Biochemical Methane Potential of Mechanically and Enzymatically Pretreated Solid Olive Mill Waste

Round 1

Reviewer 1 Report

This is a nice and very interesting work on biomethane production from mechanically destoned and enzymatically pretreated olive cake obtained from three-phase centrifugation olive oil production. The manuscript is well written and structured, with proper references, and experimental results are of technical and scientific interest. It is worth to be published in Processes journal. However, there are several points that need to be addressed and explained before acceptance.

1) Page 9, lines 350-356. I agree with the authors that the result obtained is unexpected. Do the authors have any explanation for the non-significant effect of DOC soaking time on the TS or % mass stones removed?

2) Page 11, lines 417-419. The authors indicate that “the addition of the inoculum to the enzymatically pretreated olive cakes (EDOC, DEDOC) resulted in pH > 7 in the serum bottle digesters”. A substrate:inoculum (S:I) ratio of 0.5 by VS was used. Considering the large pH difference between the inoculum (7.9) and the enzymatically pretreated olive cakes (about 4.1, as shown in Table 2), What is the S:I volume ratio used in these experiments?

3) Page 17, lines 590-599. The last paragraph of the Conclusions section could be improved by indicating the proposed treatment and/or use of the olive pits removed in the destoned stage.

Author Response

The authors thank the reviewer for the kind comments and agree the experimental results are of technical and scientific interest.

• Page 9, lines 350-356. I agree with the authors that the result obtained is unexpected. Do the authors have any explanation for the non-significant effect of DOC soaking time on the TS or % mass stones removed?

The fresh olive cake was roughly 48.5% total solids, and each replicate was diluted with 1500 mL of tap water. This would be a 5:1 w/w water/olive cake (db) dilution. With an excess of tap water and at 56 °C, we hypothesize the olive cake was completely rehydrated within an hour. The olive cake was majority fiber: hemicellulose degrades at 220–315 °C (cellulose and lignin at higher temperatures). The additional 23 hours should not lead to a higher degradation of these components which make up the majority of the olive cake.  

We expected the higher rehydration time and continuous agitation would allow more olive pomace to detach from the olive pit fragments. Our results suggest that either the olive pomace was already loosely attached or too tightly attached to the stones. There was not a significant amount of olive pomace that was loose enough that the additional 23 h made a difference to the destoning process.

• Page 11, lines 417-419. The authors indicate that “the addition of the inoculum to the enzymatically pretreated olive cakes (EDOC, DEDOC) resulted in pH > 7 in the serum bottle digesters”. A substrate:inoculum (S:I) ratio of 0.5 by VS was used. Considering the large pH difference between the inoculum (7.9) and the enzymatically pretreated olive cakes (about 4.1, as shown in Table 2), What is the S:I volume ratio used in these experiments?

Due to the high solids content of the diluted olive cake, the S:I volume ratio was roughly 1:9 v/v. Thank you very much for bringing this up. This was previously identified as a potential limitation of using olive cake (even when diluted) as a feed material; however, our discussion on the topic never made it into the paper. The following has been added to the manuscript as line 419 to 420

The near-neutral pH was due to low high S:I volume ratio (1:9 v/v) necessary to achieve the S:I volatile solids ratio of 0.5.

And from line 522 to 527

One severe limitation to this approach [co-digestion] is the high organic load (and therefore volatile solids) of olive mill waste products like olive cake and OMWW. As seen in this study, to achieve a S:I (volatile solids) of 0.5 which is quite regular to BMP studies, a low S:I volume ratio was required which is undesirable from a waste management standpoint. The higher the S:I ratio, the more of the byproduct can be used for anaerobic digestion.

• Page 17, lines 590-599. The last paragraph of the Conclusions section could be improved by indicating the proposed treatment and/or use of the olive pits removed in the destoned stage.

The authors wholeheartedly agree this would make a meaningful addition to the manuscript. The conclusion section has been modified. See addition in red of the seconf section of the conclusion

 

Energy production in the form of biomethane is only one aspect of the olive mill waste biorefinery. The biorefinery concept that has been gaining traction as an approach integrates waste management and valorization [47, 48]. If the polyphenols can be extracted, the wastewater from the three-phase centrifugation system can be used to substitute tap water for olive cake dilution. We are currently exploring this approach and how the removal of the polyphenols impacts the production of biomethane from the olive pomace. Furthermore, the ability to easily separate the digestible pulp from the olive stones with a mechanical screen engenders various avenues of research centering on the physical properties of olive stones. For example, the potential of the olive stone for biochar production is currently evaluated and a techno-economic and life cycle analysis will determine the feasibility of implementing a biorefinery concept to olive pomace. of various analysis of s another avenue that is currently explored. This should give It is suggested that the same destoning experiments be applied to olive mill wastes originating from two-phase centrifugation systems to investigate whether a similar amount of stones can be removed.

Reviewer 2 Report

To find a viable treatment for olive oil industrial waste, this paper compared the destoning efficiency of HSP and centrifuge, and the BMP of olive cake with 4 different pretreatment methods were tested. This topic is interesting, but there are some perplexities in the paper. My comments are as follows:

1. Abstract needs refining.

2. The data of global olive oil production should be up to date.

3. Some of the reference are out of date, more references of recent 3 years should be cited.

4. Figure 1 is not canonical enough, should be redrew.

5. The introduction section should be further condensed focus on the topic of this research.

6. Considering the stone removal rate, why all subsequent destoning was done with the use of HSP? It is confusing,

7. The title of 3.2 doesn’t summarize this part clear.

8. The result of L339-340 requires further explanation with experimental data or references.

9. The content of protein and lipid was described, but their role in anaerobic digestion is not described. In other words, what is their relationship to the topic of this paper.

10. Whether C/N is taken into account in anaerobic digestion?

11. Content of L408-416 is already covered in the introduction, and there is no need to discuss in the results and discussion section.

12. Please note the use of abbreviations.

13. How to determine the amount of glucose in anaerobic digestion? How to ensure the comparability with other substrates?

14. Is there any evidence for L444-447?

15. It is not convincing to explain L452-454 in this way, As can be seen form Figure 2, DEDOC group didn’t have obvious biomethane production advantage before 5 day. How can we get such a conclusion that the anaerobic digestion of DEDOC was inhibited? And inhibited by what? Have pH and VFAs yields been measured in this process? Is it inconsistent with the statement in L476 that no inhibition was received?

16. How does the description of the paragraph L483-492 relate to the topic of this paper?

17. Similarly, the paragraph L506-513 has little to do with the topic of this paper.

18. The content of L590-599 should be discussed in detail in the discussion part, rather than in conclusion section.

Author Response

The authors thank the reviewer for the valuable comments provided.

1. Abstract needs refining.

Without knowing what the reviewer was exactly meaning by “refining” the following changes were made: lines 12 and 13 have been combined and on line 18, “and” has been changed to “or” to better emphasize the two different treatments.

2. The data of global olive oil production should be up to date.

Data for global olive oil production and reference has been updated in line 30. 2020/2021 is the last season where data is currently publicly available.

3. Some of the reference are out of date, more references of recent 3 years should be cited.

One-quarter of the references (12/48) are from within the last three years (Including 2019 because 2023 only started). Over one-third of the references (17/48) are from within the last six years. From the remaining older references, 16 % (8/48) are established methods. The authors believe it is reasonable for half of the references to be from before 2017 if the information is relevant to the experiment at hand. The authors wholly welcome any input on information in the paper that may be outdated. If the reviewer is looking for specific reference(s), we welcome any recommendations.

4. Figure 1 is not canonical enough, should be redrew.

The authors are unsure what canonical means in this context. The figure has been modified to be more aesthetically pleasing.

5. The introduction section should be further condensed focus on the topic of this research..

Lines 53 to 55 which introduced the challenge of sustainability in the 21^st century have been excised. Other than these lines, the authors believe the introduction succinctly summarizes the complex topic at hand. The first paragraph introduces the scale of the problem, the second introduces olive cakes as a waste material, and the third and fourth paragraphs briefly discuss anaerobic digestion in the context of olive cake. The fifth paragraph explores a central novelty of the paper, removing the olive pits before subsequent anaerobic digestion. The last paragraph introduces the planned experiment, research goals, and states a hypothesis. The authors welcome any further clarification on which of these parts could use condensing. 

6. Considering the stone removal rate, why all subsequent destoning was done with the use of HSP? It is confusing,

As seen in Figure 2. stone removal by centrifugation results in a lower Total Solids. This would mean that more digestible pulp would be part of the pellet with the stones, not in the supernatant that will under anaerobic digestion. The following clarification has been added to lines 321 – 322

When the olive cakes were centrifuged, more of the pulp partitioned with the stones becoming part of the pellet. This is reflected in the lower %TS in the supernatant (Fig 2).

7. The title of 3.2 doesn’t summarize this part clear.

The authors agree the title could be better worded and have changed it to: 3.2 Olive Cake Destoning Efficiency of a Horizontal Screw Press (HSP) compared to Enzymatic Pretreatment

8. The result of L339-340 requires further explanation with experimental data or references.

The following has been added to lines 344-348 to explain the result.

Total solids are defined as the sum of the dissolved, suspended and settleable solids i.e. material minus the water. As enzymes catalyze reactions and do not remove material from the system, the total solids between DOC and EDOC should not be significantly different. As aforementioned this was shown to be the case. 

9. The content of protein and lipid was described, but their role in anaerobic digestion is not described. In other words, what is their relationship to the topic of this paper.

Protein and lipid content was included in the compositional analysis since a high level of protein can lead to an inhibitory level of ammonia. A high amount of lipids can lead to the accumulation of fatty acids, depressing the pH of the system. The compositional data demonstrates the possible hurdles of a feedstock. Olive cake is low in protein, has substantial fat, and is high in carbohydrates.

10. Whether C/N is taken into account in anaerobic digestion?

C/N was not taken into account for the anaerobic digestion. Instead, a pilot experiment was performed using various volatile solids loadings to determine the S:I (volatile solids) ratio and g VS/L loading. The authors agree subsequent experiments testing different C/N ratios may be beneficial for optimizing a semicontinuous or continuous system.

11. Content of L408-416 is already covered in the introduction, and there is no need to discuss in the results and discussion section.

The authors agree this repetition may be excessive and have taken out that section.

12. Please note the use of abbreviations.

All abbreviations are in parenthesis after the first use of the full name. Please advise specific abbreviations that not explained.

13. How to determine the amount of glucose in anaerobic digestion? How to ensure comparability with other substrates?

The volatile solids content of glucose powder was determined and loaded according to the S:I (volatile solids) ratio. The exact loading is mentioned in 2.3.3 to be 7.5 g VS/L.

14. Is there any evidence for L444-447?

The evidence can be found in Table 4. The total monomeric sugar of the EDOC, and DEDOC digestate at Day 0 is significantly higher than the control at the same time point. 

15. It is not convincing to explain L452-454 in this way, As can be seen form Figure 2, DEDOC group didn’t have obvious biomethane production advantage before 5 day. How can we get such a conclusion that the anaerobic digestion of DEDOC was inhibited? And inhibited by what? Have pH and VFAs yields been measured in this process? Is it inconsistent with the statement in L476 that no inhibition was received?

This is a good question and one we are still working on understanding. The inhabitation stated in line 476 is about pH and alkalinity. “With a high alkalinity and a pH within the recommended limits of anaerobic digestion, there was no evidence of inhibition due to acidification.” It does not preclude polyphenol inhibition. From our understanding and based on the HPLC performed, DEDOC should have produced an similar amount of methane to the DDOC and the EDOC, it produced less. All things equal, there must have been an inhibitory anti-synergy between the destoned olive matrix and enzymes.

16. How does the description of the paragraph L483-492 relate to the topic of this paper?

The paragraph aims to establish the S:I (volatile solids) ratio as an important parameter to consider when comparing biomethane potential assays. In the authors’ opinion it is erroneous to compare BMPs without reporting the S:I ratios.

17. Similarly, the paragraph L506-513 has little to do with the topic of this paper.

The following has been added to Line 517 to provide context on why this is relevant to the paper.

Although the SMY of three-phase olive cake co-digested with OMWW is lower than the SMY diluted olive cake, co-digestion simultaneously converts olive cake and OMWW, which is another environmentally detrimental byproduct of olive oil production. One future consideration could be extracting the polyphenols from OMWW and then co-digesting the resulting liquor with enzymatically pretreated olive cakes.

18. The content of L590-599 should be discussed in detail in the discussion part, rather than in conclusion section.

Discussion can be found L480-504 and the authors feel like L590-599 is appropriate in the conclusion. Based on the comment from reviewer 1, possible future approach was added to the conclusion. The modification of the conclusion is indicated in red.

Energy production in the form of biomethane is only one aspect of the olive mill waste biorefinery. The biorefinery concept that has been gaining traction as an approach integrates waste management and valorization [47, 48]. If the polyphenols can be extracted, the wastewater from the three-phase centrifugation system can be used to substitute tap water for olive cake dilution. We are currently exploring this approach and how the removal of the polyphenols impacts the production of biomethane from the olive pomace. Furthermore, the ability to easily separate the digestible pulp from the olive stones with a mechanical screen engenders various avenues of research centering on the physical properties of olive stones. For example, the potential of the olive stone for biochar production is currently evaluated and a techno-economic and life cycle analysis will determine the feasibility of implementing a biorefinery concept to olive pomace. of various analysis of s another avenue that is currently explored. This should give It is suggested that the same destoning experiments be applied to olive mill wastes originating from two-phase centrifugation systems to investigate whether a similar amount of stones can be removed.

 

 

 

Round 2

Reviewer 2 Report

After revision, the manuscript is scientifically and technically sound. But there are still a few things could be improved.

1. Once we have defined an abbreviation, we should ues the abbreviation in the rest of the paper instead of its full name.

2. If possible, embelish the Fig. 1. (The start-spot positions of the arrows are not very uniform. )

Author Response

We thank the reviewer for the additional comments. We have modified the article as instructed. 

Author Response File: Author Response.docx