Microwave-Hydrogen Peroxide Assisted Anaerobic Treatment as an Effective Method for Short-Chain Fatty Acids Production from Tannery Sludge

Round 1

Reviewer 1 Report

This manuscript investigated VFA production from tannery sludge, which is meaningful in the context of circular economy to recover more valuable resources from wastes than biogas. The writing is generally good and easy for understanding. However, there are some points that the authors need to clarify before I can recommend it for the publication.

The major points:

1.       The authors highlighted this work is the first for tannery sludge, however, there is no information how tannery sludge is different from municipal sewage sludge in terms of sludge characteristics and why different results might be expected from tannery sludge. As we know, there are many different types of industrial wastewater which might generate different types of sludge. Do we need to try them one by one or we could estimate based on their sludge characteristics? I think that the authors need to supplement detailed information about the characteristics of tannery sludge and compare it with municipal sewage sludge (mixture of primary and secondary sludge or either of them) for the explanation how they are different.

2.       Although the authors mentioned that they tried to avoid evaporation during MW-H2O2 pretreament experiment, it would be better to give more detailed information how the water evaporation could be avoided during MW pretreament and heating periods to 80 and 90C, respectively.

3.       It is interesting to note that all pH come to 7 regardless of initial pH. It might be easier to understand that the production of VFA could reduce pH when initial pH was 9 and 11, but it is not easy to understand how initial pH of 5 could increase to 7 or maintain 7 along the production of VFA. Could you pls discuss this?  

4.       The results showed that thermophilic condition were not beneficial for VFA production, and the authors concluded that the thermophilic temperature did not give any benefits, but the authors did not provide explanation. From the methods, it can be seen that the authors used mesophilic anaerobic digestate as inoculum. Thus, I doubt that the experimental design in this manuscript with mesophilic digestate as inoculum is problematic to compare between mesophilic and thermophilic fermentation unless the authors could prove or justify that mesophilic microbial community does not need time (or only need very short time) to acclimatize to thermophilic condition to enrich thermophilic microbial community. This point is the most serious because it is directly related with the conclusion that the authors drew from the batch study.  

5.       You stressed several times this batch work could provide useful information to continuous fermentation. It would be better to add one more paragraph to discuss how batch fermentation is different continuous fermentation and what information could be obtained from batch fermentation to guide continuous fermentation and what information has to be obtained directly from continuous fermentation.

The minor points:

L107-108, 8% and 12 % TS were chose to do the study and the authors justified in L116-117 that 8% and 12% TS is from gravity sludge thickening. Typically, the TS from the gravity sludge thickening in municipal wastewater treatment plant is ranging from 3% to 6%, and rarely over 8%.  Is 8-12% TS from tannery wastewater treatment plant or a typical value from Italian municipal wastewater plants? If possible, pls provide references for this 8-12% value.

L130. What is the working volume of the glass bottle?

L131: what is the inoculum to the sludge ratio? Is this ratio fixed for 8% and 12% TS sludge or you only fixed 50 ml inoculum to both 8% and 12% TS. Pls provide detailed information in the manuscript.

L164: tannery sludge could act as a buffer and you explained the presence of lime. Did you analyse alkalinity of tannery sludge fermentation liquor? How is that different from municipal sewage sludge?

 

Author Response

The authors would like to thank the Reviewer for the careful review. All the comments, suggestions and questions for revision have been considered. The text below lists the answers to each of them with reference to the revised manuscript.

Reviewer 1:

1. The authors highlighted this work is the first for tannery sludge, however, there is no information how tannery sludge is different from municipal sewage sludge in terms of sludge characteristics and why different results might be expected from tannery sludge. As we know, there are many different types of industrial wastewater which might generate different types of sludge. Do we need to try them one by one or we could estimate based on their sludge characteristics? I think that the authors need to supplement detailed information about the characteristics of tannery sludge and compare it with municipal sewage sludge (mixture of primary and secondary sludge or either of them) for the explanation how they are different.

Reply. Thank you for the comment. We have made some adjustments in the manuscript regarding the description of utilized tannery sludge, as suggested. Overall, the characteristics of a sludge depends on two aspects: a) the origin of the waste, which in turn affect the physico-chemical features of the waste/sludge (domestic waste are extensively different from many type of industrial waste); b) the type of waste treatment of processes in the facilities. For the case of this study, the industrial-tannery sludge undergoes both primary treatments (both chemical and physical) and biological treatment because of the presence of very high TS content, much higher than TS level of domestic sludge which usually requires only physical primary treatment followed by biological treatments.

Moreover, the sludge that has been used for this work came from a WWTP that collects wastewater from 23 tanneries, whose final products had different applications (e.g., automotive industry, furniture, footwear). Therefore, even the leather processing was not the same for all 23 tanneries, especially in the last step of the processing where the leather gets the characteristics of final product. As a results, the characteristics (such as pH, TS, metals and organic compounds) of a wastewater and primary sludge are also different, depending on the processing used by the tannery. For all these reasons, the tannery sludge could be considered substantially different from municipal sludge coming from the domestic WWTP. In addition, it could be also useful for the development of this new research line to test the tannery sludge coming from the different tannery industries available in this territorial context, since they may be different in terms of nutrients and metals level, as well as biodegradable matter and compounds.

A concise clarification has been provided in paragraph 2.1.

 

2. Although the authors mentioned that they tried to avoid evaporation during MW-H2O2 pretreament experiment, it would be better to give more detailed information how the water evaporation could be avoided during MW pretreament and heating periods to 80 and 90C, respectively.

Reply. We simply avoided water evaporation during the MW heating by not allowing our sludge to reach the boiling temperature and by interrupting the heating frequently, as suggested in Ambrose et al., 2020 [23]. We also performed a visual check of the level of the sludge in the bottles during the heating to make sure it didn’t evaporate. A sentence has been added in the paragraph 2.2 to clarify this aspect.

 

3. It is interesting to note that all pH come to 7 regardless of initial pH. It might be easier to understand that the production of VFA could reduce pH when initial pH was 9 and 11, but it is not easy to understand how initial pH of 5 could increase to 7 or maintain 7 along the production of VFA. Could you pls discuss this?  

Reply. Thank you for your point, we have modified the manuscript to make this concept clearer (paragraph 3.1). The dry tannery sludge we have used contains residues of Calcium Hydroxide (Ca(OH)₂), as this chemical was used during the primary treatment of the tanning process. Our thesis is that during the fermentation the dry sludge releases OH^- ions from the Ca(OH)₂ that increase the pH up to a value of around 7, balancing the acidification caused by the SCFAs.

 

4. The results showed that thermophilic condition were not beneficial for VFA production, and the authors concluded that the thermophilic temperature did not give any benefits, but the authors did not provide explanation. From the methods, it can be seen that the authors used mesophilic anaerobic digestate as inoculum. Thus, I doubt that the experimental design in this manuscript with mesophilic digestate as inoculum is problematic to compare between mesophilic and thermophilic fermentation unless the authors could prove or justify that mesophilic microbial community does not need time (or only need very short time) to acclimatize to thermophilic condition to enrich thermophilic microbial community. This point is the most serious because it is directly related with the conclusion that the authors drew from the batch study.  

Reply. We realize that this aspect was not clearly stated in the manuscript, and it has therefore been added. The inoculum for the mesophilic tests was used as-is from the full-scale anaerobic digestion reactor (located in Treviso WWTP). For what regards the thermophilic tests, the same digestate is kept in a thermophilic pilot reactor fed with thickened sludge from Treviso WWTP, meant for the production of biogas, which can also provide digestate acclimated at 55°C when needed for the experiments. This method was described in previous papers from our research group such as Valentino et al., 2021 [14].

 

5. You stressed several times this batch work could provide useful information to continuous fermentation. It would be better to add one more paragraph to discuss how batch fermentation is different continuous fermentation and what information could be obtained from batch fermentation to guide continuous fermentation and what information has to be obtained directly from continuous fermentation.

Reply. Since tannery sludge is a substrate that has never been tested before, some initial screening was necessary. For this purpose, the batch tests represent the best approach to investigate many different conditions at the same time, in order to assess the best parameters to maximize the SCFAs production (namely in this case temperature, initial pH, TS and the MW+H₂O₂ pretreatment). However, once these preliminary analyses have been performed, and the best working parameters have been confirmed, it is preferable to shift to a continuous process, as in terms of a process scale-up, batch tests are more costly compared to the continuous fermentation. Moreover, through a continuous process further information can be obtained, such as the optimal HRT and OLR, which are fundamental for a technical and economic feasibility assessment of the process in large scale.

 

The minor points:

1. L107-108, 8% and 12 % TS were chose to do the study and the authors justified in L116-117 that 8% and 12% TS is from gravity sludge thickening. Typically, the TS from the gravity sludge thickening in municipal wastewater treatment plant is ranging from 3% to 6%, and rarely over 8%.  Is 8-12% TS from tannery wastewater treatment plant or a typical value from Italian municipal wastewater plants? If possible, pls provide references for this 8-12% value.

Reply. We have accordingly corrected the manuscript since it was actually referred to a dynamic thickening, as the static one reaches lower values (as you said, not higher than 6%). The values of 8-12% represent a wide range of variability, within the typical values obtained with dynamic thickening of this tannery sludge, based on data from the tannery-waste WWTP we obtained the sludge from. Moreover, the TS content directly affects the OLR that can be used in a continuous process; a higher TS content would allow higher energy efficiency and less water usage, which can improve the economic feasibility of the process overall.  

 

130. What is the working volume of the glass bottle?

Reply. 200 mL working volume. This information has been added in the manuscript.

1. L131: what is the inoculum to the sludge ratio? Is this ratio fixed for 8% and 12% TS sludge or you only fixed 50 ml inoculum to both 8% and 12% TS. Pls provide detailed information in the manuscript.

Reply. We added 50 mL of inoculum to both 8% and 12%, obtaining S/I values of 8 for the 8% tests, and 12 for the 12% ones. The manuscript has been accordingly modified.  

1. L164: tannery sludge could act as a buffer and you explained the presence of lime. Did you analyse alkalinity of tannery sludge fermentation liquor? How is that different from municipal sewage sludge?

Reply. The presence of lime ensured a good control of the pH as can be also confirmed by previous literature investigations, where CaO was dosed in the fermentation tests of different kind of substrates (a- Campanari et al., 2017, Enhancing a multi-stage process for olive oil mill wastewater valorization towards polyhydroxyalkanoates and biogas production, Chemical Engineering Journal 317, 280-289; b- Valentino et al., 2015, Fate of b-hexachlorocyclohexane in the mixed microbial cultures (MMCs) three-stage polyhydroxyalkanoates (PHA) production process from cheese whey, Bioresource Technology 192, 304-311). Based on this reviewer’s request, the fermentation liquids coming from the different series M8, M12, M8-P, T8, T12 and T8-P have been analyzed for alkalinity measurements. The results showed that the alkalinity was higher in thermophilic tests; moreover, at constant temperature, the alkalinity was slightly higher in the tests conducted with pretreated sludge. In mesophilic series, the alkalinity was equal to 2.51, 2.48 and 2.62 gCaCO₃/L in M8, M12 and M8-P respectively (average data related to the different tests conducted at different initial pH). In thermophilic series, the alkalinity was equal to 2.71, 2.75 and 2.89 gCaCO₃/L in T8, T12 and T8-P respectively (average data related to the different tests conducted at different initial pH). The alkalinity values in each single test have been added in the table of supplementary material. These data revealed a good buffering capacity in a fermented stream. As an example, in Valentino et al., 2021 ([14]), a fermented stream was obtained from food waste and subjected to a digestate recirculation (usually rich in alkalinity); this buffered fermented streams had lower alkalinity if compared to the results obtained in this study. A short discussion has been also added in paragraph 3.1.

At the moment, we don’t have a fermented sewage sludge stream to do such comparison, but these data can confirm the good buffering of the tannery sludge fermentation liquids.

 

Reviewer 2 Report

1.       The author introduced the tannery sludge treatment influence factors such as pH, temperature, initial total solids (TS) content, and MW-H2O2 in the abstract, however, only the Hydrogen peroxide and Microwave were introduced in the introduction, it was inadequate, please appropriate supplements.

2.       The Microwave (MW) irradiation was introduced in the line 78-80, it was confused that the relationship of MW between H 2 O 2 and MW, please modifying the part.

3.       Methods were unclear and puzzled. Such as range of initial pH was adjusted by NaOH or H2SO4, but in the 2.3 part line140, the pH was adjusted, please clear the method.

4.       Author showed that the SCFAs production was maximal with the M W -H 2 O 2 pretreatment in the 40℃ or 50℃. It was well known that high temperature could enhance the hydrolysis and acidification of sludge. However, the mechanism of M W -H 2 O 2 on enhancing SCFAs was not mentioned, it was lacking.

5.       The SCFAs production was discussed in the different conditions, it was Lack of lateral contrast with others.

6.       TKN, N-NH₄⁺,total phosphorus, PO₄^3--P , VS, TS, [Cr(VI)], and COD were analyzed in this test, but only SCFAs, SCFAs composition and COD were reflected in the paper, other indicators were in Supplementary File, but the discussion of those indicators were to too little.

7.       The main objective of this paper discussed an effective method for short-chain fatty acids production from tannery sludge. Microwave-hydrogen peroxide was proved in this test. Many tests and results were displayed in the paper, but it was too confusion to weakening theme. I think the Structure of the manuscript should be appropriate adjusted.

8.       Appropriate reaction mechanism should be discussed.

9.       Appropriate microbial and enzymatic analysis could increase persuasiveness of manuscripts.

Comments for author File: Comments.pdf

Author Response

The authors would like to thank the Reviewer for the careful reviews. All the comments, suggestions and questions for revision have been considered. The text below lists the answers to each of them with reference to the revised manuscript.

REVIEWER  2

1. The author introduced the tannery sludge treatment influence factors such as pH, temperature, initial total solids (TS) content, and MW-H2O2 in the abstract, however, only the Hydrogen peroxide and Microwave were introduced in the introduction, it was inadequate, please appropriate supplements.

Reply. We have revised and improved the manuscript according to your suggestion. Overall, we decided to give more emphasis to the pretreatment as it offers many advantages for our tannery sludge in vision of following development of this research line. The benefits of such pretreatment have been related to the improved SCFAs production; however, a significant reduction of the solids to be disposed of at the end of the process can be also obtained and will be evaluated in future experiments. Tannery waste is very costly to dispose of and a reduction in the volume and weight of the waste can represent a very efficient improvement for the industry, as it would greatly reduce the disposal costs and reduce the landfills’ use. 

 

2. The Microwave (MW) irradiation was introduced in the line 78-80, it was confused that the relationship of MW between H 2 O 2 and MW, please modifying the part.

Reply. The combination of oxidation and thermal treatment has been chosen according to previous literature where this pretreatment has been successfully used on other matrices (Ambrose et al., 2020, Liu et al., 2017 – 2018, Özön et al. 2019 [23-25]). Both pretreatments have the effect of disrupting the sludge floc structure and causing microbial cell rupture, hence solubilizing particulate organics, resulting in a more efficient degradation process. Since our matrix is characterized by complex molecules, we decided to try the combined methods to increase as much as possible their effect. We have modified the manuscript (in the introduction) to make this aspect clearer. 

 

3. Methods were unclear and puzzled. Such as range of initial pH was adjusted by NaOH or H2SO4, but in the 2.3 part line140, the pH was adjusted, please clear the method.

Reply. We have revised the materials and methods according to this comment.

 

4. Author showed that the SCFAs production was maximal with the M W -H 2 O 2 pretreatment in the 40°C or 50°C. It was well known that high temperature could enhance the hydrolysis and acidification of sludge. However, the mechanism of M W -H 2 O 2 on enhancing SCFAs was not mentioned, it was lacking.

Reply. Hydrogen peroxide (H2O2) is an oxidizing agent (E0 = 1.78 V) and therefore it has the ability to oxidize some organic compounds directly. For instance, H2O2 attacks the double bond of an alkene, producing a hydroperoxide. Then, alcohols and ketones are produced from hydroperoxide. Hydrogen peroxide can also produce OH free radicals by the breakage of the oxygen-oxygen single bond. The free radical can attack a molecule of organic matter and, in so doing, produce another free radical. During the H2O2 treatment, the substrate was treated with MW to support the mechanism cited above.  In conclusion, pretreatment with hydrogen peroxide and MW was aimed to reduce the molecular complexity (for example by breaking the double bonds) of organic compounds and thus support the subsequent biological process. The paragraph 3.2 has been accordingly modified.

 

5. The SCFAs production was discussed in the different conditions, it was Lack of lateral contrast with others.

Reply. In the manuscript we have described the production of SCFAs in different conditions, namely pH, temperature, TS and pretreatment. In general, it has been found that the pretreated tests were the ones that gave higher acidification yields (of 0.28-0.30 g COD_SCFA/g VS₀ and 0.28-0.31 g COD_SCFA/g VS₀ respectively in M8-P and T8-P) and they were compared with non-pretreated tests that did not reach such high values. These values have been also compared with another substrate (municipal sludge) to contextualize our results. We have modified the manuscript to make these comparisons clearer.

 

6. TKN, N-NH4 ,total phosphorus, PO4^3- -P , VS, TS, [Cr(VI)], and COD were analyzed in this test, but only SCFAs, SCFAs composition and COD were reflected in the paper, other indicators were in Supplementary File, but the discussion of those indicators were to too little.

Reply. In response to this comment, we have added a new paragraph regarding nutrients’ release and their possible recovery. According to the obtained data and the absence of significative concentration of Cr(VI), we have preferred to briefly discuss this parameter in the paragraph 3.7, and adding Cr(VI) results in the summary tables of supplementary file. For what regards TS and VS, their effect has been discussed in connection to the SCFAs production and yield (paragraph 3.4). Further development of this research will be also focused on the quantification of TS and VS reduction in continuous experiments (probably CSTR reactors) and such knowledge will be discussed in terms of economics and sludge disposal costs, as replied to first question (above).  

 

7. The main objective of this paper discussed an effective method for short-chain fatty acids production from tannery sludge. Microwave-hydrogen peroxide was proved in this test. Many tests and results were displayed in the paper, but it was too confusion to weakening theme. I think the Structure of the manuscript should be appropriate adjusted.

Reply. As tannery sludge is a not-yet-investigated substrate for anaerobic fermentation, we found necessary to perform several tests with a wide variety of conditions, in order to have a clear idea of its potential. In particular we organized the manuscript to give initial emphasis to the pH with a dedicated paragraph, then a comparison of the different tests through the parameters related to AD (SCFAs production, yields and SCFAs/CODsol) and a concluding one on Cr(VI) and our sludge’s valorization. We have then added a paragraph regarding nutrients in liquid phase (according to comment 6) and we have tried to change some parts to make it more straightforward.

 

8. Appropriate reaction mechanism should be discussed.

Reply. In the manuscript we decided to put more emphasis on the H₂O₂ reaction mechanisms, which has now been implemented, also according to comment 4. For what regards the other reaction mechanisms, such as those regarding AD in general, as there is a wide array of literature focusing solely on the explanation of these reactions, we preferred to not go into detail. Our research group is still focused on the investigation of tannery sludge and ways to improve and increase its application potential, and we will investigate more in depth the anaerobic fermentation of this substrate, deepening the analysis of its behavior and the microbial communities responsible for its degradation.

 

9. Appropriate microbial and enzymatic analysis could increase persuasiveness of manuscripts.

Reply. We are definitely in agreement; even though in this phase the batch tests did not include microbial and enzymatic analyses, as we concentrated on the preliminary tests of this new substrate, we will definitely focus on this aspect in the future. Also, the length of each test as well as the use of inoculum could affect the growth of a fermentative consortium representative for our process. We believe that continuous tests of longer duration are more appropriate to evaluate the effect of important parameters like OLR, HRT (and eventually SRT) on the different microbial community enrichment (generally, OLR and HRT are not consistent with batch tests). In the close future, such aspect has to be certainly taken into account, as it represents a very important step to fully investigate and understand the use of this tannery sludge in a context of circular economy. This is another aspect we are going to assess in addition to those stated in our answer to question 5 from reviewer 1.  

 

 

Round 2

Reviewer 1 Report

suggest to accept

Reviewer 2 Report

Author have  modified the manuscript according comments, the necessary discussion have been supplemented. Therefore, it has reached publication level.