Bioconversion of Some Agro-Residues into Organic Acids by Cellulolytic Rock-Phosphate-Solubilizing Aspergillus japonicus

Round 1

Reviewer 1 Report

Fermentation

Bioconversion of Some Agro-residues into Organic Acids by Alocal Cellulolytic Rock Phosphate Solubilizing Aspergillus japonicas

Comment to the Authors

Global warming is dire concern and whole world is in urgent search for possible options and solution to address this issue. In particular, agricultural waste incineration is one of the major contributors for carbon emission. Agricultural processing industries also results in agricultural waste, which needs further processing for its disposal, which further put economic burden and increase pollution. Converting agricultural waste into bio-active materials with biological process would be a great solution. In this interesting article authors Samir A. Mahgoub, Elmadawy G. A. Kedra, Hassan I. Abdelfattah, Howaida M.L Abdelbasit, Soha A. Alamoudi, Diana A. Al-Quwaie, Samy Selim, Salam S. Alsharari, and Wesam I. A. Saber have studied bioconversion of agricultural residues into organic acids using Aspergillus japonicas. Out of 15 fungal isolates they investigated A. Japonicas with mixture of sugarcane bagasse and faba beans straw (3:7) with 7.5% (v/w) fungal inoculation was the best for such conversion of agricultural waste to seven organic acids. A. Niger also found to have such conversion abilities.

Overall, this is interesting study in the field of fermentation, green chemistry, synthesis, and catalysis. Findings provided are reasonable and backed up by experimental results and references. However, the results are not properly described, experimental model studied are not properly described, there are several errors in the results description. All these issues need needs to be addressed. This article needs to be reconsider after major revision before publication, to make this article more helpful for the readers of Fermentation:

1.     In introduction it would be great if authors can discuss more about different bio-conversion processes currently used. Are there any other alternate microorganism to fungi, which can be used for such processes? If yes, what are the advantages and disadvantages of using fungi as compared to other means (biological/chemical processes) for such bio-degradation? What is the difference between this study and previous studies which used fungi (including Aspergillus) for this process, their specific advantages, and disadvantages? Such discussion will further help enhance significance of this study.

2.     It would be very helpful for the readers if authors can describe more about the reason for selecting Aspergillus fungi based on their characteristics and morphology in this study, when the mentioned “these fungi were selected preliminary based on the distinguished characteristics of their colonies and microscopic morphology”. How are these morphological characteristics related to degradation of agricultural waste into organic acids as compared to other fungi?

3.     What is the characteristic difference between the 15 isolates studied by the authors? The morphological characteristics of each individual isolate out of 15 isolates is not discussed in the paper.

4.     Reference of Fig. 3 to show the quantitative activity is incorrect as Figure numbers are incorrect. Fig 3 shows Molecular phylogenetic tree and Fig 2 shows the activity. This needs to be corrected. Figure shown on Page 8 line 268, the labels or subtitles a), b) and c) are not discussed in the figure caption or in the main text. Authors few comments about the results observed and their significance would be very informative and helpful for the readers.

5.     Authors mentioned that T4 recorded best treatment for the production of enzymes as compared to other treatments. What could be the reason behind it? Authors comment would be very helpful for the readers. It would be very helpful for the readers if authors can show graphical representation of the data (as they have shown for the activity graphs) for the results in Table 3, 4 and 5 to clearly see the effect of each treatment with the properties they have discussed in the tables.

6.     The Figure 4 needs to be well labeled for the organic acid in the graphs. The organic acid formation order mentioned in the abstract doesn’t match in the Table 4. As per the table 4 results, A. Niger produces more organic acids than A. Japonicas. This also makes title of the paper different with respect to the results seen in Table 4. Authors comments and discussion on it would be very helpful for the readers to address such confusion.

There are several typos, grammatical and formatting errors need to be addressed. Figure numbers are not in sequence. After Figure 1, Figure 3 followed by Figure 2 and 4. Please see below for some corrections to be addressed:

Corrections:

       i.          Page 1, line 26: Please remove additional space “Aspergillus japonicas  .”

     ii.          Page 2, line 61: Authors used unit “feddan”, it would be great for broad readers if authors use SI units or provide conversion of feddan to metric scale.

    iii.          Page 2, line 80: Please correct spelling “funges”

    iv.          Page 4, line 157: proper superscripts were not used for the units: “ml-1 min-1”. Needs to be checked and corrected throughout the paper.

     v.          Page 4, line 168: Please add space after period “.PCR”

    vi.          Page 4, line 173: Please correct the typo: “contasted”

  vii.          Please correct proper chemical name format for e.g.: Page 5, line 204 and 210 “H2SO4” subscript format needs to be updated for the number. Needs to be checked and corrected throughout the paper.

 viii.          Page 8, line 278 Table 2. The footnotes or labels for a and b in Final culture pH are not described in figure caption or foot note. In next three columns there is no description for a, b, c, d, e, f, g. It would be helpful for readers if authors add such descriptions.

Comments for author File: Comments.pdf

Author Response

Reviewer 1#

Comment to the Authors

Global warming is dire concern and whole world is in urgent search for possible options and solution to address this issue. In particular, agricultural waste incineration is one of the major contributors for carbon emission. Agricultural processing industries also results in agricultural waste, which needs further processing for its disposal, which further put economic burden and increase pollution. Converting agricultural waste into bio-active materials with biological process would be a great solution. In this interesting article authors have studied bioconversion of agricultural residues into organic acids using Aspergillus japonicas. Out of 15 fungal isolates they investigated A. Japonicas with mixture of sugarcane bagasse and faba beans straw (3:7) with 7.5% (v/w) fungal inoculation was the best for such conversion of agricultural waste to seven organic acids. A. Niger also found to have such conversion abilities.

Overall, this is interesting study in the field of fermentation, green chemistry, synthesis, and catalysis. Findings provided are reasonable and backed up by experimental results and references. However, the results are not properly described, experimental model studied are not properly described, there are several errors in the results description. All these issues need needs to be addressed. This article needs to be reconsider after major revision before publication, to make this article more helpful for the readers of Fermentation:

Response: Thanks for the reviewer. All comments and suggestions have considered accordingly

1. In introduction it would be great if authors can discuss more about different bio-conversion processes currently used. Are there any other alternate microorganism to fungi, which can be used for such processes? If yes, what are the advantages and disadvantages of using fungi as compared to other means (biological/chemical processes) for such bio-degradation? What is the difference between this study and previous studies which used fungi (including Aspergillus) for this process, their specific advantages, and disadvantages? Such discussion will further help enhance significance of this study.

 

Response: Thanks for the suggestion, the discussion was enhanced

Bioconversion of biomass has important advantages over other alternative energy strategies because biomass is the most abundant and also the most renewable biomaterial on our planet. Bioconversion of lignocellulosic residues is initiated primarily by microorganisms such as fungi and bacteria which are capable of degrading lignocellulolytic materials. Fungi such as Trichoderma reesei and Aspergillus niger produce large amounts of extracellular cellulolytic enzymes, whereas bacterial and a few anaerobic fungal strains mostly produce cellulolytic enzymes in a complex called cellulosome, which is associated with the cell wall. In filamentous fungi, cellulolytic enzymes including endoglucanases, cellobiohydrolases (exoglucanases) and β-glucosidases work efficiently on cellulolytic residues in a synergistic manner. In addition to cellulolytic/hemicellulolytic activities, higher fungi such as basidiomycetes (e.g. Phanerochaete chrysosporium) have unique oxidative systems which together with ligninolytic enzymes are responsible for lignocellulose degradation (Dashtban, et al., 2009).

2. It would be very helpful for the readers if authors can describe more about the reason for selecting Aspergillus fungi based on their characteristics and morphology in this study, when the mentioned “these fungi were selected preliminary based on the distinguished characteristics of their colonies and microscopic morphology”. How are these morphological characteristics related to degradation of agricultural waste into organic acids as compared to other fungi?

 

Response: Thanks for the reviewer for the suggestion we added the following

The genus Aspergillus is characterized by the formation of flask-shaped or cylindrical phialides either in a single or double series on the surface of a vesicle at the apex of a conidiophore (Raper and Fennell 1965). Conidia are deciduous and globose, oblong to elliptical in shape, and present various colors. In 1985, Gams and coinvestigators revised the groups and assigned them to 18 sections as a formal taxonomic status (Gams et al. 1985). Currently, there are approximately 250 species assigned to 17 sections in the family Aspergillaceae (Houbraken and Samson 2011) and this number will continue to grow as new species are described. Filamentous fungi from the genus Aspergillus are of high importance for biobased organic acid production. So far, a number of Aspergillus strains belonging to phylogenetically distantly related species have been successfully applied in industrial production of organic acids due to their excellent capabilities of secreting high amounts of desired organic acids (Yang et al., 2017). Aspergillus is a diverse genus containing many species recorded as efficient organic acid producers. In general, the reported aspergilli for organic acid production can be divided into two major groups: industrial workhorses and species with potential for organic acid production. The classification and identification of Aspergillus has been based on phenotypic characters but in the last decades was strongly influenced by molecular and chemotaxonomic characterisation.

Raper, K.B.; Fennell, D.I. The genus Aspergillus. The genus Aspergillus. Williams and Wilkins, Philadelphia, 1965.

Gams, W.; Christensen, M.; Onions, A.H.; Pitt, J.I.; Samson, R.A. Infrageneric taxa of Aspergillus. In Advances in Penicillium and Aspergillus systematics; Springer: 1986; pp. 55-62.

Samson, R.A.; Houbraken, J. Phylogenetic and taxonomic studies on the genera Penicillium and Talaromyces; CBS-KNAW Fungal Biodiversity Centre: 2011.

Yang, L.; Lübeck, M.; Lübeck, P.S. Aspergillus as a versatile cell factory for organic acid production. Fungal Biol. Rev. 2017, 31, 33-49.

 

3. What is the characteristic difference between the 15 isolates studied by the authors? The morphological characteristics of each individual isolate out of 15 isolates is not discussed in the paper.

Response: The discussion part was enhanced

Aspergillus (Plural Aspergilli) is a genus of fungi that consists of about 300 identified species of mold (mould). Aspergillus can be found in a variety of environments throughout the world given that their growth is largely determined by the availability of water. The rate at which they grow is largely determined by the temperature range in the environment they grow in. Regardless, studies have shown Aspergillus to be able to tolerate extreme conditions only if all of the other conditions are ideal. Given that a majority reproduce asexually, they are often described as conidial fungi. However, studies are yet to determine how a good number of others reproduce while some have been shown to reproduce sexually. Aspergillus obtain their nutrients by releasing enzymes that break down food material into smaller constituents that can be easily absorbed. This mechanism has proved particularly beneficial in various industries where these organisms are being used for their enzymes to break down various proteins and other compounds. Thus, we selected only all the Aspergillus that have the maximum cellulolytic activity on agar plates. Then classification and identification of Aspergillus has been based on phenotypic characters.

4. Reference of Fig. 3 to show the quantitative activity is incorrect as Figure numbers are incorrect. Fig 3 shows Molecular phylogenetic tree and Fig 2 shows the activity. This needs to be corrected. Figure shown on Page 8 line 268, the labels or subtitles a), b) and c) are not discussed in the figure caption or in the main text. Authors few comments about the results observed and their significance would be very informative and helpful for the readers.

Response: It has been changed and corrected.

5. Authors mentioned that T4 recorded best treatment for the production of enzymes as compared to other treatments. What could be the reason behind it? Authors comment would be very helpful for the readers. It would be very helpful for the readers if authors can show graphical representation of the data (as they have shown for the activity graphs) for the results in Table 3, 4 and 5 to clearly see the effect of each treatment with the properties they have discussed in the tables.

Response: It has been corrected and highlighted in the manuscript with yellow color.

This part has been added to the discussion” The C/N ratio is significant for any fermentation process. A proper C/N ratio for pure culture is necessary to optimize aerobic fermentation in submerged condition from organic substrate. It is therefore, necessary to maintain proper composition of the feedstock for efficient scale up (Adour et al. 2002). Also, at lower C/N ratio, the fermentation by Aspergillus giganteus, filamentous form of growth was maintained with the hyphae being scarcely branched without bulbous cells. Membrane perturbation due to the induction of intracellular oxidative stress was noticed at higher C/N ration (Dutta and Das, 2017).

6. The Figure 4 needs to be well labeled for the organic acid in the graphs. The organic acid formation order mentioned in the abstract doesn’t match in the Table 4. As per the table 4 results, A. Niger produces more organic acids than A. Japonicas. This also makes title of the paper different with respect to the results seen in Table 4. Authors comments and discussion on it would be very helpful for the readers to address such confusion.

Response: Thanks for your observation and suggestion, the chromatogram was labeled and the HPLC chromatogram and calibration curves have been transferred to supplementary also the data of A. niger with HPLC have been deleted, we will use these data in future study. we concentrate on A. japonicas.

7. There are several typos, grammatical and formatting errors need to be addressed. Figure numbers are not in sequence. After Figure 1, Figure 3 followed by Figure 2 and 4. Please see below for some corrections to be addressed:

Response: The manuscript was revised and Figures order was adjusted

 

Corrections:

1. Page 1, line 26: Please remove additional space “Aspergillus japonicas.”

Response: It has been removed and corrected in the manuscript.

1. Page 2, line 61: Authors used unit “feddan”, it would be great for broad readers if authors use SI units or provide conversion of feddan to metric scale.

Response: We converted all units to SI units

    iii.          Page 2, line 80: Please correct spelling “funges”

Response: Done accordingly

1. Page 4, line 157: proper superscripts were not used for the units: “ml-1 min-1”. Needs to be checked and corrected throughout the paper.

Response: It has been corrected in all manuscript.

1. Page 4, line 168: Please add space after period “.PCR”

Response: It has been added.

1. Page 4, line 173: Please correct the typo: “contasted”

Response: It has been changed

  vii.          Please correct proper chemical name format for e.g.: Page 5, line 204 and 210 “H2SO4” subscript format needs to be updated for the number. Needs to be checked and corrected throughout the paper.

Response: It has been corrected and changed

 viii.          Page 8, line 278 Table 2. The footnotes or labels for a and b in Final culture pH are not described in figure caption or foot note. In next three columns there is no description for a, b, c, d, e, f, g. It would be helpful for readers if authors add such descriptions.

Response: it has been done in the manuscript and highlight with yellow color

Reviewer 2 Report

The authors demonstrated the bioconversion of sugarcane bagasse and faba bean into organic acids under fermentation conditions. The paper sounds good scientifically. However, there are several critical questions that should address before publication.

1.       Introduction is very formal. Include the novelty of your work  and some scientific papers having discussion on bioconversion of agro-residues into organic acids.

2.       Transfer the HPLC chromatogram to supplementary.

3.       Add calibration curves to supplementary.

4.       Conclusions are very limited. Expand it with some critical numerical results.

5.       Eliminate typos and grammar errors.

6.       Add a pictorial presentation of the used process/methodology.

 

7.       Correct the figure numbers.

Author Response

Reviewer 2#

The authors demonstrated the bioconversion of sugarcane bagasse and faba bean into organic acids under fermentation conditions. The paper sounds good scientifically. However, there are several critical questions that should address before publication.

Response: Thanks for the reviewer. All comments and suggestions have considered accordingly

1. Introduction is very formal. Include the novelty of your work  and some scientific papers having discussion on bioconversion of agro-residues into organic acids.

Response: We added some studies to introduction as requested

          The global organic acid market stood at $6.94 billion in 2016 and is projected to reach $12.54 billion by the end of 2026. They constitute a significant portion of the fermentation market in the world, and microbiological production is an economic alternative using pre-treated agricultural wastes (Sharma, et al., 2020). Aspergillus is one of the most important industrial filamentous fungal species, non-toxic, safe for the production and offers several advantages such as higher productivity, yields, and lower contamination risk. Several industrial wastes, due to their organic and nutrient-rich composition, have been utilized as a resource for the production of value-added products such as organic acid, biofuels, biopesticides, biohydrogen, enzymes, and bioplastics via microbial fermentation processes (Chavan, et al., 2022). Yeast and fungi can convert the simpler carbohydrates such as the sugars and malic acid in grape and apple pomace to ethanol and high-value carboxylic acids, such as lactic, fumaric, succinic and citric acid (Njokweni, et al., 2021).

Sharma, N., Sahota, P.P., Singh, M.P. (2020). Organic Acid Production from Agricultural Waste. In: Kashyap, B.K., Solanki, M.K., Kamboj, D.V., Pandey, A.K. (eds) Waste to Energy: Prospects and Applications. Springer, Singapore. https://doi.org/10.1007/978-981-33-4347-4_17.

Chavan S, Yadav B, Atmakuri A, Tyagi RD, Wong JWC, Drogui P. Bioconversion of organic wastes into value-added products: A review. Bioresour Technol. 2022 Jan;344(Pt B):126398. doi: 10.1016/j.biortech.2021.126398.

Njokweni, S.G.; Steyn, A.;Botes, M.; Viljoen-Bloom, M.;van Zyl, W.H. Potential Valorization of Organic Waste Streams to Valuable Organic Acids through Microbial Conversion: A South African Case Study. Catalysts 2021, 11, 964. https://doi.org/10.3390/catal11080964

2. Transfer the HPLC chromatogram to supplementary.

Response: Done accordingly

3. Add calibration curves to supplementary.

Response: Done accordingly

4. Conclusions are very limited. Expand it with some critical numerical results.

Response: The conclusion has been expanded with some critical results and it has been changed to be “The obtained result indicated that the suitability of using cheap and abundantly available sugarcane bagasse and faba bean straw residuals as a mixture under solid-state fermentation (SSF) technique in the presence of A. Japonicus MN960315 as suitable convertors of cellulosic biomass into organic acids (OAs). Out of 15 fungal isolates investigated A. japonicas with mixture of sugarcane bagasse and faba beans straw (3:7) with 7.5% (v/w) fungal inoculation was the best for this fermentation of agricultural waste for producing seven organic acids (i.e., ascorbic acid, oxalic acid, lactic acid, formic acid, succinic acid, citric acid and malic acid).  A. Japonicus can be used as a promising fungal strain as well as would be a valuable approach in reducing the environmental contamination. However, further optimization studies may be suggested for exploring the optimum operation conditions that maximize OAs green biosynthesis and minimize the fermentation period. Consequently, improvement and utilization of both traditional and modern novel techniques for new strain to construct super producer, together with finding the compatible and affordable substrates. Hopefully, fermentation by Aspergillus especially novel industrial filamentous fungal species, nontoxic, safe for the production and offers several advantages such as higher productivity, yields, and lower contamination risk will be the conventional technique used for commercial producing natural organic acids in the not too long future for all the purpose.

5. Eliminate typos and grammar errors.

Response: The manuscript has been revised by expert

6. Add a pictorial presentation of the used process/methodology.

Response: Done accordingly

7. Correct the figure numbers.

Response: Done accordingly

 

 

Round 2

Reviewer 1 Report

Fermentation

Bioconversion of Some Agro-residues into Organic Acids by Alocal Cellulolytic Rock Phosphate Solubilizing Aspergillus japonicas

Comment to the Authors

Authors Samir A. Mahgoub, Elmadawy G. A. Kedra, Hassan I. Abdelfattah, Howaida M.L Abdelbasit, Soha A. Alamoudi, Diana A. Al-Quwaie, Samy Selim, Salam S. Alsharari, and Wesam I. A. Saber have addressed most of the quarries and suggestions. Authors also corrected typos and errors.

In introduction authors added discussion about various microorganisms (fungi and bacteria) can be use for bioconversion of biomass and discussed advantages of fungi over bacteria. Authors gave examples of Trichoderma reesei and Aspergillus niger (page 2 line 53). However, authors didn’t described why they chose Aspergillus Japonicus and not A. niger in this study? as they have mentioned in the introduction. What are advantages of using A. Japonicus? Such discussion will provide proper backup for the title authors chose.

Also, in Table 2 it is still confusing use of the letters a, b, c, d, e, f and g. They are not well described in the text or in the foot note. It is also confusing why authors have some of these letters in superscript and some aren’t.

Organic acid production sequence in decreasing order given in abstract and in results and discussion “3.7 Organic acids production: page 13 and 14 line 431, 432 and 433” are different. Authors comment and explanation would be very helpful to clear such confusion about the inconsistencies of the results shown in this article.

I am not sure what authors trying to mean in title by “Alocal”? Is it typo?

All these issues need needs to be addressed before publication, to make this article more helpful for the readers of Fermentation.

Comments for author File: Comments.pdf

Author Response

Reviewer 1 comments

In introduction authors added discussion about various microorganisms (fungi and bacteria) can be use for bioconversion of biomass and discussed advantages of fungi over bacteria. Authors gave examples of Trichoderma reesei and Aspergillus niger (page 2 line 53). However, authors didn’t described why they chose Aspergillus Japonicus and not A. niger in this study? as they have mentioned in the introduction. What are advantages of using A. Japonicus? Such discussion will provide proper backup for the title authors chose.

Response: Thanks for the reviewer for enhancing our manuscript, this part has been added in the Introduction:

Aspergills japonicus can release soluble phosphate from rock phosphate (RP) and excreted multiple organic acids, the main mechanism of RP solubilization is the production of organic acids (Xiao, et al., 2017).  Additionally, Nasr, et al., (2021) found that A. japonicus was the most fungi in solubilizing insoluble phosphate in rock that solubilized during phosphate solubilization, producing different organic acids (i.e., salicylic, ascorbic, citric, formic, lactic, oxalic and malic acid) during this process comparing the other fungi. On the other hand, found that A. japonicus produce high content of oxalic acid, which suppress the growth of Sclerotinia sclerotiorum

References

Xiao, C., Tingting, L., Guang, X and Ruan, C. Characteristics and Mechanisms of Biosolubilization of Rock Phosphate by Aspergillus japonicus. Brazilian Archives of Biology and Technology (2017), 60:  e16160541.  https://doi.org/10.1590/1678-4324-2017160541

Nasr, S.H., Mousa, A.M., Marzouk, M.A. and Yasser, M. M. Quantitative and Qualitative Analysis of Organic Acids Produced by Phosphate Solubilizing Fungi. Egypt. J. Bot. Vol. 61, No. 1, pp. 167-176 (2021).

Atallah, O., & Yassin, S. (2020). Aspergillus spp. eliminate Sclerotinia sclerotiorum by imbalancing the ambient oxalic acid concentration and parasitizing its sclerotia. Environmental microbiology, 22(12), 5265-5279.‏

Also, in Table 2 it is still confusing use of the letters a, b, c, d, e, f and g. They are not well described in the text or in the foot note. It is also confusing why authors have some of these letters in superscript and some aren’t.

Response: It has been cleared accordingly.

Organic acid production sequence in decreasing order given in abstract and in results and discussion “3.7 Organic acids production: page 13 and 14 line 431, 432 and 433” are different. Authors comment and explanation would be very helpful to clear such confusion about the inconsistencies of the results shown in this article.

Response: The sequences of organic acids were unified 

I am not sure what authors trying to mean in title by “Alocal”? Is it typo?

Response: The title was reformulated and “Alocal” was deleted

All these issues need needs to be addressed before publication, to make this article more helpful for the readers of Fermentation.

Response: Thank for the reviewer for enhancing the manuscript. All issues have been addressed in the manuscript.

Reviewer 2 Report

The authors improved the quality. Revised version can be accepted 

Author Response

Reviewer 2 comments

The authors improved the quality. Revised version can be accepted 

Response: Thanks a lot for the reviewer. We appreciate the valuable effort in enhancing our manuscript.