Application of Biogas Digestate with Rice Straw Mitigates Nitrate Leaching Potential and Suppresses Root-Knot Nematode (Meloidogyne incognita)
Round 1
Reviewer 1 Report
More clear data on digestate are needed. Please, clarify what you mean by wet digestate (digestate from wet anaerobic digestion? or liquid fraction of digestate?) and similarly, dry digestate (from dry anaerobic digestion? or solid fraction after drying?). Further, each digestate sample was taken from a different biogas plant or the same one? It is not clear once you mention that once pig slurry (line 82) is treated in the biogas plant, while then pig manure is mentioned later (line 83), but referring only to one biogas plant (line 82).
I recommend to add the following information about each types of digestate:
o Coming from wet/dry proces of anaerobic digestion
o Temperature of digestion
o Hydraulic retention time of digestion
o Whether the digestate was matured (for how long, at what temperature) or taken directly from the effluent of the digestor („fresh“). This might be reflected in the conclusion/recommendation – whether to use „fresh“ digestate or matured one to keep balanced nutritious (and nematode management) values and potential emmisions of methane, nitrate, carbon dioxide while applied in the soil.
Line 117 and 118 – please explain what you mean by fermentation of DBD20 and DBD30 samples and why you have done it (aerobic or anaerobic fermentation? for how long? what temperature?).
Author Response
Please refer to “track changes file” line number.
Responses to the comments of Reviewer #1
1. More clear data on digestate are needed, such as temperature, hydraulic retention time. Please, clarify what you mean by wet digestate (digestate from wet anaerobic digestion? or liquid fraction of digestate?) and similarly, dry digestate (from dry anaerobic digestion? or solid fraction after drying?).
Response: Thank you very much for the useful comment. According to the comments, we added more details on the digestates used in Line 86-91.
Original: The wet digestate was collected from a biogas plant in Aichi Prefecture, Japan, which treated pig slurry. Dry digestate was obtained from a thermophilic (55 °C) digester that primarily used pig manure and was supplemented with rice straw to adjust its C/N ratios to 20:1 or 30:1 [31].
Corrected: The wet digestate was collected from a biogas plant in Aichi Prefecture, Japan, in which pig slurry was anaerobically digested at 35°C with a hydraulic retention time of 15 to 20 days. Dry digestate was obtained from a dry thermophilic (55 °C) anaerobic digestion pilot plant, that primarily used pig manure and was supplemented with rice straw to adjust its C/N ratios to 20:1 or 30:1, with a sludge retention time of 40 days, in Tokyo University of Agriculture and Technology, Institute of Engineering, Japan [34].
2. Further, each digestate sample was taken from a different biogas plant or the same one? It is not clear once you mention that once pig slurry (line 82) is treated in the biogas plant, while then pig manure is mentioned later (line 83), but referring only to one biogas plant (line 82).
Response: Thank you very much for the useful comment. Each digestate sample was taken from different biogas plants. For further details, please see the above comment 1.
3. Whether the digestate was matured (for how long, at what temperature) or taken directly from the effluent of the digestor („fresh“). This might be reflected in the conclusion/recommendation – whether to use „fresh“digestate or matured one to keep balanced nutritious (and nematode management) values and potential emissions of methane, nitrate, carbon dioxide while applied in the soil.
Response: Thank you for the comments. In Hokkaido, digestate is stores in the winter season and stored digestate is applied to fields at spring. But in the other region of Japan, digestate is treated immediately after anaerobic digestion. Thus, we used “fresh” digestate. The following description was added to the end of the explanation on digestate.
Original: Collected digestates were stored at 5°C until use.
Corrected: Both digestates were directly taken from the effluent of the digester and stored at 4°C until use. Line 92.
4. Line 117 and 118 – please explain what you mean by fermentation of DBD20 and DBD30 samples and why you have done it (aerobic or anaerobic fermentation? for how long? what temperature?).
Response: Our main target for anaerobic digestion is pig slurry. Pig slurry contains a too high concentration of ammonia which inhibits anaerobic digestion. Thus, we mixed pig slurry with rice straw to avoid ammonia toxicity and get a more amount of biogas. However, since availability of rice straw is limited, we tested two levels of rice straw as a model experiment. This concept is fully explained in the previous paper (31) and here we described the fermentation conditions (please see comment 1).
Reviewer 2 Report
The paper describes the nitrogen transformation in two soils after addition of different digestates, digestate mixed with straw and chemical fertilizer as well as the relationship of nitrogen transformation to the C/N ratio and labile organic carbon content to evaluate the effect of these fertilizers on nitrate leaching potential. The second part of the paper concentrates on the results of the evaluation the suppressive effect of different digestates and their mixtures with rice straw on root-knot nematode population. This is the innovative connection of two important issues in soil management, the limiting of nitrate leaching together with decreasing root-knot nematode population in soil by addition of valuable fertilizer. Digestate is getting more and more attention not only because of its eco-friendly origin and favorable influence on soil quality and crop production but also because of potential negative impact on environment. Another novelty of the research is usage of digestate from dry fermentation as one of soil amendments. Dry digestion is still less popular system used in commercial biogas production and studies on the dry digestion and especially on the digestate from dry digestion are still conducted in smaller amount.
The introduction is well organized and informative, points out the lack of presented investigations. The aim of the study is clear. However, there is a need to add some short information on the digestate quality as fertilizer and the nitrification rates.
The methods are quite standard and appropriate for the study. However the number of replicates is missed in description of methods used to determine the chemical properties of digestate and rice straw. The SD is also missed in the Table 1. The methods used to determine the chemical properties of digestate and rice straw also could be described in more detailed way, especially the extraction method. In the description of incubation experiment the time period of experiment needs the explanation as well as the temperature of incubation. Statistical methods are appropriate.
The results are well organized and comprehensible. However, I miss the comparison of NH4-N content in control and soils amended with organic fertilizers and with chemical fertilizer. I miss also the comparison of NH4-N content at day 0 in both soils with amendments and without them.
The discussion is easy to follow and is explanatory. However, I miss comments to other treatments. The discussion is mainly focused on one treatment which decreases potential NO3-N leaching from soil. Perhaps short comment on other treatments will be valuable. I would also underline that final NO3-N content in both soils at the end of experiment in case of treatment Mix2 was at the level of NO3-N content in control soils. There is also confusing usage of terms net nitrate conversion and nitrate conversion, which should be checked in the text. The conclusions are well supported by the results. The detailed comments are given below.
Line 30 – anaerobic digestion converts into biogas not only organic wastes but for example energy crops such as maize silage, therefore I would use different words
Line 31 – it would be worth to add information if the digestate is better or similar to other organic fertilizers. I realize that in line 37 and 39-42 the main properties of digestate and its influence on soil properties is given, however a short information at the beginning of the paragraph would be valuable
Line 43 – it would be worth to add if the nitrification rate is similar or higher in soils fertilized with digestate comparing to other organic amendments and chemical fertilizers.
Line 74 – please use the abbreviation for root-knot nematodes. I would use the RKN in the whole text instead of mixing abbreviation with the full name. However, if you wish to use the full name, please, unify the writing. I suggest root-knot nematodes, as it is written for example in lines 56 and 78.
Line 82 – please specify the temperature of anaerobic digestion process
Line 88 – please describe in details the method of extraction eg. time of mixing, filtering method
Line 94 Table 1 – water content of rice straw is “4.?”, please check this value. Please explain in text if the properties of organic soil amendments were measured in triplicates and add the SD values to the results shown in table.
Line 111 please check if there are two spaces between words “soil” and “were”
Line 109-112 – I assume that values of chemical properties are given in kg of dry soil therefore I miss the description of measuring the moisture of fresh soil.
Line 118 – please check the spaces between “30” and “and”
Line 119 – please check the spaces between “16” and (DBD20….)
Line 118-119 – in figure 1 captions the phrase “mixed with a low amount of…” and “mixed with a high…” I would suggest using these phrases also in methods
Line 120 – please explain the application rate in terms of agricultural practices eg., is this rate commonly used in agriculture?
Line 127 – please explain the rates of rice straw added; use the same units as it was in case of digestate and the values in Mg ha-1
Line 131 – please check the sentence: …. With described (??? missing word) above…
Line 133 – please give the explanation for the incubation temperature
Line 146 – please specify why the 35th day of incubation was used for determination of net N-mineralization and nitrate conversion. In results it is suggested that these values was calculated also for other days eg. day 90
Line 183 – only incubation experiment was done in triplicate
Line 189-190 – I would rather use the phrase “…from day 0 to the 7th day of incubation…”
Lines 189-194 – I miss in this part of results any information on the CF treatment
Line 192 – day 7 or 7th day of incubation. I would change this in the whole text.
Line 194 – please add short information on ammonium content in the period from day 35 to the end of the incubation
Line 196 – please use the abbreviation for net N-mineralization in the whole text
Line 196 and 200 – please add the p level after word “significantly”
Lines 195-201 – this is a confusing paragraph, because in methods net N-mineralization was described as calculated for 35 days of incubation and in this paragraph it is suggested that it was calculated for 90 days. In Line 197 should be at day 7 of incubation.
Line 203 Figure 1 – please specify where the NC is shown on the chart or remove it from the figure caption
Line 218 Figure 2 and methods – please unify the term net nitrate conversion. In methods it is named nitrate conversion.
Line 218 Figure 2 – please unify the term “net N-mineralization” (this is how it is written in methods, later in the text it is written net N mineralization).
Line 217 Figure 2 – please specify the * in values of R2 on the charts
Lines 221-224 – please change to day 30 or 30th day
Line 227 – please refer to table 2, because table 3 should be numbered as table 2
Line 228 Table 3 – this table should be numbered as table 2 and in line 227 the reference should be done to table 2
Line 237 Figure 3 – please check the caption “rootroot-knot”. I would use “amended” instead of “applied”
Lines 245-251 – please change to day 0, etc.; please refer to table 3 not to table 2
Line 245 – please add the p level after word “significantly”
Line 249 – I would use term extractable organic C (EOC) content
Line 250 – please change to: EOC content at day 0 was the….
Line 252 Table 2 – it should be table 3. In this table I would give units below EOC not next to it; please change to at day 0, in title after (EOC) I would add word “content”
Lines 255-259 – change please to day 0, day 90 or end of experiment
Line 261 – on the chart and in the caption pleas unify the “root knot nematode” term with used at the beginning “root-knot…”, please give the explanation to ** in values of R2 on the chart
Line 271 – please refer to other figure or show the NC on the charts in figure 1
Line 285 – please refer to table 3
Lines 294 and 295 – please change to day 14, day 35, etc.
Line 308 – please add the p level after word “significantly”
Lines 312 and 313 and 314 – please change to C/N ratio
Line 387 – space is missing between words “application” and “mixed”
Author Response
Please refer to “track changes file” line number.
Responses to the comments of Reviewer #2
1. There is a need to add some short information on the digestate quality as fertilizer and the nitrification rates.
Response: Thank you very much for the useful comment. We already mention the effect of digestate on nitrification rate (line 45 to 46), but according to the comment, we added one sentence for nitrification rate as well as descriptions on fertilizer effects. Line 40-49
Original: The use of digestate as an organic amendment in agriculture can be an effective management practice not only as a biofertilizer but also for improving soil physical properties such as aggregate formation and moisture retention, sustaining soil organic matter concentrations, enhancing biological activities, and suppressing pathogenic organisms [4,5]. However, when digestate is applied to soil, ammonium in biogas digestate can be readily nitrified [6]. Thus, the use of biogas digestate may increase nitrate leaching risks [7-11], as our previous study [12] reported that biogas digestate increased nitrate contents in soil through a stimulatory effect on nitrification.
Corrected: Biogas digestate is a highly valuable nutrient-rich and humus-rich fertilizer [4]. In addition to the biofertilizer effect, the use of digestate can be an effective management practice as an organic amendment in agriculture for improving soil physical properties such as aggregate formation and moisture retention, sustaining soil organic matter concentrations, enhancing biological activities, and suppressing pathogenic organisms [4,5]. However, when digestate is applied to soil, ammonium in biogas digestate can be readily nitrified [6]. Previous studies have demonstrate that biogas digestate application led to a higher soil nitrification rate than manure [7, 8] and compost [9]. Thus, the use of biogas digestate may increase nitrate leaching risks [10-14], as our previous study [15] reported that biogas digestate increased nitrate contents in soil through a stimulatory effect on nitrification.
2. The number of replicates is missed in description of methods used to determine the chemical properties of digestate and rice straw.
Response: Thank you for the comment. We measured the chemical properties of digestate and rice straw in triplicate and added this information. Line 95-96
Original: The chemical properties of digestates and rice straw are shown in Table 1.
Corrected: The chemical properties of digestates and rice straw are shown in Table 1 and each sample was analyzed with three replicates.
3. The SD is also missed in the Table 1.
Response: We can show SD values in Table 1. But we do not focus on the differences in each parameter among samples. In addition, too many numbers make tables complex and less understandable. Thus, we decided not to show them.
4. The methods used to determine the chemical properties of digestate and rice straw also could be described in more detailed way, especially the extraction method.
Response: We have addressed this comment by adding a few lines on Line 95-99.
Original sentence: Ammonium-N content was measured by extracting from 5 ml of wet digestate or 5 g of dry digestate and rice straw with 25 ml of de-ionized water and by using the indo-phenol blue method.
Corrected: “Ammonium-N content was measured by using the indo-phenol blue method [35]. Extraction was done by i) mixing 5 ml of wet digestate or 5 g of dry digestate and rice straw with 25 ml of 2M KCl, ii) shaking for 1 h at 120 rpm, and iii) filtering through No.5C filter paper (ADVANTEC Toyo Kaisha, Ltd.)
5. In the description of incubation experiment the time period of experiment needs the explanation as well as the temperature of incubation.
Response: we decided the time period depending on our previous study (Sawada and Toyota 2015). However, NO3-N of Mix2 in Fuchu soil was negligible even at day 35 and thus we extended incubation period to 60 and 90 days to better know the mineralization of once immobilized N. Incubation temperature was decided based on the average temperature in our summer season and the availability of our incubator at 25 °C to 27 °C. The information was added in the revised manuscript.
Original: The vials were covered with aluminum foil and incubated for 0, 7, 14, 35, 60, 90 days at 27 °C.
Corrected: Line 146-148.
The vials were covered with aluminum foil and incubated for 0, 7, 14, 35, 60, 90 days at 27 °C to analyze N immobilization and subsequent N mineralization rates. Average temperatures in our summer period ranged from 25 to 30°C and thus our incubator was set up at 25 to 27 °C.
6. I miss the comparison of NH4-N content in control and soils amended with organic fertilizers and with chemical fertilizer.
7. I miss also the comparison of NH4-N content at day 0 in both soils with amendments and without them.
Response: According to this comment we added a few lines.
Original: Ammonium concentration markedly decreased to less than 20 mg NH4–N kg-1 soil from 0 day to 7 days of incubation in all of the treatments in Kikugawa soil, indicating the readily occurrence of nitrification (Figure 1). In Fuchu soil, it was still higher than 20 mg NH4–N kg-1 soil in CF, WBD, DBD20, DBD30 at 7 days of incubation, while it was less than 10 mg NH4–N kg-1 soil in Mix1 and Mix2 (Figure 1). After 35 days of incubation, the NH4–N concentration was lower than 10 mg NH4–N kg-1 soil in both soils.
Line 206-217
Corrected: Ammonium concentrations of Kikugawa and Fuchu soil at day 0 were 29 and 44 mg NH4–N kg-1 soil and increased to 159 to 244 and 216 to 267 in the amended treatments, respectively, which nearly corresponded to the application rate (200 mg NH4–N kg-1 soil). With incubation NH4–N concentration markedly decreased and it was less than 20 mg NH4–N kg-1 soil at day 7 in all of the treatments in Kikugawa soil, indicating the readily occurrence of nitrification (Figure 1). In Fuchu soil, it was still higher than 20 mg NH4–N kg-1 soil in CF, WBD, DBD20, DBD30 at day 7, while it was less than 10 mg NH4–N kg-1 soil in Mix1 and Mix2 (Figure 1). After day 35, NH4–N concentration was consistently lower than 10 mg NH4–N kg-1 soil in both soils. Through 90 days of incubation periods, there were no significant (p < 0.05) differences between CONT and the other amended treatments in Fuchu soil, and also no significant (p < 0.05) differences between CONT and CF, DBD20, DBD30 and Mix1 treatments in Kikugawa soil.
8. I miss comments to other treatments. The discussion is mainly focused on one treatment which decreases potential NO3-N leaching from soil. Perhaps short comment on other treatments will be valuable.
Response: Thank you very much for your useful advice. We newly added some short comments on the comparison between wet and dry digestion, as described below.
Original: Nitrate concentration in CONT increased by 466 and 141 mg N kg-1 soil at day 90, in Kikugawa soil and Fuchu soil, respectively (Figure 1). This result indicated that Nm was higher in Kikugawa soil than in Fuchu soil. At day 7 of incubation, NO3-N was significantly (p < 0.05) higher in WBD, DBD20 and Mix1 treatment, followed by CF and DBD30 treatment in Fuchu soil, indicating that the net nitrification rate was the highest in WBD, DBD20 and Mix1 treatment. Nitrate concentration was significantly (p < 0.05) lower in Mix2 treatment in both soils than in other treatments throughout 90 days.
Line 219-229-
Corrected. Nitrate concentration in CONT increased by 466 and 141 mg N kg-1 soil at day 90, in Kikugawa soil and Fuchu soil, respectively (Figure 1). This result indicated that Nm was higher in Kikugawa soil than in Fuchu soil. At day 7 of incubation, NO3-N was significantly (p < 0.05) higher in WBD, DBD20 and Mix1 treatment, followed by CF and DBD30 treatment in Fuchu soil, indicating that the net nitrification rate was the highest in WBD, DBD20 and Mix1 treatment. Nitrate concentration was significantly (p < 0.05) lower in Mix2 treatment in both soils than in other treatments throughout 90 days. In comparison between wet and dry digestate, NO3-N concentration was significantly (p < 0.05) lower in DBD30 than in WBD in Fuchu soil at day 14 to day 90 and similar tendencies were observed in Kikugawa soil.
Original: Populations of root-knot nematodes were significantly lower (p < 0.05) in Mix2 than in CF at day 30. On day 60 and 90, populations were significantly (p < 0.05) lower in DBD30, Mix1 and Mix2 than in CF. Populations significantly (p < 0.05) decreased in DBD30 and Mix2 from day 30 to day 90 (Figure 3).
While plant growth was not significantly different among treatments, gall formation was significantly (p < 0.05) lower in Mix2 than in CF, WBD, DBD20 and DBD30, and it was significantly (p < 0.05) lower in Mix1 than in CF and WBD (Table 2).
Line 251-260
Corrected: Populations of root-knot nematodes were significantly lower (p < 0.05) in Mix2 than in CF at day 30. On day 60 and 90, populations were significantly (p < 0.05) lower in DBD30, Mix1 and Mix2 than in CF. Populations significantly (p < 0.05) decreased in DBD30 and Mix2 from day 30 to day 90 (Figure 3). In comparison between wet and dry digestate, populations significantly lower in DBD30 than in WBD at day 60 and 90.
While plant growth was not significantly different among treatments, gall formation was significantly (p < 0.05) lower in Mix2 than in CF, WBD, DBD20 and DBD30, and it was significantly (p < 0.05) lower in Mix1 than in CF and WBD (Table 2). In comparison between wet and dry digestate, gall formation tended to be lower by 10% to 20% in DBD30 and DBD20 than in WBD, although there were no significant differences.
Original: According to Jothi et al. [28], a type of wet biogas digestate reduced damage severity to tomato as well as population of M. incognita. In addition, Westphal et al. [29] found that the amendment with a wet digestate to soil reduced infection of Heterodera shcachtii to the host plant and improved the plant growth. However, in this study, populations of root-knot nematodes did not decrease in WBD and DBD20, compared with those of CONT and CF, for 60 days to 90 days of incubation (Figure 3). The exact reason why WBD20 and DBD20 did not decrease populations of M. incognita was unclear, although the lower EOC in these two treatments could be involved (Figure 4). Several studies have already reported that not all types of organic amendments were beneficial in the suppression of root-knot nematodes [59,60].
Line 364- 377
Corrected: According to Jothi et al. [28], a type of wet biogas digestate reduced damage severity to tomato as well as population of M. incognita. In addition, Westphal et al. [29] found that the amendment with a wet digestate to soil reduced infection of Heterodera shcachtii to the host plant and improved the plant growth. However, in this study, populations of root-knot nematodes did not decrease in WBD and DBD20, compared with those of CONT and CF, for day 60 to day 90 (Figure 3). The exact reason why WBD20 and DBD20 did not decrease populations of M. incognita was unclear, although the lower EOC in these two treatments could be involved (Figure 4). Several studies have already reported that not all types of organic amendments were beneficial in the suppression of root-knot nematodes [59,60]. In contrast, DBD30 significantly decreased populations of root-knot nematodes at day 60 to day 90 than WBD. DBD30 showed a significantly higher EOC at day 0 than WBD and therefore, NO3-N concentration was consistently lower in DBD30 than in WBD at day 14 to day 90. These results may suggest that a dry biogas digestate DBD30 is better than a wet biogas digestate WBD in terms of nitrate leaching risk and root-knot nematode management.
9. I would also underline that final NO3-N content in both soils at the end of experiment in case of treatment Mix2 was at the level of NO3-N content in control soils.
Response: Thank you very much for the useful comment. As the reviewer suggested, the final NO3-N concentration in Mix2 was in the same level of control. This indicates most of N added to Mix2 was microbially immobilized. We newly described this finding.
Original: Nitrate concentration in CONT increased by 466 and 141 mg N kg-1 soil for 90 days, in Kikugawa soil and Fuchu soil, respectively (Figure 1). This result indicated that net N mineralization rate was higher in Kikugawa soil than in Fuchu soil. At 7 days of incubation, NO3-N was significantly higher in WBD, DBD20 and Mix1 treatment, followed by CF and DBD30 treatment in Fuchu soil, indicating that the net nitrification rate was the highest in WBD, DBD20 and Mix1 treatment. Nitrate concentration was significantly lower in Mix2 treatment in both soils than in other treatments throughout 90 days.
Line 219-229
Corrected: Nitrate concentration in CONT increased by 466 and 141 mg N kg-1 soil at day 90, in Kikugawa soil and Fuchu soil, respectively (Figure 1). This result indicated that Nm was higher in Kikugawa soil than in Fuchu soil. At day 7 of incubation, NO3-N was significantly (p < 0.05) higher in WBD, DBD20 and Mix1 treatment, followed by CF and DBD30 treatment in Fuchu soil, indicating that the net nitrification rate was the highest in WBD, DBD20 and Mix1 treatment. Nitrate concentration was significantly (p < 0.05) lower in Mix2 treatment in both soils than in other treatments throughout 90 days. The final NO3-N concentration in Mix2 was in the same level of control in both soils, indicating that most of N added to Mix2 was microbially immobilized even at day 90. In comparison between wet and dry digestate, NO3-N concentration was significantly (p < 0.05) lower in DBD30 than in WBD in Fuchu soil at day 14 to day 90 and similar tendencies were observed in Kikugawa soil.
10. There is also confusing usage of terms net nitrate conversion and nitrate conversion, which should be checked in the text.
Response: Thank you very much for the comment. We should have unified net nitrate conversion throughout the text, but we did “nitrate conversion” in one place. In the revised manuscript, we unified net nitrate conversion (NC) or NC. Line 161, 249, 320, 321.
Line 30 – anaerobic digestion converts into biogas not only organic wastes but for example energy crops such as maize silage, therefore I would use different words
Reply: Line 30-31
Original: Anaerobic digestion is a process which converts various organic wastes into biogas and digested substrate, which is commonly named digestate.
Corrected: Anaerobic digestion is a process which converts organic substrates, such as organic waste and energy crops, into biogas and digested residue, which is commonly named digestate.
Line 31 – it would be worth to add information if the digestate is better or similar to other organic fertilizers. I realize that in line 37 and 39-42 the main properties of digestate and its influence on soil properties is given, however a short information at the beginning of the paragraph would be valuable
Reply: Same with question NO.1.
Line 43 – it would be worth to add if the nitrification rate is similar or higher in soils fertilized with digestate comparing to other organic amendments and chemical fertilizers.
Reply: Same with question NO.1.
Line 74 – please use the abbreviation for root-knot nematodes. I would use the RKN in the whole text instead of mixing abbreviation with the full name. However, if you wish to use the full name, please, unify the writing. I suggest root-knot nematodes, as it is written for example in lines 56 and 78.
Reply: According to the comment, we changed all the corresponding parts into root-knot nematodes.
Line 82 – please specify the temperature of anaerobic digestion process
Reply: According to the comments, we added more details in Line 86-93.
Original: The wet digestate was collected from a biogas plant in Aichi Prefecture, Japan, which treated pig slurry. Dry digestate was obtained from a thermophilic (55 °C) digester that primarily used pig manure and was supplemented with rice straw to adjust its C/N ratios to 20:1 or 30:1 [31].
Corrected: The wet digestate was collected from a biogas plant in Aichi Prefecture, Japan, in which pig slurry was anaerobically digested at 35°C with a hydraulic retention time of 15 to 20 days. Dry digestate was obtained from a dry thermophilic (55 °C) anaerobic digestion pilot plant, that primarily used pig manure and was supplemented with rice straw to adjust its C/N ratios to 20:1 or 30:1 with a sludge retention time of 40 days, in Tokyo University of Agriculture and Technology, Institute of Engineering, Japan [31].
Line 88 – please describe in details the method of extraction eg. time of mixing, filtering method
Reply: Same with question NO.4.
Line 94 Table 1 – water content of rice straw is “4.?”, please check this value. Please explain in text if the properties of organic soil amendments were measured in triplicates and add the SD values to the results shown in table.
Reply: Thank you for the comments. Table 1-water content of rice straw is “4.0 g water/100 g fresh weight. The samples were analyzed in triplicates to measure the digestate and rice straw samples. We did not pay attention to the differences in the chemical properties among the samples, and thus for better readability, we did not show SD values in Table1.
Line 111 please check if there are two spaces between words “soil” and “were”
Reply: Line 122 Thank you very much for the comment. We deleted the space.
Line 109-112 – I assume that values of chemical properties are given in kg of dry soil therefore I miss the description of measuring the moisture of fresh soil.
Reply: We apologize for this inconvenience. We added the relevant information.
Line 117-118
Original: Freshly collected soil samples were sieved to 5 mm and stored at field moisture at 5 °C until use.
Corrected: Freshly collected soil samples were sieved to 5 mm and stored at field moisture (fresh soil moisture content was 28% and 14% in Kikugawa and Fuchu soil, respectively) at 5 °C until use.
Line 118 – please check the spaces between “30” and “and”
Reply: Line 129 Thank you very much for the comment. We properly amended.
Line 119 – please check the spaces between “16” and (DBD20….)
Reply: Line 130 Thank you very much for the comment. We properly amended.
Line 118-119 – in figure 1 captions the phrase “mixed with a low amount of…” and “mixed with a high…” I would suggest using these phrases also in methods
Reply: Thank you very much for the useful comment. We amended based on the comment.
Line 129-131
Original: vi) DBD20 mixed with rice straw to adjust the C/N ratio to 16 (DBD20 : rice straw = 1 : 0.06) (Mix1), vii) DBD20 mixed with rice straw to adjust the C/N ratio to 30 (DBD20 : rice straw = 1 : 0.4) (Mix2).
Corrected: vi) DBD20 mixed with a low amount of rice straw to adjust the C/N ratio to 16 (DBD20 : rice straw = 1 : 0.06) (Mix1), vii) DBD20 mixed with a higher amount of rice straw to adjust the C/N ratio to 30 (DBD20 : rice straw = 1 : 0.4) (Mix2).
Line 120 – please explain the application rate in terms of agricultural practices eg., is this rate commonly used in agriculture?
Reply: Thank you very much for the comment. Based on the practical tomato cultivation, we decided the fertilization rate. Line 133-134:
Original: Their application rates were adjusted to 200 mg ammonium (NH4+-N) kg-1 dry soil (equivalent to ~300 kg NH4-N ha-1), except for CONT.
Corrected: Their application rates were adjusted to 200 mg ammonium (NH4+-N) kg-1 dry soil (equivalent to ~300 kg NH4-N ha-1) (roughly corresponds to a commonly used N level for tomato cultivation), except for CONT.
Line 127 – please explain the rates of rice straw added; use the same units as it was in case of digestate and the values in Mg ha-1
Reply: In general, 6 Mg ha-1 of rice straw is obtained from 1 ha of paddy field. Thus, we used this level. With an increasing amount of rice straw, an amount of biogas increases in the anaerobic digestion of pig slurry and rice straw. However, it is more difficult to obtain an enough amount of rice straw. To compromise better biogas production and availability of rice straw, we decided 40 Mg ha-1.
Line 138-141:
Original: The actual added amounts of WBD, DBD20, DBD30, Mix1 and Mix2 were 48, 74, 125, 74 and 74 mg g-1 dry soil (equivalent to 72, 111, 187, 111, and 111 Mg ha-1), respectively. In Mix1 and Mix2, rice straw was added at rates of 4.4 and 29.7 g kg-1, respectively.
Corrected: The actual added amounts of WBD, DBD20, DBD30, Mix1 and Mix2 were 48, 74, 125, 74 and 74 mg g-1 dry soil (equivalent to 72, 111, 187, 111, and 111 Mg ha-1), respectively. In Mix1 and Mix2, rice straw was added at rates of 4.4 and 29.7 g kg-1 dry soil (equivalent to 6.6 and 44.6 Mg ha-1), respectively.
Line 131 – please check the sentence: …. With described (??? missing word) above…
Reply: Line 144
Original: Supplemented soil samples with described above were mixed thoroughly with a spatula and each 5 g (60 °C oven dry basis) was dispensed into a 50 ml glass vial.
Corrected: Supplemented soil samples with i) to vii) described above were mixed thoroughly with a spatula and each 5 g (60 °C oven dry basis) was dispensed into a 50 ml glass vial.
Line 133 – please give the explanation for the incubation temperature
Reply: Same question with NO.5.
Line 146 – please specify why the 35th day of incubation was used for determination of net N-mineralization and nitrate conversion. In results it is suggested that these values was calculated also for other days eg. day 90
Reply: Thank you for your comments, we changed our calculation from day 35 to day 90 and showed in Figure2, and accordingly changed descriptions in results and discussion.
Original: Nm was the lowest in Mix2 (-61% and -63%) of Kikugawa and Fuchu soil, and lower in DBD30 (-21% and -9%) and Mix1 (-35% and -22%) than in WBD (-26% and -8%) of Kikugawa and Fuchu soil, respectively (Figure 2). While Nm was -16% in DBD20 of Kikugawa soil, it decreased to -35% in Mix1 to which 4 mg rice straw g-1 of soil was added to DBD20 (data not shown).
NC (-20% and -17%) was the lowest in Mix2 of Kikugawa and Fuchu soils, respectively (Figure 2). NC was lower in Mix1 (20% and 31%) than in DBD20 (44% and 52%) of Kikugawa and Fuchu soil, respectively.
Corrected: Line238-245
Nm was the lowest in Mix2 (-34% and -53%) of Kikugawa and Fuchu soil, and lower in DBD30 (-15% and -8%) and Mix1 (-23% and -24%) than in WBD (-14% and -1%) of Kikugawa and Fuchu soil, respectively (Figure 2). While Nm was -12% in DBD20 of Kikugawa soil, it decreased to -23% in Mix1 to which 4 mg rice straw g-1 of soil was added to DBD20 (data not shown).
NC (8% and -7%) was the lowest in Mix2 of Kikugawa and Fuchu soils, respectively (Figure 2). NC was lower in Mix1 (33% and 29%) than in DBD20 (48% and 47%) of Kikugawa and Fuchu soil, respectively.
Original: There were significant relationships between the Nm (R2 = 0.646, p < 0.05 in Kikugawa soil and R2 = 0.702, p < 0.05 in Fuchu soil, Figure 2) or NC (R2 = 0.927, p < 0.01 in Kikugawa soil and R2 = 0.724, p < 0.05 in Fuchu soil, Figure 2) and EOC at 0 day.
Corrected: Line 288-290
There were significant relationships between the Nm (R2 = 0.593, p < 0.05 in Kikugawa soil and R2 = 0.678, p < 0.05 in Fuchu soil, Figure 2) or NC (R2 = 0.750, p < 0.01 in Kikugawa soil and R2 = 0.762, p < 0.05 in Fuchu soil, Figure 2) and EOC at day 0.
Original: The large negative values of Nm in Mix 2 of both soils (-63% and -61% in Kikugwa and Fuchu soils, respectively) supported the negative net mineralization rates.
Corrected: Line 314-315
The large negative values of Nm in Mix 2 of both soils (-34% and -53% in Kikugwa and Fuchu soils, respectively), supported the negative net mineralization rates.
Original: Alburquerque et al. [6] reported digestate with a higher C/N ratio (18.5) induced net N immobilization (Nm = -96%) while digestate with a lower C/N ratio (1.5) induced net mineralization rate (Nm = -8%) when they were mixed to soil and incubated. In our study, C/N ratio (12) of DBD20 was increased to 16 in Mix1 by adding rice straw and their Nm were decreased from -25% and -31% in DBD20 (Kikugawa and Fuchu soils) to -60% and -38% in Mix1, respectively. In addition, NC was consistently lower in Mix1 than in DBD20. These results suggested that the increase in the C/N ratio of biogas digestate by 4 stimulated N immobilization, and that the application of dry biogas digestate together with rice straw would be a proper strategy to mitigate the nitrate leaching potential.
Corrected: Line 310-318
Alburquerque et al. [6] reported digestate with a higher C/N ratio (18.5) did not induced net nitrate conversion (NC= -29%) while digestate with a lower C/N ratio (1.5) induced net nitrate conversion (NC= 84%) when they were mixed to soil and incubated. In our study, C/N ratio (12) of DBD20 was increased to 16 in Mix1 by adding rice straw and NC decreased from 47% and 48% in DBD20 (Kikugawa and Fuchu soils) to 29% and 33% in Mix1, respectively. These results suggested that the increase in the C/N ratio of biogas digestate by 4 stimulated N immobilization, and that the application of dry biogas digestate together with rice straw would be a proper strategy to mitigate the nitrate leaching potential.
Line 183 – only incubation experiment was done in triplicate
Reply: In addition to incubation experiments, we did chemical analysis in triplicate also for the materials used.
Line 199-203
Original: All results are expressed as means and standard deviations for three replicate measurements. The effects of all fertilizer treatments and incubation time as well as their interactions on NO3-N and NH4-N and nematodes number were tested by a two-way ANOVA followed by a Tukey HSD mean comparison (p < 0.05) using the software SPSS version 22.
Corrected: All the data in inorganic nitrogen, EOC content, pH and the number of root-knot nematodes were obtained using triplicate and expressed as means and standard deviations. The effects of all fertilizer treatments and incubation time as well as their interactions on NO3-N and NH4-N and nematodes number were tested by a two-way ANOVA followed by a Tukey HSD mean comparison (p < 0.05) using the software SPSS version 22.
Line 189-190 – I would rather use the phrase “…from day 0 to the 7th day of incubation…”
Reply: Thank you very much for the useful comment. We properly amended all into day 0 and day 7, 35, 60, 90. (Line 2, 24, 159, 176, 209, 212, 213, 219, 221, 232, 247, 252, 254, 279, 280, 282, 284, 286, 291, 292, 294, 328, 329, 331, 368. .
Lines 189-194 – I miss in this part of results any information on the CF treatment
Reply: Please see comment NO.6.
Line 192 – day 7 or 7th day of incubation. I would change this in the whole text.
Reply: According to the comment, we changed the relevant parts. Please see Line 189-190.
Line 194 – please add short information on ammonium content in the period from day 35 to the end of the incubation
Reply: Please see comment NO.6.
Line 196 – please use the abbreviation for net N-mineralization in the whole text
Reply: Thank you very much for the useful comment. We have changed the corresponding parts. Line 161, 221, 238, 240, 247, 248, 288.
Line 196 and 200 – please add the p level after word “significantly”
Reply: Thank you very much for the useful comment. We properly amended. Line 215, 216, 222, 224, 255, 278, 342.
Lines 195-201 – this is a confusing paragraph, because in methods net N-mineralization was described as calculated for 35 days of incubation and in this paragraph it is suggested that it was calculated for 90 days. In Line 197 should be at day 7 of incubation.
Reply: Thank you very much for the useful comment. We properly amended. Please see the above Line 146.
Line 203 Figure 1 – please specify where the NC is shown on the chart or remove it from the figure caption
Reply: Line 231 Thank you very much for the useful comment. We deleted NC from figure 1.
Line 218 Figure 2 and methods – please unify the term net nitrate conversion. In methods it is named nitrate conversion.
Reply: Thank you very much for the useful comment. We properly amended. We unified term to net nitrate conversion. Line 161, 249, 320, 321.
Line 218 Figure 2 – please unify the term “net N-mineralization” (this is how it is written in methods, later in the text it is written net N mineralization).
Reply: Thank you very much for the useful comment. We properly amended.
Line 161, 248, 313.
Line 217 Figure 2 – please specify the * in values of R2 on the charts
Reply: Line 249: *: p < 0.05.
Lines 221-224 – please change to day 30 or 30th day
Reply: Thank you very much for the useful comment. We properly amended. Please see the above Line 146.
Line 227 – please refer to table 2, because table 3 should be numbered as table 2
Reply: Thank you very much for the useful comment. We properly amended.
Line 285: Changed original table 2 to table 3.
Line 228 Table 3 – this table should be numbered as table 2 and in line 227 the reference should be done to table 2
Reply: Thank you very much for the useful comment. We properly amended
Line 258, 261, original table 3 to table 2. Line 279, 283, 285, 318 changed to table 3.
Line 237 Figure 3 – please check the caption “rootroot-knot”. I would use “amended” instead of “applied”
Reply: Thank you very much for the useful comment. We properly amended. Line 270
Lines 245-251 – please change to day 0, etc.; please refer to table 3 not to table 2
Reply: Thank you very much for the useful comment. We properly amended. Same question with line 228.
Line 245 – please add the p level after word “significantly”
Reply: Thank you very much for the useful comment. We properly amended. Same question with Line 196 and 220.
Line 249 – I would use term extractable organic C (EOC) content
Reply: Thank you very much for the useful comment. We properly amended. Line 282
Line 250 – please change to: EOC content at day 0 was the….
Reply: Thank you very much for the useful comment. We properly amended. Lind 284.
Line 252 Table 2 – it should be table 3. In this table I would give units below EOC not next to it; please change to at day 0, in title after (EOC) I would add word “content”
Reply: Thank you very much for the useful comment. We properly amended. Lin 285-286.
Lines 255-259 – change please to day 0, day 90 or end of experiment
Reply: Thank you very much for the useful comment. We properly amended. Same question with above Line 189-190.
Line 261 – on the chart and in the caption pleas unify the “root knot nematode” term with used at the beginning “root-knot…”, please give the explanation to ** in values of R2 on the chart
Reply: Thank you very much for the useful comment. We properly amended. Line 294-295, unified to root-know nematode, **: p < 0.01,
Line 271 – please refer to other figure or show the NC on the charts in figure 1
Reply: We apologize our mistake. The figure referred is figure 2, not figure 1. Line 304
Line 285 – please refer to table 3
Reply: Thank you very much for the useful comment. We properly amended. Line 318 changed to table 3.
Lines 294 and 295 – please change to day 14, day 35, etc.
Reply: Thank you very much for the useful comment. We properly amended. Same question with above Line 189-190.
Line 308 – please add the p level after word “significantly”
Reply: Thank you very much for the useful comment. We properly amended. Same question with above Line 196 and 220.
Lines 312 and 313 and 314 – please change to C/N ratio
Reply: Thank you very much for the useful comment. We properly amended. Line 346-349: Changed all to C/N ratio.
Line 387 – space is missing between words “application” and “mixed”
Reply: Thank you very much for the useful comment. We properly amended. Line 435.
