Toward a New Way for the Valorization of Miscanthus Biomass Produced on Metal-Contaminated Soils Part 1: Mesocosm and Field Experiments

Round 1

Reviewer 1 Report

The paper of C. Waterlot et al on the valorization of miscanthus biomass for remediation of contaminated soil is well performed and gave interesting results. However, some mitigation of potential commercial use of biomass is necessary.

 

Some comments:

• Why did the authors use the concept of “potentially toxic elements”? Cd and Pb are toxic elements, and they are present in the soil, leaves of miscanthus or ashes form miscanthus is a problem. As such, the authors should mitigate the potential of marketable of Cd and Pb contaminated biomass.

 

• I don’t know why the presence of toxic elements (Cd, Pb) among nutrients has potential for the miscanthus biomass valorization. The presence of these toxic elements must be further discussed.

 

• Miscanthus was used for bioremediation, but no information on the bioconcentration factor of metal was discussed. We could only have table 2 difficult to read. No comparison with other biomass is made …

 

• The authors use monocalcium phosphate (MCP) and dicalcium phosphate (DCP), but the same results were obtained. The chemical properties of these phosphates salts are different. The authors should comment on why no difference could be detected in section 3.3. In this section, the authors mentioned the effect of DCP on Fe and Zn immobilization, but no comparison could be found in the text with MCP (and neither from the results in Tables 3 and 4).

 

• Phosphate influences biomass (Figures 5 and 6), but what is the influence of phosphate on bioconcentration factor ?

Author Response

Response to Reviewer 1 Comments

 

 

The authors would like to thank the reviewers for their interest in our research, their remarks recommendations and comments to improve the quality of the manuscript.

The paper of C. Waterlot et al on the valorization of miscanthus biomass for remediation of contaminated soil is well performed and gave interesting results. However, some mitigation of potential commercial use of biomass is necessary.

 Some comments:

• Why did the authors use the concept of “potentially toxic elements”? Cd and Pb are toxic elements, and they are present in the soil, leaves of miscanthus or ashes form miscanthus is a problem. As such, the authors should mitigate the potential of marketable of Cd and Pb contaminated biomass.

I agree with the reviewer. The concept of potentially toxic elements is quite ambiguous in our study since Cd et Pb are known as carcinogenic, mutagenic and reprotoxic compounds. Therefore, potentially toxic metals was replaced by metals throughout the manuscript.

As recommended by the reviewer the concept of potential marketable biomass was removed from the summary and keywords. (In fact, the percentage of Cd and Pb in our miscanthus is below 0.05% if I consider only the concentration of metals).  

• The authors use monocalcium phosphate (MCP) and dicalcium phosphate (DCP), but the same results were obtained. The chemical properties of these phosphates salts are different. The authors should comment on why no difference could be detected in section 3.3. In this section, the authors mentioned the effect of DCP on Fe and Zn immobilization, but no comparison could be found in the text with MCP (and neither from the results in Tables 3 and 4).

The results from Table 3 show that the effects of DCP and MCP on the extractable-metal concentrations are not the same for Fe (in mesocosm) and Zn, Fe in field experiment (10-25 cm). That’s why we focused on DCP. Regarding the concentration in leaves and stems of miscanthus, no statistical difference was highlighted between modalities. In mesocosm, this result is explained by the high standard deviation (Fe and Zn). One again, for our application, DCP is more attractive sonce for us, the average concentration 139 mg Zn / kg + 56 mg Fe / kg instead of 119 mg Zn / kg + 34 mg Fe / kg may change the activity or the future catalyst.   

• Miscanthus was used for bioremediation, but no information on the bioconcentration factor of metal was discussed. We could only have table 2 difficult to read. No comparison with other biomass is made. Phosphate influences biomass (Figures 5 and 6), but what is the influence of phosphate on bioconcentration factor ?

The following section was added to the manuscript.

The average bioconcentration factors (BCF) of Cd, Pb, Zn, Cu, Mn, Fe, K, Ca in the leaves and stems of miscanthus were summarized in Table 5. The BCFs allowed ranking the accumulation of metals in mesocosm experiment as follows: K>Ca>Mg>Cu>Cd>Zn>Mn> Fe>Pb in leaves and K>Ca>Cd>Mg>Cu>Zn>Mn>Pb>Fe for each modalities. Results from field experiments showed that these orders are: K>Ca>Mg>Cu>Cd>Mn>Zn>Fe>Pb in leaves and K>Ca>Mg>Cu>Cd>Zn=Mn>Fe=Pb in stems. These results correlated well with those of Nsanganwimana et al. for Cd, Pb and Zn [22]. On the other hand, the bioconcentration factors are generally less than 1 suggesting that metal transfer from the soil to miscanthus is limited. This result confirms that Miscanthus x giganteus is an excluder species suitable for phytostabilization [15,42]. However, it is worth mentioning that K-BCF and Ca-BCF in leaves were greater than 1. As shown in Table 5, DCP enhanced the transfer of Ca from the soil to the leaves of miscanthus and positively influenced the biomass of leaves. Consequently, potential utilization of leaves of miscanthus to catalyse organic reactions could be investigated in the future.

Table 5. Bioconcentration factors (BCF) of metals in the studied organs of miscanthus produced in mesocosm and field experiments in presence or not of P-fertilizers.

* Ø: without phosphate; ** MCP: Monocalcium phosphate; *** DCP: Dicalcium phosphate.

Author Response File: Author Response.pdf

Reviewer 2 Report

It is an interesting paper on the valorisation of miscanthus biomass produced on metal contaminated soils. This paper demonstrates the novelty and importance of new potential valorisation ways for leaves and stems biomass. The work presented here is of relevance to other studies or how to extend methodological approaches beyond the state-of-the-art in sustainable use of P-fertilizers in soils. To the best of my knowledge the experimental labor has been carried out with care and the conclusions have been inferred prudently.  Nevertheless, I am not sure that the title is the most appropriate. Please justify how soil was sampled on sub-plots, and were considered as pseudo-replicates. Authors should explain better why they are selected this soil and its classification. I suggest to improve the figures and captions. Please use only a decimal in the data or pH and organic carbon. Finally, I suggest to delete the content of calcium carbonate as data and naturally their method of determination: you only must to point out “without carbonates “.

Author Response

Response to Reviewer 2 Comments

 

 

The authors would like to thank the reviewer for their interest in our research, their remarks recommendations and comments to improve the quality of the manuscript.

 

It is an interesting paper on the valorisation of miscanthus biomass produced on metal contaminated soils. This paper demonstrates the novelty and importance of new potential valorisation ways for leaves and stems biomass. The work presented here is of relevance to other studies or how to extend methodological approaches beyond the state-of-the-art in sustainable use of P-fertilizers in soils. To the best of my knowledge the experimental labor has been carried out with care and the conclusions have been inferred prudently.

Nevertheless, I am not sure that the title is the most appropriate. Please justify how soil was sampled on sub-plots, and were considered as pseudo-replicates. Authors should explain better why they are selected this soil and its classification. I suggest to improve the figures and captions. Please use only a decimal in the data or pH and organic carbon. Finally, I suggest to delete the content of calcium carbonate as data and naturally their method of determination: you only must to point out “without carbonates “.

0 – I don’t know if the reviewer will review the second part 2 of this study. If not, the general title “Toward a new way for the valorisation of miscanthus biomass valorisation produced on metal-contaminated soils” is common to the first and second part. In this last part, miscanthus was used to synthesize Moclobemide, a main ingredient of a drug used to treat depression.

1 - This soil was chosen for our experiments due to the fact that it is one of our in-situ sites in which experiments began in 2010. That’s why, at the end of the introduction and just before ginving the aim of this study, the context is described :

In 2010, an in situ rehabilitation project (PHYTENER) was initiated with the aim of (i) developing a phytostabilisation process of metals and (ii) producing plant biomass for energy applications. Indirect planting of rhizomes from a perennial grass (Miscanthus x giganteus) was conducted on a 0.8 ha agricultural plot (named M700) with different modalities (i.e. density from 15,000 up to 20,000 plants ha^-1) [20]. Rhizomes of miscanthus (hybrid plant) were supplied from Novabiom (formely Bical Biomass). The experimental agricultural plot (M700; latitude: N 50° 26' 15.9''; longitude: E 3° 01' 04.9'') was located at Evin-Malmaison in the north of France at about 1 km north from the former lead smelter (Metaleurop Nord) (Figure 1). M700 is a silty loam and alkaline soil, poorly carbonated (0.4%) and with low organic matter content (2.7%) [20,21]. The study of Nsangawimana et al. [22] showed that concentrations of Pb, Zn, Ca and Na were higher in leaves than in stems in summer and autumn, whereas concentrations of N, P, K and Mg decreased. Consequently, for best practices in our phytomanagment, we decided to harvest the stems of miscanthus after the senescent period and to let leaves of miscanthus on soil. Recently, impact of leaf decomposition and the consequences on metal behaviour and on the succeeding culture was studied [23]. If no persistent impacts of the leaf litter on the soil-miscanthus system was highlighted, the authors showed a significant increase of Cd and Pb CaCl₂-extractable concentrations. This result means that the availability of Cd and Pb increases in time when contaminated areas are harvested late (after senescence). Consequently, the current study aimed at evaluating the sustainable use of phosphates on the (i) leaves and stems biomass of miscanthus and (ii) bioavailability of metals and nutrients in order to propose a potential new valorisation method of the aerial organs of miscanthus.

2 - The quality of figure depends on their size that will be corrected on the final version of the manuscript if accepted for publication.

3 - Tittle and legend of Figures and Tables were checked.

4 - Title of axes were added in figures 2, 4, 5, 6.

5 - One decimal was used in the data of pH and organic matter as recommended.

6 - The content of calcium carbonate was removed from Table 1 since he concentration was below 1 mg kg^-1 for each modality.

7 - The method determination of calcium carbonate was removed from the section 2.3.1 title “Physicochemical parameters and metal concentrations”.

 

Reviewer 3 Report

After carefully reading, the current manuscript needs major revision.

1. L27: "from 0.022% to 0.026% w:w" The addition rate has never mentioned in the text, please add into the revised text and describe it.
2. L30: "... since Ca concentration was up to 9000 mg kg^-1 DW." The concentration of Ca in Table 4 or any Table is not up to 9000 mg kg^-1 DW.
3. L30-31: unclear description, the study results of current study cannot prove it.
4. L98, section 2.1: Please rephrase this section. Field treatment is very important in this study, but the authors just use citations to describe it (reference 24 and 25). Please describe more clearly, including the amount of MCP and DCP, how to apply, adding other fertilizer or not, soil water control (irrigation or not), field management, etc.
5. L182, section 3.1: Please supply much more soil physico-chemical characteristics in the main text or as the supplemental materials. The introduction of raw metal-contaminated soi is important for reader to understand understand the in situ soil charcateristics.
6. L200-201: uncleaer sentence, "significant differences"? from which table or figure?
7. All tables have no significant difference analysis. In order to compare the difference between treatments, and before and after treatment, the significant difference analysis is necessary in this study.
8. Figure 2, 5, and 6 are lack of title of X; Figure 4 is lack of title of X and Y axis.
9. L230-231: :OM....; FDAH...." should be deleted.
10. L276: repeated sentence, delete
11. L389 and L394:"OM.." delete
12. L397, section 4: Similar with the abstract, please rephrase. In addition, L402-405 should be clearly described in the text and prove it.

Author Response

Response to Reviewer 3 Comments

 

 

The authors would like to thank the reviewers for their interest in our research, their remarks recommendations and comments to improve the quality of the manuscript.

 

1. L27: "from 0.022% to 0.026% w:w" The addition rate has never mentioned in the text, please add into the revised text and describe it.

The following sentence was added into the manuscript (Line 104-106 in the new version).

Each phosphate-calcic compound was added to the soil surface with respect to the molar ratio P/(Pb+Cd) = 3/5 using Pb₅(PO₄)₃(F,Cl,OH) as reference [25]. Thus, each amended subplot received 73.8 g MCP (0.022% w:w) or 85.8 mg DCP (0.0.26% w:w).

2. L30: "... since Ca concentration was up to 9000 mg kg^-1" The concentration of Ca in Table 4 or any Table is not up to 9000 mg kg^-1DW.

The concentrations of Ca in Table 4 are expressed as mean ± standard deviation (n=6). The average concentrations of Ca in the leaves of miscanthus grown on soils under mesocosm experiments were higher 7462-7976 mg/kg. Among the eighteen Ca concentrations (6 replicates*3 modalities), some of them were up to 9000 mg/kg.

3. L30-31: unclear description, the study results of current study cannot prove it.

Among the Ca concentrations measured in the stems of miscanthus, some of them were higher than 9000 mg/kg :

8 760; 5 789; 6 538; 7 471; 9 006; 7 146; 8 314; 7 529; 7 279; 8 521; 7 744; 8 468; 7 599; 8 535; 8 035; 8 104; 9 239; 6 166 mg Ca Kg^-1.  

 

4. L98, section 2.1: Please rephrase this section. Field treatment is very important in this study, but the authors just use citations to describe it (reference 24 and 25). Please describe more clearly, including the amount of MCP and DCP, how to apply, adding other fertilizer or not, soil water control (irrigation or not), field management, etc.

The following sentences were added into the manuscript (Line 104-106 in the new version).

Each phosphate-calcic compound was added to the soil surface with respect to the molar ratio P/(Pb+Cd) = 3/5 using Pb₅(PO₄)₃(F,Cl,OH) as reference [25]. Thus, each amended subplot received 74 g MCP (0.022% w:w) or 86 g DCP (0.0.26% w:w). No other fertilizer was added and the rainfall was 203 L m² from the beginning to the end.

5. L182, section 3.1: Please supply much more soil physico-chemical characteristics in the main text or as the supplemental materials. The introduction of raw metal-contaminated soil is important for reader to understand the in situ soil characteristics.

The following sentences were added into the manuscript.

Clay, silt and sand in the studied soil were 19.5%, 53% and 27.5%, respectively [20]. The cation exchange capacity was 14.9 ± 1.6 cmol⁺ kg^-1, the concentration of phosphorus was 0.16 mg P₂O₅ ± 1.6 cmol kg^-1, mineralisation index was C/N = 15.2 ± 0.4 and the total carbonate was below 1 mg kg^-1 for the 9 modalities. The most important physico-chemical parameters for the study and the microbial activity. The most important physicochemical parameters for the study and the microbial activity of the soil used in mesocosm and field experiments were summarized in Table 1.

 

6. L200-201: unclear sentence, "significant differences"? from which table or figure?

The sentence was re-phrased as recommended by the reviewer.

No significant effect of P-fertilizers was highlighted on soil pH and organic matter (p<0.05; Table 1).

7. All tables have no significant difference analysis. In order to compare the difference between treatments, and before and after treatment, the significant difference analysis is necessary in this study.

Data from Tables 1 and 2 were described in section 3.1 in which no statistical difference between data from each table was highlighted. This was similar for pH and organic matter before and after treatments. In contrast, difference in FAH was added for each modality (Table 1). Modifications were reported in Tables 3, 4

 

 

Round 2

Reviewer 3 Report

Dear authors:

In the abstract, you metioned about "The effects P-fertilizers (mono- and di-calcium phosphates) on the bioavailability of metals (Cd, Pb) and nutrients in leaves and stems of Miscanthus x giganteus were studied in mesocosm and field experiments in order to propose a new way for the miscanthus biomass valorisation.", but the conclusion has changed to "Leaves of the investigated perennial grass may be of great interest as catalyst in organic chemistry since Ca concentration was up to 9000 mg kg-1 DW. Stems represent a potential biomass that can be used as renewable resource of Lewis acids, currently used in organic syntheses (the sum of Zn, Cu, Mn, Fe, Mg, Si and Al was near 1000 mg kg-1 DW).".

1. How about the Cd and Pb in the current study?
2. The objectives of the current study are unclear: the authors try to reduce the bioavailability  concentration of Cd and Pb in studied contaminated soil or try to increase the concentration of Ca and sum of Zn, Cu, Mn, Fe, Mg, Si, Al and Ca concentration in plant for the valorisation of miscanthus  biomass valorisation produced on metal-contaminated soils ?
3. My comment #12 "L397, section 4: Similar with the abstract, please rephrase. In addition, L402-405 should be clearly described in the text and prove it.", the authors have no reply. The contents of abstract and conclusions are almost the repetition, please clearly indicate the main objective of the current study and re-write the conclusions.

Author Response

Response to Reviewer Comments

 

 

The authors would like to thank the reviewer for the three remarks and questions.

In the abstract, you metioned about "The effects P-fertilizers (mono- and di-calcium phosphates) on the bioavailability of metals (Cd, Pb) and nutrients in leaves and stems of Miscanthus x giganteus were studied in mesocosm and field experiments in order to propose a new way for the miscanthus biomass valorisation.", but the conclusion has changed to "Leaves of the investigated perennial grass may be of great interest as catalyst in organic chemistry since Ca concentration was up to 9000 mg kg-1 DW. Stems represent a potential biomass that can be used as renewable resource of Lewis acids, currently used in organic syntheses (the sum of Zn, Cu, Mn, Fe, Mg, Si and Al was near 1000 mg kg-1 DW).".

The authors thank the reviewer. Information about Cd and Pb was added at the end of the abstract and “(Cd, Pb)” was removed from the abstract since metals studied in this study are not only two.

The effects P-fertilizers (mono- and di-calcium phosphates) on the bioavailability of metals and nutrients in leaves and stems of Miscanthus x giganteus were studied in mesocosm and field experiments in order to propose a new way for the miscanthus biomass valorisation. The concentration of potentially toxic elements were generally higher in stems than in leaves. Although P-fertilizers was added to contaminated soils under sustainable conditions (from 0.022% to 0.026% w:w), the average of leaves and stems biomass generally increased in the presence of P-fertlizers due to the changes in the speciation of phosphorus. Leaves of the investigated miscanthus may be of great interest as catalyst in organic chemistry since Ca concentration was up to 9000 mg kg^-1 DW. Stems represent a potential biomass that can be used as renewable resource of Lewis acids, currently used in organic syntheses (the sum of Zn, Cu, Mn, Fe, Mg, Si and Al was near 1000 mg kg^-1 DW). The percentage of Cd and Pb in leaves and stems of miscanthus did not significantly change with P-fertilizers. Depending on the mesocosm and field experiments, it ranged from 0.004% to 0.016% and from 0.009% and 0.034% for Cd in leaves and stems, respectively and from 0.004% to 0.015% and from 0.009% and 0.033% for Pb in leaves and stems, respectively.

 .

 

1. How about the Cd and Pb in the current study?

The percentage of Cd and Pb in leaves and stems of miscanthus did not significantly change with P-fertilizers. Depending on the mesocosm and field experiments, it ranged from 0.004% to 0.016% and from 0.009% and 0.034% for Cd in leaves and stems, respectively and from 0.004% to 0.015% and from 0.009% and 0.033% for Pb in leaves and stems, respectively.

 

1. The objectives of the current study are unclear: the authors try to reduce the bioavailability  concentration of Cd and Pb in studied contaminated soil or try to increase the concentration of Ca and sum of Zn, Cu, Mn, Fe, Mg, Si, Al and Ca concentration in plant for the valorisation of miscanthus  biomass valorisation produced on metal-contaminated soils ?

 

The current study aimed at evaluating the sustainable use of phosphates on the (i) leaves and stems biomass of miscanthus and (ii) bioavailability of metals and nutrients in order to propose a potential new valorisation method of the aerial organs of miscanthus. To do this, it is possible (i) to reduce the bioavailability of cd and Pb and increase the bioavailability of other metals, (ii) to have unchanged Cd and Pb bioavailability and to increase the bioavaiability of the other metals and (iii) to increase the bioavailability of all metals but to reduce the fraction [Cd]/[metals] and [Pb]/[metals]. The strategy depends on the biomass. In the current study, leaves of miscanthus seem interesting in organic synthesis in view of the Ca concentration. Stems are interesting regarding the sum of metal concentrations (Zn, Cu, Mn, Fe, Mg, Si, Al and Ca). The great interest in sing miscanthus is due to the fact that miscanthus is known as an excluder plant of Cd and Pb and so the concentration of both metals are very low. At the stage of our research, we have explored the use of stems of miscanthus in organic synthesis (Part 2 submitted in this Journal). More later we will focused on the use of leaves and we will try to extract Ca from these leaves. 

 

1. My comment #12 "L397, section 4: Similar with the abstract, please rephrase. In addition, L402-405 should be clearly described in the text and prove it.", the authors have no reply. The contents of abstract and conclusions are almost the repetition, please clearly indicate the main objective of the current study and re-write the conclusions.

Modification was reported in the last version of the manuscript (lines 361-363) but it is not possible to add values in detail (like 8 760; 5 789; 6 538; 7 471; 9 006; 7 146; 8 314; 7 529; 7 279; 8 521; 7 744; 8 468; 7 599; 8 535; 8 035; 8 104; 9 239; 6 166 mg Ca Kg^-1). Modification of sentence is below :

Finally, it is worth mentioning the high average concentration of Ca in the leaves of miscanthus from microcosm and field experiments (up to 7300 mg kg^-1 DW and 9000 mg kg^-1, respectively and depending on the replicates (n=18)) and thus, their potential interest in organic synthesis.

 

The conclusion was re-phrased in part as follow:

 

Sustainable amount of P-fertilizers (MCP and DCP) were added to soil to evaluate their effects on (i) the accumulation of metals and nutrients in leaves and stems of miscanthus, (ii) the biomass of the aerial parts (leaves and stems) and (iii) the length and the diameter of stems. Organic phosphorus increased overtime and biomass production was favoured in presence of P-fertilzers. Significant effects of phosphates were registered on LMWOAs-extractable concentrations of Zn, Fe and K and allowed an increase of the sum metal concentrations (Zn, Cu, Mn, Fe, Mg, Si and Al) in stems of miscanthus making them suitable in organic synthesis. Otherwise, very high concentration of Ca have been measured in the leaves of miscanthus, which can be used as a renewable resource of this essential element in organic synthesis or in other applications.

Author Response File: Author Response.pdf