Vermi-Conversion of Anaerobic Sludges by Eisenia fetida Earthworms

Round 1

Reviewer 1 Report

Statistical analysis of the data should be carried out to compare the different treatments with each other.

 

Comments for author File: Comments.pdf

Author Response

Statistical analysis of the data should be carried out to compare the different treatments with each other.

It is now corrected.

 

REVIEW

According to zoological nomenclature the correct species name is Eisenia fetida. To be corrected throughout the manuscript

It is now corrected.

Materials and Methods

The statistical analyses carried out must be indicated in the material and methods section. At no point in the manuscript is it indicated that any statistical analysis was performed, but the different samples are compared throughout the text. This point should be clarified.

A new figure was added: “Figure 1. Comparison of A) pH and EC; B) TKN; C) TP and D) TK in cow dung (control 1A, 1B) and anaerobic sludge mixtures after vermicomposting by E. fetida” for results comparison, with the indication that “(Values are mean, n=2, error bars indicate SD).”

Line 99: Indicate the origin of the earthworms.

It is added in the Manuscript (lines 107-108): “..(purchased from a worm farm in Corinth, south-central Greece, originated in New Zealand)..”

Results

Statistical analysis should be carried out to check whether the differences found are significant or due to random chance alone.

A new figure was added: “Figure 1. Comparison of A) pH and EC; B) TKN; C) TP and D) TK in cow dung (control 1A, 1B) and anaerobic sludge mixtures after vermicomposting by E. fetida” for results comparison, with the indication that “(Values are mean, n=2, error bars indicate SD).”

Lines 160-161: “have been due to the loss of weight of organic matter”. This parameter was not measured, so this assertion cannot be made.

The organic matter decrease was monitored through the VS measurement. Therefore, the sentence is now corrected as: “The increase in EC might have been due to the loss of weight of organic matter (indicated by the VS decrease)…”

Lines 166-171: Comparisons should be made after the same days of vermicomposting, while in CD1 vermicomposting is 54 days in CD1* vermicomposting is 75 days. why these differences?

As indicated in (new) lines 131-132, the presented results in Table 2 refer to vermicompost samples obtained at the termination of experiments. So, the differences in sampling time are due to the different duration of each experiment.

Line 181: To affirm this, ammonium had to be measured.

Ammonium was measured indirectly through Total Kjeldahl Nitrogen (TKN) analysis. The sentence is added: “TKN analysis measures both organic nitrogen and ammonia nitrogen.” Moreover, at the pH levels measured in our samples almost the entire amount of ammonia is in the form of ammonium.

Lines 180-185: The pH is very high in the initial waste, after vermicomposting this pH decreases. If the starting pH was low, after vermicomposting the pH would increase, as the earthworms would add calcium to the waste.

It is correct. The sentence is added: “The increase of phosphorus, calcium and potassium concentration in the vermicomposted sludge sample may be attributed to the action of the earthworms during vermicomposting [13,28].”

Lines 186-187: It is not possible to mention a significant increase, if no statistical analysis has been carried out.

It has been corrected. The word ‘significant’ has been removed.

Line 188: Indicate any reference that affirms this: “Comparable results were found in literatura”.

The references are added: “Comparable results were found in literature [4,25-27].”

Lines 193-196: We do not know if we are comparing number of days of vermicompost or number of worms. It is not the same a 36 days vermicompost, than a 54 or 75 days vermicompost It is necessary to unify variables.

The analyses should have been carried out at the same time on all samples, because in this way it is not possible to know whether the effect is due to time or to the sample.

The low observed TP values may be attributed either to the low number of earthworms used (in case of CD1) or to the limited time of vermicomposting (in case of CD2). Both factors could have the same result in TP.

Lines 200-203: To clarify this, because it is not understood how after 75 days of vermicomposting the decrease of VS is lower than after 36 days of vermicomposting.

The sentence is corrected: “In our cases VS was reduced by only solely 4%, 7% and 4% by the end of 54, 75 and 36 days, respectively thus the resulting vermicompost was not yet enough stabilized, even after such a long time. The differences in the VS reduction were within experimental error.”

Lines 205-209: The only sample in which the TK content decreases is CD2 in which there has only been a 36-day vermicomposting, and the decrease is 17%.

It is now corrected: “TK concentrations were also higher in the final product compared to the initial CD1 and CD1* feeds, an increase of 39% and 36%, respectively. However, TK analysis in the final CD2 showed a decrease of 17%.”

Lines 219-220: It is not possible to mention significant changes in the absence of statistical analysis.

The word “significant” is now deleted. Moreover, a new figure has been added: “Figure 1. Comparison of A) pH and EC; B) TKN; C) TP and D) TK in cow dung (control 1A, 1B) and anaerobic sludge mixtures after vermicomposting by E. fetida” for comparison of obtained results, with the indication that “(Values are mean, n=2, error bars indicate SD).”

Lines 243-244: This contradiction may be due to the sampling for analysis - how was the sample homogenised for analysis? Clarify this in methodology

The following sentence has been added in the ‘Material and Methods’: “Prior to analysis a homogenized (via manual mixing) sample was obtained from the (vermi)reactors.”

Lines 248-250: Was earthworm mortality taken into account? This is a very important parameter in this type of studies.

The following sentence has been added: “For the feed mixture of 90% CD1- 10% AS1, 90% CD1- 10% AS2* and 85% CD2- 15% AS2 the mortality was 80% (after 47 days), 80% (after 40 days) and 85% (after 56 days), respectively.”

The growth and reproduction of E. fetida in different vermireactors are described in §3.3. The mortality was taken into account indirectly through the mean individual biomass measurements (mg/ number of earthworms) in Figure 2.

Lines 257-263: It is curious that only the TP content of the 85% CD2-15% AS1 and the 80% CD1- 20% AS1 samples decreases and that these are the ones with the shortest vermicomposting time (32 and 48 days respectively), it could be because of the time, or because of the mixture with AS1. It is not possible to compare different variables and try to draw conclusions.

The reviewer is correct. So the sentence has been added: “..and also, to the high amount (15 and 20%) of anaerobic sludge AS1 used.”

Lines 272-273: In material and methods it should be explained how this measurement was carried out and which parameters were taken into account.

The following sentence has been added at the Materials and Methods section: “Thus the biomass production by E. fetida in different vermireactors were measured as mean individual biomass (mg per earthworm) during vermicomposting.”

Lines 272-304: It is essential to perform a statistical analysis to see the differences in earthworm growth over time on the one hand and on the other to see if there have been differences in growth between the beginning and the end of the experiment by calculating the % increase in weight, taking into account the number of earthworms at the beginning and at the end of the experiment.

Both the individual earthworm weight and the number of earthworms has been taken into account into the combined parameter used in this work which refers to the mean individual weight (mg) per earthworm. Moreover, the presentation of results is based on absolute values and no percentages of growth are reported. Thus, no statistical analysis is really needed at this point since no comparisons between results are involved.

Lines 288-289: According to the legend of fig. 1, there is no CD2 sample.

The Figure (Figure 2) has been corrected.

Author Response File: Author Response.pdf

Reviewer 2 Report

1.     Please add one introductory line in the beginning of the abstract, for example you can start by stating the problem or background of the study

2.     I suggest the authors separate the materials form the experimental designs and analytical techniques used.

3.     The materials and methods have some results and discussion mentioned, line 108 onwards, it’s a bit confusing.

4.     In table 1 some of the number of reactors are marked with * but the explanation for it does not correspond. Check line 135-137.

5.     The evaluated Physicochemical analysis has different duration, why was different duration chosen as the ending point.

6.      Line 145-147 t stands for the duration of earthworms’ activity time, however in line 148 t refers to the experimental sampling time following earthworms’ addition. What’s the consistency?

7.     The Physicochemical parameters i.e. pH and EC, what’s its importance in vermicompost.

8.     What’s the ideal pH and EC for vermicompost?

9.     In most cases sludge contains some amount of toxic heavy metals, therefore why didn’t authors examine the presence of heavy metals in their samples.

10.   In this experiment, significant aspects such as C:N ratio of vermicompost for all substrates should be highlighted, followed by the ideal C:N ratio of all substrates.

need revision

Author Response

Reviewer 2

Please add one introductory line in the beginning of the abstract, for example you can start by stating the problem or background of the study

The sentence is added:  “Sludge management is considered a difficult and challenging task and is a priority of environmental policy.”

2. I suggest the authors separate the materials form the experimental designs and analytical techniques used.

It is now corrected.

3. The materials and methods have some results and discussion mentioned, line 108 onwards, it’s a bit confusing.

It is now corrected. The part that presents results has been now transferred to the Results and Discussion section.

4. In table 1 some of the number of reactors are marked with * but the explanation for it does not correspond. Check line 135-137.

It is explained in the footnote: “90% CD1-10%AS1* and 90% CD1-10%AS2* feed mixtures were derived from 90% CD1-10%AS1 and 90% CD1-10%AS2, respectively after the mortality of sole five adult earthworms”

5. The evaluated Physicochemical analysis has different duration, why was different duration chosen as the ending point.

The sentence is added: “The time of final vermicompost refers to the time that mortality of all E. fetida earthworms was recorded.”

6. Line 145-147 t stands for the duration of earthworms’ activity time, however in line 148 t refers to the experimental sampling time following earthworms’ addition. What’s the consistency?

It is now corrected.

7. The Physicochemical parameters i.e. pH and EC, what’s its importance in vermicompost.

The sentences are added: “Earthworms are susceptible to pH, thus pH of vermicompost may be a factor that limits the distribution and numbers of earthworms [18].”

And

“EC shows the variation of salt and mineral ion concentrations [9]. In practice, EC measurement in vermicompost is to check its safe use in agricultural systems [19]. EC values in vermicomposts increase over time.”

8. What’s the ideal pH and EC for vermicompost?

The sentences were added: “The pH of the worm bed should be maintained within 6.8–8 and decreases over time of vermicomposting”; “Previous study revealed that the survival of the worms was affected at values of 0.92 dS/m [13]”

 

9. In most cases sludge contains some amount of toxic heavy metals, therefore why didn’t authors examine the presence of heavy metals in their samples.

Unfortunately, no heavy metal content in neither the cow dung nor the anaerobic sludges was measured in this study. We thank however the reviewer for his/her comment which will definitely be taken into account in our further studies.

10. In this experiment, significant aspects such as C:N ratio of vermicompost for all substrates should be highlighted, followed by the ideal C:N ratio of all substrates.

Unfortunately, no carbon content, and thus no C:N ratio was measured in this study. Instead of C, the organic matter was monitored using the VS content.

Author Response File: Author Response.pdf