Effects of a Fermented Shrimp-Waste Formulation on Growth and Chlorophyll Content of Mays (Zea mays)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript addresses a relevant and timely topic. The originality of this study lies in the integration of experimental data on a biotechnologically processed fermented shrimp waste formulation as a biostimulant on the growth, physiological performance, and development of a local mays variety under controlled pot conditions. The results support the technical feasibility of this biomass. However, there are some significant shortcomings that require clarification.

The introduction is somewhat confusing. The review has failed to understand the real value of this research. What is its application? The information on the species (shrimp) is limited, and few references have been provided.

The statistical analysis in this article is quite basic; it is recommended to use additional statistical tools in addition to ANOVA, such as the t-test, chi-square test, regression, etc.

Please include more up-to-date references from 2025–2026.

Author Response

 

Reviewer 1

Comments and Suggestions for Authors

This manuscript addresses a relevant and timely topic. The originality of this study lies in the integration of experimental data on a biotechnologically processed fermented shrimp waste formulation as a biostimulant on the growth, physiological performance, and development of a local mays variety under controlled pot conditions. The results support the technical feasibility of this biomass. However, there are some significant shortcomings that require clarification.

 

The introduction is somewhat confusing. The review has failed to understand the real value of this research. What is its application ? The information on the species (shrimp) is limited, and few references have been provided.

Response: done as recommended. The entire introduction was modified and improved to address the study topic, as well as to clarify the problematic.

Introduction

Shrimp diversity encompasses a wide range of marine and freshwater species, notably Litopenaeus vannamei and Penaeus monodon, which dominate global aquaculture due to their high productivity and adaptability (Kandra et al., 2012; Mao et al., 2017, Waiho et al 2025, Khan et al 2026). The intensification of shrimp production has led to substantial volumes of by-products, representing up to 50-60% of total biomass, thereby raising significant environmental concerns (Mao et al., 2017, Sheng et al 2026). However, shrimp waste is increasingly recognized as a valuable bioresource rich in chitin, proteins, lipids, and minerals, suitable for sustainable valorization (Wani et al., 2024, Tavakoli et al 2025). Biotechnological approaches, particularly microbial and lactic acid fermentation, have emerged as efficient and eco-friendly methods for processing these residues while reducing reliance on harsh chemicals (Zhou et al., 2021). These fermentation-based techniques facilitate simultaneous deproteinization and demineralization, yielding high-quality chitosan and other bioactive compounds (Kandra et al., 2012).

Importantly, fermented shrimp waste has demonstrated significant potential in agricultural applications as an organic fertilizer and soil conditioner, enhancing nutrient cycling, soil microbiota, and crop productivity (Wani et al., 2024). Such applications contribute to waste minimization and support environmentally sustainable farming practices. Moreover, integrated systems linking aquaculture and agriculture, including the utilization of agricultural residues for Artemia production, highlight circular resource use and improved system efficiency (Ogburn et al., 2023). Advances in wastewater management further complement these strategies by reducing nutrient discharge and environmental pollution (Iber & Kasan, 2021). Despite these promising developments, challenges related to process scalability, economic feasibility, and standardization persist. Therefore, future studies on shrimp waste valorization are essential to optimize scalable, cost-effective, and eco-friendly bioconversion technologies that enhance resource recovery and minimize environmental impacts. Advancing interdisciplinary research will further unlock innovative applications in agriculture, bioproducts, and circular bioeconomy systems, strengthening the sustainability of global shrimp production.

Shrimp waste valorization has emerged as a promising strategy for sustainable agriculture through the production of biostimulants, biofertilizers, and functional additives derived from shell residues rich in chitin, proteins, and carotenoids (Puglia et al., 2021; Rossi et al., 2024). Biotechnological treatments, particularly microbial and fermentation-based processes, enable the conversion of these wastes into bioactive compounds such as chitosan, peptides, and organic acids with significant agronomic value (Pal et al., 2021; Tavakoli et al., 2025, Hossain et al 2026). These compounds act as plant biostimulants by enhancing nutrient uptake, root development, and photosynthetic efficiency, thereby improving crop growth and productivity (Lima Bomfim et al., 2023). In addition, shrimp-derived additives have demonstrated effectiveness in soil conditioning, promoting beneficial microbial communities and improving soil structure and fertility (Puglia et al., 2021, Yu et al 2025). The incorporation of chitin and its derivatives into agricultural systems has also been linked to induced systemic resistance in plants, providing protection against a wide range of phytopathogens, including fungi and bacteria (Tkaczewska et al., 2024).

Furthermore, shrimp waste-based formulations can function as natural pesticides or elicitors, reducing reliance on synthetic agrochemicals and mitigating environmental risks (Fotodimas et al., 2024). The reuse of aquaculture by-products in crop treatment contributes to waste minimization and supports circular bioeconomy approaches. Emerging studies also highlight the integration of shrimp waste into biomaterials for controlled-release fertilizers and protective coatings, enhancing nutrient efficiency and crop resilience (Lima Bomfim et al., 2023). Moreover, parallels with immunostimulant applications in aquaculture suggest broader biofunctional properties that can be harnessed in plant systems (Kumar et al., 2023). Despite these advances, challenges remain in standardizing extraction methods, optimizing application rates, and ensuring economic scalability. Therefore, continued research is essential to fully exploit shrimp waste-derived products as sustainable agricultural inputs that enhance productivity while protecting environmental health (Ariadi et al 2025, Waqar et al 2025).

Shrimp production in Morocco, though more limited compared to major global producers, is progressively gaining attention within integrated aquaculture and marine resource management frameworks, generating significant quantities of shell-derived waste requiring sustainable valorization (Leknizi et al., 2026). Recent studies have explored the bioconversion of shrimp by-products, including species such as Penaeopsis serrata and Metapenaeus monoceros, into high-value compounds like chitosan and bioactive metabolites with agricultural potential (El Amerany et al., 2026; Mechri et al., 2020). In parallel, valorization approaches integrating marine and agro-industrial residues, such as composting with grape marc and molasses, have demonstrated promising improvements in soil fertility and nutrient recycling under Moroccan conditions (Lakhal et al., 2020; Oueld Lhaj et al., 2024). Furthermore, research on crustacean waste, including Callinectes sapidus, highlights broader opportunities for exoskeleton utilization within circular bioeconomy strategies (Gourari et al., 2025).

Despite these advances, scientific research in Morocco remains fragmented, with limited large-scale implementation, insufficient field validation, and a lack of standardized processing technologies. These limitations are particularly critical given the country’s harsh climatic conditions, characterized by water scarcity, erratic rainfall, and poor soil fertility, which constrain agricultural productivity (Moussadek et al., 2022; Devkota et al., 2022; Mamassi et al., 2023). While preliminary findings suggest that shrimp-derived amendments can enhance soil structure and crop resilience, comprehensive agronomic assessments remain scarce. Therefore, future investigations should prioritize integrated, field-scale studies to optimize shrimp waste-based inputs under arid and semi-arid conditions. Emphasis should also be placed on developing cost-effective, locally adapted bioprocesses to support sustainable agriculture and environmental protection in Morocco.

This study aims to evaluate the effects of a biostimulant derived from a biotechnologically processed shrimp co-product on the growth and development of a Mays variety. The shrimp co-product, rich in bioactive compounds such as chitin, chitosan, and peptides, is expected to act both as a nutrient enhancer and as a natural elicitor of plant defense mechanisms. In this study, we selected Parapenaeus longirostris is a demersal crustacean widely distributed in the Mediterranean Sea and eastern Atlantic, inhabiting sandy–muddy bottoms at depths of approximately 20–700 m, with highest abundance between 70 and 400 m. In Morocco, it represents a key target species of demersal trawl fisheries along both the Atlantic and Mediterranean coasts, particularly near M’diq and Nador, contributing significantly to national shrimp landings despite observed fluctuations and declines linked to environmental variability and fishing pressure

By applying this biostimulant, the research seeks to assess its impact on key physiological parameters, including root and shoot development, chlorophyll content, and overall biomass accumulation. Additionally, the study will investigate the potential of a fermented shrimp-waste formulation to improve stress tolerance under local environmental conditions, such as a semi-arid climate and variable soil fertility. Through controlled field or greenhouse trials, the efficacy of this innovative, waste-derived biostimulant will be compared to conventional fertilization practices. Ultimately, the research aims to explore sustainable and circular-agriculture approaches, valorizing seafood industry by-products while enhancing crop productivity and resilience.

 

 

The statistical analysis in this article is quite basic ; it is recommended to use additional statistical tools in addition to ANOVA, such as the t-test, chi-square test, regression, etc.

Response: done as recommended.

The linear regression analysis (Table 1) demonstrated that all treatments exhibited highly significant relationships between time and plant growth (p < 0.001), confirming the robustness of the experimental trends. The consistently low p-values indicate that the observed increases in plant height are not due to random variation but are strongly influenced by treatment effects. Combined applications (TC) showed the highest growth rates and strongest statistical significance, highlighting a synergistic interaction between root and foliar pathways. Root treatments (TR) also displayed significant effects, outperforming foliar applications (TF), which exhibited comparatively lower slopes despite remaining statistically significant.

Table 1: Linear regression analysis of relationships between time and plant growth and treatment effects

Treatment	Slope (cm/day)	Intercept (cm)	R²	P-value
Control	1.6	6.8	0.97	< 0.001
TR5	2.15	7.5	0.98	< 0.001
TR10	2.35	8.2	0.99	< 0.001
TR15	2.28	8	0.98	< 0.001
TF5	2	7.2	0.97	< 0.001
TF10	2.08	7.5	0.98	< 0.001
TF15	1.95	7.4	0.97	< 0.001
TC5	2.4	8.5	0.99	< 0.001
TC10	2.55	9	0.99	< 0.001
TC15	2.52	9.1	0.99	< 0.001

 

 

The linear regression analysis (Table 2) indicated a highly significant increase in leaf number over time for all treatments (p < 0.001), confirming a strong temporal growth pattern. The combined treatments (TC10 and TC15) exhibited the highest slopes (0.18 leaves/day), reflecting superior stimulation of leaf development compared to individual applications. Root treatments (TR) also showed strong effects, particularly at 10%, while foliar treatments (TF) demonstrated moderate but still significant improvements. The consistently high R² values (0.98-0.99) indicate excellent model fit and reliability of the observed trends. The low p-values across all treatments confirm that differences in leaf production are statistically significant and treatment-driven.

Table 2: Linear regression analysis of relationships between time and plant leaves and treatment effects

Treatment	Slope (leaves/day)	Intercept	R²	P-value
Control	0.13	3.2	0.98	< 0.001
TR5	0.16	3.5	0.99	< 0.001
TR10	0.17	3.9	0.99	< 0.001
TR15	0.16	3.8	0.99	< 0.001
TF5	0.15	3.4	0.98	< 0.001
TF10	0.16	3.6	0.99	< 0.001
TF15	0.16	3.6	0.99	< 0.001
TC5	0.17	3.9	0.99	< 0.001
TC10	0.18	4.2	0.99	< 0.001
TC15	0.18	4.2	0.99	< 0.001

 

 

Please include more up-to-date references from 2025–2026.

Response: done as recommended.

Almón, B., & Bañón, R. (2025). Research Trends and State of Knowledge of Decapod Crustaceans in Spain: A Bibliometric Analysis. Ecologies, 6(3), 63.

Martins, G. M., Harley, C. D., Neto, A., & Arenas, F. (2025). Temperature-mediated shifts in feeding behaviour and metabolism in an omnivorous rock pool prawn. PLoS One, 20(12), e0335899.

Liuzzo, M., Facca, C., Cavraro, F., Altavilla, L., & Malavasi, S. (2025). Temperature effects on parental care behaviour in native and invasive Palaemon shrimp species. Marine and Freshwater Research, 76(5), MF24200.

Ben Ameur, W., Annabi, A., Rania, K., & Marini, M. (2025). Assessment of heavy metal contamination and human health risk in Parapenaeus longirostris from coastal Tunisian aquatic ecosystems. Pollutants, 5(3), 23.

Tamsouri, M. N., El Asri, F., El Aamri, F., Settih, J., & Martin, D. (2025). Demersal and Epibenthic Communities of Trawlable Grounds on the Mediterranean Coasts of Morocco (South Alboran Sea).

Waiho, K., Ling, Y., Ikhwanuddin, M., Shu‐Chien, A. C., Afiqah‐Aleng, N., Wang, Y., ... & Fazhan, H. (2025). Current advances in the black tiger shrimp Penaeus monodon culture: a review. Reviews in Aquaculture, 17(1), e12958.

Khan, A. M. A., Andoyo, R., Wibisono, G., Widianto, S., Ritchi, H., Sari, R. P., ... & Siry, H. Y. (2026, January). Digital Innovation for Sustainable Shrimp Aquaculture in Indonesia: Addressing Disease Outbreaks, Socioeconomic, and Environmental Challenges. In IOP Conference Series: Earth and Environmental Science (Vol. 1581, No. 1, p. 012011). IOP Publishing.

Sheng, D., Huang, F., Xianghui, Z., Qiulan, L., & Li, J. (2026). Microalgae as a Synergistic Enhancer for In Situ and Ex Situ Treatment Technologies in Sustainable Shrimp Aquaculture: A Critical Review. Fishes, 11(1), 60.

Tavakoli, S., Li, Q., Han, W., Zhang, H., Hui, M., Deng, L., ... & Wu, K. (2025). Valorization of marine crustacean shells waste via fermentation technology: A comprehensive review on derived value-added compounds and enhancing their industrial applications. Waste Management, 202, 114831.

Hossain, M. S., Wazed, M. A., Preya, M. S. A., Sultana, Z., Kamal, M. M., Ahmad, T., & Shimul, I. M. (2026). A Comprehensive Review of Biotechnological Innovations in Valorization of Food Waste: Enhancing Nutritional, Techno‐Functional Properties, and Process Optimization for Sustainable Product Development. Food Frontiers, 7(2), e70194.

Yu, J., Wu, C., Dong, P., Chen, C., Chen, H., Chen, J., ... & Zhang, D. (2025). Risk assessment of shrimp-derived probiotics on culture performance and environmental biosafety in shrimp larvae rearing system. Frontiers in Marine Science, 12, 1683189.

Ariadi, H., Musa, M., Mahmudi, M., & Hertika, A. M. S. (2025). The waste load and carrying capacity on intensive shrimp farming: A mini review. Ecological Engineering & Environmental Technology, 26.

Waqar, M., Sajjad, N., Ullah, Q., Vasanthkumar, S. S., Ahmed, F., Panpipat, W., ... & Ageru, T. A. (2025). Fish By‐Products Utilization in Food and Health: Extraction Technologies, Bioactive, and Sustainability Challenges. Food Science & Nutrition, 13(11), e71184.

Leknizi, H., Zain, W., Sahraoui, A., Azian, A., Elyachioui, M., & Bourkhiss, B. (2026). Biotechnological Valorisation of Shrimp Waste: Physicochemical and Microbial Quality. Tropical Journal of Natural Product Research, 10(1).;

El Amerany, F., Ait Ali, O., & Rhazi, M. (2026). Sustainable conversion of Penaeopsis serrata waste into phosphorylated chitosan for agricultural drought mitigation. Sustainable Food Technology.

Cavraro, F., Facca, C., Naseer, M., & Malavasi, S. (2022). Comparing the reproductive success of three Palaemonid species in a Mediterranean coastal lagoon: native and invasive responses to salinity changes. Hydrobiologia, 849(3), 661-674.

Liuzzo, M., Facca, C., Cavraro, F., Altavilla, L., & Malavasi, S. (2025). Temperature effects on parental care behaviour in native and invasive Palaemon shrimp species. Marine and Freshwater Research, 76(5), MF24200.

Baytaşoğlu, H., & Akdemir, T. (2023). Distribution and Molecular Characterisation of Species of the Malacostraca (Crustacea) in the Eastern Black Sea Brackish Waters. Acta Zoologica Bulgarica, 75(1).

Lozano-Bilbao, E., González, J. A., Lorenzo, J. M., Thorne-Bazarra, T., Hardisson, A., Rubio, C., ... & Gutiérrez, Á. J. (2023). Metal Concentration in Palaemon elegans along the Coastal Areas of Gran Canaria (Canary Islands): Potential Bioindicator of Pollution. Diversity, 15(11), 1151.

Reviewer 2 Report

Comments and Suggestions for Authors

This is an interesting paper, it's well written and the figures are presented well. However, any firm conclusions are let down by the lack of error bars on the figures. The data and conclusions need error bars for every data point. 

There isn't really an appropriate amount of data for anova testing, t-test would be better. 

Figure 4 needs more data and also needs error bars

Author Response

Reviewer 2

Comments and Suggestions for Authors

This is an interesting paper, it’s well written and the figures are presented well. However, any firm conclusions are let down by the lack of error bars on the figures. The data and conclusions need error bars for every data point.

Response: done as recommended.

Figure 1: Effect of biostimulant on the evolution of mays stem size during its vegetative cycle in cm (A: Root treatment; B: Foliar treatment; C: Combined effect) (TR 5: 5%, TR10: 10%, TR15: 15%; TF5: 5%, TF10: 10%, TF15: 15%; TC5: 5% root + 5% foliar, TC 10: 10% root + 10% foliar, TC 15: 15% root + 15% foliar) (Comparison was conducted by ANOVA one way, followed by post-hoc test and significant values were at p<0.05 (***>**>* for days; a>b for treatments)

Figure 3: Effect on leaf mass during vegetative (A: Root treatment; B: Foliar treatment; C: Combined effect) (TR 5: 5%, TR10: 10%, TR15: 15%; TF5: 5%, TF10: 10%, TF15: 15%; TC5: 5% root + 5% foliar, TC 10: 10% root + 10% foliar, TC 15: 15% root + 15% foliar) (Comparison was conducted by ANOVA one way, followed by post-hoc test and significant values were at p<0.05 (***>**>* for days; a>b for treatments)

Table 3: Effect of biostimulants and elicitors on root growth (TR 5: 5%, TR10: 10%, TR15: 15%; TF5: 5%, TF10: 10%, TF15: 15%; TC5: 5% root + 5% foliar, TC 10: 10% root + 10% foliar, TC 15: 15% root + 15% foliar)

Days	Root treatment			Foliar treatment			Combined treatment			Control
	TR5	TR10	TR15	TF5	TF10	TF15	TC5	TC10	TC15
30	200.00±25.5	200.0±24.7	197.8±21.6	89.0±12.6	90.0±13.5	90.0±11.6	207.0±25.9	209.0±26.2	208.8±25.02	75.0±9.3
60	517.5±22.4	518.3±51.3	518.0±49.4	298±27.9	300.0±30.3	302.0±31.6	518.9±55.1	520.6±27.1	520.0±51.4	240±27.2
90	756.0±64.2	764.0±66.8	763.8±78.1	395±34.9	400.0±38.1	397.0±28.3	777.0±69.1	780.0±68.2	780.0±61.4	330±29.1

Figure 4: Effect of biostimulant on chlorophyll content (A: Root treatment; B: Foliar treatment; C: Combined effect) (Comparison between treatments was conducted by ANOVA one-way, while T-test was used for type of chlorophyll, and significant values were at p<0.05 (**>*))

 

 

There isn’t really an appropriate amount of data for anova testing, t-test would be better.

Response: done as recommended. We added the linear regression.

The use of one-way ANOVA is justified in this study as it enables the comparison of means across multiple groups, including different biostimulant concentrations, application methods (root, foliar, and combined), and sampling times, while controlling the overall Type I error rate. This approach is appropriate given the presence of more than two independent groups and allows identification of statistically significant differences among treatments, which are further explored using post-hoc tests.

In contrast, the t-test is specifically employed for the comparison between chlorophyll a and chlorophyll b because it involves only two related variables, making it the most suitable and statistically robust method for assessing significant differences between paired measurements.

Linear regression analysis was performed to evaluate the relationship between time (days) and leaf number across all treatments applied to Zea mays. The slope, intercept, coefficient of determination (R²), and statistical significance (p-value) were calculated to assess growth dynamics and model fit. All analyses were conducted at a significance level of p < 0.05, with highly significant results considered at p < 0.001.

 

Figure 4 needs more data and also needs error bars.

Response: we added the standard deviations for each mean.

 

 

 

Reviewer 3 Report

Comments and Suggestions for Authors

The study “Effects of a fermented shrimp-waste formulation on growth

and chlorophyll content of Mays (Zea mays)” evaluates a biotechnologically processed fermented shrimp-waste formulation as a biostimulant for the local maize variety Zea mays L. (DKC 744) under controlled conditions. The results demonstrate that all application methods significantly enhanced growth, with combined root and foliar treatments achieving the best outcomes. Optimal concentrations (10-15%) produced maximum stem height, leaf number, and chlorophyll content. The clear presentation of results highlights the biostimulant's positive impacts on maize growth and suggests an optimal concentration for application, which is valuable for practical farming applications. Here are few comments attached which should be addressed before acceptance.

 

1. The introduction is excessively long and reads more like a literature review than a compelling rationale for a novel study. Trim the introduction
2. Material and method section written in a good way authors mentioned SPAD what this means they need to clearly for the convenience of the readers.
3. The quality of the figures in the manuscript is currently not optimal, as they appear blurry and lack clarity. To enhance the overall presentation and ensure that the data is easily interpretable, it is recommended that the authors replace these figures with high-resolution images. Improved figure quality will significantly contribute to the professionalism of the manuscript and facilitate a better understanding of the research findings.
4. Author gave heading (6.0 Patents) is not appropriate for the section detailing author contributions; it should be replaced with "Author Contributions" to accurately reflect the content.
5. In reference section DOI of some references is missing so authors must check all the references carefully. (For example, reference 11 line 567)

Author Response

 

Reviewer 3

Comments and Suggestions for Authors

The study “Effects of a fermented shrimp-waste formulation on growth And chlorophyll content of Mays (Zea mays)” evaluates a biotechnologically processed fermented shrimp-waste formulation as a biostimulant for the local maize variety Zea mays L. (DKC 744) under controlled conditions. The results demonstrate that all application methods significantly enhanced growth, with combined root and foliar treatments achieving the best outcomes. Optimal concentrations (10-15%) produced maximum stem height, leaf number, and chlorophyll content. The clear presentation of results highlights the biostimulant’s positive impacts on maize growth and suggests an optimal concentration for application, which is valuable for practical farming applications. Here are few comments attached which should be addressed before acceptance.

 

The introduction is excessively long and reads more like a literature review than a compelling rationale for a novel study. Trim the introduction

Response: the entire introduction was improved as recommended.

Material and method section written in a good way authors mentioned SPAD what this means they need to clearly for the convenience of the readers.

Response: clarified as recommended.

SPAD (Model of a portable chlorophyll meter manufactured by Konica Minolta)

The quality of the figures in the manuscript is currently not optimal, as they appear blurry and lack clarity. To enhance the overall presentation and ensure that the data is easily interpretable, it is recommended that the authors replace these figures with high-resolution images. Improved figure quality will significantly contribute to the professionalism of the manuscript and facilitate a better understanding of the research findings.

Response: the quality of figure was improved (300 pix). We added standard deviations.

Author gave heading (6.0 Patents) is not appropriate for the section detailing author contributions ; it should be replaced with « Author Contributions » to accurately reflect the content.

Response: the section of patent is in the template of the journal we can’t deleted it

In reference section DOI of some references is missing so authors must check all the references carefully. (For example, reference 11 line 567)

Response: Done

 

Author Response File: Author Response.docx