Aspergillus niger Enhances the Efficiency of Sewage Sludge Biochar as a Sustainable Phosphorus Source

Round 1

Reviewer 1 Report

The study demonstrated in this manuscript contributes the effect of sludge biochar on the solubilization of phosphate minerals as well as the potential of P release from this biochar into soil solution by A. niger. Study is interesting but still needs improvement.

1.      Authors should justify how the P solubilization was higher in sewage sludge biochar produced at 300 °C. However as per given data, P content is higher in sewage sludge biochar produced at 500 °C.

2.      A. niger showed proficient efficacy for P release from sewage sludge biochar. What is the mechanism behind it?

3.      Clear the objectivity of this study at the end of introduction without incorporating unwanted details there.

4.      After discounting the amount of P solubilized from the SS biochars, the sources AlPO4 and Araxá RP showed average percentages of P solubilization of 73 and 83%, respectively, which were higher than the treatment with no biochar. Justification

5.      A. niger also enhances the efficiency of TSP, authors suggested the further implementation of this study for agronomic performance, what about the elemental toxicity release from P source. Is there any role of HMs removal by such fungi.

6.      Lines 127-129. The experiment was carried out under a CRD with four repetitions. Data subjected to ANOVA and treatments were compared using Tukey test (p < 0.05). Revise  and incorporate the statistical details in separate section

7.      I didn’t find variability in data in fig.1 over the treatments.

8.      Revise the conclusion in terms of future prospects

9.      References are not in the same format. Revise

 

Author Response

1. Authors should justify how the P solubilization was higher in sewage sludge biochar produced at 300 °C. However as per given data, P content is higher in sewage sludge biochar produced at 500 °C.

Authors: This subject is discussed in the third paragraph of the Discussion section. We have reformulated the whole paragraph as follows:

The pyrolysis temperature did not affect the amount of P solubilized from biochar by A. niger. Even though the biochar produced at 500 °C presented P content nearly 50% higher, the soluble P concentration at the end of incubation was similar for biochar produced at 300 and 500 °C. Likewise, in a soil-plant study, soil available P level was not affected by different pyrolysis temperatures employed to produce biochar from SS [14]. More stable forms of P are formed in SS biochar made at higher pyrolysis temperatures [24], which may have impaired further fungal solubilization of biochar produced at 500 °C. Indeed, extremely stable P-Al compounds presenting low solubility in citric acid have been found in SS biochar produced at 500 °C [25]. Also, in a previous study, SS biochar produced at the lowest temperature (300 °C) presented P more readily available by distinct extractant solutions, including neutral ammonium citrate + water and Mehlich-1, as influenced by higher levels of carbon and nitrogen in the biochar.

 

2. A. niger showed proficient efficacy for P release from sewage sludge biochar. What is the mechanism behind it?

Authors: We improved the discussion to better explain the mechanism of P solubilization. The following sentences were included and/or improved:

The mechanism of phosphate solubilization by A. niger is the production of organic acids such as citric, oxalic, and gluconic acid [29,30,37]. These acids supply the protons (H⁺) that react with the sparingly-soluble phosphate mineral and release soluble phosphate ions. Moreover, organic acids form stable complexes with the metal cations present in the mineral structure, favouring the forward reaction by removing this reaction product [38,39].

[…]

Most of P contained in SS biochars is in the form of insoluble calcium-phosphate compounds [26,27], similar to the P minerals found in rock phosphates [46]. Calcium phosphates are promptly solubilized by A. niger as a result of medium acidification [29,44,47]. In the present research A. niger acidified the medium to a pH of 2.1, resulting in the solubilization of nearly 50% of P contained in the SS biochar after 7 days.

3. Clear the objectivity of this study at the end of introduction without incorporating unwanted details there.

Authors:  We rewritten the last paragraph of introduction as follows:

Developing sustainable agricultural inputs from urban waste is an increasing demand from society. In this sense, the complete use of P from pyrolyzed SS still lacks detailed studies. The use of phosphate solubilizing microorganisms is an alternative to enable the complete adoption of SS biochar as a P source for plants. Furthermore, elucidating the effects of combining biochar and microorganisms can create technological routes for the development of novel fertilizers. The fungus Aspergillus niger can solubilize Ca-, Al-, and Fe-phosphates [29,30] and therefore could be an alternative to increase the effectiveness of biochar as a P fertilizer. Moreover, it was demonstrated that biochar with low P content boosted mineral phosphate solubilization by A. niger [31,32]. Thus, we hypothesized that a P-rich biochar could both boost P release from phosphate minerals and act as a P source itself. Therefore, this research aimed at evaluating the effect of SS biochar on the solubilization of phosphate minerals by A. niger and testing the potential of P release from this biochar into soil solution by the fungus.

 

4. After discounting the amount of P solubilized from the SS biochars, the sources AlPO4 and Araxá RP showed average percentages of P solubilization of 73 and 83%, respectively, which were higher than the treatment with no biochar. Justification

Authors: We presented a better discussion of these results as follows:

As previously reported, biochar can increase fungal phosphate solubilization by promoting organic acid production and by mitigating toxicity of elements released from the P source [31]. Ionic forms of Al, F, and HMs are released concomitantly with P during the solubilization of Araxá RP [40]. Likewise, Al³⁺ is one of the reaction products of AlPO₄ solubilization. Biochar can adsorb such ions [31,41,42] and therefore remove reaction products potentially inhibitory for A. niger, allowing for increased solubilization of AlPO₄ and Araxá RP.

5. A. niger also enhances the efficiency of TSP, authors suggested the further implementation of this study for agronomic performance, what about the elemental toxicity release from P source. Is there any role of HMs removal by such fungi.

Authors: TSP does not have significant amounts of HMs since it is a P source derived from rock phosphate processing. Also, it must meet legal requirements of absence significant amounts of HMs. Therefore, HMs release would not be a problem in this case.

6. Lines 127-129. The experiment was carried out under a CRD with four repetitions. Data subjected to ANOVA and treatments were compared using Tukey test (p < 0.05). Revise  and incorporate the statistical details in separate section

Authors: A new section has been added to the text including statistical analyses details. The following sentences have been added to the text:

For both experiments, data normality was validated by the Kolmogorov-Smirnov test and the homogeneity of variance by Brown-Forsythe test (p < 0.05). Data were subjected to analysis of variance (ANOVA) and the Fisher’s Least Significant Difference (LSD) test was used to compare treatment means (p < 0.05). In experiment II, data were square-root transformed to fit a normal distribution.

 

7. I didn’t find variability in data in fig.1 over the treatments.

Authors: We improved the figure legend to clarify the comparisons between treatments.

 

8. Revise the conclusion in terms of future prospects

Authors: The following sentences have been provided in the conclusion section:

As a perspective, these in vitro findings need to be further evaluated in a soil-plant system. Therefore, for agronomic and environmental applications, an additional evaluation must be performed in field experiments. In addition, the scaling-up process and economic analysis need to be better understood.

 

9. References are not in the same format. Revise

Authors: We checked and corrected the reference format.

Reviewer 2 Report

The authors investigated the effect of SS biochar on the solubilization of phosphate minerals by A. niger. They also investigated the potential of P release of this biochar into the soil solution by fungus. The topic taken up by the authors and the data obtained are interesting. The publication requires a  correction. Detailed comments:

1.       Introduction - briefly explain the motivation for undertaking this research, its relevance and originality, where it fits into the development of the field, and why it should be of interest to readers.

2.       Introduction - The utility of this study should be clearly highlighted in the manuscript.

3.       The weaknesses and limitations of this study compared to other studies should be considered.

4.       It is important to check that the writing text clearly expresses and explains each idea and result obtained.

5.       A better discussion would be necessary in order to emphasize the main findings. The discussion section of the article is poorly written. Must be upgraded with new articles.

6.       Line 138 - Figure 1 - no explanation letters (a and b) above the error bars.

7.       Line 163 - Figure 2 a and Figure 2 b- no explanation letters (a - ef) above the error bars.

8.       The conclusions needs improvement - highlight the most important findings and identify the added value of the main finding.

9.       It is strongly recommended to add more latest citations (of 2021 and 2022) throughout the manuscript.

10.   Recommends adding more items from the IJERPH journal to which the authors have submitted their manuscript, in order to emphasize that it fits the subject of the journal.

Author Response

1. Introduction - briefly explain the motivation for undertaking this research, its relevance and originality, where it fits into the development of the field, and why it should be of interest to readers.

Authors: The introduction section has been improved according to the reviewer’s suggestion

2. Introduction - The utility of this study should be clearly highlighted in the manuscript.

Authors: The utility of our study has been highlighted in the introduction section. Please see the attached file with all changes marked.

3. The weaknesses and limitations of this study compared to other studies should be considered.

Authors: The weaknesses and limitations of this study has been considered in the Discussion and Conclusions sections. The following sentences have been added to the text.

Discussion: These results are limited to in vitro conditions, remaining the need to expand the evaluations to include soil and plant components.

Conclusions: As a perspective, these in vitro findings need to be further evaluated in a soil-plant system. Therefore, for agronomic and environmental applications, an additional evaluation must be performed in field experiments. In addition, the scaling-up process and economic analysis need to be better understood.

 

4. It is important to check that the writing text clearly expresses and explains each idea and result obtained.

Authors: The results section has been substantially improved.

5. A better discussion would be necessary in order to emphasize the main findings. The discussion section of the article is poorly written. Must be upgraded with new articles.

Authors: The Discussion section has been improved to address the issues raised by both reviewers.

6. Line 138 - Figure 1 - no explanation letters (a and b) above the error bars.

Authors: Explanation letters have been provided.

7. Line 163 - Figure 2 a and Figure 2 b- no explanation letters (a - ef) above the error bars.

Authors: Explanation letters have been provided.

8. The conclusions needs improvement - highlight the most important findings and identify the added value of the main finding.

Authors: The conclusions section has been improved highlighting the most important findings and presenting some perspectives. The following sentences have been added:

The results of the present study show for the first time the action of A. niger in the solubilization of P from SS biochar. In addition, the study demonstrated that the SS biochar potentiates the P solubilization from rock phosphate by A. niger.

As a perspective, these in vitro findings need to be further evaluated in a soil-plant system. Therefore, for agronomic and environmental applications, an additional evaluation must be performed in field experiments. In addition, the scaling-up process and economic analysis need to be better understood.

9. It is strongly recommended to add more latest citations (of 2021 and 2022) throughout the manuscript.

Authors: Citations of 2021 and 2022 have been added to the text. Some examples are shown below:

Figueiredo, C.C.; Reis, A.S.P.J.; Araujo, A.S.; Blum, L. E. B.; Shah, K.; Paz-Ferreiro, J. Assessing the potential of sewage sludge-derived biochar as a novel phosphorus fertilizer: Influence of extractant solutions and pyrolysis temperatures. Waste Manag. 2021, 124, 144-153.

Zhou, Y.; Li, D.; Li, Z.; Guo, S.; Chen, Z.; Wu, L.; Zhao, Y. Greenhouse Gas Emissions from Soils Amended with Cornstalk Biochar at Different Addition Ratios. Int. J. Environ. Res. Public Health 2023, 20, 927. https://doi.org/10.3390/ijerph20020927

Bashir, M.A.; Wang, X.; Naveed, M.; Mustafa, A.; Ashraf, S.; Samreen, T.; Nadeem, S.M.; Jamil, M. Biochar Mediated-Alleviation of Chromium Stress and Growth Improvement of Different Maize Cultivars in Tannery Polluted Soils. Int. J. Environ. Res. Public Health 2021, 18, 4461. https://doi.org/10.3390/ijerph18094461

 

 

10. Recommends adding more items from the IJERPH journal to which the authors have submitted their manuscript, in order to emphasize that it fits the subject of the journal.

Authors: Articles from the IJERPH have been considered and cited throughout the manuscript. See below some examples:

Zhou, Y.; Li, D.; Li, Z.; Guo, S.; Chen, Z.; Wu, L.; Zhao, Y. Greenhouse Gas Emissions from Soils Amended with Cornstalk Biochar at Different Addition Ratios. Int. J. Environ. Res. Public Health 2023, 20, 927. https://doi.org/10.3390/ijerph20020927

Bashir, M.A.; Wang, X.; Naveed, M.; Mustafa, A.; Ashraf, S.; Samreen, T.; Nadeem, S.M.; Jamil, M. Biochar Mediated-Alleviation of Chromium Stress and Growth Improvement of Different Maize Cultivars in Tannery Polluted Soils. Int. J. Environ. Res. Public Health 2021, 18, 4461. https://doi.org/10.3390/ijerph18094461

Zhu, Y.; Ma, J.; Chen, F.; Yu, R.; Hu, G.; Zhang, S. Remediation of Soil Polluted with Cd in a Postmining Area Using Thiourea-Modified Biochar. Int. J. Environ. Res. Public Health 2020, 17, 7654. https://doi.org/10.3390/ijerph17207654

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The comments are addressed properly and necessary corrections have been done. The manuscript can be accepted.

Author Response

Thank you for the the time dedicated to our work.