Ethanol and Xylitol Co-Production by Clavispora lusitaniae Growing on Saccharified Sugar Cane Bagasse in Anaerobic/Microaerobic Conditions

Round 1

Reviewer 1 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

1. The important experimental data should be supplemented and the significance of this paper should be explained.
2. The differences between this paper and previous reports are compared, and the novelty of this study is elaborated.
3. Results and Discussion. This part requires more in-depth analysis and discussion, especially mechanism analysis.
4. The shortcomings of the techniques used in this paper need to be summarized and future research should be prospected.

Author Response

Answer to Review 1. (Please see attached document)

Revised manuscript (RMS) fermentation-3490554: Ethanol and xylitol co-production by Clavispora lusitaniae growing on saccharified sugar cane bagasse in anaerobic/microaerobic conditions.

 

The authors thank Reviewer #1 for her/his suggestions and comments to improve this manuscript.

Answer to comments (A). We highlighted all the revisions in the RMS with red underlined characters.

1. “The important experimental data should be supplemented and the significance of this paper should be explained”.

(A.1): Thank you very much for the suggestion. The results obtained in the different fermentation conditions and their statistical analysis were included as supplementary material in their respective sections.

(A.1.1): This document has relevance because to the best of our knowledge, this work marks the initial report of the biosynthesis of ethanol and xylitol by a non-conventional yeast strain, Clavispora lusitaniae, which was isolated from mezcal must and grown on saccharified sugarcane bagasse (SSCB). This Mexican native strain performed best in producing high ethanol concentration under both anaerobic and a combination of anaerobic and microaerobic conditions. In contrast, xylitol synthesis was the best under microaerobic conditions.

Several studies have explored various fermentation strategies for the co-production of ethanol and xylitol from saccharified SCB. These strategies involve two separate fermentation stages. In the first stage, Saccharomyces cerevisiae produces ethanol from glucose. In contrast, in the second stage, xylose-rich syrups are inoculated with unconventional yeasts such as C. tropicalis (Raj and Krishnan, 2020) or C. guilliermondii TISTR 5068 to produce xylitol (Hor et al., 2022). Another approach involves the production of xylitol from xylose in the first stage using Pichia guilliermondii RLV-04 (MH588234.1) followed by ethanol production in the second stage using S. cerevisiae (Ahuja et al., 2024). However, to the best of our knowledge, no studies have reported the co-production of ethanol and xylitol by Clavispora lusitaniae from saccharified lignocellulosic biomass (LCB) using a single system that includes an initial anaerobic phase followed by a microaerophilic phase.

It is important to note that anaerobic conditions were not suitable for xylitol production. Furthermore, the activities of xylose reductase and xylitol dehydrogenase were investigated in the SSCB-grown cultures. The results showed that glucose suppressed the expression of these enzymes to a large extent.

These outcomes suggest that C. lusitaniae has the potential to be employed for producing ethanol and xylitol from SSCB, with specific aeration strategies that enhance the yield of each metabolite/product. This study further highlights the need to optimize fermentation conditions for producing specific products, especially in non-conventional yeast strains such as C. lusitaniae.

REFERENCES:

-K. Raj and C. Krishnan, “Improved co-production of ethanol and xylitol from low-temperature aqueous ammonia pretreated sugarcane bagasse using two-stage high solids enzymatic hydrolysis and Candida tropicalis,” Renew. Energy, vol. 153, pp. 392–403, Jun. 2020, doi: 10.1016/j.renene.2020.02.042.

-S. Hor, M. B. Kongkeitkajorn, and A. Reungsang, “Sugarcane Bagasse-Based Ethanol Production and Utilization of Its Vinasse for Xylitol Production as an Approach in Integrated Biorefinery,” Fermentation, vol. 8, no. 7, p. 340, Jul. 2022, doi: 10.3390/fermentation8070340.

-V. Ahuja, S. Chinnam, and A. K. Bhatt, “Yeast based biorefinery for xylitol and ethanol production from sugarcane bagasse,” Process Saf. Environ. Prot., vol. 191, pp. 676–684, Nov. 2024, doi: 10.1016/j.psep.2024.08.122.

2. “The differences between this paper and previous reports are compared, and the novelty of this study is elaborated.”

      (A.2): Indeed, the authors made an exhaustive review of the state of the art and knowledge related to the subject, basing the discussion of the results on the most recent reports.

-K. O. Barros et al., “Oxygenation influences xylose fermentation and gene expression in the yeast genera Spathaspora and Scheffersomyces,” Biotechnol. Biofuels Bioprod., vol. 17, no. 1, p. 20, Feb. 2024, doi: 10.1186/s13068-024-02467-8.

-C. I. D. G. Bonan et al., “Redox potential as a key parameter for monitoring and optimization of xylose fermentation with yeast Spathaspora passalidarum under limited-oxygen conditions,” Bioprocess Biosyst. Eng., vol. 43, no. 8, pp. 1509–1519, Aug. 2020, doi: 10.1007/s00449-020-02344-2.

-B. C. Bolzico, S. Racca, J. N. Khawam, R. J. Leonardi, A. H. Tomassi, M. T. Benzzo, and R. N. Comelli, “Exploring xylose metabolism in non-conventional yeasts: kinetic characterization and product accumulation under different aeration conditions,” J. Industrial Microbiol. Biotechnol., 51, Jun. 2024, doi.org/10.1093/jimb/kuae023.

-K. Manjarres-Pinzón, D. Mendoza-Meza, M. Arias-Zabala, G. Correa-Londoño, and E. Rodriguez-Sandoval, “Effects of agitation rate and dissolved oxygen on xylose reductase activity during xylitol production at bioreactor scale,” Food Sci. Technol., vol. 42, p. e04221, 2022, doi: 10.1590/fst.04221.

3. “Results and Discussion. This part requires more in-depth analysis and discussion, especially mechanism analysis.”

(A.3): Thank you for your valuable suggestion. We have included further analysis and discussion in the manuscript. The following paragraph has been included in the RMS on Pp 10-11, L 300-338:

Pp 10-11 L 300-338: Some studies have explored a variety of fermentation strategies for the co-production of ethanol and xylitol from saccharified lignocellulosic biomass, with a particular focus on SCB. These strategies generally involve two separate fermentation stages to optimize the conversion of sugars to both products. In the first anaerobic stage, Saccharomyces cerevisiae, a well-known ethanol-producing yeast, ferments glucose derived from the hydrolysis of the SCB. This stage is highly efficient due to S. cerevisiae's well-established metabolic pathways and tolerance to ethanol, making it the yeast of choice for ethanol production in industrial processes. In the second stage, xylose-rich syrups obtained from the hydrolysis of hemicellulose are inoculated with unconventional yeasts such as Candida tropicalis (Raj and Krishnan, 2020) or Candida guilliermondii TISTR 5068, which are capable of converting xylose to xylitol (Hor et al., 2022).

An alternative two-stage strategy involves the use of Pichia guilliermondii RLV-04 (MH588234.1), which ferments xylose to xylitol in the first stage, followed by the production of ethanol from glucose in the second stage, again using S. cerevisiae (Ahuja et al., 2024). While these strategies are effective, they often face challenges related to the coordination of two separate fermentation phases, which can increase the process's overall complexity, time, and cost.

In contrast, the co-production of ethanol and xylitol in a single fermentation system would greatly simplify the process by eliminating the need for separate fermentation stages. This approach, which could integrate the anaerobic and microaerophilic phases into a single bioreactor and microorganism, would streamline the entire process and offer several potential advantages. A single reactor system would reduce the need for complex fermenter configurations, simplify the overall process design, and potentially reduce operational costs.

To the best of our knowledge, there are no published studies on the co-production of ethanol and xylitol by C. lusitaniae from saccharified lignocellulosic biomass (LCB) using a unified fermentation system that includes both anaerobic and microaerophilic phases. C. lusitaniae uses xylose as a carbon source and has potential tolerance to both the fermentation environment and ethanol accumulation.

This research could fill a significant gap in the current literature by demonstrating the feasibility of a single-phase fermentation process that simultaneously produces both ethanol and xylitol, thereby simplifying the bioconversion of lignocellulosic biomass. Such an integrated process could offer substantial advantages in terms of efficiency and cost-effectiveness, particularly for large-scale industrial applications. Furthermore, this work may contribute to advancing the sustainability of biofuel and bioproduct production by utilizing waste biomass more efficiently and economically viable.

Future research could focus on optimizing the fermentation conditions, scaling up the process, and evaluating such a co-production system's economic and environmental benefits.

 

4. “The shortcomings of the techniques used in this paper need to be summarized and future research should be prospected.”

(A.4): We appreciate the suggestion. Rather than viewing the loss of ethanol from the culture medium as a disadvantage, we consider it an opportunity. Since the two metabolites of interest, ethanol and xylitol, need to be separated and recovered at the end of the process, removing ethanol can be beneficial. Ethanol can be recovered via distillation, while xylitol can be recovered through crystallization. By removing ethanol early in the process, its recovery will be more cost-effective. However, the primary objective of the current study is not focused on ethanol recovery but on establishing the conditions that enable the metabolic co-production of both metabolites.

The text in the RMS has been revised to prevent any confusion for the readers and now reads as follows on Pp 9, L 262-271:

Pp9 L-262-271:  It was observed that ethanol produced in the C1 condition and during the aeration phase in the C3 condition was removed from the culture medium via venting. This loss was substantial, with ethanol titers decreasing to 71.23% and 67.09% in the C3 and C1 conditions, respectively. However, this situation presents two potential advantages. First, removing ethanol from the culture medium reduces its toxicity to the yeast. Second, ethanol can be effectively recovered from the vent stream through pervaporation, a separation process that employs selective polydimethylsiloxane (PDMS) membrane, thereby facilitating the recovery of this metabolite [21,22]. Future research will focus on utilizing this technique to recover ethanol, thereby facilitating the subsequent recovery of xylitol through crystallization.

Author Response File: Author Response.docx

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

The authors of the manuscript with title "Ethanol and xylitol co-production by clavispora Iusitaniae growing on saccharified sugar cane bagasse in anaerobic/microaerobic conditions" reports important research results useful for researchers working in the research fields of ethanol and xylitol production.

However, there are several are several drawbacks in the manuscript. The manuscript can be revised taking into consideration following comments of the reviewer.  

1. Introduction section of the manuscript sholud be modified with the latest state of the art research results reported in the open literature regarding production of ethanol and xylitol using sugar cane bagasse. 
2. Figure 1. Metabolic pathways involved in the production of ethanol, xylitol and arabitol from SSCB by non-conventional yeast.  The figure should also include pathways other than metabolic for the production of ethanol, xylitol and arabitol.
3. 2.2 Pretreatment and saccharification of sugar cane bagasse. It is suggested that authors discuss the effect of pretreatment temperature, time and concentration of NaOH on sugar cane baggase.
4. 2.3 Batch reactor fermentation, It is mentioned that bioreactor was stirred at 100 rpm at 300 0C for 80 h. Have the authors studied the effect of stirring rate, time and temperature other than than 30 0C ?
5. Figure 2. Production of ethanol, xylitol and D-arabitol C. lusitaniae growing in SSCB under microaerobic conditions. Authors are suggested to explain the reasons for substantial differences in the production of ethanol, xylitol and D-arabitol with changes in time
6. Figure 4. Production of ethanol, xylitol and D-arabitol by C.lusitaniae in SSCB under sequential anaerobic / microaerobic phases (C3). How much the amount of sugars and metabolits effect production of ethanol, xylitol and D-arabitol
7. Conclusion should be rewritten with the most important results from the research.

 

 

Author Response

Answer to Review 2.

Revised manuscript (RMS) fermentation-3490554: Ethanol and xylitol co-production by Clavispora lusitaniae growing on saccharified sugar cane bagasse in anaerobic/microaerobic conditions.

 

The authors thank Reviewer #2 for her/his suggestions and comments to improve this manuscript.

Answer to comments (A). We highlighted all the revisions in the RMS with red underlined characters.

The authors of the manuscript with title "Ethanol and xylitol co-production by clavispora Iusitaniae growing on saccharified sugar cane bagasse in anaerobic/microaerobic conditions" reports important research results useful for researchers working in the research fields of ethanol and xylitol production.

However, there are several are several drawbacks in the manuscript. The manuscript can be revised taking into consideration following comments of the reviewer.  

1. “Introduction section of the manuscript sholud be modified with the latest state of the art research results reported in the open literature regarding production of ethanol and xylitol using sugar cane bagasse.” 

(A.1): Thank you very much for the suggestion. We have modified introduction with the latest state of the art research results reported in the open literature regarding production of ethanol and xylitol using sugar cane bagasse.

The text in the RMS now reads as follows Pp 2 L63-72

Pp 2 L 63-72: In contrast, non-conventional yeasts like Kluyveromyces, Candida, and Scheffersomyces, among others, can ferment hexoses and pentoses, with some species also producing xylitol as a byproduct from xylose metabolism. Clavispora lusitaniae has emerged as a promising candidate for co-production due to its ability to efficiently metabolize cellobiose, glucose and xylose and its tolerance to diverse fermentation conditions. The simultaneous production of ethanol and xylitol enables the efficient utilization of LCB, offering a sustainable pathway for biofuel and biochemical production.

Studies have shown that aerobic or microaerobic conditions favor xylitol production, while anaerobic conditions are more conducive to ethanol production. One of the key bottlenecks in the co-production of ethanol and xylitol with non-conventional yeasts is optimizing the aeration conditions required for the efficient accumulation of each metabolite. The production of ethanol from glucose is most efficient when the process is operated under anaerobic conditions. While the production of xylitol from xylose occurs to a greater extent under aerobic or microaerobic conditions [12,13].

Pp 2-3, L77-88:

Several studies have explored various fermentation strategies for the co-production of ethanol and xylitol from saccharified SCB. These strategies involve two separate fermentation stages. In the first stage, Saccharomyces cerevisiae produces ethanol from glucose. In contrast, in the second stage, xylose-rich syrups are inoculated with unconventional yeasts such as C. tropicalis (Raj and Krishnan, 2020) or C. guilliermondii TISTR 5068 to produce xylitol (Hor et al., 2022). Another approach involves the production of xylitol from xylose in the first stage using Pichia guilliermondii RLV-04 (MH588234.1) followed by ethanol production in the second stage using S. cerevisiae (Ahuja et al., 2024). However, to the best of our knowledge, no studies have reported the co-production of ethanol and xylitol by Clavispora lusitaniae from saccharified lignocellulosic biomass (LCB) using a single system that includes an initial anaerobic phase followed by a microaerophilic phase.

Pp 3, L97-99:

It is worth mentioning that xylitol accumulation results from an imbalance between the cofactors NADPH and NAD⁺ during microaerobic xylose metabolism through the oxidoreduction pathway (Figure 1), [10,11]. Then, XR and XDH activities were detected in C. lusitaniae when exposed to xylose. The expression levels of the corresponding genes are closely correlated with the observed enzyme activities.

 

 

2. “Figure 1. Metabolic pathways involved in the production of ethanol, xylitol and arabitol from SSCB by non-conventional yeast.  The figure should also include pathways other than metabolic for the production of ethanol, xylitol and arabitol.”

(A2). Thank you very much for the suggestion.

We would have liked to include all metabolic pathways in Figure 1, but they would be lost in the diagram. We want to highlight the main pathways for producing ethanol from glucose and xylitol from xylose.

3. "2.2 Pretreatment and saccharification of sugar cane bagasse. It is suggested that authors discuss the effect of pretreatment temperature, time and concentration of NaOH on sugar cane baggase".

(A.3): Thank you for your valuable suggestion. Sugarcane bagasse (SCB) was subjected to alkaline pretreatment (NaOH) under optimal conditions designed to maximize delignification, increase the cellulose content, and minimize energy and NaOH consumption. The effect of pretreatment temperature, time, and concentration of NaOH on SCB were discussed by de la Torre (1981); de la Torre & Casas-Campillo (1984) and Ponce-Noyola & de la Torre, (1995). Our research group has consistently applied these conditions, ensuring the same pretreatment protocol is followed with each new batch of SCB to maintain its consistent composition (Table 1).

The following paragraph has been included in the RMS on Pp 4, L123-126:

The text in the RMS now reads as follows:

Pp4 L123-127. Ground SCB (10%) was alkali-pretreated under optimal conditions of 2% NaOH at 80 °C for 15 minutes (de la Torre, 1981; de la Torre & Casas-Campillo, 1984; Ponce-Noyola & de la Torre, 1995) to maximize lignin removal, increase the cellulose content, and minimize energy and NaOH consumption. The same pretreatment protocol is followed with each new batch of SCB to maintain its consistent composition (Table 1).

Table 1 was included in RMS

Table 1 Composition of sugar cane bagasse

 

SCB Treatment	Lignin (%)	Cellulose (%)	Hemicellulose   (%)
Raw material	23.27±0.39	47.4±1.54	17.98±1.27
NaOH (2%)	15.37±0.66	65.83±3.64	12.52±1.52

 

4. "2.3 Batch reactor fermentation, It is mentioned that bioreactor was stirred at 100 rpm at 300 0C for 80 h. Have the authors studied the effect of stirring rate, time and temperature other than than 30 0C ?”

Thank you very much for your comments.

(A.4): Before this study, a central composite design (CCD) was performed, in which agitation, temperature, substrate concentration and other variables were assayed in response to xylitol production. It was observed that 100 rpm provided adequate mass transfer, allowing for optimal growth and ethanol and xylitol production per C. lusitaniae. In addition, 30°C was observed to be the optimum temperature for the growth of this yeast. The cultures were incubated for 80 hours, with particular attention to the depletion of xylose as substrate. The above results will be in a manuscript that is being prepared.

5. “Figure 2. Production of ethanol, xylitol and D-arabitol C. lusitaniae growing in SSCB under microaerobic conditions. Authors are suggested to explain the reasons for substantial differences in the production of ethanol, xylitol and D-arabitol with changes in time”

We greatly appreciate your suggestions.

(A.5): The substantial variations in ethanol, xylitol, and D-arabitol production over time are attributed to the dynamic nature of the process. This study was conducted in a batch bioreactor, where the initial sugar concentration is fixed. Upon inoculation, C. lusitaniae first consumes glucose, leading to ethanol production. Once glucose is depleted, the yeast shifts to xylose consumption, initiating xylitol production. The rates of consumption and product formation depend on the cell's metabolism.

6. “Figure 4. Production of ethanol, xylitol and D-arabitol by C.lusitaniae in SSCB under sequential anaerobic / microaerobic phases (C3). How much the amount of sugars and metabolits effect production of ethanol, xylitol and D-arabitol

Thank you very much for your comments.

(A.6): The amount of sugars and metabolites significantly influences the production of ethanol, xylitol, and D-arabitol, as they are directly involved in the metabolic pathways that lead to the formation of these compounds. Ethanol production is highly dependent on the availability of fermentable sugars such as glucose, xylose, or other sugars. When sugar concentrations are high, fermentation rates are faster, leading to higher ethanol yields up to a certain threshold. Beyond this threshold, high sugar concentrations may inhibit the fermentation process due to osmotic stress or feedback inhibition on enzymes involved in fermentation.

Xylitol is typically produced from xylose, and D-arabitol is produced from xylose or arabinose. Their production is sensitive to the initial concentration of these pentoses. High pentose concentrations can enhance xylitol or arabitol production by increasing the flux through the pentose phosphate pathway. However, high xylose concentrations may also lead to the accumulation of undesirable byproducts, which could divert the metabolic flow away from xylitol synthesis.

 

7. “Conclusion should be rewritten with the most important results from the research.”

Thank you very much for the suggestion. The authors have rewritten the conclusions including some of the most important results.

The text now reads as follows on Pp 20, L579-597

(A.7): Pp20 L579-597: Clavispora lusitaniae is a non-conventional yeast with significant potential in lignocellulosic biorefineries co-producing ethanol and xylitol from saccharified sugarcane bagasse (SSCB). When cultivated in SSCB, the yeast achieves its highest ethanol titer of 31.8 g/L under anaerobic conditions, compared to 18.1 g/L under microaerobic conditions. In contrast, the highest xylitol titer of 14.3 g/L is attained under microaerobic conditions, a value 3.5 times greater than that observed under anaerobic conditions. These results emphasize the critical role of aeration conditions in optimizing xylose metabolism and balancing the co-production of ethanol and xylitol from SSCB.

A strategy involving an initial anaerobic phase followed by a microaerophilic phase promotes the co-production of ethanol and xylitol by C. lusitaniae, as the titers of both metabolites under this two-phase approach are like those obtained under pure anaerobic (C2) and microaerobic (C1) conditions, respectively. Furthermore, the higher accumulation of xylitol in C. lusitaniae is attributed to increased xylose reductase activity (XR) relative to xylitol dehydrogenase activity (XDH) under microaerophilic conditions, which is consistent with the higher expression of the XYL1 gene compared to XYL2.

These findings underscore the potential of C. lusitaniae for efficient co-production of biofuels and biochemicals from lignocellulosic feedstocks, providing valuable insights into optimizing aeration conditions for enhanced metabolic flux toward both ethanol and xylitol production.

 

References that support our arguments:

 

 

Raj and C. Krishnan, “Improved co-production of ethanol and xylitol from low-temperature aqueous ammonia pretreated sugarcane bagasse using two-stage high solids enzymatic hydrolysis and Candida tropicalis,” Renew. Energy, vol. 153, pp. 392–403, Jun. 2020, doi: 10.1016/j.renene.2020.02.042.

 

Hor, M. B. Kongkeitkajorn, and A. Reungsang, “Sugarcane Bagasse-Based Ethanol Production and Utilization of Its Vinasse for Xylitol Production as an Approach in Integrated Biorefinery,” Fermentation, vol. 8, no. 7, p. 340, Jul. 2022, doi: 10.3390/fermentation8070340.

 

Ahuja, S. Chinnam, and A. K. Bhatt, “Yeast based biorefinery for xylitol and ethanol production from sugarcane bagasse,” Process Saf. Environ. Prot., vol. 191, pp. 676–684, Nov. 2024, doi: 10.1016/j.psep.2024.08.122.

 

Torre M. de la, " SCP production from cellulosic wastes". Conser Recycling vol. 5 pp. 41-45

 

De La Torre and C. C. Campillo, “Isolation and characterization of a symbiotic cellulolytic mixed bacterial culture,” Appl. Microbiol. Biotechnol., vol. 19, no. 6, pp. 430–434, Jun. 1984, doi: 10.1007/BF00454383.

 

Ponce-Noyola and M. De La Torre, “Isolation of a high-specific-growth-rate mutant of Cellulomonas flavigena on sugar cane bagasse,” Appl. Microbiol. Biotechnol., vol. 42, no. 5, pp. 709–712, Jan. 1995, doi: 10.1007/BF00171949.

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

 Accept in present form

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

The authors of the revised version of the manuscript with title "Ethanol and xylitol co-production by clavispora lusitspora growing on saccharified sugar cane baggase in anaerobic/microaerobic conditions" have taken into consideration comments of the reviewer.

The manuscript is recommended for publication.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. Section 2.2: Did you adjust the pH before saccharification of sugar cane bagasse? Please provide detailed information.
2. Section 2.3: Why did you measure the cell growth at OD660?

3. I noticed that the error bars for ethanol concentrations in Figure 4 appear identical across all time points, which raises my concerns about the accuracy of the reported variability. It is unusual for fermentation processes to exhibit perfectly uniform standard deviations, as both biological and technical replicates typically show variability, especially considering the authors mentioned ethanol loss due to venting.

   I recommend that the authors clarify the error bars, whether they represent biological or technical replicates and verify the calculations for standard deviations to ensure the variability is accurately reflected.

4. Please revise the format of Table 2

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript 'Ethanol and xylitol co-production by Clavispora lusitaniae growing on saccharified sugar cane bagasse in anaerobic/microaerobic conditions' investigated the effect of aeration on the co-production of ethanol and xylitol from saccharified sugar cane bagasse. The topic is interesting and some suggestions are shown to improve this work.

1. It is suggested to add some important experimental data and describe the significance of this paper.
2. It is suggested to add some research cases on bioenergy production and compare the differences between this paper and previous reports to further elaborate on the novelty of this paper.
3. Materials and Methods. It is suggested to explain why Clavispora lusitaniae is used in this article.
4. Results and Discussion. It is suggested that the experimental results should be analyzed and discussed in depth in combination with literature analysis to better demonstrate the main conclusions and mechanisms of this paper.
5. Whether a single culture will degenerate or be easily contaminated in practice, especially if there are many sugars in the medium. Please discuss it.
6. To improve this study, some work on biofuel such as ‘Antioxidants alleviated low-temperature stress in microalgae by modulating reactive oxygen species to improve lipid production and antioxidant defense’ could be considered.
7. Results and Discussion. It is suggested that the shortcomings of the methods used in this paper are summarized, and the future technological development is analyzed and prospected.