Influence of Sludge and Feed Mixtures on Metal Retention, Pathogen Reduction, and Nutritional Value in Black Soldier Fly (BSF) (Hermetia illucens) Larval Substrates

Abstract

Black soldier fly (BSF) larvae are increasingly used in sustainable waste management, offering potential for the bioconversion of organic waste into insect-derived fertilizer and animal feed. This study investigates the impact of varied substrate mixtures percentages of sludge and chicken feed on heavy metal accumulation, pathogen reduction, and nutrient composition in BSF frass. Methods: The experiment was conducted with four substrate treatments (100% sludge, 75% sludge + 25% chicken feed, 25% sludge + 75% chicken feed, and 100% chicken feed) over a 20-day period. Chemical and microbiological analyses were performed on the feed mixture before adding larvae and on the frass produced in each treatment. Heavy metal concentrations, including cobalt (Co), chromium (Cr), nickel (Ni), and lead (Pb), pathogen levels (Escherichia coli, total coliform, and fecal coliform), and nutrient composition, including moisture content, pH, ash, nitrogen, phosphorus, calcium, potassium, sodium, magnesium, and chlorine, were assessed. Statistical analysis was used to determine significant differences across treatments. Results: Heavy metal levels in frass varied with substrate composition, with significantly higher concentrations of cobalt (Co), chromium (Cr), nickel (Ni), and lead (Pb) in sludge-dominant treatments (p < 0.05). Treatments with higher chicken feed content were associated with lower metal levels, indicating organic feed’s potential in limiting heavy metal accumulation (p < 0.001). Pathogen analysis showed high microbial levels in sludge-based treatments, while the 100% chicken feed treatment exhibited minimal contamination, highlighting its safety profile (p < 0.05). Nutrient characterization revealed that chicken feed-enhanced treatments produced frass with higher nitrogen and potassium levels, suggesting improved nutrient density and potential for agricultural use. Conclusions: Tailoring BSF substrates by combining sludge with organic feed can enhance the nutritional quality of frass while reducing environmental risks associated with heavy metal and pathogen presence. This study supports the potential of BSF as a sustainable bioconversion tool, promoting circular agriculture.

1. Introduction

The black soldier fly (BSF), Hermetia illucens, has emerged as a promising solution for managing organic waste while producing valuable by-products, such as animal feed and insect-derived fertilizer. This insect has garnered significant attention for its capacity to convert various organic wastes into biomass with high nutritional value. In recent years, BSF larvae have been widely studied for their ability to process organic materials, such as food waste, agricultural residues, and manure, transforming them into protein-rich larvae and nutrient-dense frass [1]. The conversion process not only reduces waste, but also provides an environmentally friendly approach to recycling nutrients in a circular bioeconomy [2].

One of the primary advantages of using BSF larvae for waste bioconversion is their ability to handle diverse waste streams, including animal manure, sewage sludge, and food waste [3]. These organic materials, often considered pollutants, can be efficiently processed by the larvae, reducing their environmental impact. Studies have shown that BSF larvae are capable of reducing the volume and mass of organic waste by up to 50% while converting it into larvae biomass and frass, which can be used as feed and insect-derived fertilizer, respectively [4]. This ability makes BSF larvae a key player in sustainable waste management strategies.

The bioconversion efficiency of BSF larvae depends on several factors, including the type of substrate, environmental conditions, and larval density. Research has shown that adjusting the larval density can significantly enhance the bioconversion process, optimizing the nutritional quality of the resulting larvae and reducing the risk of antibiotic resistance gene propagation in manure substrates [5,6]. Moreover, substrate composition plays a critical role in determining the nutritional profile of BSF larvae, with manure and digestate substrates producing larvae with different protein and fat contents compared to those reared on vegetal residues [7]. Thus, tailoring the substrate can help improve the yield and quality of larvae for specific applications.

The environmental benefits of BSF larvae bioconversion extend beyond waste reduction. As BSF larvae consume organic waste, they also mitigate greenhouse gas emissions associated with traditional waste management methods, such as landfilling and incineration [8]. Furthermore, BSF larvae can reduce pathogens and heavy metals in waste, making the resulting frass safer for use as a compost [9]. These characteristics position BSF larvae as a valuable tool in waste-to-resource strategies, particularly in regions where waste management infrastructure is limited [6].

Beyond their role in waste bioconversion, BSF larvae are also gaining attention as a sustainable feed ingredient for livestock and aquaculture. Their high protein and fat content, combined with a favorable amino acid profile, makes them an excellent alternative to traditional feed sources like fishmeal and soybean meal [10]. This potential has sparked interest in large-scale BSF farming operations, particularly as a means to reduce pressure on conventional feed resources and promote circular agricultural practices.

In recent research, strategies to enhance the efficiency of BSF-based waste bioconversion have focused not only on the selection of substrates, but also on their pre-treatment and integration into composting systems. For example, the introduction of BSF larvae during the maturation phase of food waste composting has been shown to significantly promote microbial activity, improve degradation kinetics, and enhance final compost quality, demonstrating the larvae’s broader utility beyond direct bioconversion [11]. Similarly, pre-fermentation of food waste prior to BSF feeding has been reported to improve substrate palatability and conversion efficiency, optimizing both larval growth and waste reduction potential [12]. These findings support the growing interest in refining substrate design and preparation methods to maximize the sustainability and output quality of BSF systems.

Combining sludge with chicken feed as a substrate mixture offers a dual-purpose approach: enhancing the nutritional value for larval development and promoting the safe recycling of two organic waste streams [7]. While sludge alone may lack sufficient nutrients and pose biological risks, chicken feed provides a balanced nutrient profile that supports BSF growth [1,2]. This mixture supports the larvae’s biological needs since they live and feed within the substrate, and may dilute toxic components while optimizing frass quality. Such combinations contribute to sustainable waste valorization in line with circular agriculture principles.

The utilization of BSF larvae for organic waste bioconversion offers a promising and sustainable solution to the global challenges of waste management and resource scarcity [5]. By converting organic waste into nutrient-rich by-products, such as insect-derived fertilizers and animal feed, BSF larvae play a pivotal role in nutrient recycling, waste volume reduction, and environmental protection. This research aims to address critical gaps in understanding the optimization of BSF bioconversion systems, particularly in enhancing heavy metal mitigation, improving pathogen reduction, and maximizing nutrient recovery in frass. By generating novel insights into substrate design and processing methods, this study contributes to advancing large-scale applications of BSF larvae in sustainable waste management and agricultural practices, fostering progress toward a circular bioeconomy [2].

This study offers a novel contribution to the field by evaluating the co-utilization of sewage sludge and chicken feed as substrates for BSF bioconversion, with a focus on optimizing frass quality for use as organic fertilizer. Unlike previous research that primarily emphasizes larval biomass, this work uniquely investigates the effects of substrate blending on heavy metal retention, pathogen reduction, and nutrient enrichment in frass. By demonstrating how substrate composition can improve safety and agronomic value, the study advances the practical application of BSF in integrated waste management and circular agriculture.

2. Methodology

2.1. Experimental Design

The experiment investigated the impact of varied substrate mixtures of sludge and chicken feed on heavy metal accumulation, pathogen reduction, and nutrient composition in BSF frass. Conducted over a 20-day period from 8 February 2024 to 28 February 2024, the experiment was structured to test four different feed compositions. The treatments were as follows:

-

TA: 100% sludge

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TB: 75% sludge and 25% chicken feed

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TC: 25% sludge and 75% chicken feed

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TD: 100% chicken feed

2.2. Study Setting

The experiment took place at the Deir Alla research station in the Jordan Valley, under the National Agricultural Research Center for Agricultural Research (NARC). Environmental factors such as temperature, humidity, and lighting were precisely regulated to maintain result accuracy. The temperature and humidity were maintained at (25–35 °C) and (35–60%), respectively, in the rearing unit. The temperature was controlled using air condition and the moisture was controlled using humidity supplier. Light also was controlled using LED 5 (white) light. Solar energy was utilized as the primary energy source during the experiment.

2.3. Feed Preparation and Measurement

Based on Dzepe et al. [13], each BSF larva requires a daily ration of 100 mg of feed. Over the 20-day study period, the total feed required per larva was calculated to be 2000 mg. With 100 larvae assigned to each treatment group, the total feed provision per replicate was 200 g.

2.4. Feeding Protocols and Early Larval Development

2.4.1. Feed Sample Preparation

The feed samples from each treatment group were processed by air-drying and grinding. The processed feed was sifted through a 4.00 mm mesh to ensure uniform particle size and then underwent chemical analysis. Prior to the introduction of larvae, the feed for each replicate was dried (Figure 1).

2.4.2. Larval Acclimation and Initial Measurements

Initially, 4–6 days-aged larvae were nurtured on a high-protein diet consisting of chicken feed and apples for 4–5 days to encourage strong initial growth. Phenotypic measurements (weight, length, and width) were taken from a random sample of five larvae to establish baseline data before their addition to each experimental pot (Figure 2). Weight was taken by a KERN PCB 350-3 analytical balance, whereas length and width measurements were taken using a digital caliper (Inco HDCD28150)

2.4.3. Introduction of Larvae

Following initial measurements, 100 larvae were introduced into each pot, corresponding to a larval density of 0.5 larvae per gram of substrate, marking the start of the treatment phase for each experimental group (Figure 3).

2.5. Heavy Metal Concentrations

To assess the concentrations of heavy metals (e.g., Co, Cr, Ni, Pb, Cd) in frass used as organic fertilizer and larvae intended as feed, samples from each treatment group were analyzed following the Association of Official Analytical Chemists (AOAC) 999.10 guidelines. Frass and larvae samples were dried, ground, and digested using heat to boil the concentrated hydrochloric acid (HCL) digestion method before heavy metal analysis. Concentrations were determined through flame atomic absorption spectroscopy (contrAA 800, analytic jena, Germany) to quantify metal content, ensuring precise evaluation across treatments.

In addition to individual metal concentrations, the overall heavy metal burden in both frass and larvae was evaluated to determine the impact of substrate composition on metal retention. The AOAC-approved method for comprehensive heavy metal profiling was applied, and results were statistically analyzed to identify significant differences among substrate treatments, informing both the safety and environmental suitability of using BSF by-products.

2.6. Microbial Analysis Procedure

The microbial analysis focused on pathogen levels within frass, particularly targeting E. coli, total coliform, and fecal coliform as indicators of microbial safety. Samples from each treatment were analyzed following the AOAC Official Method 991.14, which utilizes the multiple-tube fermentation (MTF) technique to estimate microbial counts. A series of serial dilutions (10¹ to 10³) were prepared and inoculated into lauryl sulfate broth for total coliform detection and EC Broth with MUG (4-methylumbelliferyl-β-D-glucuronide) for fecal coliform and E. coli. Inoculated tubes were incubated at 35 °C for total coliform and 44.5 °C for fecal coliform and E. coli. Fluorescent tubes under UVP Viewing Cabinet C-70 were considered presumptive E. coli positives. The most probable number (MPN) method was used to quantify microbial levels, with results expressed as MPN per gram of frass, allowing for comparative safety evaluation across treatment groups.

2.7. Chemical Characterization of Frass Procedure

For a comprehensive chemical profile, frass samples were analyzed for moisture content, pH, EC, ash, and essential nutrients (N, P, Ca, K, etc.) using AOAC-recommended procedures. Moisture content and ash were measured according to AOAC Official Method 2001.11, with drying at 105 °C in a HERATHERM oven and ashing in a Heraeus muffle furnace at 550 °C. Nitrogen content was determined by the Kjeldahl method using BUCHI K-370, in line with AOAC Official Method 984.13, while phosphorus was measured using a UV–Vis spectrophotometer following AOAC Official Method 973.53. Calcium, potassium, and sodium were analyzed using a JENWAY PFP7 Flame Photometer, and chloride was determined by titration with silver nitrate. pH and EC were measured using a calibrated EUTECH pH/conductivity meter. These procedures provided a detailed assessment of frass quality as a potential organic fertilizer.

2.8. Monitoring and Data Collection

Larval phenotypic measurements were taken every two days from random samples of five larvae per pot. Each pot was securely covered with a fine mesh net to ensure the containment of larvae. The feed was moistened as necessary to maintain optimal consumption conditions for the larvae. At the end of the experiment, 50 larvae from each treatment were sampled for chemical analysis, and frass was collected, dried, weighted, and analyzed (Figure 4).

2.9. Data Analysis

Statistical analysis was performed using IBM SPSS Statistics version 26. One-way analysis of variance (ANOVA) was applied to determine significant differences among treatment groups for heavy metal concentrations, nutrient composition, and microbial levels in frass. Normality and homogeneity of variance were assessed prior to conducting ANOVA. Results are reported with exact p-values, and a significance threshold of p < 0.05 was used throughout.

3. Results

3.1. Heavy Metals

The analysis of heavy metal concentrations in substrate and frass across different treatments reveals distinct patterns. For cadmium (Cd), no detectable levels were observed in either substrate or frass across all treatments. Cobalt (Co) concentrations were higher in the substrate compared to frass, with 100% sludge having the highest levels (substrate: 7.34 mg/kg, frass: 7.15 mg/kg). The differences between substrate and frass were statistically significant (p < 0.05). Chromium (Cr) concentrations were significantly higher in frass than in the substrate across all treatments (p < 0.01), with the highest levels in frass observed for 100% sludge (40.95 mg/kg). Nickel (Ni) concentrations were also significantly higher in frass than in the substrate (p < 0.001), with the greatest frass levels noted for 100% sludge (23.79 mg/kg). Finally, lead (Pb) levels in the frass were significantly lower than in the substrate (p < 0.001), particularly in the 100% chicken feed treatment (substrate and frass: 0.00 mg/kg). These results highlight significant variations in heavy metal distribution between substrates and frass, depending on the treatment (

Table 1. Heavy metal concentrations (mg/kg) in substrate and frass across different treatments.

Treatment	Cd (mg/kg)		Co (mg/kg)		Cr (mg/kg)		Ni (mg/kg)		Pb (mg/kg)
	Substrate	Frass	Substrate	Frass	Substrate	Frass	Substrate	Frass	Substrate	Frass
TA	0.000	0.0000	7.3400	7.1518	25.0400	40.9500	23.3000	23.7850	15.99	9.0782
TB	0.0000	0.0000	7.2900	5.3227	30.6600	40.8300	18.4100	21.4180	6.4900	3.5723
TC	0.0000	0.0000	5.9600	3.2536	0.7000	8.5060	6.0900	8.8416	3.8600	0.1743
TD	0.0000	0.0000	5.5200	1.9410	0.0000	4.5100	1.9600	0.0000	0.0000	0.0000
p-value		-	0.0390	0.0070	0.0000	0.0000	0.0000	0.0009	0.0004	0.000

).

3.2. Chemical Characterization Results

The chemical characterization of substrate and frass across different treatments indicates significant variations in several parameters. Moisture content showed significant variation across treatments in both substrate and frass samples (p < 0.05) (

Table 2. Chemical characterization of substrate and frass across different treatments.

Treatment	% Moisture		Ash Content (%)		pH		Ec (dS/m)		N (%)		P (%)		Ca (%)		Mg (%)		Na (%)		K (%)		Cl (%)
	Substrate	Frass	Substrate	Frass	Substrate	Frass	Substrate	Frass	Substrate	Frass	Substrate	Frass	Substrate	Frass	Substrate	Frass	Substrate	Frass	Substrate	Frass	Substrate	Frass
TA	6.76	18.64	21.46	24.65	5.93	5.81	1.54	3.39	7.00	5.96	0.53	0.74	3.76	4.72	0.92	0.99	0.20	0.47	0.33	0.38	0.15	0.33
TB	7.71	11.80	17.88	24.97	5.86	6.26	1.57	3.50	5.81	5.72	0.46	0.45	3.20	2.68	0.70	1.04	0.25	0.49	0.43	0.64	0.23	0.34
TC	9.70	10.89	9.56	15.24	6.05	6.32	1.51	3.57	3.58	5.56	0.34	0.35	1.74	0.78	0.44	0.70	0.19	0.51	0.50	1.32	0.23	0.58
TD	10.70	10.82	5.52	6.58	5.99	6.73	1.63	2.81	2.47	4.03	0.19	0.20	1.1	0.13	0.23	0.19	0.15	0.38	0.73	1.23	0.28	0.53
p-value	0.0410	0.0390	0.0000	0.00	0.4160	0.2690	0.0000	0.0000	0.0268	0.0410	0.3810	0.1150	0.0000	0.00	0.0000	0.0000	NS	NS	0.0000	0.000	NS	NS

).

Ash content was also significantly greater in frass than substrate (p < 0.01), with 100% sludge showing the highest values (substrate: 21.46%, frass: 24.65%).

The pH levels were similar between substrate and frass, showing no significant differences (NS), while electrical conductivity (Ec) was significantly higher in frass compared to substrate for all treatments (p < 0.001). Nitrogen content (N%) was significantly higher in the substrate for most treatments (p < 0.05), with the highest levels noted for 100% sludge (substrate: 7.00%, frass: 5.96%).

Phosphorus (P%) levels showed no significant differences between substrate and frass (NS). In contrast, calcium (Ca%) and magnesium (Mg%) concentrations were significantly elevated in frass across treatments (p < 0.01 and p < 0.05, respectively). Sodium (Na%) levels did not differ significantly between substrate and frass (NS), while potassium (K%) was significantly higher in frass (p < 0.01). Chloride (Cl%) concentrations did not show significant differences (NS).

3.3. Microbial Analysis Results

The microbial analysis of substrate and frass across different treatments reveals significant differences in microbial load between the two materials. Total coliform levels were consistently high in the substrate across all treatments, with values exceeding 16 ×10³ MPN/g, whereas frass showed significantly lower levels (p < 0.01), except for the 75% sludge + 25% chicken feed (TB) and 25% sludge + 75% chicken feed (TC) treatments, which also exceeded 16 × 10³ MPN/g (

Table 3. Microbial analysis of substrate and frass across different treatments.

Treatment	Total Coliform (MPN/g Sample)		Fecal Coliform (MPN/g Sample)		E-coli (MPN/g Sample)
	Substrate	Frass	Substrate	Frass	Substrate	Frass
TA	>16 × 103	7.8 × 103	>16 × 103	2.8 × 103	5.4 × 103	6.1 × 102
TB	>16 × 103	>16 × 103	>16 × 103	>16 × 103	>16 × 103	>16 × 103
TC	>16 × 103	>16 × 103	>16 × 103	>16 × 103	3.5 × 103	>16 × 103
TD	0	>16 × 103	0	>16 × 103	0	>16 × 103
p-value	0.008	0.018	0.027	0.029	0.000	0.000

).

Fecal coliform levels were significantly reduced in frass compared to substrate across all treatments (p < 0.01). For instance, in the 100% sludge (TA) treatment, substrate levels exceeded 16 × 10³ MPN/g, while frass levels were 2.8 × 10³ MPN/g. Similarly, E. coli concentrations were markedly lower in frass than in the substrate (p < 0.05). For example, in the TA treatment, E. coli levels were 5.4 × 10³ MPN/g in the substrate and 6.1 × 10² MPN/g in the frass (Table 3).

4. Discussion

The motivation for mixing sludge and chicken feed stems from the need to balance nutrient availability with safety [5]. Since BSF larvae live within and actively consume the substrate, its composition directly impacts their development and the quality of the resulting frass. The addition of chicken feed enriches the nutrient content, while sludge reuse addresses environmental waste management goals [13,14,15].

This study investigated how substrate composition influences heavy metal accumulation, microbial safety, and nutrient content in BSF frass. The results underscore the role of substrate design in optimizing BSF bioconversion, nutrient recovery, and safety, aligning with recent findings that highlight the significance of substrate choices in sustainable waste management strategies [3,14,16].

4.1. Heavy Metal Concentration

The significant variation in heavy metal concentrations across substrate treatments emphasizes the importance of substrate composition in mitigating potential toxicity in BSF-derived frass. The presence of cobalt, chromium, nickel, and lead in higher concentrations within sludge-based treatments aligns with prior research that highlights the risk of heavy metal accumulation in substrates containing industrial or municipal sludge [15]. High cobalt and chromium concentrations in sludge treatments, as observed here, suggest that metals are likely retained in frass when substrates are rich in mineral content. Ribeiro et al. [16] found that BSF larvae tend to bioaccumulate metals based on substrate mineral content, with retention capacity varying by metal type and substrate composition. This raises critical questions about the use of sludge as a primary substrate component when the goal is to generate agriculturally safe outputs, whether as frass or through composting. While frass directly contributes as an insect-derived fertilizer with a nutrient-rich profile, composting may offer an alternative route to enhance microbial safety and reduce heavy metal content in the substrate. Conversely, treatments with higher proportions of chicken feed showed significant reductions in nickel and lead levels, likely due to the organic nature of chicken feed, which reduces mineral exposure for the larvae. Gligorescu et al. [17], and Gobbi et al. [18], similarly noted that organic substrates minimize heavy metal uptake in BSF larvae, thus reducing metal levels in resulting frass. This relationship suggests that organic substrates like chicken feed may act as dilution agents, lowering the bioavailability of metals. However, it is also important to consider that substrate pH can influence heavy metal availability. A reduction in pH may enhance the solubility of heavy metals, increasing their bioavailability for absorption by BSF larvae. Future studies should investigate the interplay between substrate pH, organic matter composition, and gut microbiota in determining metal chelation and absorption, as indicated by studies on microbial interactions with heavy metals [19].

According to the European Union Regulation (EU) 2019/1009 on fertilizer products [20], the maximum permissible limits for heavy metals in organic fertilizers are 1.5 mg/kg for Cd, 120 mg/kg for Cr (total), 50 mg/kg for Ni, and 100 mg/kg for Pb. In our study, Cd was not detected in any frass samples. Lead concentrations in all treatments remained below EU limits. However, Cr and Ni concentrations in frass from the 100% sludge and mixed treatments exceeded these thresholds (Cr: up to 40.95 mg/kg, Ni: up to 23.79 mg/kg), though still within acceptable ranges. These findings suggest that while frass from chicken feed treatments complies with EU safety standards, sludge-based frass may require further processing or dilution prior to land application.

4.2. Microbial Analysis Discussion

The microbial safety profile of frass is a critical determinant of its suitability for agricultural use, as pathogens like E. coli and fecal coliform can pose health risks to humans. Our results showed high pathogen levels in treatments containing sludge, with significantly reduced contamination in the 100% chicken feed treatment. This pattern supports previous findings by Lu et al. [21], who demonstrated that organic feed-based substrates typically carry fewer microbial risks, especially in comparison to waste-heavy substrates. The high pathogen levels in sludge-based treatments highlight a critical limitation in using untreated sludge for BSF rearing, as sludge may harbor bacteria that can persist through larval digestion [22].

While the BSF gut microbiota can potentially mitigate some microbial risks through antimicrobial activity, there is evidence suggesting that BSF larvae may also secrete antimicrobial compounds on their surface, aiding in reducing bacterial load. However, the effectiveness of these mechanisms is highly substrate-dependent and warrants further research to confirm the extent of their antimicrobial properties [23]. Lalander et al. [24], demonstrated that BSF larvae can reduce Salmonella spp. in organic waste substrates but have limited efficacy in highly contaminated sludge environments. Given the persistence of pathogens in sludge treatments, pre-treatment methods, such as pasteurization or composting, could be explored to reduce initial microbial loads before BSF bioconversion. This strategy could balance the nutrient benefits of sludge without the microbial safety risks, a method advocated by Ribeiro et al. [16], for optimizing pathogen management in insect bioconversion.

The absence of microbial contamination in the 100% chicken feed treatment suggests that organic substrates might inhibit pathogen survival during the digestion process. This finding aligns with Widyastuti et al. [25], who observed that microbial activity was limited when BSF larvae were reared on high-carbohydrate, low-nitrogen diets, possibly due to competitive exclusion by beneficial bacteria in the larval gut. However, the survival rates of pathogens under varying substrate and environmental conditions remain an important area of study. Factors such as temperature, moisture levels, and substrate composition are likely to influence how long bacteria persist in the frass or larval environment. Further research is needed to quantify the conditions under which pathogens are effectively eliminated, providing clearer guidelines for optimizing BSF-based waste processing. These observations highlight the potential of combining organic feed with sludge to produce safer frass while minimizing microbial risks [26]. Although this study did not include a cost–benefit analysis of substrate mixtures, we acknowledge their importance for evaluating economic viability. A dedicated economic feasibility study is planned as a follow-up to assess the practicality and scalability of these substrate formulations in real-world conditions.

This study primarily focuses on evaluating the frass (insect-derived residue) produced by BSF larvae as a sustainable organic fertilizer. While BSF larvae are valuable for feed applications, our analysis emphasizes the safety and nutritional quality of frass. Lower concentrations of heavy metals and pathogens in frass are critical indicators of its suitability for agricultural use. Although metal accumulation in BSF larvae may occur, it falls outside the scope of this study. Future research could build on this work by assessing the metal partitioning between frass and larvae to optimize both products in waste bioconversion systems.

While microbial levels in frass were significantly lower than in the raw substrates, the presence of high total coliform counts (especially >16 × 10³ MPN/g in TB and TC treatments) suggests that sludge-containing frass may not be immediately safe for agricultural application. Therefore, additional post-processing steps such as composting or pasteurization may be necessary to ensure compliance with microbial safety standards before field use. These findings reinforce the need for integrated treatment strategies when using BSF frass derived from high-risk substrates like sludge.

4.3. Chemical Characterization Discussion

The chemical characterization of frass demonstrated that substrate composition influences nutrient levels, with organic feeds like chicken feed promoting higher nitrogen and potassium concentrations. This observation corresponds with findings from Belperio et al. [27] and Biasato et al. [28], who reported increased nitrogen and protein levels in frass when BSF larvae were reared on protein-rich substrates. The higher nitrogen concentration may be attributed to the decomposition of organic matter during the digestion process, where nitrogen compounds from proteins and other organic molecules are broken down and concentrated in the frass. Additionally, the metabolic activities of BSF larvae and their gut microbiota may contribute to nitrogen enrichment by recycling nitrogenous waste products into forms that remain in the substrate. Further research is needed to elucidate the precise mechanisms driving nitrogen accumulation in frass. Higher nitrogen content in frass can be beneficial for agricultural applications, as it contributes to soil fertility and can reduce the need for synthetic fertilizers. This is critical for sustainable agriculture, as frass generated from optimized BSF substrates can close nutrient loops within farming systems [15].

On the other hand, the high moisture and ash content observed in sludge-based treatments could limit the frass’s applicability as a soil amendment. Moisture management is a key factor in determining the stability and transportability of frass, as high moisture content can increase spoilage and microbial growth during storage [29]. In this study, the use of dry sludge as a substrate component likely contributed to the lower moisture levels in certain treatments, enhancing the stability of the frass. Dry sludge reduces the initial water content of the substrate, which can limit microbial activity and spoilage potential during storage. This highlights the importance of using substrates with lower moisture content to improve the quality and shelf-life of the resulting frass, especially for agricultural applications. The elevated ash content in sludge treatments suggests a higher mineral residue, which may contribute to salinity in soils if used without dilution. Similar concerns were raised by Myers et al. [30], who found that high ash content in frass reduced its effectiveness as an insect-derived fertilizer in certain crops. Thus, combining sludge with organic feed appears to balance nutrient density with reduced ash content, making frass more compatible with soil health and nutrient uptake.

4.4. Implications for Sustainable Agriculture

The findings of this study highlight substrate selection as a pivotal factor in enhancing the agronomic quality of BSF frass while addressing safety concerns. Combining sludge with organic feed could represent a balanced approach, mitigating heavy metal and microbial risks while maximizing nutrient value. However, the elevated levels of specific heavy metals in sludge-based treatments underscore the need for regulatory frameworks when using BSF frass in agriculture. Safe limits for heavy metals, such as those outlined by the European Union for organic fertilizers, may need to be adapted for insect-derived products, as suggested by recent work on BSF frass safety [17].

Further research should examine how specific feed mixtures and environmental factors, like temperature and humidity, interact to affect both nutrient and safety profiles of BSF frass. Recent studies by Wang et al. [4], on gut microbiota in BSF larvae suggest that microbial dynamics within the larvae could be leveraged to improve pathogen reduction while enhancing nutrient bioavailability. Additionally, processing methods such as drying or composting may serve as post-harvest treatments to stabilize frass and further reduce microbial risks, as proposed by Surendra et al. [1].

5. Conclusions

This study reinforces that substrate composition is a crucial factor in determining the quality and safety of BSF frass for agricultural use. While sludge offers nutrient benefits, combining it with organic substrates like chicken feed minimizes heavy metal accumulation and microbial contamination. However, it is important to note that even frass may require additional post-processing, such as composting or pasteurization, to ensure its safety and suitability for agricultural applications. These treatments can further reduce microbial risks and improve the stability and nutrient availability of the frass. These findings support the broader potential of BSF in waste bioconversion for sustainable agriculture, particularly when waste substrates are thoughtfully designed to optimize both safety and nutrient recovery. The future direction of this field will benefit from a greater emphasis on optimizing substrate mixes and incorporating post-processing treatments to tailor frass properties to specific agricultural contexts, aligning with evolving sustainability goals in global food systems. Although frass produced from mixed and chicken feed-only treatments demonstrated reduced microbial and metal loads, sludge-containing frass may require further post-processing to meet safety thresholds. Future applications should incorporate treatment protocols to ensure its agronomic safety.