removal. Therefore, it provides a detailed analysis of nutrient uptake (N and P), pigment production, and lipid yield in mixed cultures. When the manuscript discusses the valorisation of biomass and metabolite production, this paper could be highly useful, as it quantifies lipid and pigment yields from microalgal biomass, highlighting biofuel and bioproduct potential.

Thank you for the suggested bibliography.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The paper explores how to treat wastewater from honey processing using two biological steps. First, it applies a yeast called Candida ethanolica, which was found in the wastewater. Then, it adds the microalga Chlorella vulgaris, along with a second type of wastewater from the same facility. This second step provides nutrients and helps the algae grow. The study presents an interesting approach, but several points need to be addressed:

• The abstract is too dense. It should be simplified. Try cutting unnecessary detail and clearly explaining the two-stage process in plain terms.

• The introduction is generally fine, but it would benefit from a final paragraph that clearly outlines the research aims and hypothesis.

• The total sugar content in the honey wastewater is around 3690 ± 275 mg/L. This level of sugar could have other uses, such as for producing ethanol fuel. Why was this particular treatment approach chosen over other options? The paper does not clearly explain the advantage of this method.

• What are the current treatment practices for honey-processing wastewater? This context is missing and would help justify the need for a new approach.

• Is the treated water from this process safe to release into the environment? The paper should compare the final water quality to national discharge guidelines.

• What are the byproducts of this process, such as sludge, and how can they be used or disposed of?

• The feasibility of this method should be discussed. Are there examples of similar processes used in real-life conditions?

• How practical is it to apply this process on a full scale, especially considering the need to maintain specific microbial strains?

Comments on the Quality of English Language

The quality of the English language is generally acceptable; however, the document is difficult to follow in several places. I recommend that the author review the entire paper to improve its readability and overall flow.

Author Response

The paper explores how to treat wastewater from honey processing using two biological steps. First, it applies a yeast called Candida ethanolica, which was found in the wastewater. Then, it adds the microalga Chlorella vulgaris, along with a second type of wastewater from the same facility. This second step provides nutrients and helps the algae grow. The study presents an interesting approach, but several points need to be addressed:

• The abstract is too dense. It should be simplified. Try cutting unnecessary detail and clearly explaining the two-stage process in plain terms.

Thank you for the suggestion. The abstract has been revised.

• The introduction is generally fine, but it would benefit from a final paragraph that clearly outlines the research aims and hypothesis.

We appreciate your feedback. The introduction has been revised.

• The total sugar content in the honey wastewater is around 3690 ± 275 mg/L. This level of sugar could have other uses, such as for producing ethanol fuel. Why was this particular treatment approach chosen over other options? The paper does not clearly explain the advantage of this method.

Thank you for the comment. The primary goal of this manuscript is to propose a sustainable biological solution for effluent management. This is the first reported integrated approach for treating honey industry wastewater using a native yeast–microalgae system, with in situ effluent recycling and dual waste valorization potential. The focus was on remediation rather than biofuel production, aiming to improve environmental impact at the source.

• What are the current treatment practices for honey-processing wastewater? This context is missing and would help justify the need for a new approach.

Thank you for the observation. The introduction has been expanded.

• Is the treated water from this process safe to release into the environment? The paper should compare the final water quality to national discharge guidelines.

Thank you for the comment. This study was conducted at the laboratory scale, setting the stage for future optimization. Large-scale honey fractionation generates highly contaminated effluents that require efficient treatment before reuse or discharge. While the proposed system showed substantial potential in reducing COD and sugar levels, the final water quality did not meet national discharge standards (e.g., COD thresholds). Therefore, additional post-treatment processes are necessary. This clarification has been included in the Conclusions section.

• What are the byproducts of this process, such as sludge, and how can they be used or disposed of?

Thank you for the question. We are currently investigating the characteristics and potential applications of the byproducts generated, including sludge.

• The feasibility of this method should be discussed. Are there examples of similar processes used in real-life conditions?

This is the first integrated approach reported for this specific application, which reinforces its value as a nature-based solution with potential for broader adoption in similar contexts.

• How practical is it to apply this process on a full scale, especially considering the need to maintain specific microbial strains?

Thank you for the comment. We are familiar with the use of raceway systems employing Chlorella and other microalgae for large-scale effluent treatment, such as in municipal wastewater facilities. In fact, the proposed system is currently being implemented on a full scale at a honey fractionation plant in Buenos Aires Province (Argentina), demonstrating its practical feasibility.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Accept. The authors have addressed the reviewers' comments to a reasonable extent.