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    Review of 'Plastic agriculture using worms: Augmenting polystyrene consumption and using frass for plant growth towards a zero-waste circular economy.'

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    Plastic agriculture using worms: Augmenting polystyrene consumption and using frass for plant growth towards a zero-waste circular economy.

    Polystyrene (PS) is one of the major plastics contributing to environmental pollution with its durability and resistance to biodegradation. Recent research has found mealworms ( Tenebrio molitor ) and superworms ( Zophobas morio ) to be able to utilize PS as a carbon food source and degrade them without toxic effects. In this study, the effects of food additives on plastic consumption augmentation were studied, with small additions of sucrose and bran found to increase PS consumption. To close the plastic carbon cycle, we also evaluated the use of worm frass for dragon fruit cacti ( Hylocereus undatus ) growth and found that superworm frass supported rooting and growth better than mealworm frass and control media over a fortnight. Superworms, apart from being known fish and poultry feed, have been shown to be a suitable natural solution to the PS plastic problem that can support plant growth towards a zero-waste sustainable bioremediation cycle.
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      Review information

      10.14293/S2199-1006.1.SOR-EARTH.ABOH45.v1.RPPACK

      This work has been published open access under Creative Commons Attribution License CC BY 4.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Conditions, terms of use and publishing policy can be found at www.scienceopen.com.

      Keywords:

      Review text

      Review

      “Plastic agriculture using worms: Augmenting polystyrene consumption and using frass for plant growth towards a zero waste circular economy”

      Darius Wen-Shuo Koh et al

       

      Recommendation

      Major revision required within 60 days or paper will be rejected.

      Any revised paper will be subject to further review as it will be a substantially different paper.

      Reasons for recommendation

      Overview provided here. Detailed points provided in the attached copy of a pdf of the paper.

      Overview:

      I think the paper seeks to determine whether:

      1. “worms” (actually insect larvae, as the authors acknowledge) can consume and breakdown a recalcitrant waste product – polystyrene.
      2. Frass, from larvae fed on this material could, as part of a more circular economy, be used in plant production.

      The paper is within scope of the journal in that in covers a number of disciplinary areas – waste management, sustainability (circular economy), biology (animal and plant), chemical analysis in a broadly ecotoxicological context.

      The paper addresses an important topic and may represent an early study designed to help develop  more detailed studies in future (if so, this must be stated) but to be published it needs substantial revision in the areas set out below:

      Throughout the paper attention needs to be paid to this general point: Such complex studies need a very clear set of aims and objectives and well-considered experimental designs and protocols if they are to succeed. The paper does not set out these aims, objectives, designs and protocols at all clearly and does not explain why, when such a rich set of information is potentially available from the studies, such a limited set of data is presented, very little of which has been subject to an appropriate statistical analysis. More detail is provided on the comments in the text of the paper which has been returned to the authors direct.

      All the points mentioned here and on the text of the paper need to be addressed in the revision.

      Methodology

      The paper must include a rationale for and a description of the experimental design for both the studies on “worms” and those on cacti. These descriptions must include an outline of experimental conditions (for example the temperature at which studies were conducted) sufficient to enable the study to be reproduced by other workers, a description of the nature of any control and treatment samples, together with some information on the consistency with which treatments were applied (e.g. what media were used to rear the worms used in the study; and what time was allowed for this media to pass through the worms before the polystyrene study began so as to minimise its influence on the results; the weights of materials included in each group of worms; how many worms were used in each test sample; by how much did any of these factors vary; how any changes were measured – for example did this involve batches of worms or measures on individuals).

      Although innovative use of materials for studies is welcomed, it seems odd to state that wine glasses were used to grow cacti without stating why this choice was made. Linked to this was a passing mention of an office environment. Were these studies not conducted in a laboratory equipped for plant growth studies? If not, then there may be questions about the suitability of the data derived from the cacti experiments. More information is required about how the cacti were raised as this could influence how they reacted in the experiment. For example, were they produced for the experiment in/on other growth media or were they grown on the experimental media only? Likewise, the sources of some of the materials would appear to be retail outlets. If so, information on how the authors ensured consistency between such commercial batches is required (such information is also relevant of course to materials purchased form more standard laboratory suppliers). This is important as there are many different kinds of bran for example.

      Data presentation

      Data presented in the Tables refers mostly to changes in variables in larvae or plants and, puzzlingly, there is little data about the way in which the larvae breakdown the polystyrene material. This is a major weakness. It is unclear how much polystyrene material was actually consumed and whether this led to simple physical fragmentation of the material or its real chemical breakdown to simpler chemical components. Simple fragmentation of the waste might not represent a real advance in the management of polystyrene waste. This is because merely breaking the waste into smaller physical components could just move issues from one part of the environment to another and create new risks and issues without really reducing previous ones.

      Absolute and not just change data is required in the Tables if the reader is to be able to judge the value of the work (for example, data is needed on how much “worms” grew, how much cacti grew – just giving percentage change data is inadequate). It is important to establish how the larvae responded to consuming polystyrene in this study and how this was affected by the additions of other material (again how much other material, such as bran on sugar, was used needs to be stated clearly).

      Data analysis has used a mixed selection of statistical tests and the choice of tests has not been justified. Some of the tests are demonstrably inappropriate. For example, a one sided T-test is employed when it is clear that the data show a two-sided test would have been appropriate. The use of p values is at least on one occasion inappropriate. Very surprisingly, there is little or no indication of the number of samples used in each of the experimental groups. Without this information the validity of the statistics cannot be judged and thus the validity of the studies.

      The data on cacti growth seems to be presented in a partial fashion. Photographs are presented that are difficult to interpret without supporting quantitative data or at least some more descriptive information that a reader could interpret with respect to the qualitative information (e.g. on rooting).

      The GCMS studies on the chemical composition of some elements of the various pieces of experimental work leaves a good deal of detail out and the choice of diagrams is set out without justification. No attempt seems to have been made to determine which of the chemicals listed in one of the supplementary tables is actually present in the samples analysed and no listing or other information is provided as to the differences between experimental control and treatment groups. This makes it very difficult for a reader to understand how the consumption of polystyrene by the worms might impact a circular economy waste cycle. If such an approach to managing polystyrene waste is to be effective, then the ability of the worms to break down the polystyrene chemically needs to be clearly demonstrated. It is not sufficient in such studies to simply present sample traces without making it clear how representative these are of the overall study results. If the worms are simple breaking down the polystyrene physically then it is important to know this. The GCMS studies could have provided vital information on this very important element of the study.

      Discussion/interpretation of results

      This is very limited and it is not clear what the authors believe their study has demonstrated and this arises because this complex study appears to have been undertaken with a number of aims in mind not all of which have been accounted for in the study design. A separate section of the paper is required that interprets the results of these studies. Several of the result appear to be “negative” in nature and whilst the presentation of such results is welcome it is important that their meaning for the circular economy potential of worms and polystyrene waste processing is explained.

      Detailed points per line

      Line 138 - How were additives removed from PS before weighing?

      Line 139 - Mortality rate required. There will be missing data if this information is not included.

      Line 142 - Were these therefore just the control samples that were used? If so why were the samples from treatments that had additives not investigated?

      Line 143 - Why is this reference made to an office environment. Were these studies not conducted in a laboratory with a controlled environment?

      Line 144 - Can the authors explain why wineglasses were used to grow cacti?

      Line 149 - The last sentence seems to contradict the first sentence. Please clarify

      Line 152 - What is a technical replicate?

      Line 160 - What does this mean? The extent to which samples were diluted at this stage could have a substantial effect on the results.

      Line 163 - Please say why this was done.

      Line 166 - Please say why this temperature pattern was used.

      Line 170 - If other additives were used please say which ones. It is otherwise not clear because of the phrase "such as".

      Line 176 - How much is “small amounts"? This is important to know in the interests of reproducibility.

      Line 180 - What does "outperform" mean? Does this refer to PS consumption or to some other characteristics of the worms?

      Line 181 - Is the paper then testing the efficacy of additives or the fact that these insect larvae eat PS? This is not clear in the paper from this section.

      Line 182 - when were worms weighed? Please indicate when this happened in the experimental protocol.

      Line 184 - The experimental design used in the study design is very unclear. Some basic details are needed. Some of what is required is suggested in the overview of this review.

      Line 191 – Line 192 - What type of bran was used (for example, the plant the bran was derived from needs to be stated)? Was it the same batch throughout the studies? Did the bran used for the cacti differ from the bran used for the larvae?

      Line 193 - Again, because the experimental design Lacks sufficient detail it is difficult to interpret these results. Much greater clarity is required on, say, the total numbers of cacti used in the experiment so that the reader can understand how clear (or not) the result is.

      Fig 1: shows that the authors had access to very rich sources of information, such as the growth rates of larvae (length and weight data), rates of consumption of polystyrene, generation of frass, various aspects of plant growth etc. It is a pity that the opportunity that such a rich source of data provided has not been exploited fully. Also an opportunity seems to have been missed to properly determine whether the larvae have actually simple broken the polystyrene down physically (basically they have simply ground the material into smaller units) or whether the larvae have actually decomposed the polystyrene molecules into simpler chemical components. The GCMS data, as presented, fails to do this.

      Fig 2: shows the results from this study are very variable. Many reviewers would say the statistical analysis is wrong. With this kind of experimental design the best statistical analysis is some form of analysis of variance especially as the underlying variability in these results is very high. A p value of 0.1 is of little or no interest if it arises from a one-sided T-test especially when the choice of a one-sided test is inappropriate as the results themselves demonstrate. One-sided statistical tests should be used with great caution and cannot really ever be used in circumstances where responses could be higher or lower than controls. The use of a one-sided test needs a great deal of justification. If a two sided test were use these result might not be significant in the normal sense of that word.

      Fig 4: What is a “technical” replicate? What does technical mean in this sense? Please explain this term. It is clear some rooting occurred in B (right photo) was this detail recorded or not?

      Fig 5: Because the experimental design of the study is unclear the source of the samples subject to GCMS analysis is unclear. More information on the samples used for GCMS analysis is needed.

      Table S1: This table should not be in supplementary information. The data here suggest that only in one instance did worms gain weight. Weight loss occurred in all other groups suggesting the worms were not thriving. Is this not a problem for their use in a circular economy?

      Table S2: The data in this table cannot be judged by a reviewer unless absolute heights are provided. Just supplying change data is insufficient. Other tables have the same problem. The table seems to suggest that the only significant result is from the group where most cacti died. This factor needs accounting for in some additional manner. Also, the Table is difficult to interpret as it is not possible to use a growth media that is a control and a treatment in the same analysis. Were any cacti grown on a standard growth medium? This could have been a real control.

      Table S3: This table should not be in supplementary information and again is in any case inadequate as the authors do not indicate in which samples these chemicals were found.

       

       

       

       

      Comments

      21 Dec 2020

       

      Dear Editor,

       

      We thank you and the reviewer for the helpful comments to our paper entitled “Plastic agriculture using worms: Augmenting polystyrene consumption and using frass for plant growth towards a zero-waste circular economy”.

                 We have done our best to incorporate the many helpful suggestions, and have given our responses in bold as below.

       

      Thank you.

                                                               

      Detailed points per line

      Line 138 - How were additives removed from PS before weighing?

      Response: We used a sieve to isolate the PS from the frass as much as possible, but we do not have the methods to ensure completely removal. As the difference between the initial weight (including PS and additives) are known, as well as the final weight were used, we did take the additive weight into account and there is thus minimal impact on the analysis of the consumption rate.

       

      Line 139 - Mortality rate required. There will be missing data if this information is not included.

      Response: As the consumption experiments was within 4 days, we did not observe any death of worms. It is also probably too short for drastic effects if any. It was rather the cacti that had death. (Some pictures where no dead worm were observed after 4 days of feeding on PS are provided in link: https://bit.ly/3nDvYlk )

      Line 142 - Were these therefore just the control samples that were used? If so why were the samples from treatments that had additives not investigated?

      Response: Only frass from the control samples were used. We did not investigate the frass that were treated with the additives since the main concern was plastic fed worm frass on plant growth, rather than the  were  of cinnamon, sucrose and bran on plant growth. The additives were for the earlier separate segment of augmenting PS consumption, upon which, would guide future augmentation with the optimized additive if necessary in the future.

      Line 143 - Why is this reference made to an office environment. Were these studies not conducted in a laboratory with a controlled environment?

      Response: Studies were conducted in a closed office environment that is also environmentally controlled. There were control plants with the same environmental controls set up as internal controls. Part of the project was performed over the lockdown in Singapore. The worms were bought as pet food and are common home pet feed, we did not introduce them into the laboratory.

       

      Line 144 - Can the authors explain why wineglasses were used to grow cacti?

      Response: The single use plastic wine glasses were recycled and they were used for 1) transparency to observe and ensure that the plants were inserted to the frass with the funnel shape to hold up the cacti graft, and for monitoring; 2) demonstration of conceptual circular economy and recycling.

       

      Line 149 - The last sentence seems to contradict the first sentence. Please clarify

      Response: Sentence has been rewritten for clarity (lines 148 to 150).

       

      Line 152 - What is a technical replicate?

      Response: We have changed this to just replicates (line 153).

       

      Line 160 - What does this mean? The extent to which samples were diluted at this stage could have a substantial effect on the results.

      Response: Sentence has been rewritten for clarity (polystyrene or bran were dissolved in a consistent manner by gram of frass to fixed volume of gas chromatography grade dichloromethane solvent for standardized comparisons). 

       

      Line 163 - Please say why this was done.

      Response: To ensure a fair comparison between different samples in comparing peaks across the samples so that we have a standardised comparison for quantification of compounds by per gram of frass across the different conditions.

       

      Line 166 - Please say why this temperature pattern was used.

      Response: It was previously reported that 25°C is the ideal temperature for worms to consume PS and we have added the reference. It is also the air-conditional temperature in our office space where the experiment were conducted. This also allows us to demonstrate that we do not need special conditions for worm consumption and frass plant growth such as green house, etc.

       

      Line 170 - If other additives were used please say which ones. It is otherwise not clear because of the phrase "such as".

      Response: Sentence has been rewritten for clarity (lines 174). No other additives were used.

       

      Line 176 - How much is “small amounts"? This is important to know in the interests of reproducibility.

      Response: Sentence has been rewritten for clarity (lines 177).

       

      Line 180 - What does "outperform" mean? Does this refer to PS consumption or to some other characteristics of the worms?

      Response: Sentence has been rewritten for clarity (lines 181). It refers to the context of this part of the experiment, which is PS consumption.

       

      Line 181 - Is the paper then testing the efficacy of additives or the fact that these insect larvae eat PS? This is not clear in the paper from this section.

      Response:  We have a few areas to investigate and demonstrate to show the circular economy. The first is the the role of food additives in plastic degradation by the two types of worms, followed by the evaluation of mealworm vs superworm frass to grow dragon fruit (Hylocereus undatus) plant (written in introduction, lines 112 to 115). We have added areas of clarifications.

       

      Line 182 - when were worms weighed? Please indicate when this happened in the experimental protocol.

      Response: The experiment was over 4 days in the materials and method section. The worms were weighed on the day the experiment started where food additives and PS were then added to their confined living environment. They were subsequently weighed after four days separately.

       

      Line 184 - The experimental design used in the study design is very unclear. Some basic details are needed. Some of what is required is suggested in the overview of this review.

      Response: Due to the intended demonstration of the circular economy, we have various parts. The first part is to evaluate the augmentation of PS consumption by both mealworms and superworms by the addition of additives.  Following which, is the evaluation of  cacti growth and rooting,on superworm and mealworm frass fed on PS. We have added these details to the section.

       

      Line 191 – Line 192 - What type of bran was used (for example, the plant the bran was derived from needs to be stated)? Was it the same batch throughout the studies? Did the bran used for the cacti differ from the bran used for the larvae?

      Response: Methods section has been re-written to include the brand of bran used (line 149).

       

      Line 193 - Again, because the experimental design Lacks sufficient detail it is difficult to interpret these results. Much greater clarity is required on, say, the total numbers of cacti used in the experiment so that the reader can understand how clear (or not) the result is.

      Response: 48 budding offshoots from cacti grown as described in the materials and methods section were used. We have added the details as suggested (Line 147).

       

      Fig 1: shows that the authors had access to very rich sources of information, such as the growth rates of larvae (length and weight data), rates of consumption of polystyrene, generation of frass, various aspects of plant growth etc. It is a pity that the opportunity that such a rich source of data provided has not been exploited fully. Also an opportunity seems to have been missed to properly determine whether the larvae have actually simple broken the polystyrene down physically (basically they have simply ground the material into smaller units) or whether the larvae have actually decomposed the polystyrene molecules into simpler chemical components. The GCMS data, as presented, fails to do this.

      Response: The ability of the worms to mineralize the plastic though gut microbes have been extensively studied (See: Yang et al., 2018a, 2018b, 2020, 2015a, 2015b), thus we skipped the details of how the PS were consumed, and in fact focussed on the demonstration of the circular economy aspects of speeding up the consumption of PS as well as the use of worm frass for plant growth. For practical usage for implementation, we adopted the use of weight rather than measuring individual worms parameters as in large scale implementation of the plastic waste, the use of weight to overcome individual worm variations were more feasible.

       

      Fig 2: shows the results from this study are very variable. Many reviewers would say the statistical analysis is wrong. With this kind of experimental design the best statistical analysis is some form of analysis of variance especially as the underlying variability in these results is very high. A p value of 0.1 is of little or no interest if it arises from a one-sided T-test especially when the choice of a one-sided test is inappropriate as the results themselves demonstrate. One-sided statistical tests should be used with great caution and cannot really ever be used in circumstances where responses could be higher or lower than controls. The use of a one-sided test needs a great deal of justification. If a two sided test were use these result might not be significant in the normal sense of that word.

      Response: We used one-tailed because bran was previously demonstrated to improve PS consumption (See: Yang et al., 2018a, 2018b, 2020, 2015a) , but we also acknowledge that there is the need to test the possible lower consumption and have switched to two-tailed, while discussing also as one-tail where suitable. We did consider ANOVA, but as the key was improvement from no additive condition rather than comparing with other additives, we opted for T-test.

       

      Fig 4: What is a “technical” replicate? What does technical mean in this sense? Please explain this term. It is clear some rooting occurred in B (right photo) was this detail recorded or not?

      Response: We have reworded the “technical replicate” to just replicates to avoid confusion.  While there is rooting in B, these were not below the soil line and existed prior to the grafting as it is common for the cacti to grow aerial roots. We only classified rooting to be induced if it occurred at below the soil line as those are the roots that demonstrate growing in the soil media and would allow the plant to be planted. The natural aerial roots that occur are pre-existing roots (evident from its length), and such aerial roots do not allow assessment of suitability of the frass as media.

       

      Fig 5: Because the experimental design of the study is unclear the source of the samples subject to GCMS analysis is unclear. More information on the samples used for GCMS analysis is needed.

       

      Response: We have included Table 3 which shows the list of predicted chemicals corresponding to the respective peaks.

       

      Table S1: This table should not be in supplementary information. The data here suggest that only in one instance did worms gain weight. Weight loss occurred in all other groups suggesting the worms were not thriving. Is this not a problem for their use in a circular economy?

      Response: Yang (Yang et al., 2018a) showed that the worms can grow solely on it. We also have worms fed solely in PS that have pupated into beetles and had offspring, all fed on PS alone.

      In real-life circular economy practice, this may be remediated by co-feeding the worms with other organic materials (See: Yang et al., 2018a, 2018b, 2020, 2015a).

       

      Table S2: The data in this table cannot be judged by a reviewer unless absolute heights are provided. Just supplying change data is insufficient. Other tables have the same problem. The table seems to suggest that the only significant result is from the group where most cacti died. This factor needs accounting for in some additional manner. Also, the Table is difficult to interpret as it is not possible to use a growth media that is a control and a treatment in the same analysis. Were any cacti grown on a standard growth medium? This could have been a real control.

      Response: We updated Table S2 containing the absolute heights of the plants.  We did not use soil as the plants in the office were already grown solely on used tea leaves for 3 years, so to ensure consistency, we continued using use tea leaves. The use of used tea leaves is also a common known method for planting, and this also allowed to limit the media support of other chemicals that can vary on soil for better comparison with frass.

       

      Table S3: This table should not be in supplementary information and again is in any case inadequate as the authors do not indicate in which samples these chemicals were found.

      Response: We have included Table 3 which lists the predicted chemicals corresponding to the respective peaks.

       

      Thank you for your comments.

       

      Sincerely yours,

      Samuel Ken-En Gan

      On behalf of all the authors

       

       

      2020-12-21 05:36 UTC
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