The Decomposition Dynamics and Substrate Component Potential of Biomass from the Seagrass Posidonia oceanica (L.) Delile

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Strengths:

This study presents valuable insights into the management strategies for Posidonia oceanica debris, particularly through innovative approaches such as introducing forest microbiomes for accelerated decomposition and utilizing debris as a growth substrate for plants.

For possible improvement:

1. Description of Experimental Environment:

   Detailed information about the simulated experimental environment, encompassing temperature, humidity, and light conditions, is essential for comprehending result reproducibility in laboratory settings and its extrapolation to natural conditions.

2. Soil Characterization:

   Providing a more detailed description of soil characteristics, including texture and organic matter content, would contribute to a better overall understanding of the experimental conditions.

3. Temporal Chemical Analysis:

   The inclusion of samples at various time points for chemical analysis would enrich the understanding of microbial impacts on the decomposition process.

4. Consideration of Growth Stages:

   If the study considered different growth stages of plants, presenting relevant data and engaging in further discussion would bolster the robustness of the findings.

5. Detailed Explanation of Flower/Fruit/Leaf Ratios:

   Offering a more detailed explanation and further discussion regarding observed differences in flower/fruit/leaf ratios would underscore the ecological and physiological significance of these variations.

In conclusion, this study significantly contributes to the field of Posidonia oceanica debris management. Addressing the suggested improvements will not only enhance the quality and transparency of the research but also amplify its broader impact.

Author Response

1. Description of Experimental Environment:

Detailed information about the simulated experimental environment, encompassing temperature, humidity, and light conditions, is essential for comprehending result reproducibility in laboratory settings and its extrapolation to natural conditions.

Done. We thank the reviewer for their valuable comments. We added the mentioned information regarding the experimental environment: “Subsequently, the containers were relocated to the department's greenhouse to emulate in situ conditions, characterized by daily average environmental parameters measured with an OFYLIA 4.0-C sensor, indicating a temperature range of 14°C to 28°C and a daylight intensity of 1100 ppfd/umol/s/m².”. Lines 141-145.

 

2. Soil Characterization:

Providing a more detailed description of soil characteristics, including texture and organic matter content, would contribute to a better overall understanding of the experimental conditions.

Done. We added the characteristics of the soil used in the experiment as follows: “In detail, the sandy soil comprises 79.3% sand, 19.4% silt, and 1.3% clay. Additional properties include a 3.08% organic matter content, a pH of 7.53, an electrical conductivity (EC) of 1.1 dS/m, an NH₄ (ammonium) concentration of 0.19 mg/Kg, a total nitrogen (N) content of 16.8 g/Kg, and a phosphorus pentoxide (P₂O₅) content of 96.65 mg/Kg.”. Lines 165-168.

 

3. Temporal Chemical Analysis:

The inclusion of samples at various time points for chemical analysis would enrich the understanding of microbial impacts on the decomposition process.

We sincerely thank the reviewer for their valuable suggestion. However, within the specific scope of this study, we opted not to conduct chemical analyses of Posidonia oceanica tissue at various time points to understand the impact of soil microbiota on the decomposition process. Our primary focus was on identifying the most effective microbiota for facilitating rapid decomposition, rather than delving into the nuanced aspects of microbiota influence on decomposition dynamics. Nevertheless, in future perspectives, we plan to employ high-throughput sequencing to identify and characterize the microbial communities present in each of the soil types used. Additionally, we aim to correlate their abundance with the observed mass loss. This approach aligns with our commitment to exploring comprehensive microbial aspects in subsequent research. Moreover, the investigations carried out in prior research by Bonanomi et al. (2022), have already focused on the chemical analyses of Posidonia oceanica residues during decomposition.

Bonanomi, G.; Cesarano, G.; Iacomino, G.; Cozzolino, A.; Motti, R.; Idbella, M. Decomposition of Posidonia oceanica (L.) Delile Leaf Blade and Rhizome in Terrestrial Conditions: Effect of Temperature and Substrate Fertility. Waste Biomass Valorization 2023, 14, 1869–1878, doi:10.1007/s12649-022-01990-9.

 

4. Consideration of Growth Stages:

If the study considered different growth stages of plants, presenting relevant data and engaging in further discussion would bolster the robustness of the findings.

We would like to clarify to the reviewer that, for the paper's objectives, it was not essential to examine the intermediate stages of growth to evaluate the plants' response during the experiment. Consequently, we opted to assess the entire crop cycle of the plants.

 

5. Detailed Explanation of Flower/Fruit/Leaf Ratios:

Offering a more detailed explanation and further discussion regarding observed differences in flower/fruit/leaf ratios would underscore the ecological and physiological significance of these variations.

Done. We would like to thank the reviewer for his suggestion. Recognizing the significance of incorporating flower/fruit/leaf ratios to enhance the ecological and physiological implications of our study, we regret to inform that such data is available solely for Tomato plants. Consequently, its application to other plants in our study is not feasible. However, considering the importance of presenting a comprehensive view of eco-physiological responses, we conducted a thorough analysis. While unable to include flower/fruit/leaf ratios for all plants, we have introduced an alternative ratio that encapsulates the overall eco-physiological dynamics. This is reflected in a newly added table (Table 5), specifically illustrating the shoot-to-root ratios for all tested plants. The obtained results have been presented in lines 285-290: “The shoot-to-root ratios of the cultivated species exhibited distinct responses. Both ornamental species, S. vira vira and S. chamaecyparissus, experienced a reduction in the ratio with increasing P. oceanica application rate. O. basilicum maintained a consistent trend in shoot-to-root ratio, while L. sativa demonstrated a higher ratio in treatments with decomposed P. oceanica compared to fresh P. oceanica. Lastly, in C. pepo and S. lycopersicum, the fertilized treatments yielded higher results than their unfertilized counterparts (Table 5)” and discussed in lines 397-405: “The analysis of the shoot-to-root ratio for all species considered in the experiment provides an eco-physiological perspective on the obtained results. The root, responsible for various functions including mineral nutrition, plays a crucial role in supplying essential mineral elements for the growth of the shoot [42]. Numerous studies have investigated plant responses to nutrient availability over the years. It has been demonstrated that deficiencies in nutrients such as N, P, and K lead to an increase in root biomass and a decrease in shoot-to-root ratios [43,44]. Additionally, shoot-to-root ratios tend to rise with increasing fertilizer concentration [45]. Our results, presented in the Table 5, align with these findings.”

Reviewer 2 Report

Comments and Suggestions for Authors

There are two parts of the paper, The first part which deals with the decomposition dynamics of two different types of  P. 0ceanica  (according to age).The work on the dynamics of the breakdown of  P. 0ceanica is very innovative specially the introduction of ligninolytic microbes from the forest microbiomes. The second part where the authors are using the material as a growth medium to my mind lacks credibility at least from the growing media point  of view despite the fact they got fairly good results. It is good they looked crops with different sensitivity to EC.

Let me explain. First the nutrient profile of the materials (called chemical traits) used is not given. They refer to a previous paper (ref 17). In the previous paper there is little or no information of the materials for total nutrients and  extractable e.g at least P, K,Ca, and Mg. See for instance like the Table 2 of the publication below which gives such information.

Mininni, C.; Santamaria, P.; Abdelrahman, H.M.; Cocozza, C.; Miano, T.; Montesano, F.; Parente, A. Posidonia-Based 472 Compost as a Peat Substitute for Lettuce Transplant Production. HortScience, 2012, 47, 1438-1444.

Information on bulk density and particle size of the material is essential when formulating a growing media.No information presented

There are now standard methods available at an European level (CEN).The lack or excessive  presence of total and extractable nutrients can affect the performance of the plants.Hence data on total and extractable nutrients are essential for this type of work i.e peat substitution.

 See EN methods for soil improvers and growing media.

EN 13037-2002, 2002 Soil Improvers and Growing Media-Determination of pH.

EN 13038-2002, 2002 Soil Improvers and Growing Media-Determination of Electrical Conductivity.

EN 13651-2002, 2002. Soil Improvers and Growing Media-Extraction of Calcium Chloride/DTPA (CAT).

There also EN methods for, pH bulk density and particle size.

The authors then tried to add an N,P and K compound fertilizer(Nitraphoska) There are different types of Nitrophoske.  Please give details which one is used in their trial.  But I ask the authors  what if the material already had high levels of say P and K why add these nutrients as Nitrophoska? In a paper a methodology is used where different quantities of N, P and K are used to equalise levels of nutrients as far as possible. . Idealy they should have analysed the peat and soil for extractable nutrients as well as the two  P. 0ceanica materials and tried to equalise  at least the N, P and K up to a point where it was possible. The equalization of nutrients as far as practicable is essential when the  materials being added to peat. Peat  has a clean sheet as regards nutrients and the new material P. 0ceanica  contains nutrients as we assume to be the case A paper that has used this methodology, see table 1 is worth looking at: Biochar Type, Ratio, and Nutrient Levels in Growing Media Affects Seedling Production and Plant Performance. Agronomy 2020, 10(9), 1421; https://doi.org/10.3390/agronomy10091421 see table 1.

 

 In addition for the Electrical conductivity method they used  was not the a standard method but a somewhat  peculiar method. The authors also used the w/v rather than v/v method. They do not give the bulk density of the materials used as mentioned earlier but I assume the BD of the material would be low and by adding at 10, 30, 50 and 100 % to peat and soil they are changing the nutrient profile of each treatment here. Please  note that more details of the soil used should have been given . Peat is not just one type, what was the peat, young or older peat (on von Post Scale)? Soil can be more variable than peat and details of the soil should be given especially nutrients. However, soil now is very seldom used in container growing at least in Europe.

The authors mention the (phyto) toxicity of Na. Usually in growing media research, the term  phytotoxicity  refers to organic acids etc in the material and not excessive levels of nutrients say e.g Na.It is well known what levels of Na in growing media is likely to be toxic. In the EN method for phytotoxicity the effect of EC is eliminated by dilution before the test is done. The  EN method is "Soil improvers and growing media - Determination of plant response - Petri dish test using cress. prEN 16089"It may be of interest to the authors.

 

Author Response

Let me explain. First the nutrient profile of the materials (called chemical traits) used is not given. They refer to a previous paper (ref 17). In the previous paper there is little or no information of the materials for total nutrients and extractable e.g. at least P, K, Ca, and Mg. See for instance like the Table 2 of the publication below which gives such information.

Mininni, C.; Santamaria, P.; Abdelrahman, H.M.; Cocozza, C.; Miano, T.; Montesano, F.; Parente, A. Posidonia-Based 472 Compost as a Peat Substitute for Lettuce Transplant Production. HortScience, 2012, 47, 1438-1444.

Done. We extend our appreciation to the reviewer for providing this valuable comment. In addition to building upon the insights presented by Bonanomi et al. in 2022, we conducted new analyses on both fresh and decomposed P. oceanica debris, exploring parameters such as CSC, P, K, Ca, Na, Mg, N, C, C/N, C/P, B, Fe, Mn, Zn, Se, Mo, Al, Cu, Pb, Hg, Cr, Ni, Cd, and As. The outcomes of these new analyses have been integrated into the manuscript and are presented in a new Table 1. The text in M&M was integrated as follows: “Further chemical analyses, encompassing major nutrients and heavy metals, were conducted to enhance our understanding of the chemical profile of the utilized P. oceanica (Table 1).”, lines 131-133. The new results have been opportunely integrated in the Discussion.

 

Information on bulk density and particle size of the material is essential when formulating a growing media. No information presented.

Done. We incorporated details about the soil utilized in the experiment and revised the sentence as follows: “In detail, the sandy soil comprises 79.3% sand, 19.4% silt, and 1.3% clay. Additional properties include a 3.08% organic matter content, a pH of 7.53, an electrical conductivity (EC) of 1.1 dS/m, an NH₄ (ammonium) concentration of 0.19 mg/Kg, a total nitrogen (N) content of 16.8 g/Kg, and a phosphorus pentoxide (P₂O₅) content of 96.65 mg/Kg”. Lines 165-169.

In the revised version, we have included the bulk density values for P. oceanica peat and soil respective mixtures in the new Table 2. The text was integrated as follows: “It is widely recognized that the density of P. oceanica is particularly low, surpassing even that of peat (Table 2). As a result, the addition of P. oceanica into both soil and peat caused a reduction in bulk density, proportionate to the amount added to the mixture.”. Lines 172-175.

 

There are now standard methods available at a European level (CEN). The lack or the excessive presence of total and extractable nutrients can affect the performance of the plants. Hence, data on total and extractable nutrients are essential for this type of work i.e peat substitution.

See EN methods for soil improvers and growing media.

EN 13037-2002, 2002 Soil Improvers and Growing Media-Determination of pH.

EN 13038-2002, 2002 Soil Improvers and Growing Media-Determination of Electrical Conductivity.

EN 13651-2002, 2002. Soil Improvers and Growing Media-Extraction of Calcium Chloride/DTPA (CAT).

There also EN methods for, pH bulk density and particle size.

 

Done. We thank the reviewer for the insightful comment, which has highlighted a potential lack of clarity in one of the objectives of our paper. Our main goal was to establish a methodology for the sustainable management of Posidonia oceanica debris, specifically exploring its application as a substrate or as part of a substrate. Our focus was on environmentally friendly uses, particularly in urban gardens and green spaces. It is crucial to clarify that our intention was not to advocate for the replacement of conventional substrates like peat, nor were we seeking to identify a new substrate for efficient use in horticulture or floriculture cultivation systems. Therefore, we have modified the Introduction as follows: “The second strategy evaluated the viability of P. oceanica debris as a growth substrate for ornamental and horticultural plants, directly without undergoing expensive processes like composting. This assessment underscores its potential for eco-friendly applications, including integration into urban gardens and green spaces.”. Lines 89-93.

 

The authors then tried to add an N, P and K compound fertilizer (Nitraphoska) There are different types of Nitrophoske.  Please give details which one is used in their trial.  But I ask the authors what if the material already had high levels of say P and K why add these nutrients as Nitrophoska? In a paper a methodology is used where different quantities of N, P and K are used to equalise levels of nutrients as far as possible... Ideally, they should have analysed the peat and soil for extractable nutrients as well as the two P. oceanica materials and tried to equalise at least the N, P and K up to a point where it was possible. The equalization of nutrients as far as practicable is essential when the materials being added to peat. Peat has a clean sheet as regards nutrients and the new material P. Oceanica contains nutrients as we assume to be the case A paper that has used this methodology, see table 1 is worth looking at: Biochar Type, Ratio, and Nutrient Levels in Growing Media Affects Seedling Production and Plant Performance. Agronomy 2020, 10(9), 1421; https://doi.org/10.3390/agronomy10091421 see table 1.

Done. We thank the reviewer for their suggestion. We added the information about the type of the fertilizer and modified as follows: “…including the application of a ternary fertilizer Nitrophoska® Special 12-12-17, in dosage N (60 Kg / Ha); P (60 Kg / Ha) and K (85 Kg / Ha).” Lines 185-187.

The use of Nitrophoska is rationalized by the limited availability of nutrients in P. oceanica debris and their partial sequestration within the tissue, posing challenges for their release (Table 1). This necessity prompted the incorporation of fertilizer, supported by our results, which demonstrate enhanced plant growth in substrates enriched with the mentioned fertilizer.

 

In addition, for the Electrical conductivity method they used was not a standard method but a somewhat peculiar method. The authors also used the w/v rather than v/v method. They do not give the bulk density of the materials used as mentioned earlier but I assume the BD of the material would be low and by adding at 10, 30, 50 and 100 % to peat and soil they are changing the nutrient profile of each treatment here. Please note that more details of the soil used should have been given. Peat is not just one type, what was the peat, young or older peat (on von Post Scale)? Soil can be more variable than peat and details of the soil should be given especially nutrients. However, soil now is very seldom used in container growing at least in Europe.

Done. We appreciate the reviewer for bringing the oversight to our attention, and we acknowledge the mistake. Primarily, we want to update the reviewer that we have incorporated a new Table 2, including data on the bulk density of peat and soil following the addition of various concentrations of P. oceanica debris. We recognize the significance of this information and concur with the reviewer on its importance. Moreover, we have rectified the unit of measurement of the electrical conductivity as follows:" The pH and salinity (EC) of P. oceanica debris were measured. For this purpose, we used two instruments, the METTLER TOLEDO pH meter for pH and the BASIC 30 CRISON conductivity meter for salinity. Measurements were performed on 2 dm³ dry weight samples of both types of P. oceanica mixed in 3 dm³ of distilled water inside a 5-l beaker after 24 hours of stirring at 200 RPM. This process was repeated three times, and the averages were calculated based on the measurements.”. Lines 123-128.

Soil information have been added in lines 165-169: “In detail, the sandy soil comprises 79.3% sand, 19.4% silt, and 1.3% clay. Additional properties include a 3.08% organic matter content, a pH of 7.53, an electrical conductivity (EC) of 1.1 dS/m, an NH₄ (ammonium) concentration of 0.19 mg/Kg, a total nitrogen (N) content of 16.8 g/Kg, and a phosphorus pentoxide (P₂O₅) content of 96.65 mg/Kg.”

Characteristics concerning peat has also been added in lines 169-172: “The utilized peat is classified with a H5 value on the von Post Humification Scale, with properties comprising a pH of 5.87, electrical conductivity (EC) of 0.95 dS/m, NH₄ content of 58.3 mg/kg, N (as Kjeldahl nitrogen) of 0.98 g/kg, P of 0.93 mg/kg, K of 1.96 mg/kg, and Ca of 1.45 mg/kg.”.

 

The authors mention the (phyto) toxicity of Na. Usually in growing media research, the term phytotoxicity refers to organic acids etc in the material and not excessive levels of nutrients say e.g Na. It is well known what levels of Na in growing media is likely to be toxic. In the EN method for phytotoxicity the effect of EC is eliminated by dilution before the test is done. The EN method is "Soil improvers and growing media - Determination of plant response - Petri dish test using cress. prEN 16089"It may be of interest to the authors.

We would like to thank the reviewer for their comment. In the literature, the term "phytotoxicity" is often associated with the harmful effects on plant growth caused by various factors, including excessive levels of nutrients such as sodium. While organic acids are commonly discussed in growing media research, it is essential to recognize that the term can be applied more broadly to encompass any substance or condition that negatively affects plant development at certain concentrations. Moreover, we would like to thank the reviewer for his insights on the importance of dilution, which aims to minimize the confounding effects of EC, allowing for a more focused evaluation of the potential phytotoxicity of sodium itself. We appreciate this clarification and will certainly consider this method when conducting our Petri dish test for growing media, as it aligns with our goal of thoroughly investigating the impact of sodium on plant growth.

 

Reviewer 3 Report

Comments and Suggestions for Authors

Be sure to be clear that the % mix (with peat or soil) is stated as on a dry wt basis.

On pg 3 line 117 and 136 by means of (needs the s). in l 137 I think the correct word is beaker, not backer or bucket. in materials Mettler has two t.

In table 1,   Ocimum basilicum needs to be spelled out more fully.

 

A fundamental concern with this project is the economics of converting the beach waste into a useful culture medium. The difficulty in removing salinity, and the dispersed nature of the banquettes does not favor its use for most purposes.

Comments on the Quality of English Language

Other than the  points noted above, the English language is quite adequate. The above comments are not about language usage but mostly typing or formatting errors

Author Response

Be sure to be clear that the % mix (with peat or soil) is stated as on a dry wt basis.

Done. We thank the reviewer for pointing out this point. We have, thus, clarified the sentence as follows: “For ornamental species, P. oceanica debris were mixed with peat at different application rates, in terms of dry weight, namely: 10%, 30%, 50%, and 100%. Instead, for horticul-tural species, the debris were mixed with a sandy soil, as previously characterized by [23].”, lines 162-165.

 

On pg 3 line 117 and 136 by means of (needs the s). in l 137 I think the correct word is beaker, not backer or bucket. in materials Mettler has two t.

Done. We have corrected the terms indicated: “by mean of” to “by means of” lines 112 and 149; and “backer” to “beaker” line 149; “MELTER” in “METTLER” line 124.

 

In table 1, Ocimum basilicum needs to be spelled out more fully.

Done. We have corrected O. basilicum in the Table 3.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The Version 2 of the paper is now suitable for publication.

Author Response

We thank the reviewer for this comment.