Ichu Valorization by Pleurotus spp. Cultivation and Potential of the Residual Substrate as a Biofertilizer
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors- Pleae, provide more valuable keywords. Please replace the keywords, which are repeated with the tilte of the manuscript.
- The study uses only three replicates per Pleurotus species, which limits statistical power and reliability. A larger sample size would improve confidence in the reported lack of significant differences (p > 0.05) in productivity indicators (CC, BE, PR) among species.
- While the 19.8% BE is compared to other substrates (e.g., coffee husk at 60%), the analysis of lignin’s inhibitory role is underdeveloped. The authors should elaborate on mechanistic links between lignin content and fungal metabolism, and explore pre-treatment methods (e.g., chemical or microbial delignification) to enhance BE.
- The use of a conversion factor of 4.38 for protein content, justified by chitin presence, lacks sufficient validation. The study should reference peer-reviewed methodologies specific to Pleurotus spp. to justify this factor, as standard conversion factors (e.g., 6.25 for plants) may not apply.
- Germination tests were conducted only on radish seeds (Raphanus sativus) under controlled conditions. Field trials and testing across diverse plant species are necessary to assess broader agronomic applicability. Additionally, the residual substrate’s long-term effects on soil health and crop yields are not evaluated.
- The projected selling price of mushrooms ($6.58/kg) lacks empirical validation from local or regional market data. Without context-specific pricing analysis, the profitability claims may be optimistic. A sensitivity analysis incorporating variable costs/prices would strengthen the economic model.
- While ANOVA is mentioned, details on post-hoc tests (e.g., Tukey HSD) and validation of assumptions (normality, homogeneity of variances via Kolmogorov-Smirnov/Levene tests) are absent. Transparent reporting of statistical methods is essential for reproducibility.
- Clarify abbreviations (e.g., "EB" vs. "BE" for biological efficiency).
Author Response
Q1: Please provide more valuable keywords. Replace keywords that are repeated with the title of the manuscript.
R1: Keywords: Pleurotus cultivation; sustainable substrate; Valorization of Andean grasslands
Q2: The study uses only three replicates per Pleurotus species, which limits statistical power and reliability. A larger sample size would increase confidence in the absence of significant differences (p > 0.05) in productivity indicators (CC, BE, PR) between species.
R2: We appreciate your comment on sample size. In this study, three replicates per Pleurotus species were used, a design that has been employed in previous studies on fungal production on alternative substrates. While we recognize that a larger number of replicates could increase statistical power, the results obtained were consistent and showed limited variability between replicates, suggesting that differences in productivity indicators were not substantial between species. In addition, the focus of the study was to explore the viability of Jarava ichu as a substrate, and the biological efficiency and production values obtained support this possibility within the conditions evaluated. References were added as:
Pathmashini, L., Arulnandhy, V., & Wijeratnam, R. W. (2009). Cultivation of oyster mushroom (Pleurotus ostreatus) on sawdust. Ceylon Journal of Science (Biological Sciences), 37(2).
Q3: Although 19.8 % EB compares with other substrates (e.g., 60 % coffee husk), the analysis of the inhibitory role of lignin is underdeveloped. The authors should further explore the mechanistic links between lignin content and fungal metabolism, and explore pretreatment methods (e.g., chemical or microbial delignification) to improve EB.
R3: We appreciate your comment on the analysis of the inhibitory role of lignin on biological efficiency (BE) and the suggestion to explore pretreatment methods to improve it. We address these points below:
Interaction between lignin content and fungal metabolism
Lignin is a complex, recalcitrant polymer that forms a physical barrier around structural polysaccharides in plant biomass, making it difficult for fungi to access the cellulose and hemicellulose necessary for their growth. Pleurotus fungi possess ligninolytic enzymes, such as laccases, lignin peroxidases and manganese peroxidases, which allow them to degrade lignin and access fermentable carbohydrates. However, the efficiency of this process may vary depending on the specific composition and structure of the lignin present in the substrate (Sarita et al., 2012).
Saritha, M., Arora, A., & Lata. (2012). Biological pretreatment of lignocellulosic substrates for enhanced delignification and enzymatic digestibility. Indian journal of microbiology, 52, 122-130.
Pretreatment methods to improve biological efficiency
To improve lignin degradation and thus increase the BE in Pleurotus culture, several pretreatment methods have been proposed:
1. Chemical pretreatments: The use of chemical agents, such as combinations of acids and oxidants, has proven to be effective in disrupting the aromatic structures of lignin, facilitating its removal and increasing the accessibility of cellulose. For example, pretreatment with phosphoric acid and hydrogen peroxide has been successful in removing up to 70% of lignin in agricultural residues.
Saritha, M., Arora, A., & Lata. (2012). Biological pretreatment of lignocellulosic substrates for enhanced delignification and enzymatic digestibility. Indian journal of microbiology, 52, 122-130.
Biological pretreatment: The application of white rot fungi, such as Phanerochaete chrysosporium, can selectively degrade lignin without significantly affecting cellulose. These fungi produce specific enzymes that break down lignin, improving the digestibility of the substrate for other microorganisms.
Saritha, M., Arora, A., & Lata. (2012). Biological pretreatment of lignocellulosic substrates for enhanced delignification and enzymatic digestibility. Indian journal of microbiology, 52, 122-130.
3. Combined pretreatments: The combination of physical, chemical and biological methods can result in more efficient lignin removal. For example, alkaline pretreatment followed by the action of ligninolytic fungi has been shown to increase the efficiency of lignin degradation in various lignocellulosic substrates.
Applicability to Jarava ichu
Considering the lignocellulosic nature of Jarava ichu, the implementation of appropriate pretreatments could improve its suitability as a substrate for Pleurotus cultivation. However, it is essential to evaluate the economic and environmental feasibility of these methods in the specific context of Jarava ichu and local production conditions.
In future research, it is recommended to explore and optimize these pretreatments to determine their effectiveness in improving the BS and sustainability of the Pleurotus cultivation process in Jarava ichu.
Q4: The use of a conversion factor of 4.38 for protein content, justified by the presence of chitin, lacks sufficient validation. The study should cite peer-reviewed methodologies specific to Pleurotus spp. to justify this factor, as standard conversion factors (e.g., 6.25 for plants) may not be applicable.
R4: We appreciate your comment on the nitrogen to protein conversion factor used in our study. The choice of the factor 4.38 is based on the need to correct for the overestimation of protein content in edible mushrooms, due to the significant presence of non-protein nitrogen, mainly derived from chitin in mushroom cell walls. This approach is supported by peer-reviewed studies that have addressed this issue.
For example, in the article “Circular reuse of bio-resources: the role of Pleurotus spp. in the development of functional foods” published in Food & Function, it is stated that the protein content of foods is generally determined as a function of the total nitrogen content evaluated by the Kjeldahl method, multiplied by the conversion factor 6.25. However, in the case of edible mushrooms, numerous studies have indicated a probable digestibility of 60% to 70% for proteins calculated as N × 6.25, due to their remarkable amount of non-protein nitrogen in the form of glucosamine in their chitinous cell walls. Therefore, a conversion factor of 4.38 (i.e., 0.7 × 6.25) has been proposed to obtain a more accurate approximation of protein content in Pleurotus species.
Saritha, M., Arora, A., & Lata. (2012). Biological pretreatment of lignocellulosic substrates for enhanced delignification and enzymatic digestibility. Indian journal of microbiology, 52, 122-130.
Likewise, in the study “Physiochemical analysis and centesimal composition of Pleurotus ostreatus mushroom grown in residues from the Amazon” published in SciELO Brazil, it is mentioned that the N to protein conversion factor used to evaluate the protein content was 4.38, considering the exclusion of the non-protein nitrogen coming from the chitin of the mushroom cell wall, instead of the 6.25 normally used for most foods, thus avoiding an overestimation of the protein content.
Sales-Campos, C., Araujo, L. M., Minhoni, M. T. D. A., & Andrade, M. C. N. D. (2011). Physiochemical analysis and centesimal composition of Pleurotus ostreatus mushroom grown in residues from the Amazon. Food Science and Technology, 31, 456-461.
These studies provide adequate validation for the use of the conversion factor of 4.38 in the determination of protein content in Pleurotus spp. justifying its application in our analysis.
Q5: Germination tests were conducted only with radish (Raphanus sativus) seeds under controlled conditions. Field trials and tests with various plant species are required to evaluate its broader agronomic applicability. In addition, long-term effects of the residual substrate on soil health and crop yield are not evaluated.
R5: We appreciate your observation and acknowledge that our study focused on germination tests using only radish (Raphanus sativus) seeds under controlled conditions. This approach provided a first approximation of the potential of residual substrate as a biofertilizer.
We add as a recommendation:
It is necessary to further evaluate its agronomic applicability, it is essential to conduct field trials with various plant species and to analyze the long-term effects on soil health and crop yields.
Previous studies have shown that residual substrate from fungal cultures can be beneficial to different plant species. For example, research has indicated that the use of Pleurotus ostreatus residual substrates improved the development of tomato (Lycopersicum esculentum) seedlings when mixed with fertilized soil, providing the nutrients necessary for optimal growth (Fontalvo et al., 2013).
Fontalvo, J. A. L., López, L. S. C., Pertuz, K. I. G., & Borja, I. M. R. (2013). Effect of agroforestry residues partially biodegraded by Pleurotus ostreatus (Pleurotaceae) on tomato seedlings development. Acta Biológica Colombiana, 18(2), 365.
In addition, it has been observed that the incorporation of residual fungal substrates into the soil can increase nutrient availability and improve microbial activity, potentially benefiting the growth of various plant species (Martín et al., 2023).
Although our initial results are promising, we recognize the importance of future research addressing these aspects to validate and expand the use of residual substrate as a biofertilizer in sustainable agriculture towards field trials, crop diversity and long-term impact.
Q6: The projected selling price of mushrooms ($6.58/kg) lacks empirical validation from local or regional market data. Without context-specific price analysis, profitability claims could be optimistic. A sensitivity analysis incorporating variable costs/prices would strengthen the economic model.
R6: We recognize that the estimated figure of US$6.58 per kilogram lacks empirical validation based on current local or regional market data. Edible mushroom prices in Peru have shown variability in different regions and at different times. For example, in 2008, the Suillus luteos mushroom was reported to sell for more than US$8.17 per kilogram in the domestic market and up to US$50 in the international market.
In 2014, producers in Lambayeque were selling dehydrated mushrooms for between US$3.27 and US$4.09 per kilogram, following improvements in product quality.
These figures indicate significant fluctuations in prices depending on the species, product presentation and market conditions.
https://andina.pe/agencia/noticia-cultivo-hongos-comestibles-otorgaria-hasta-50-rentabilidad-su-potencial-exportador-166492.aspx#:~:text=El%20cultivo%20de%20hongos%20comestibles,Conservaci%C3%B3n%20de%20Suelos%20(Pronamachcs).
https://gestion.pe/economia/hongos-comestibles-duplican-precio-benefician-500-productores-lambayeque-152246-noticia/
We recognize that without a detailed price analysis specific to the current context, claims of profitability may be optimistic. Therefore, we recommend conducting updated market research to obtain accurate data on mushroom sales prices in the target regions.
Q7: Although ANOVA is mentioned, no details are provided on post-hoc tests (e.g., Tukey's HSD) or on the validation of assumptions (normality, homogeneity of variances by Kolmogorov-Smirnov/Levene tests). Transparency in the presentation of statistical methods is essential for reproducibility.
R7: Yes we say so:
As there were no significant differences there is a reason why it is called post hoc, after the analysis of variance, i.e. if it is not significant there is no need to do it like Tuckey, perhaps to indicate if the data are normal and homogeneous after the tests.
Treatment comparisons were conducted using analysis of variance (ANOVA) for a completely randomized design (CRD) with SPSS software (version 26) at a 95% confidence level. Significant differences between treatments were identified through multiple comparisons using the Tukey HSD test as a post hoc analysis. Additionally, ANOVA assumptions, including residual normality and homogeneity of variances, were assessed using the Kolmogorov-Smirnov and Levene tests to ensure the statistical validity of the results.
Q8: Clarify abbreviations (e.g., “EB” vs. “BE” for biological efficiency).
R8: abbreviations were added
Reviewer 2 Report
Comments and Suggestions for AuthorsSeveral questions and remarks regarding the article by Solorzano et al. “Ichu valorization by Pleurotus spp. cultivation and potential of the residual substrate as a biofertilizer”.
The study's practical originality is well validated. Nevertheless, the scientific novelty of this study is still unclear. In the Introduction, please provide a clear explanation of the scientific uniqueness of your research. Additionally, the Conclusions section should include more new scientific findings (rather than practical results as now).
“…this study aimed to evaluate the efficiency of the Ichu as a substrate for Pleurotus spp. cultivation…” (Lines 99-100) To assess the effectiveness of a substrate, it is important to compare numerous substrates. Only by examining the distinctions between substrates can we determine whether a substrate is effective or not. Why did you not compare the Ichu-based substrate with other substrates?
“The fungal species Pleurotus citrinopileatus, Pleurotus djamor, and Pleurotus ostreatus, known for their ability to degrade lignocellulosic materials…” (Lines 112-113) Why have you not determined the ability of Pleurotus spp. to degrade lignocellulosic materials in Ichu. The cellulose and lignin content of Ichu was assessed only before the experiment began (subsection 3.2) and not after the experiment had concluded.
Subsec. “2.1 Literature search and dataset construction.” Literature search? The title of this subsection does not accurately reflect the content.
Keywords should be revised. Avoid using words from the article title as keywords (to minimise duplication).
Add research limitations and perspectives for future exploration to the Discussion section.
Author Response
Q1: The practical originality of the study is well validated. However, its scientific novelty is not yet clear. In the introduction, provide a clear explanation of the scientific uniqueness of your research. In addition, the conclusion section should include more new scientific findings (rather than practical results, as is currently done).
R1: We appreciate your comments and suggestions to clarify the scientific novelty of our research.
In the introduction it has been highlighted:
Although there are previous studies on the cultivation of Pleurotus on various substrates, such as agricultural residues and invasive species, our study is a pioneer in using Jarava ichu, a high Andean grass, as a substrate. This innovation not only proposes a sustainable alternative to the traditional management of this species, but also explores its dual potential in the production of edible mushrooms and as a biofertilizer, areas that have not been previously integrated in scientific research.
As for the conclusions, emphasize the novel scientific findings, such as:
- The confirmation that Jarava ichu is a viable substrate for the cultivation of Pleurotus spp. evidenced by an average biological efficiency of 19.8%.
- The high protein content of the mushrooms cultivated in this substrate, which ranges between 24.1% and 30.41% on a dry basis, indicating its significant nutritional value.
- The improvement in phosphorus and potassium levels in the residual substrate compared to unprocessed Jarava ichu, suggesting its potential as a biofertilizer.
These results provide unprecedented knowledge on the valorization of Jarava ichu in agricultural biotechnology, opening new avenues for its sustainable use in high Andean ecosystems.
Q2: “...this study aimed to evaluate the efficiency of Ichu as a substrate for the cultivation of Pleurotus spp.” (Lines 99-100) In order to evaluate the effectiveness of a substrate, it is important to compare several substrates. Only by examining the differences between substrates can we determine whether a substrate is effective or not. Why didn't they compare the Ichu-based substrate with other substrates?
R2:
The main objective of this study was to demonstrate, in a preliminary way, that ichu can be used as a substrate for Pleurotus spp. cultivation, given its abundance in high Andean ecosystems and its scarce utilization. Unlike other agricultural production systems, there is no universal “gold standard” for the cultivation of Pleurotus spp. since its development depends on a wide variety of lignocellulosic materials that may differ according to the region and the local availability of resources.
While comparing ichu with other substrates could provide information on its relative efficiency, this study focused on validating its viability as a sustainable alternative in the specific conditions of the Peruvian Andes. Future studies could expand this research to include comparisons with other agroindustrial wastes used in the production of edible mushrooms, which would allow evaluating their relative performance in terms of productivity and nutritional quality.
Q3: The fungi species Pleurotus citrinopileatus, Pleurotus djamor and Pleurotus ostreatus, known for their capacity to degrade lignocellulosic materials..." (Lines 112-113). Why has the ability of Pleurotus spp. to degrade lignocellulosic materials in ichu not been determined? The cellulose and lignin content of ichu was evaluated only before the start of the experiment (subsection 3.2) and not after its conclusion.
R3: The purpose of the study was to evaluate the viability of ichu as a substrate for the cultivation of Pleurotus spp. focusing on fungal biomass production and the adaptation of selected species to this material. Although it is recognized that Pleurotus spp. have the ability to degrade lignocellulosic materials, the study did not include a specific evaluation of ichu degradation because the focus was on the productive response of the fungus, rather than on the enzymatic processes involved.
Analysis of cellulose and lignin content prior to the experiment allowed characterization of the initial substrate and its chemical composition prior to fungal colonization. However, the quantification of these compounds after fungal cultivation could provide additional information on the degradation efficiency of Pleurotus spp. in ichu. This aspect is relevant and can be addressed in future studies to better understand the transformation dynamics of ichu as a substrate in the production of edible mushrooms.
Q4: Subsection “2.1 Bibliographic search and construction of data sets”. Bibliographic search? The title of this subsection does not accurately reflect the content.
R4: For clarity and consistency, the title of the subsection has been changed to something more precise
Q5: Keywords should be reviewed. Avoid using words from the article title as keywords (to minimize duplication).
R5: Keywords: Pleurotus cultivation; sustainable substrate; Valorization of Andean grasslands
Q6: Add research limitations and prospects for future exploration to the Discussion section.
R6: Lack of comparison with other substrates: Although the objective of the study was to preliminarily evaluate ichu as a substrate for Pleurotus spp. the absence of a comparison group with other commonly used substrates limits the possibility of establishing its relative efficiency. Future studies could include comparisons with traditional substrates, such as agricultural residues or wood, to better contextualize the results.
Limited evaluation of the lignocellulosic degradation process: Although the properties of ichu were analyzed prior to the experiment, no post-culture analysis was performed to determine the extent to which Pleurotus spp. degraded the lignocellulosic components. Future research could include degradation trials at different stages of cultivation to better understand the dynamics of ichu decomposition.
Scalability and applicability in real conditions: This study was conducted under controlled conditions, so its applicability in large-scale production systems has not yet been validated. Future studies should address the viability of ichu as a substrate in different environmental conditions and its impact on mushroom productivity in the field.
Limitations in the evaluation of the residual substrate as a biofertilizer: Although the residual substrate was tested on radish germination, its long-term effects on soil fertility and other crops were not evaluated. Further studies could explore its impact on different plant species and its potential as a biofertilizer in sustainable agricultural systems.
Reviewer 3 Report
Comments and Suggestions for AuthorsComment 1: In the introduction, it is suggested to supplement the literature to illustrate the current research gaps in the field of SMS as bioferfertilizer.
Comment 2: There are many abbreviations in the manuscript, which are easy to confuse. A separate explanation of abbreviations is recommended.
Comment 3: Lines 299-301: It was mentioned in the manuscript that ANOVA results showed no significant difference, but no significant analysis was found in Table 2. Please express the data in the manuscript as variance ± standard deviation and label their significance.
Comment 4: Most of the data in the manuscript exists in the form of tables, and it is recommended to replace them with bar charts or line charts to reflect data changes.
Comment 5: Lines 428-429: Please explain why nitrogen levels are lower than without mycelium
Comment 6: Practical application limitations should be mentioned.
Comment 7: Table 5: A blank control group should be added to verify the relative effect of SMS on germination rate.
Comment 8: Section 3.5: Limited to the plant growth phenotype (germination rate), it is recommended to add some crop indicators to further indicate the biological potential of SMS.
Comment 9: It is suggested to add simple cost-benefit analysis to compare with traditional fertilizers.
Comment 10: Lines 447-460: Lack of mechanism discussion Please discuss the possible mechanism of SMS in the light of existing research.
Author Response
Q1: In the introduction, it is suggested to complement the literature to illustrate the current research gaps in the field of SMS as a biofertilizer.
R1: added references to show the knowledge gap
Q2: The manuscript contains numerous abbreviations that can be easily confused. A separate explanation of the abbreviations is recommended.
R2: The abbreviations were improved and the most relevant ones were explained.
Q3: Lines 299-301: It was mentioned in the manuscript that ANOVA results showed no significant differences, but no significant analysis was found in Table 2. Express the data in the manuscript as variance ± standard deviation and label its significance.
R3: deviations were aggregated and significance was indicated in each case.
Q4: Most of the data in the manuscript exist in tabular form and it is recommended to replace them with bar charts or line graphs to reflect changes in the data.
R4: some tables in figures have been modified as requested by
Q5: Lines 428-429: Explain why nitrogen levels are lower than without mycelium.
R5: Added: Nitrogen levels in Pleurotus spp. post-culture substrate (SMS) are lower than in uninoculated ichu, which can be explained by several factors related to fungal metabolism and substrate composition.
During growth, Pleurotus spp. use the nitrogen present in the substrate to synthesize proteins and other essential compounds. This consumption reduces the amount of nitrogen available in the residual substrate after the mushrooms are harvested.
Q6: The limitations of the practical application should be mentioned.
R6: The practical application of mushroom post-crop substrate (SMS) as a biofertilizer presents several limitations that must be considered: Variability in the composition of SMS, Possible presence of pathogens or contaminants, Limited availability and logistics of application, Interactions with other agricultural inputs.
Q7: Table 5: A blank control group should be added to verify the relative effect of SMS on germination rate.
R7: Soil only, if we have it that is chemical fertilization.
Q8: Section 3.5: Limited to the plant growth phenotype (germination rate), it is recommended to add some crop indicators to further indicate the biological potential of the MSS.
R8: No further data is available, it is a preliminary test to obtaining the spent substrate in any case if the evidence is not very robust it may be considered to remove it.
Q9: It is suggested to add a simple cost-benefit analysis to compare with traditional fertilizers.
R9: It does not replace chemical fertilizers, the idea is only to have a standard of comparison, the benefits of the spent substrate go beyond fertilization as a source of organic matter and soil amendment.
Q10: Lines 447-460: Lack of discussion of mechanism. Discuss the possible mechanism of SMS in light of existing research.
R10:
The application of spent mushroom substrate (SMS) as a biofertilizer can influence seed germination through several mechanisms:
Contribution of essential nutrients: SMS enriched with nutrients such as nitrogen, phosphorus and potassium can improve the availability of these elements to developing plants, promoting more efficient germination.
Medina, E., Paredes, C., Pérez-Murcia, M. D., Bustamante, M. A., & Moral, R. (2009). Spent mushroom substrates as component of growing media for germination and growth of horticultural plants. Bioresource technology, 100(18), 4227-4232.
Osmotic effects: Incorporation of SMS into the soil can increase electrical conductivity, affecting the osmolarity of the medium and potentially influencing the ability of seeds to absorb water, which impacts germination rate.
Aamlid, T. S., & Landschoot, P. J. (2007). Effect of spent mushroom substrate on seed germination of cool-season turfgrasses. HortScience, 42(1), 161-167.
Release of bioactive compounds: During the decomposition of SMS, compounds such as humic and fulvic acids are released that can stimulate physiological processes in seeds, favoring their germination.
Aamlid, T. S., & Landschoot, P. J. (2007). Effect of spent mushroom substrate on seed germination of cool-season turfgrasses. HortScience, 42(1), 161-167.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsI have received sufficient responses to each question posed during the earlier review rounds. The manuscript has been carefully revised.
Reviewer 3 Report
Comments and Suggestions for Authors.
