Sequential Bioprocess with Gluconobacter oxydans and Candida tropicalis for Gluconic Acid and Single-Cell Protein Production from Enzymatic Hydrolysate
Round 1
Reviewer 1 Report
Comments
The manuscript titled “Gluconic acid and single-cell protein production from enzymatic hydrolysate by a sequential bioprocess with Gluconobacter oxydans and Candida tropicalis” aims to evaluate the application of some bacterial species for the gluconic acid production from lignocellulose. Although, the manuscript has presented an interesting topic, there are some major issues/concerns that deserve thorough attention for possible publication. The language of the article is kind of weak and needs a careful revision by a native English speaker. Thus, authors have an opportunity to improve the manuscript’s readability, merits as well as its understanding for the journal readers.
Abstract
1. The abstract lacks quantitative data to support your conclusion/statement.
2. Abstract needs elaboration to support the research hypothesis and its conclusions.
Introduction
- The introduction section of the manuscript is kind of weak and needs a careful revision with proper organization in terms of the key concept development/problem identification towards the lignocellulose degradation processing.
- In literature, a number of species/organisms are reported to degrade lignocellulose. Authors should highlight why a particular organism is more promising than other agents. When the authors narrow down the literature survey to the target bacterium, G. oxydans, there is a gap in literature. Add a paragraph about the disadvantages or lacunae of other agents/methods like consolidating bioprocessing used for the bioconversion of lignocellulosic biomass. Further, Authors have missed relevant studies published in recent years such as listed below, among others.
Host-specific diversity of culturable bacteria in the gut systems of fungus-growing termites and their potential functions towards lignocellulose bioconversion. Insects (2023) 14, 403. https://doi.org/10.3390/insects14040403
Exploring the region-wise diversity and functions of symbiotic bacteria in the gut-system of wood-feeding termite, Coptotermes formosanus, towards lignocellulose degradation. Insect Science (2022) 29, 0-19. https://doi.org/10.1111/1744-7917.13012
Evaluation and characterization of the cellulolytic bacterium, Bacillus pumilus SL8 isolated from the gut of oriental leafworm, Spodoptera litura: an assessment of its potential value for lignocellulose bioconversion. Environmental Technology & Innovation 27 (2022) 102459. https://doi.org/10.1016/j.eti.2022.102459
Purification of a cellulase from cellulolytic gut bacterium, Bacillus tequilensis G9 and its evaluation for valorization of agro-wastes into added value byproducts. Biocatalysis and Agricultural Biotechnology 20 (2019)101219. https://doi.org/10.1016/j.bcab.2019.101219
Valorization potential of a novel bacterial strain, Bacillus altitudinis RSP75, towards Lignocellulose Bioconversion: An assessment of symbiotic bacteria from the stored grain pest, Tribolium castaneum. Microorganisms 9, 1952. https://doi.org/10.3390/microorganisms9091952
Evaluation of cellulose degrading bacteria isolated from the gut-system of cotton bollworm, Helicoverpa armigera and their potential values in biomass conversion. PeerJ: Life & Environment 9, e11254. https://doi.org/10.7717/peerj.11254
Formulation of synthetic bacterial consortia and their evaluation by principal component analysis for lignocellulose rich biomass degradation. Renewable Energy 148, 467-477. https://doi.org/10.1016/j.renene.2019.10.053
Exploring the gut of Helicoverpa armigera for cellulose degrading bacteria and evaluation of a potential strain for lignocellulosic biomass deconstruction. Process Biochemistry 73, 142-153. https://doi.org/10.1016/j.procbio.2018.08.001
Isolation of cellulolytic bacteria from the gastro-intestinal tract of Achatina fulica (Gastropoda: Pulmonata) and their evaluation for cellulose biodegradation. International Biodeterioration and Biodegradation 98, 73-80. http://dx.doi.org/10.1016/j.ibiod.2014.11.016
Statistical optimization of lignocellulosic waste containing culture medium for enhanced production of cellulase by Bacillus tequilensis G9. Waste Disposal & Sustainable Energy 1, 213-226. https://doi.org/10.1007/s42768-019-00016-w
3. At the end of the Introduction section, Authors have missed to specify the need of the objectives of this investigation.
4. Although authors have estimated ethanol production also, but the relevant description is missing in the introduction section.
Material and methods
1. How was the inoculum prepared for the co-fermentation? Did you culture the bacteria and yeast species separately and then mixed them? What was the ratio of the two species used for the inoculation?
2. The methodology related to the measurement and calculation of the dry weight the bacterial and yeast cells needs to be elaborated.
3. The methodology related to the sample preparation for the HPLC analysis is lacking, therefore authors should add more details about the sample collection, and processing.
4. Did author perform replicates of the experiments? The statistical analysis applied to the observed data is not mentioned in the methodology.
Results and discussion
1. Lines 174, 175 and so on, replace the word, “solids loadings” with “solid loading”.
2. Lines 188-200; the relevant discussion supported by previous studies is missing.
3. Line 204, 206, 211 & throughout the manuscript; italicize the species name.
4. Line 212; 81.4% of what, theoretical yield? Please explain.
5. The legend of the Figures needs to be elaborated and the applied statistics can be mentioned also.
6. What is simulation medium?
7. What are the possible inhibitors present in the enzymatic hydrolysate obtained from the lignocellulose?
8. Since authors applied HPLC analysis, what were the intermediate compounds detected in the study? Authors can also propose the possible pathways mechanisms of the conversion of pretreated lignocellulose into GA and thereof.
9. Line 257; which machine was used to measure an OD as high as 12.4?
10. It is highly advised to support your results with proper citation of some previous descriptions from other researchers. Overall a proper discussion for the observed results is lacking.
11. Line 297; which previous study? Authors should either mention about previous findings or cite a study that they are talking about.
12. Line 315; down regulation or diminished production?
13. The data of the Figure 2 can be presented in the tabular form.
Conclusion
- The conclusion section should be rewritten in terms of your innovations or new discoveries. I wonder why authors didn’t provide the limitations as well as future directions of the research at least in the conclusion section.
The manuscript's language is weak and needs careful revision by a native English speaker. Some of the sentences are very long and confusing. Further, the taxonomic names of the bacteria and yeasts are not presented properly.
Author Response
Abstract
Point 1: The abstract lacks quantitative data to support your conclusion/statement.
Response: Thanks for the reviewer’s suggestion. We have added the data in the abstract.
Point 2: Abstract needs elaboration to support the research hypothesis and its conclusions.
Response: Special thanks for reviewer’s suggestion and we have added the elaboration in the abstract.
Introduction
Point 1: The introduction section of the manuscript is kind of weak and needs a careful revision with proper organization in terms of the key concept development/problem identification towards the lignocellulose degradation processing.
Response: Thanks for the reviewer’s suggestion. We have added the problem of the lignocellulose degradation processing. “The high cost of pretreatment and enzymatic hydrolysis during lignocellulose degradation is still a problem that needs to be solved.”
Point 2: In literature, a number of species/organisms are reported to degrade lignocellulose. Authors should highlight why a particular organism is more promising than other agents. When the authors narrow down the literature survey to the target bacterium, G. oxydans, there is a gap in literature. Add a paragraph about the disadvantages or lacunae of other agents/methods like consolidating bioprocessing used for the bioconversion of lignocellulosic biomass. Further, Authors have missed relevant studies published in recent years such as listed below, among others.
Response: Thanks for the reviewer’s suggestion. We have added the reason for choosing GO in the introduction. “The current production process of GA is relatively mature, and the commonly used bacteria is Aspergillus niger. However, in the presence of inhibitors, the conversion ability of Aspergillus niger will be inhibited and the fermentation time will be extended [16].”(Reference: Zhang, H.; Zhang, J.; Bao, J. High titer gluconic acid fermentation by Aspergillus niger from dry dilute acid pretreated corn stover without detoxification. Bioresour. Technol. 2016, 203, 211-219.)
We have also added the above recommended literature relevant to this paper. “Some cellulolytic bacteria have been identified and used to break down lignocellulosic biomass. However, the mechanisms involved and the processes used for large-scale production are yet to be explored [7,8]”(Reference: Dar, M.A.; Shaikh, A.A.; Pawar, K.D.; Pandit, R.S. Exploring the gut of Helicoverpa armigera for cellulose degrading bacteria and evaluation of a potential strain for lignocellulosic biomass deconstruction. Process Biochem. 2018, 73, 142-153.
Dar, M.A.; Dhole, N.P.; Xie, R.; Pawar, K.D.; Ullah, K.; Rahi, P.; Pandit, R.S.; Sun, J. Valorization potential of a novel bac-terial strain, Bacillus altitudinis RSP75, towards lignocellulose bioconversion: an assessment of symbiotic bacteria from the stored grain pest, Tribolium castaneum. Microorganisms. 2021, 9, 1952.)
Point 3: At the end of the Introduction section, Authors have missed to specify the need of the objectives of this investigation.
Response: Thanks for the reviewer’s suggestion. We have made clear the need of our objectives in the last part of the introduction. The goal of this study was to maximize the economic benefits of enzymatic hydrolysate from the abundant and inexpensive lignocellulosic biomass by a two-step process.
Point 4: Although authors have estimated ethanol production also, but the relevant description is missing in the introduction section.
Response: Thanks for the reviewer’s suggestion. We have added the relevant description of ethanol in the introduction section.
“C. tropicalis may also convert xylose to ethanol, which can affect the purity of gluconic acid in the fermentation broth; therefore, C. tropicalis is introduced to simultaneously remove xylose and obtain SCP as value-added product by controlling the conditions.”
Material and methods
Point 1: How was the inoculum prepared for the co-fermentation? Did you culture the bacteria and yeast species separately and then mixed them? What was the ratio of the two species used for the inoculation?
Response: The G. oxydans and C. tropicalis stored frozen in glycerol tubes were activated and value added for inoculation, respectively. We cultured the bacteria and yeast species separately and then mixed them. The ratio of the two species used for the inoculation was 1:1.
Point 2: The methodology related to the measurement and calculation of the dry weight the bacterial and yeast cells needs to be elaborated.
Response: Thank the reviewers for their careful evaluation. In the process of bacterial dry weight measurement, 6 equal volume bacterial culture medium were first taken, of which 3 samples were measured turbidity by ultraviolet spectrophotometer at the wavelength of 600 nm, and the other 3 were measured for bacterial dry weight using an infrared moisture analyzer, and the average value was taken respectively. Then, linear fitting was performed according to the turbidities and dry weights, and the standard curve was drawn.
Point 3: The methodology related to the sample preparation for the HPLC analysis is lacking, therefore authors should add more details about the sample collection, and processing.
Response: Thanks for the reviewer's subtle observation. We have added more details about the sample collection, and processing.
“At each interval, 1 mL of fermentation broth samples were taken for the detection of fermentation products. All fermentation broth samples were centrifuged (10,000 rpm for 5 min) and the supernatant was diluted and used for analysis by HPLC and Dionex ICS-3000.”
Point 4: Did author perform replicates of the experiments? The statistical analysis applied to the observed data is not mentioned in the methodology.
Response: We performed replicates of the experiments. We have added a description of the replicated experiment.
“All the above experiments were performed three times and average results are shown.”
Results and discussion
Point 1: Lines 174, 175 and so on, replace the word, “solids loadings” with “solid loading”.
Response: Thanks for the reviewer’s suggestion. We have replaced it.
Point 2: Lines 188-200; the relevant discussion supported by previous studies is missing.
Response: Sorry for the references missing, and we have cited the literatures in this section.
Point 3: Line 204, 206, 211 & throughout the manuscript; italicize the species name.
Response: Thanks for the reviewer’s suggestion. We have revised them accordingly.
Point 4: Line 212; 81.4% of what, theoretical yield? Please explain.
Response: We are sorry for the unclear description. 81.4% refers to the yield of GA in the experiment.
Point 5: The legend of the Figures needs to be elaborated and the applied statistics can be mentioned also.
Response: Thanks for the reviewer's brilliant suggestion. We have revised the legend of the Figures in the manuscript.
Point 6: What is simulation medium?
Response: The simulation medium refers to media synthesized by simulating glucose and xylose reagents based on the content of glucose and xylose in the enzymatic hydrolysate.
Point 7: What are the possible inhibitors present in the enzymatic hydrolysate obtained from the lignocellulose?
Response: The possible inhibitors present in the enzymatic hydrolysate include acetic acid, furfural and phenols.
Point 8: Since authors applied HPLC analysis, what were the intermediate compounds detected in the study? Authors can also propose the possible pathways mechanisms of the conversion of pretreated lignocellulose into GA and thereof.
Response: Thanks for the reviewer’s suggestion. The intermediate products may have gluconolactone. But I am sorry that we did not measure the specific values and study the pathway mechanism of the conversion of pretreated lignocellulose to GA.
Point 9: Line 257; which machine was used to measure an OD as high as 12.4?
Response: OD was measured by Amersham Biosciences UV/Visible spectrophotometer (Spectrumlab 752 s) at 600 nm.
Point 10: It is highly advised to support your results with proper citation of some previous descriptions from other researchers. Overall a proper discussion for the observed results is lacking.
Response: As your suggestion, we tried our best to improve the manuscript and made some changes in the manuscript.
Point 11: Line 297; which previous study? Authors should either mention about previous findings or cite a study that they are talking about.
Response: We are sorry for the unclear description. The previous study refers to the conclusions drawn in 3.2 and 3.3 and not to the conclusions obtained in other articles. We have revised the description.
Point 12: Line 315; down regulation or diminished production?
Response: We are sorry for our incorrect writing. We have changed “down-regulation” to “diminished production” in the article.
Point 13: The data of the Figure 2 can be presented in the tabular form.
Response: Thanks for the reviewer’s suggestion. We have used a graphical format in order to show more visually the increase and decrease of the components in the fermentation medium.
Conclusion
Point: The conclusion section should be rewritten in terms of your innovations or new discoveries. I wonder why authors didn’t provide the limitations as well as future directions of the research at least in the conclusion section.
Response: Special thanks for reviewer’s suggestion. We have added the future directions of the research at least in the conclusion section. “The started sugar concentration is still lower and cannot satisfy the request of industrialization, therefore, in the future, high concentration enzymatic hydrolysate fermenta-tion also need to continue to study for reducing energy consumption and improving the utilization rate of equipment.”
Comments on the Quality of English Language
The manuscript's language is weak and needs careful revision by a native English speaker. Some of the sentences are very long and confusing. Further, the taxonomic names of the bacteria and yeasts are not presented properly.
Response: Thank you for your useful comment. English was improved by a professional language editor to improve the quality of the manuscript.
Once again, thank you very much for your comments and suggestions.
Reviewer 2 Report
This paper presents an interesting topic about lignocellulosic biomass utilisation. However, some changes are required prior to publication.
1) Abstract needs improvement, we need to see main findings in terms of numbers. Also, it's not clear that xylose can be converted into XA, it looks dubious if you're converting xylose into GA.
2)"However, G. oxydans cannot simultaneously convert glucose and xylose into corresponding sugar acids" (lines 68-70)
Please mention both acids that are produced.
3) "The initial inoculum of G. oxydans was 2 OD and the fermentation was performed at 30 ℃ and 220 rpm for 24 h." (line 119)
What does 2 OD mean? Please mention the definition when you first use the abbreviation
4) "Therefore, we used a fed-batch enzymatic hydrolysis operation (5% solids loading every 12 h) to obtain a relatively high concentration of fermentable sugars." (lines 172-173)
Where is the fed-batch graph? I can only see fig1 which is batch mode.
5) "It could be seen that glucose was completely converted into 96.7 g/L GA by G. oxydans with a yield of 90.5% in 12 h." (line 303)
If the yield was not quantitative you cannot say it was completely converted.
I suggest revising the English of the paper with either a native speaker or an online editing tool, there are some words and expressions that are not very well utilised.
Author Response
Point 1: Abstract needs improvement, we need to see main findings in terms of numbers. Also, it's not clear that xylose can be converted into XA, it looks dubious if you're converting xylose into GA.
Response 1: Thanks for the reviewer’s suggestion. We have revised the description in the abstract.
Point 2: "However, G. oxydans cannot simultaneously convert glucose and xylose into corresponding sugar acids" (lines 68-70)
Please mention both acids that are produced.
Response 2: Thanks for the reviewer’s suggestion. We have revised it accordingly.
Point 3: "The initial inoculum of G. oxydans was 2 OD and the fermentation was performed at 30 ℃ and 220 rpm for 24 h." (line 119)
What does 2 OD mean? Please mention the definition when you first use the abbreviation
Response 3: Thanks for the reviewer’s suggestion. 2 OD means 2 optical density. It was first mentioned in line 141.
Point 4: "Therefore, we used a fed-batch enzymatic hydrolysis operation (5% solids loading every 12 h) to obtain a relatively high concentration of fermentable sugars." (lines 172-173)
Where is the fed-batch graph? I can only see fig1 which is batch mode.
Response 4: We are sorry for the unclear description. In the fact, the experiment in Figure 1 adopted the method of fed-batch enzyme supplementation. For example, in a 25% enzyme addition experiment group, 5% solids were added at 0 h, 12 h, 24 h, 36 h, 48 h, and 60 h, respectively. Moreover, we have revised the figure 1 caption in the manuscript for more clearly expression.
Point 5: "It could be seen that glucose was completely converted into 96.7 g/L GA by G. oxydans with a yield of 90.5% in 12 h." (line 303).` If the yield was not quantitative you cannot say it was completely converted.
Response 5: Thanks for the reviewer’s suggestion. We have revised it accordingly.
Comments on the Quality of English Language
I suggest revising the English of the paper with either a native speaker or an online editing tool, there are some words and expressions that are not very well utilised.
Response: Thank you for your useful comment. English was improved by a professional language editor to improve the quality of the manuscript.
Round 2
Reviewer 1 Report
Authors have addressed all the concerns of the reviewers and have made significant improvement to the manuscript. Therefore, I suggest acceptance of the manuscript for publication in Fermentation.
