Applied Tests to Select the Most Suitable Fungal Strain for the Recovery of Critical Raw Materials from Electronic Waste Powder
, Grazia Cecchi 1, Michela Mazzoccoli 2, Micol Zerbini 3, Anna Maria Cardinale 3,†and Mirca Zotti 1,†Round 1
Reviewer 1 Report
Page 2 line 79: Digestion protocol for fine powder is missing. Add it.
Page 3 line 105-111: It is not described when and how the e-waste powder was added to malt extract agar. Describe it. Where and when do you place the microporous membrane mentioned in line 178 on page 5? How large are pores of that membrane?
Page 3 line 122: Is TI the tolerance index?
Page 4/5 Table3: Is the biomass shown in table 3 the Biomass on mm² after 18 days? If so the data from figure 2 don’t correspond. In fig. 2 the biomass of Penicillium glandicola is 5 times lower than in table 3 and even lower than biomass for Trichoderma harzianum. Explain the differences.
Page 5 table 4 How do you determine the pH of MEA dishes? What is the pH range of growth for the three strains? In which pH range do the CAS assay work? Show the pH sensitivity test for it.
Page 6 table 5 The solubility of metals in WEEE powder differs between the petri dishes of that 3 strains. How is that fact included in TI calculations?
Page 6/7 figure 3/4 What should I see? What is BFC Biomass?
Page 7/8 lines 224 to 245 The used methods show high uncertainness, therefor this part is not confirmed by presented results. Redo the experiments with other methods or validate the methods with further experiments.
Page 8 line 266 A test on agar dish don’t has a TRL5. Proof that claim with experiments in industrially relevant environment!
Author Response
Dear Reviewer,
Thank you very much for all the comments and suggestions that were helpfulto implement the article. Below we provide answers to comments, the details of the changes made are in the main document.
Advice: Page 2 line 79: Digestion protocol for fine powder is missing. Add it.
Reply: we clarified the protocol.
Advice: Page 3 line 105-111: It is not described when and how the e-waste powder was added to malt extract agar. Describe it. Where and when do you place the microporous membrane mentioned in line 178 on page 5? How large are pores of that membrane?
Reply: we clarified it in the text, we added where and when we placed the membrane on the plates and added the length of the pores.
Advice: Page 3 line 122: Is TI the tolerance index?
Reply: TI is the tolerance index and it has been better detailed in the text.
Advice: Page 4/5 Table3: Is the biomass shown in table 3 the Biomass on mm² after 18 days? If so the data from figure 2 don’t correspond. In fig. 2 the biomass of Penicillium glandicola is 5 times lower than in table 3 and even lower than biomass for Trichoderma harzianum. Explain the differences.
Reply: thank you for the advice. We explained more in the text how the values in table three were obtained and how the curves in figure 2 were obtained. The value in the table corresponds to the specific weight of the mycelial biomass, while the curves are obtained by multiplying the biomass by the average colony surface area measured on each plate at different time.
Advice: Page 5 table 4 How do you determine the pH of MEA dishes? What is the pH range of growth for the three strains? In which pH range do the CAS assay work? Show the pH sensitivity test for it.
Reply: the pH value was measured using pH meter equipped with special probe for direct misurations (Hanna Instrument - HI12923) first on the plates before inoculation and then was measured under the colonies after the 18-day growth period. The pH of CAS agar was buffered at 5.6. According to Machuca and Milagres 2003 the pH does not affect the CAS agar test. The change in pH is due to the production of different types of acids from the strains that can sometimes also act as siderophore.
Advice: Page 6 table 5 The solubility of metals in WEEE powder differs between the petri dishes of that 3 strains. How is that fact included in TI calculations?
Reply: The culture medium with the addition of the semi-finished product containing the metals was prepared in a single aliquot and then distributed for all the petri dishes. Due to the preparation method the TI value indicates which is the most resistant strain.
Advice: Page 6/7 figure 3/4 What should I see? What is BFC Biomass?
Reply: To describe the adsorption performance of the different strains with respect to the different metals has been chosen the Bioconcentration Factor (BFC), as described in literature (Jakubiak et al.,2013). This parameter has been better described in chapter 3.4.
Advice: Page 7/8 lines 224 to 245 The used methods show high uncertainness, therefor this part is not confirmed by presented results. Redo the experiments with other methods or validate the methods with further experiments.
Reply: thank you for the clarification, indeed the discussion was unclear and presented uncertainty we have better explained how to interpret the results obtained in order to be able to efficiently screen the strains and interpret the results.
Advice: Page 8 line 266 A test on agar dish don’t has a TRL5. Proof that claim with experiments in industrially relevant environment!
Reply: thanks for the advice. We didn't mean that this experiment has TRL 5 we meant that our process is useful to scale up the technology towards level 5. To date, there are no existing experiments in industrial use precisely because of the difficulties often encountered in finding strains with performance that make the process economically viable. Since fungal activity is often strain-specific, it is important to have method, like our, that allow test different strains quickly and rationally.
Reviewer 2 Report
The idea of the paper is good and needed. However the manuscript lacks details and some missing data should be provided.
Major concern - methods are insufficiently described. Please provide details about the producers of reagents and equipment. Add times and volumes, etc.
Using this sentence as an example:
Later, in order to remove any polymeric residue present, the powder with added sawdust undergoes a microwave treatment in absence of oxygen at 1 kW.
- is this a modification of a known method(?) (citation?)
- how much powder, how much sawdust (what kind of sawdust?), proportions?
- microwave (what equipment?)
- how was "absence of oxygen" obtained? what gas was used for the atmosphere?
- how long was the process? how its completion was verified?
This is just an example. Please go over all the materials/methods section and add the missing info. Such details are necessary for the experiments to be comparable and repeatable.
Where there any (what were) the controls for all the experiments?
Is Table 1 showing the actual composition or just the selected elements? Were any other elements analyzed (ICP-MS) than those presented? Sulfur?
Is the process indeed "adsorption" or "absorption" -- could the Authors provide some evidence? (e.g. SEM/TEM images?)
in this context please correct - Line 131 - "bioassorbed" (?)
Five replicates allow for some (basic) statistics. Indeed standard deviation is mentioned in Table 5. How it was calculated? Why statistical data is missing in other tables/figures/results? Please make the necessary statistical calculations and add them to the data presented.
Although BFC is an interesting factor for data presentation, I recommend adding raw ICP data (the concentrations of metals in the biomass) as a table (e.g. in the supplementary materials section).
Figures with BFC show only some of the elements known to be in the waste (as shown in Table 1). Please add data for all the elements in the BFC figures or indicate why they are missing in figure captions.
line 228 - (This datum confirms) - archaic, better use "data"
Although the manuscript is more focused on the method than the actual "fungal" result, I nevertheless recommend to add a comment in the conclusions which strain (of the tested) is the best.
Author Response
Dear Reviewer,
thank you for your suggestion, we have implemented information in the text about the methodologies, instruments and reagents used during the experiments.
Below we provide answers to comments, the details of the changes made are in the main document.
Advice: Where there any (what were) the controls for all the experiments?
Reply: the description of the controls was added in the text
Advice: Is Table 1 showing the actual composition or just the selected elements? Were any other elements analyzed (ICP-MS) than those presented? Sulfur?
Reply: to characterize the semi-finished product coming from the waste, attention has been focused on metals because those are the elements studied regarding absorption on fungi
Advice: s the process indeed "adsorption" or "absorption" -- could the Authors provide some evidence? (e.g. SEM/TEM images?)
Reply: The process is erroneously described and called in some paper lines as “adsorption” or “absorption”. We corrected it in the text. The process we exploited by fungi was “bioaccumulation”, active method of accumulation mediated by the fungal metabolism.
Advice: in this context please correct - Line 131 - "bioassorbed" (?)
Reply: Done.
Advice: Figures with BFC show only some of the elements known to be in the waste (as shown in Table 1). Please add data for all the elements in the BFC figures or indicate why they are missing in figure captions.
Reply: the composition of e-waste powder is very complex. We indicated the most relevant metals, rare earth elements and precious metals recovered by fungi.
Advice: line 228 - (This datum confirms) - archaic, better use "data"
Reply: the text has been edited as suggested
Advice: Although the manuscript is more focused on the method than the actual "fungal" result, I nevertheless recommend to add a comment in the conclusions which strain (of the tested) is the best.
Reply: Done.
Reviewer 3 Report
General Comments:
The authors have conducted some preliminary tests to demonstrate that 3 commonly used for metal bioremediation fungi strains can also produce siderophores (2/3) and bioaccumulate a handful of metals from electronic waste or WEEE, as they have dubbed it. However, the authors have not provided enough evidence to justify their claims that they have developed an improved or standardized method for screening fungi for WEEE recovery or that they have met tech ready level 4.
There are several typos and grammar mistakes throughout manuscript.
Specific Comments:
Introduction
- Details are sparse, would benefit from specific examples throughout introduction paragraph such as names of key metals, names of microorganisms, also better explanation of lines 32-33.
- lines 34-35: does WEEE count as raw materials? raw materials are unprocessed? It is certainly underutilized material.
- Last paragraph needs to be rewritten. In present form, it is difficult to interpret. (Lines 63-69)
- additional citations should be considered throughout manuscript. The authors only cite 22 articles, which is a little sparse for such well-studied bioremediation fungi, the methods selected and the recent literature on critical metal recovery. The authors also need additional citations to support the claim that they have developed a new selection method for WEEE recovering fungi.
Materials and Methods
- Manufacturer information was not provided for equipment nor reagents, which is important for such sensitive measurements. Model number of major equipment like exact ICP used was also not provided.
- Data for their tests of the metals concentrations in media and agar were not provided or not conducted. It is important to know if any traces of metals were also present in the growth medium and agar used that would give a false positive or additional values to the accumulation if not from e-waste.
- Similarly, the ICP values for metals in biomass for no e-waste plate (media only) was not mentioned, which is a necessary control and comparison.
- method for measuring pH was not provided nor was the removal of biomass with a filter and what that was used for, only mentioned in results.
- lines 78-9, 86-87 Insufficient detail provided about ICP-MS method.
- line 90 needs a citation and more description about why these 3 strains were chosen
Results
- In general, the results subsections are too short and do not completely describe the results presented or interpret the findings. Additionally, some tables and figures are presenting the same data.
- All captions need revision. Captions should be a stand alone description of the figure, not just a simple title. Description should also include number of replicates and method used to calculate error bars etc.
- Many figures need revision as well. Some a missing axis unit labels. Some use commas for decimals while others use periods. Sometimes the title or acronym is misspelled. The labeling in the results also does not match the results (mm vs cm; tolerance level not presented in results).
Line 139- what constitutes the semi-finished product? (Not mentioned in methods). I see no error bars, or ranges. Was this number only from one sample? Were only these metals detected? ICP usually analyzes all. How do these values compare to growth media?
Figure 1, Table 2- and lines 144-7. Where is orange halo for t har (e &f). It is not visible. I also don't understand how a measurement of 0 mm diameter is a positive, weak result for siderophore production. Did they only do one plate for each strain? At least triplicates should be performed. Un-inoculated control plate? What values define the cutoffs for calling it strong/weak. How were those cut offs determined? What does the teal color mean?
Line 161- how is stationary determined? What tool is used to scrap off the biomass? How is it determined that no agar was removed?
Lines 163-9, Table 3 & Figure 2. Are the samples in Figure 2 also from a 1 mm2 area? Are they taken from same plate everyday? How decide where to harvest on the plate? Colony growth is not a perfect circle. What data is table 3 showing that is not in figure 2? Table 3 no stnd dev or any ranges for these values- did they only measure 1 sample? Figure 2- which type of error bars are shown? Which experiments were done with the reported 5 replicates?
Lines 177-80 and Table 4: this whole part is not in the methods and seems important. Need way more info about this membrane removal and how they measured pH. Also, why is pH relevant to WEEE recovery? Again replicates for measurements?
Lines 186-9: Authors should describe interpretation of these values
Section 3.4: Is it BCF or BFC- both are used. What is BFC for biomass? commas vs periods for decimals. Fig 3 missing y-axis label. Are these averages? no error bars?
Discussion
- What is the relevance of siderophore production to other metals besides iron? Also, if siderophore halo is well beyond colony, any metals bound by siderophores would not be recovered in colony scraping. What type of siderophores are being produced? (CAS media color usually indicator and also biosynthesis genes, genomes of all 3 fungi known).
The authors make many claims in this section that have have created steps or developed a method at a certain tech ready level. However, they have provided no benchmarking data or comparisons to other methods, they have provided no cost information and ICPs are not cheap, incubation time was 18 days which is not quick. How did they arrive at their assessment of tech readiness? Their study is still on a rich media. How much better is it compared to other REE from e-waste recovery systems? or is it even better? They have used very established methods for screening bioaccumulation.
They did not mention the metals they did not recover.
Lines 218-23 citation? in this paragraph and above. What study is this paragraph even talking about?
Conclusion
Author Response
Dear Reviewer,
Thank you for your valuable comments and suggestions, which were helpful in implementing the article. Below are the replies to the comments, the details of the changes made are in the main document.
Advices:
- Details are sparse, would benefit from specific examples throughout introduction paragraph such as names of key metals, names of microorganisms, also better explanation of lines 32-33.
- lines 34-35: does WEEE count as raw materials? raw materials are unprocessed? It is certainly underutilized material.
- Last paragraph needs to be rewritten. In present form, it is difficult to interpret. (Lines 63-69)
- additional citations should be considered throughout manuscript. The authors only cite 22 articles, which is a little sparse for such well-studied bioremediation fungi, the methods selected and the recent literature on critical metal recovery. The authors also need additional citations to support the claim that they have developed a new selection method for WEEE recovering fungi.
Reply: the introduction was implemented, and some parts were rewritten. The small number of citations in the introduction is due to the fact that the cited works include some excellent recently review papers on the subject, so we thought it was redundant to include specific work again.
Materials and Methods
Advice: manufacturer information was not provided for equipment nor reagents, which is important for such sensitive measurements. Model number of major equipment like exact ICP used was also not provided.
Reply: The missing information have been added
Advice: Data for their tests of the metals concentrations in media and agar were not provided or not conducted. It is important to know if any traces of metals were also present in the growth medium and agar used that would give a false positive or additional values to the accumulation if not from e-waste.
- Similarly, the ICP values for metals in biomass for no e-waste plate (media only) was not mentioned, which is a necessary control and comparison.
Reply: all chemicals and ingredients used to prepare the culture media were free of a valuable amount of the analysed metals, furthermore all the data reported take into account the blank of the whole analitycal procedure.
Advice: method for measuring pH was not provided nor was the removal of biomass with a filter and what that was used for, only mentioned in results.
Reply: biomass was completely eliminated through the use of the microporous membrane placed over the medium prior to inoculation. This allowed the mycelium to develop, interact with the underlying soil and absorb the nutrients necessary for the colony to develop. Moreover, when development was complete, thanks to the membrane it was possible to eliminate only the part of mycelial biomass completely free of residual medium. Once the mycelial mass had been removed, the measurement was carried out with a pH meter equipped with a special probe for direct soil analysis (Hanna Instrument - HI12923) that allows measurements to be taken directly in the medium.
Advice: lines 78-9, 86-87 Insufficient detail provided about ICP-MS method.
Reply: the missing information have been added
Advice: line 90 needs a citation and more description about why these 3 strains were chosen
Reply: this is explained in lines 93-96 “In particular, P. glandicola and T. harzianum have been isolated in areas polluted by heavy metals, the third strain, A. tubingensis, was tested for its ability to produce large quantities of calcium oxalate under specific environmental conditions (data not published)”
Results
- In general, the results subsections are too short and do not completely describe the results presented or interpret the findings. Additionally, some tables and figures are presenting the same data.
- All captions need revision. Captions should be a stand alone description of the figure, not just a simple title. Description should also include number of replicates and method used to calculate error bars etc.
- Many figures need revision as well. Some a missing axis unit label. Some use commas for decimals while others use periods. Sometimes the title or acronym is misspelled. The labelling in the results also does not match the results (mm vs cm; tolerance level not presented in results).
Reply: result section has been improved
Advice: Line 139- what constitutes the semi-finished product? (Not mentioned in methods). I see no error bars, or ranges. Was this number only from one sample? Were only these metals detected? ICP usually analyses all. How do these values compare to growth media?
Reply: As described in chapter 2.1 the semi -finished powder is the product provided by company managing the WEEE. Owing to a preselection of the WEEE components, the metals selected for the adsorption are the ones analysed.
Advice: Figure 1, Table 2- and lines 144-7. Where is orange halo for t har (e &f). It is not visible. I also don't understand how a measurement of 0 mm diameter is a positive, weak result for siderophore production.
Reply: the orange halo in T. harzianum plate is not evident. In this case, the reaction is visible as weak decolorization of the CAS-agar which was blue colour and became teal colour. This phenomenon means that the reaction took partially place (partially because the halo is not orange). It cannot be considered as a completely negative result. However, we better clarified it in the text and defined the T. h. halo as not detectable.
Advice: Did they only do one plate for each strain? At least triplicates should be performed.
Reply: The experiments were conducted in five replicates for growing test and in duplicate for CAS-agar test as reported in M&M section.
Advice: Un-inoculated control plate? What values define the cutoffs for calling it strong/weak. How were those cut offs determined?
Reply: The chemical control was carried out, we added the image. The cutoff is determined as the rate among the diameters of halos and the diameters of the fungal colonies (lines 108-110). We better clarified the values of cutoffs in the Results section lines 158-161.
Advice: What does the teal color mean?
Reply: Explained above.
Advice: Line 161- how is stationary determined?
Reply: we consider the growth “stationary“ when the colony no longer increases its diameter for at least 6 days. In our case this happened to two of the three strains tested, in fact, as can be seen from figure 2, the growth of T. harzianum was stationary from day 9 while for P. glandicola from day 12. Furthermore, although not completely stationary, the growth of A. tubingesis was slowed down to a plateau
Advice: What tool is used to scrap off the biomass? How is it determined that no agar was removed?
Reply: once growth was completed, the microporous membrane with the biomass on it was removed from the surface of the medium. Subsequently, using sterile disposable plastic spatulas, the biomass is scraped off the surface of the membrane.
Advice: Lines 163-9, Table 3 & Figure 2. Are the samples in Figure 2 also from a 1 mm2 area? Are they taken from same plate every day? How decide where to harvest on the plate? Colony growth is not a perfect circle. What data is table 3 showing that is not in figure 2? Table 3 no stnd dev or any ranges for these values- did they only measure 1 sample? Figure 2- which type of error bars are shown? Which experiments were done with the reported 5 replicates?
Reply: thank you for pointing out these inaccuracies. The lack of clarity in this part of the article was also pointed out to us by reviewer 1. We have better explained in the text how the values in table 3 were obtained and how the curves in figure 2 were obtained. The surface of each colony was measured with ImageJ in orde to obtain a reliable data to calculate the biomass index mg/mm2. The value in the table corresponds to the specific weight of the mycelial biomass, while the curves are obtained by multiplying the biomass by the average colony surface area at different time.
Advice: Lines 177-80 and Table 4: this whole part is not in the methods and seems important. Need way more info about this membrane removal and how they measured pH. Also, why is pH relevant to WEEE recovery? Again replicates for measurements?
Reply: the characteristics of the microporous membrane and how it can be used have been better described. In addition, the mode and instrumentation used for direct pH measurement was better detailed.
Advice: Lines 186-9: Authors should describe interpretation of these values
Reply: Tolerance Index have been described in a better way
Advice: section 3.4: Is it BCF or BFC- both are used. What is BFC for biomass? commas vs periods for decimals. Fig 3 missing y-axis label. Are these averages? no error bars?
Reply: to describe the adsorption performance of the different strains with respect to the different metals has been chosen the Bioconcentration Factor, as described in literature (Jakubiak et al.,2013). This parameter has been better described in chapter 3.4. The BCF indication has been uniformed. The BCF values are ratios between two concentration, so we don’t report error bar.
Discussion
- What is the relevance of siderophore production to other metals besides iron? Also, if siderophore halo is well beyond colony, any metals bound by siderophores would not be recovered in colony scraping. What type of siderophores are being produced? (CAS media color usually indicator and also biosynthesis genes, genomes of all 3 fungi known).
The authors make many claims in this section that have have created steps or developed a method at a certain tech ready level. However, they have provided no benchmarking data or comparisons to other methods, they have provided no cost information and ICPs are not cheap, incubation time was 18 days which is not quick. How did they arrive at their assessment of tech readiness? Their study is still on a rich media. How much better is it compared to other REE from e-waste recovery systems? or is it even better? They have used very established methods for screening bioaccumulation.
They did not mention the metals they did not recover.
Lines 218-23 citation? in this paragraph and above. What study is this paragraph even talking about?
Reply: we worked as much as possible to fill all the gaps. With regard to the technological level achieved, we have worked mainly on optimizing the use and selection of fungal strains and the work carried out contribute to planning a method to arrive at a higher technological level.
Round 2
Reviewer 1 Report
page 2 line 80 How long was the saw dust heated with how much energy?
page 3 Fungal growth dish test: I assume that after filling the petri dishes with MEA+e-waste powder the e-waste powder sedimented at the bottom of petri dish. Is that true does it influence the kinetic of interaction or if not how did you pervent the sedimentation of e-waste powder?
page 6 table 4 The ref [20] tested the pH influence on CAS in a sodium acetate buffered media between 4.5 an 5.5
The CAS media used in this paper are not buffered, moreover pH of 1 or 8 are much outside the in [20] tested buffer range. Overall rises doubt that CAS agar assay is the right method to proof the formation of siderophores of organic acid forming funghi.
Author Response
Dear Reviewer,
thank you for your suggestions.
The changes made are described below.
Comment: page 2 line 80 How long was the saw dust heated with how much energy?
Reply: information has been added at line 79-80. The process was carried out at 1 kW power, for 2 hours.
Comment: page 3 Fungal growth dish test: I assume that after filling the petri dishes with MEA + e-waste powder the e-waste powder sedimented at the bottom of petri dish. Is that true does it influence the kinetic of interaction or if not how did you prevent the sedimentation of e-waste powder?
Reply: at line 119-121 we specified how we prevent powder sedimentation in the Petri dishes.
Comment: page 6 table 4 The ref [20] tested the pH influence on CAS in a sodium acetate buffered media between 4.5 and 5.5
The CAS media used in this paper are not buffered, moreover pH of 1 or 8 are much outside the in [20] tested buffer range. Overall rises doubt that CAS agar assay is the right method to proof the formation of siderophores of organic acid forming fungi.
Reply: In their work Machuca et al state that pH does not affect CAS agar “the CAS-reaction rate was not affected by different pH conditions”. However the pH at the beginning of our experiment was 5.6 and the values 1, 4, and 8 were recorded at the end of the experiment, so this shift is due only to the fungal metabolism.
