Enhancing chicken manure with bread waste and black soldier fly associated bacteria to increase larval biomass

TL;DR

This study shows how adding bread waste and specific bacteria to chicken manure can boost black soldier fly larvae growth and nutrient content, offering a sustainable way to recycle waste.

Contribution

The study introduces a novel method of enhancing black soldier fly larvae biomass using bread waste and selected bacteria isolated from BSF egg surfaces and guts.

Findings

A 1:1 bread waste to chicken manure mixture maximized larval biomass and feed conversion efficiency.

Five bacterial strains, including Microbacterium paraoxydans and Morganella morganii, significantly improved larval growth when added to diets.

Larvae fed with supplemented diets showed increased levels of essential and non-essential amino acids.

Abstract

Black soldier fly larvae (BSFL) represent a promising and sustainable source of protein and essential nutrients for poultry feed. This study aimed to improve BSFL growth and organic waste bioconversion by utilizing chicken manure (CM), a widely available but environmentally problematic byproduct of poultry production due to its unpleasant odor, greenhouse gas emissions, and pathogen risks. In the first trial, CM was combined with bread waste (BW) at different ratios to overcome the poor larval growth observed when reared on CM alone. The 1:1 BW: CM mixture yielded the highest larval biomass, feed conversion efficiency, and nutritional value. In the second trial, beneficial bacteria were isolated from BSF egg surfaces and larval guts (3.5 × 10⁶ and 2.4 × 10⁶ CFU/ml, respectively). Thirteen isolates were screened for urease and protease activity, and five were selected and identified using phenotypic traits and 16 S rRNA sequencing: Microbacterium paraoxydans, Bacillus proteolyticus, Priestia megaterium, Bacillus subtilis, and Morganella morganii. Supplementation of CM-based diets with these strains significantly improved BSFL dry weight and feed conversion efficiency, with the most notable enhancements achieved using M. paraoxydans and M. morganii. Amino acid analysis of larvae fed a 3CM:1BW diet supplemented with M. paraoxydans revealed elevated levels of essential (Isoleucine, leucine, Phenylalanine, valine) and non-essential (alanine, Glycine) amino acids. Overall, this study demonstrates a cost-effective and eco-friendly strategy for optimizing BSFL production while recycling organic waste in sustainable livestock systems.

The online version contains supplementary material available at 10.1038/s41598-026-39463-x.

Figures

Protease and urease detection in the best bacterial isolates on agar plates. (a) Protease assay on skimmed milk agar medium which the positive results are a clear halo zone and negative results are no clear halo zone. (b) Urease assay on a urea agar medium in which the positive results are red color and negative results are yellow color.

Different microbial colonies from BSF eggs-associated and larval guts on LB agar medium plates (A), Growth of the bacterial isolates (EO4, EO6, EO3 and GL isolates) on LB agar medium (B-E), and Gram stain and cell morphology of some of the bacterial isolates EN2, EO4, EO6 and GL (F-I).

Neighbor-joining tree based on 16 S rRNA sequences of the genus bacterial isolate codes EN2 (A), EO3 (B), EO4 (C), EO6 (D) and GL (E) obtained from BLAST search showing the position of isolate and related strains.

Retention times (min) of the amino acids analysis for each numbered peak using high-performance amino acid analyzer system. (a) After feeding on 3CM:1BW alone as a control; (b) After feeding on 3CM:1BW in combination with *M. paraoxidans* strain SR.

Full text

Introduction

The management of organic waste remains a critical global concern, especially with the rapid growth of agricultural and food industries. Chicken manure (CM), in particular, is produced in large quantities by the poultry sector and poses environmental risks due to its high nutrient load, potential for pollution, and disposal challenges. Traditional methods such as landfilling and incineration are not sustainable, prompting the need for alternative, eco-friendly solutions. Bioconversion of organic waste using Hermetia illucens (black soldier fly, BSF) has gained attention as a sustainable and efficient strategy for waste management and alternative protein production. BSF larvae are rich in protein and fat, with a nutritional profile comparable to conventional feed ingredients like fishmeal and soymeal^1,2^. Given the expected scarcity and rising cost of traditional protein sources, BSF presents a viable alternative for poultry feed while also addressing environmental concerns by converting organic waste into valuable nutrients^2^.

BSF larvae contribute to the circular economy by yielding lipids, proteins, and chitin, which can be further processed into chitosan with applications in agriculture, cosmetics, and pharmaceuticals^3–5^. Moreover, they enable more efficient and rapid improvement of fertilizing properties compared to traditional composting methods^6,7^. Studies have shown that larvae raised on diverse waste types such as chicken or pig manure, brewers’ grains, fruits, and vegetables—achieve high survival rates (up to 97.7%) and significant biomass gain (average larval weight of 134.9 mg), highlighting their capacity for efficient nutrient conversion^2,8^. Overall, BSF bioconversion supports food security, reduces environmental impact, and offers an adaptable, cost-effective method for producing high-quality protein for animal feed^9,10^.

Bread waste (BW) is an abundant and carbohydrate-rich by-product—typically containing 50–70% starch, 8–10% protein, and only 1–5% fat—which can effectively balance the nitrogen load of CM and act as a more energy-dense substrate. In many low- and middle-income countries like Egypt, BW accumulates in significant quantities and remains massively underutilized despite its suitability for inclusion in feed^11^.

In Egypt, poultry production is substantial, with over 1.4 billion birds reared annually, including approximately 320 million chickens^12^. This large-scale production generates considerable amounts of CM, which, if not properly managed, poses environmental challenges due to nutrient overload and potential pollution. Concurrently, BW represents a highly abundant and underutilized carbohydrate-rich resource, with estimates suggesting up to 20% of bakery products are discarded annually^11,13^. The integration of CM and BW as complementary substrates offers a sustainable strategy for bioconversion using BSFL, simultaneously addressing waste management, nutrient recovery, and alternative protein production. Utilizing these locally available resources leverages both environmental and economic benefits, making the proposed approach highly relevant for scaling up BSFL-based feed production.

Numerous symbiotic bacteria coexist with insects like H. illucens and contribute significantly to their physiological development, including immune system regulation^14,15^. Environmental factors influence the development of the gut microbiota and the production of digestive enzymes throughout the insect’s life cycle^16^. BSF-associated microbes have been shown to improve insect performance and development^17^. For instance, larvae fed CM enriched with Bacillus isolates showed improved weight gain and faster development^18^. Adding specific probiotic isolates to the larval diet has also been reported to significantly enhance larval growth, weight, and adult survival^19^. Moreover, the composition of gut microbiota, including bacteria and fungi, is highly influenced by the type of feeding substrate^20^. These microorganisms perform complex functions such as enhancing gut health, modulating immune responses^21^, breaking down plant polymers^22^, synthesizing essential amino acids and vitamins^23^, and producing pheromones and kairomones for communication, as well as defending against pathogens and parasites^14^. Overall, the ability of BSF larvae to thrive on microbe-rich substrates like manure is linked to a highly adaptable immune response. However, the specific role of symbiotic bacteria in enhancing nutrient production and improving the conversion rate of waste into insect biomass still requires further investigation.

In this study, BW was incorporated as a carbohydrate-rich substrate to balance the high nitrogen load of CM, creating a more favorable rearing substrate for BSF larvae. Annually, large volumes of CM and BW are generated worldwide; for example, poultry industries in developed countries produce millions of tons of CM, while bakery and household sectors generate substantial amounts of BW. CM is rich in protein and nitrogen but low in energy, whereas BW provides abundant carbohydrates that can complement CM. This synergistic use of CM and BW not only enhances larval performance and waste conversion but also valorizes locally available by-products as sustainable feed alternatives. The novelty of this work lies in integrating CM and BW with BSF-associated beneficial bacteria to improve waste reduction efficiency, larval biomass yield, and nutrient quality, providing a practical and scalable solution for sustainable waste management and protein production.

Materials and methods

Study site and insect collection

Experiments were conducted during 2023–2024 at the Faculty of Agriculture, Ain Shams University, Egypt, involving three departments: Plant Protection (Environmental Studies Laboratory), Agricultural Microbiology, and Animal Production. BSF larvae were collected in spring 2023 from a private Farm in Obour City, Qalyubia Governorate (30.21641°N, 31.58279°E), with average temperature 25 ± 2 °C and relative humidity 65 ± 2%. They were reared to adulthood to produce eggs, which were then used to establish a generation for feed production and subsequent experimental use.

Substrate Preparation

Fresh chicken manure was collected from the Faculty of Agriculture poultry farm and sun-dried for one month during the autumn season, when sunlight intensity was relatively low. This step was performed to reduce moisture content, minimize odor, and allow insects to escape from the substrate. After drying, the manure was sieved to remove large debris, weighed, and immediately used in the feeding trials without further storage or additional chemical/physical pretreatments. Bread waste was sourced from a local restaurant, dried at 50 °C to constant weight, ground, and sieved. Diets were mixed to prepare four treatments: BW alone, CM alone, CM: BW at 3:1 and 1:1 ratios.

Insect rearing

BSF adults were reared in 30 × 25 × 30 cm wooden cages lined with muslin for ventilation and fitted with transparent tops. Rolled cardboard served as an oviposition site above about 200 g from food waste diets. Eggs were incubated at 25 ± 2 °C and 60 ± 5% Relative Humidity (R.H) Larvae were reared on moistened BW diet (50% moisture) as per^23^.

Bacterial media used

Luria-Bertani (LB) agar medium^24^ was used for isolation and maintenance of bacterial strains. It contained of tryptone, 10.0; yeast extract, 5.0; NaCl, 10.0; agar, 18.0; and pH was adjusted to 7.0. Luria-Bertani (LB) broth medium was the same as LB agar medium without adding agar.

Skimmed milk (SM) agar medium^25^ was used for the protease producing bacteria. It consists (g/L) of skimmed milk, 28.0; peptone, 5.0; yeast extract, 25.0; glucose, 1.0; CaCl_2_.2H_2_O 0.1; KH_2_PO_4_ 0.5; agar, 20.0; and pH was adjusted to 7.0. Urea agar medium was used for the urea-producing bacteria. This medium was purchased from HIMEDIA.

Proximate chemical analysis of different substrates

Dried CM, BW and their mixtures (3:1 and 1:1) were analyzed in triplicate according to (AOAC, 2007)^26^ standard procedures. Dry Matter (DM) using AOAC Method 930.15, Crude Protein (CP) using AOAC Method 976.05, Crude Fat (Lipid) using AOAC Method 920.39, Ash using AOAC Method 942.05, and Crude Fiber (CF) using AOAC method 978.10, were determined. Carbohydrates were calculated by difference on a dry matter basis and gross energy was estimated using standard conversion factors.

Feed experiment

Two independent feeding trials were conducted: the first without bacterial inoculation and the second with inoculated beneficial bacteria. In each trial, four treatments were tested: 100% BW (control), 100% CM (control), CM: BW at 3:1, and CM: BW at 1:1. Each treatment was replicated three times, with 100 six-day-old larvae per replicate. Larvae (average weight 0.4 g dry and 1.2 g fresh) were transferred to plastic plates (5 cm height × 15 cm diameter). Each group was fed three times during the experimental period with 30, 25, and 25 g of diet calculated on a dry matter basis, which was subsequently adjusted to 50% moisture before feeding. Treatments were maintained at 25 ± 2 °C and 60–65% RH until the fifth larval stage (~ 14 days). At harvest, larvae were washed and weighed; half of the samples were oven-dried for biomass determination, while the remainder was allowed to pupate. Dry biomass was measured after drying larvae and pupae in an electric oven (Nüve En 500) at 65 °C for 48 h and then weighed on a digital balance (KERN ADJ 200–4) until constant weight. Bioconversion efficiency and feed conversion ratio^27^ were calculated at harvest following equations as:

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All treatments, including the different CM: BW feeding ratios and bacterial inoculations, were conducted in three biological replicates, each replicate consisting of 100 larvae of the same age.

Enumeration and isolation of bacterial diversity in BSF eggs and the larval gut

Approximately 0.5 g of BSF eggs (collected within 6 h of oviposition) was obtained in order to extract the bacteria from the egg surface. The eggs were homogenized with 2 mL of sterilized distilled water. The mixture was constantly stirred and incubated for 5 min at 28 °C ± 2. Following this, the suspension was diluted by the serial dilution method^27^ up to 10^− 8^ and they used to enumerate and isolate bacterial diversity using the pour plates method on LB agar medium. The plates were incubated at 28–30 °C for 5 days. The guts of 4th instar larvae were separated as well. The larval surface was sterilized with 75% ethanol for 3 min and then rinsed three times in sterile phosphate buffered saline (PBS) at pH 7.4. The whole gut of 10 larvae was separated aseptically, transferred to 100 µL of sterile PBS, and homogenized. The suspension was isolated based on colony selection after diluting by serial dilution method^28^up to 10^− 8^ and used to enumerate and isolate bacterial diversity. The diluted cell suspensions were plated on solid LB agar and incubated at 28–30 °C for 5 days. After incubation, the Petri dishes were examined for the appearance of individually isolated colonies across the medium. The pure colonies which are of varying size shape and color isolated and transported into test tube culture media to prepare pure cultures. The isolated bacterial colonies may be a kind of mixed culture. For the purification of bacterial cultures from the previous Petri dishes, the isolated bacterial colony may be purified following the streak plate technique^29^. Total number of colony-forming units (CFU) on the surface of an agar medium is enumerated. The Calculation of CFU/mL is done by using the following Eq. (10):

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The pure bacterial colonies were maintained on LB agar slant and then kept in a refrigerator at 5 °C for further studies.

Maintenance of cultures

Stock culture slants were maintained at 5 °C on a preservation LB agar after incubation at 30 ± 2 °C for 3–5 days. Sub-culturing was usually carried out every three months using the same medium.

Bacterial enzymes assay

Urease assay

Urease activity of bacterial isolates was tested using urea agar medium containing phenol red as a pH indicator. Inoculated plates were incubated at 30 ± 2 °C for 48 h. Urease-producing bacteria were identified by a color change in the medium from yellow to pink or red, indicating an increase in pH due to ammonia release.

Protease assay

Protease production was assessed on skimmed milk agar plates. Bacterial isolates were spot-inoculated onto the medium and incubated at 30 ± 2 °C for 48 h. Proteolytic activity was indicated by the formation of a clear halo zone around the bacterial colonies, resulting from casein hydrolysis. The diameter of the clear zone was recorded to assess the relative protease activity. Results were qualitatively expressed as (+) for presence and (−) for absence of proteolytic activity^25^. All tests were performed in triplicate.

Phenotypic and genotypic identification of bacterial isolates

Phenotypic characteristics, which included the following, were used to identify the chosen bacterial isolates: the colony’s shape and pigment diffusion determined the cultural features that could be seen with the naked eye. The shape of the cells, Gram staining, endospore formation, and motility all affected the morphological characteristics, which were viewed at 1000x magnification using a light microscope. These characteristics were derived from Bergey’s Manual of Systematic Bacteriology’s^30–32^ suggested keys.

Molecular characterization (16S rRNA sequencing) provided the basis for confirmed identification. The bacterial isolates were grown on sterile Petri plates containing autoclaved LB agar medium and incubated for 2 days at 28–30°C. The culture was extracted at the Molecular Biology Research Unit (MBRU), Assiut University, for DNA extraction using a patho-gene-spin DNA/RNA extraction kit that was provided by Intron Biotechnology Company, Korea. The bacterial DNA product was then sent to SolGent Company, Daejeon, South Korea, for polymerase chain reaction (PCR) and rRNA gene sequencing. The PCR of the bacterial isolate was performed using two universal primers (forward and reverse), which were incorporated into the reaction mixture. Primers have the following composition: 27F (5’-AGAGTTTGATCC TGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTA CGACTT-3’). The purified PCR product (amplicons) was sequenced with the same primers with the incorporation of ddNTPs in the reaction mixture^33^. The Basic Local Alignment Search Tool (BLAST) from the National Center of Biotechnology Information (NCBI) website was used to evaluate the sequences retrieved. MegAlign (DNA Star) software version 5.05 was used to perform phylogenetic analysis of sequences.

Effect of bacterial strains supplementation on larval development

Inoculum Preparation

For inoculum preparation, a single colony of each selected bacterial isolate was picked from a fresh LB agar plate and inoculated into a 250 mL conical flask containing 50 mL of sterile LB broth. The culture was incubated for 24 h at 30 ± 2 °C on a rotary shaker at 150 rpm. Following incubation, the optical density (OD) of the broth culture was measured at 600 nm and adjusted with sterile LB broth to achieve a cell density corresponding to 10^8^ CFU/mL. This concentration was periodically confirmed using the serial dilution and plate count method as described in the enumeration section. One millilitre of this standardized culture was used as the final inoculum.

Amino acids profile of BSFL

The amino acid analysis of BSFL samples was conducted at the Central Laboratory, Desert Research Center, Ministry of Agriculture and Land Reclamation, Cairo, Egypt. The amino acids were determined following the method of Pellet and Young^34^. Briefly, 0.1 g of freeze-dried BSFL sample was hydrolyzed in a sealed glass tube with 10 ml of 6 N HCl at 110 °C for 24 h. After hydrolysis, the sample was filtered, and the filtrate was evaporated to dry. The residue was dissolved in distilled water and diluted to a final volume of 100 ml. The solution was then filtered through a 0.45 μm micropore membrane before injection.

The amino acid profile was determined using a SYKAM S433 High-Performance Amino Acid Analyzer (SYKAM GmbH, Germany) equipped with a sodium-based ion exchange column (25 cm length), under standard operating conditions. A volume of 50 µl was injected, and amino acids were detected using a visible light detector following post-column derivatization with ninhydrin reagent. The detection wavelengths were 570 nm for most amino acids and 440 nm for proline. Quantification was performed using external standards and the SYKAM S2100 integrator software based on peak area and retention time. Results were expressed in mg amino acid per g dry weight. All samples were analyzed in triplicate.

Statistical analysis

Data analyses were performed with IBM^®^ SPSS^®^ Statistics software version 19.0.^35^ using one-way analysis of variance (ANOVA) followed by Tukey-Kramer Honest Significant Difference (HSD) tests at a significance level of P < 0.05. All data are presented as mean ± standard deviation (SD) for larval chemical composition and growth performance parameters.

Statement

The authors guarantee that all procedures were carried out in compliance with the rules and regulations that applied. The complete experimental protocol was approved by the Research Ethics at Ain Shams University, Agriculture Sector Committee with approval No. 14- 2024-13.

Results and discussion

Diet analysis

Table 1 summarizes the proximate composition of CM, BW, and their mixtures (1:1 and 3:1 CM: BW ratios) used as substrates for BSFL rearing. The mixing ratios of CM and BW were selected to gradually improve the nutritional properties of CM by supplementing it with BW as a carbohydrate source. The chosen proportions (75% CM + 25% BW and 50% CM + 50% BW) were designed to demonstrate a stepwise enhancement in substrate quality while still maintaining CM as the main component of the diet. The selection of these mixing ratios was based on a nutritional strategy to create diets with varying Carbon-to-Nitrogen (C: N) ratios. The 3:1 (CM: BW) ratio represented a nitrogen-rich diet primarily composed of manure, while the 1:1 ratio was intended to provide a more nutritionally balanced substrate by increasing the proportion of carbohydrates. A higher proportion of BW was not tested, as this would have resulted in an impractically low amount of CM, which was the primary focus of this study. CM showed numerically higher contents of dry matter, ash, ether extract, and crude protein compared to BW, while BW was richer in total carbohydrates and gross energy. The mixtures exhibited intermediate values, indicating that BW supplementation effectively balanced the nutrient composition of CM. For example, crude protein increased from 9.49% in BW to 14.07–16.30% in the mixtures, while total carbohydrates decreased but remained higher than in CM alone. Previous studies also support the suitability of such organic materials. For instance, Singh et al.^36^ reported protein and carbohydrate contents of 34.5% and 31% in fresh CM, compared to 27.13% and 31% in composted manure. The composition of CM varies depending on factors such as bird age, litter type, manure age and moisture, and storage conditions^37^. Similarly, Al-Dalain and Morsy^38^ reported 11.14% moisture, 1.12% ash, 1.53% fat, 12.57% protein, and 73.65% carbohydrates in CM. Siddiqui et al.^39^ found average BSFL diet values of 16.76% protein, 7.38% fat, a protein/fat ratio of 5.69, and 4.73% minerals.

This adjustment is particularly reflected in the protein + carbohydrate content (CP + TC) and the protein-to-carbohydrate ratio (CP: TC), which increased proportionally with higher CM contribution (0.12 in BW to 0.43 in the 3:1 CM: BW mixture). These findings align with previous reports highlighting the importance of the protein-to-carbohydrate (CP: TC) ratio in shaping BSFL performance. Barragán-Fonseca et al.^40^ demonstrated that diets with moderate protein and carbohydrate levels, combined with relatively low P: C ratios (≈ 1:3), enhanced larval growth and yield, whereas excessively protein-biased (≈ 1:1) or carbohydrate-biased (≈ 1:9) diets impaired survival and delayed development. Similarly, Eggink et al.^41^ reported optimal larval yield and feed conversion efficiency at P: C ratios between 1:2 and 1:3. In our study, the BW-based diet (CP: TC = 0.12) was too carbohydrate-rich, while CM alone (CP: TC = 0.74) leaned toward protein dominance. The mixtures (0.28 and 0.43) provided more balanced macronutrient profiles, which are expected to support improved larval performance compared to the single substrates. Therefore, the designed CM: BW mixtures demonstrate a practical approach to upgrading CM as a BSFL substrate by supplementing it with carbohydrate-rich BW, achieving a stepwise enhancement of nutritional quality. This balance is particularly relevant for large-scale applications, where diet formulation must optimize both larval growth and waste bioconversion efficiency.

Table 1. Proximate chemical composition of different diets used for BSFL.Contents (%)SubstratesBWCMCM: BW (with ratio 1:1)CM: BW (with ratio 3:1) Dry matter 95.1296.1795.6095.90 Ash 1.0327.1814.1020.64 Ether extracts 5.9322.2814.1018.19 Crude protein 9.4918.6514.0716.30 Crude fiber 8.246.707.478.08 Total carbohydrates 75.3025.2050.2537.72 Gross energy (kcal/100 g DM) 392.53375.92384.18379.79 CP + TC 84.7943.8564.3254.02 CP: TC ratio 0.120.740.280.43Values represent a single sample analyzed per diet; no statistical analysis was performed.Abbreviations: CM, Chicken manure; BW, Bread waste; CP, Crude protein, TC, Total carbohydrates.

Biological parameters of BSFL on different diets

As shown in Table 2, diet type had a significant effect on larval and pupal biomass (ANOVA, p < 0.001). The 1:1 CM: BW mixture supported the best performance, producing the highest larval and pupal biomass (260 mg fresh weight (FW) and 90.6 mg dry weight (DW) per larvae; 77 mg DW per pupa), significantly outperforming all other treatments (Tukey’s HSD, p < 0.05). The 3:1 CM: BW mixture also yielded good larval growth (253.3 mg FW), but the corresponding dry weights of larvae and pupae were comparable to those obtained from BW alone, with no significant differences. In contrast, CM alone resulted in the lowest biomass values (126.6 mg FW and 44.2 mg DW per larvae; 29 mg DW per pupa), likely due to its lower gross energy and total carbohydrate contents compared to BW and the mixed diets (Table 1). A similar pattern was observed for substrate conversion efficiency, with the 1:1 CM: BW mixture achieving the highest bioconversion rate (10.82%), followed by 3:1 CM: BW (8.78%) and BW alone (8.60%), while CM alone showed the poorest performance (4.94%). This was further confirmed by FCR values, which displayed the expected inverse relationship: the lowest FCRs were recorded in the 1:1 and 3:1 CM: BW mixtures (4.85 and 4.97, respectively), while the highest was in CM alone (7.46). Collectively, these findings demonstrate that blending CM with BW, particularly at a 1:1 ratio, enhances larval biomass production, bioconversion efficiency, and feed utilization. A key finding of this study is the superior performance of larvae reared on the 1:1 CM: BW substrate. This result can be attributed to the optimized nutritional profile created by this specific blend. Chicken manure (CM) is rich in nitrogen but can lead to high ammonia levels and an imbalanced C: N ratio, which can be metabolically costly for the larvae to process. Conversely, bread waste (BW) is a source of readily available carbohydrates but is comparatively poor in nitrogen and other essential nutrients. The 1:1 ratio likely established a more favorable Carbon-to-Nitrogen (C: N) ratio, which is a critical determinant of larval growth and metabolic efficiency. A balanced C: N ratio allows the larvae to efficiently allocate resources towards both energy production (from carbohydrates) and protein synthesis for biomass accumulation (from nitrogenous compounds). This nutritional synergy minimizes the metabolic energy spent on excreting excess nitrogen or compensating for nutrient deficiencies, thereby maximizing the conversion of feed into biomass. This explains not only the increased larval weight but also the enhanced bioconversion efficiency observed in this group. Therefore, the chemical composition of the substrate directly dictates the metabolic pathways and growth potential of the larvae, highlighting the importance of strategic substrate blending for optimizing BSFL production. These results are in line with previous studies. ur Rehman et al.^42^ reported lower larval weights on CM, while Danieli et al.^43^ observed higher FCRs for animal manure and municipal waste compared to fruit- and vegetable-based diets. Reported FCRs for standard BSFL diets typically range between 4.96 and 7.11, which aligns with the values obtained in the present study. More recent studies confirm the importance of substrate type: Belperio et al.^44^ found that larvae fed vegetable-based diets exhibited reduced growth and dry matter, whereas omnivorous diets promoted growth comparable to high-quality chicken feed. Likewise, Cattaneo et al.^45^ demonstrated that mixed (omnivorous) substrates not only improved growth efficiency but also provided balanced nutrient profiles without signs of catabolism, while meat-based diets were less suitable. Together, these findings suggest that the benefits observed with CM: BW mixtures may be attributed not only to improved macronutrient balance but also to the complementary nature of plant- and animal-derived components. Such mixed substrates appear to represent an optimal solution for sustainable BSFL production, although their practical application may be influenced by regional waste availability and legislative restrictions. It is also important to note that strain differences may contribute to variation in the performance of H. illucens. Generalovic et al.^46^ reported significant strain × diet interactions affecting larval weight, development time, family viability, and protein content. Moreover, their study provided evidence of high heritability for body size traits, highlighting the potential for genetic improvement through selective breeding. Although the present work was performed using a single laboratory strain under controlled conditions, future research should consider evaluating multiple genetic strains to better understand genotype-by-environment interactions and their implications for large-scale rearing systems.

Table 2. Larval and pupal weights of BSF on substrate diets and their bioconversion efficiency and feed conversion ratio.DietsFW(mg/ Larvae) ± SDDW(mg/Larvae) ± SDDW(mg/Pupae) ± SDBioconversion efficiency% ± SDFeed conversion ratio ± SD CM 126.6 ± 11.5^b^44.2 ± 2.25^c^29 ± 2.4^c^4.94 ± 0.13^c^7.46 ± 0.55^a^ BW 186.6 ± 11.54^b^72.9 ± 5.87^b^56.6 ± 0.34^b^8.6 ± 0.73^b^6.88 ± 0.43^a^ 3CM :1BW 253.3 ± 11.54^a^86.2 ± 2.61^b^69.6 ± 6.0^b^8.78 ± 0.05^b^4.97 ± 0.24^b^ 1CM:1BW 260 ± 20.0^a^90.6 ± 3.14^a^77 ± 6.4^a^10.82 ± 0.39^a^4.85 ± 0.39^b^ F value 29.995.960.595.929.4 H.S.D. 1.330.320.340.810.79 P value 0.00010.0000.0000.0000.0001Values are presented as mean ± standard deviation (n = 3). Data were analyzed using a one way ANOVA. Means within the same column followed by different letters (a, b) are significantly different at P < 0.05 level according to Tukey’s HSD test. ( P < 0.001)Abbreviations: CM, Chicken manure; BW, Bread waste, FW, Fresh weight, DW, Dry weight.

Chemical composition of BSF larval biomass

The chemical composition of BSF larvae varied significantly depending on the feeding substrate (Table 3). ANOVA results indicated highly significant differences among treatments for ash, fat, and total carbohydrates (p < 0.001), and significant effects for CP (p = 0.04) and CF (p = 0.02). Larvae reared on CM alone exhibited the poorest nutritional profile, characterized by the lowest fat content (12.77%) and crude fiber (5.70%), and the highest ash (43.33%) and carbohydrate content (13.00%). In contrast, larvae from the BW treatment had the highest lipid accumulation (48.89%) and CP (31.89%), with significantly lower ash (4.10%) compared to all other groups (Tukey’s HSD, p < 0.05). The CM: BW mixtures yielded intermediate values, with the 1:1 ratio producing the most balanced composition: moderate fat (29.20%), CP (26.06%), and reduced ash (31.72%). From a nutritional perspective, supplementing CM with BW clearly enhanced larval lipid and protein content while reducing excessive ash deposition. The improved composition in CM: BW mixtures likely reflects the higher digestibility and energy availability from BW, which supports anabolic processes such as lipid storage and protein synthesis. Conversely, the poor nutritional quality of larvae reared on CM alone may be attributed to its low energy density and unbalanced nutrient profile.

These findings are in line with earlier studies reporting substrate-dependent variation in BSFL composition. Nguyen et al.^47^ showed that kitchen waste diets, rich in available energy, produced heavier larvae with higher lipid contents compared to protein-dense animal by-products. Similarly, Jucker et al.^48^ observed that larvae on low-protein, high-carbohydrate substrates accumulated more lipids but achieved smaller sizes and slower development. Our results also agree with Zhou et al.^49^, who highlighted protein as a limiting factor for larval growth and feed conversion efficiency. The relatively higher CP and lipid levels in BW and mixed diets compared to CM suggest that macronutrient balance, rather than individual nutrient concentration, plays a central role in optimizing larval composition. On a practical level, BW alone yielded superior biomass quality, but its potential economic value as animal feed may restrict large-scale use. In contrast, combining CM with BW at a 1:1 ratio produced not only the highest larval biomass (Table 2) but also a nutritionally balanced composition, offering a cost-effective strategy that enhances both protein and lipid yields while reducing ash. This aligns with the broader literature emphasizing the need to blend substrates with complementary nutrient profiles^50,51^. Moreover, BSFL rearing offers advantages over composting, including faster waste reduction and conversion of low-value residues into high-quality protein and lipids, while producing frass as a by-product fertilizer. Finally, some variation between our results and published data may be explained by environmental conditions, substrate handling, and genetic background of the larvae. As highlighted by Generalovic et al.^46^, strain × diet interactions can significantly influence growth performance and nutrient accumulation, suggesting that future studies should integrate genetic diversity into BSF rearing optimization.

Table 3. The mean values of the chemical composition of the BSF larvae reared on different diets.DietsProximate content (%)AshFatCPCFTC BW 4.10 ± 0.23^c^48.89 ± 2.30^a^31.89 ± 1.80^a^8.12 ± 0.57^a^6.99 ± 0.54^b^ CM 43.33 ± 2.37^a^12.77 ± 0.69^c^25.20 ± 1.40^b^5.70 ± 0.17^b^13.00 ± 0.92^a^ CM: BW(with ratio 1:1) 31.72 ± 0.98^b^29.20 ± 1.65^b^26.06 ± 1.25^ab^6.34 ± 0.38^ab^6.68 ± 0.83^b^ CM: BW (with ratio 3:1) 31.42 ± 1.04^b^27.48 ± 1.73^b^25.97 ± 1.39^ab^6.29 ± 0.49^ab^8.83 ± 0.64^b^ F. value 143.976.34.435.815.1 H.S.D. 4.55.54.71.42.4 P value 0.0000.0000.040.020.001**Values are presented as mean ± standard deviation (n = 3). Data were analyzed using a one way ANOVA. Means within the same column followed by different letters (a, b) are significantly different at P < 0.05 level according to Tukey’s HSD test. (* P < 0.05, ** P < 0.01, *** P < 0.001, ns)Abbreviations: CM, Chicken manure; BW, Bread waste, CP, Crude protein, CF, Crude fiber, TC, Total carbohydrate.

Enumeration, isolation and identification of bacterial diversity in BSF eggs and the larval gut

Bacteria were enumerated and isolated from the egg surfaces and larval guts of BSF reared on BW substrate, aiming to obtain isolates with potential roles in host nutrition. The total bacterial count on the egg surface reached 35 × 10⁵ CFU/ml, while the larval gut contained 24 × 10⁵ CFU/ml (Table S1). Ten bacterial isolates were recovered from the egg surfaces, and three isolates from the larval gut. Isolates were selected based on colony morphology and purified to establish a collection of pure cultures. Of the total isolates, 5 (38.46%) were cocci, and 8 (61.54%) were bacilli, indicating a higher prevalence of rod-shaped bacteria (Table S2). Similarly, Sukmawati et al.^25^ reported 46 microbial isolates (20 yeast and 26 bacterial) from BSFL, confirming the presence of diverse microbial communities.

Proteases and ureases enzyme activity

Microbial decomposition of organic waste in a BSFL bioreactor often involves a microbial consortium. These microbes produce various hydrolytic enzymes, including proteases and ureases that help break down macromolecules such as carbohydrates, proteins, and lipids^52^. At the end of the incubation period, out of five tested bacterial isolates (EN2, EO3, EO4, EO6, and GL), three isolates—EO4, EO6, and EO3—exhibited strong protease activity, evidenced by large clear zones on skimmed milk agar plates (Fig. 1a), ranked in the order EO4 > EO6 > EO3. This demonstrates their capacity to degrade proteins and secrete proteases^53^. Proteases are valuable enzymes that convert complex protein compounds into amino acids and peptides^54^. These enzymes are typically detected on skim milk agar, where a clear zone indicates protease activity due to casein degradation^55^. A clear zone of 12 mm or more is indicative of potent proteolytic activity^53^. The isolates were also screened for their ability to uptake nitrogen. EO4, EO6, and GL demonstrated high urease activity, as seen by the rapid development of a pink coloration on urea agar plates after incubation (Fig. 1b). This indicated the isolates’ ability to hydrolyze urea and release ammonia. Urease converts urea into ammonium (NH^4+^) and carbonate (CO_3_^2−^), raising local pH and promoting calcium carbonate precipitation^56–58^. BSFL naturally produce enzymes associated with their digestive systems, including amylase, lipase, protease, and cellulase^59^. For example, Bacillus subtilis found in the BSFL gut is known for amylase production. Sukmawati et al.^25^ also reported that 12 yeast isolates from BSFL showed protease activity, with 11 isolates rated (+) and one (++), and 16 bacterial isolates showed protease activity, with 11 rated (+) and five (++). From the results, the five bacterial isolates—EN2, EO3, EO4, EO6, and GL—demonstrated urease and protease activities and were selected for further identification.

Fig. 1. Protease and urease detection in the best bacterial isolates on agar plates. (a) Protease assay on skimmed milk agar medium which the positive results are a clear halo zone and negative results are no clear halo zone. (b) Urease assay on a urea agar medium in which the positive results are red color and negative results are yellow color.

Identification of selected isolates

These five isolates were identified phenotypically and genotypically. Phenotypic identification based on cultural and morphological features followed the keys by Garrity et al.^30^, Niall and Paul^31^, and Parte et al.^32^, as summarized in Table 4; Fig. 2a-e. EO3, EO4, and EO6 were classified as Bacillus species: Gram-positive, long rod-shaped, endospore-forming, and motile. GL was identified as Morganella—short rods, Gram-negative, non-spore-forming, and non-motile. EN2 was identified as Microbacterium, appearing as Gram-positive cocci in clusters, non-spore-forming, and producing yellow pigment. Genotypic identification was confirmed by 16 S rRNA sequencing. The amplified sequences were 1374, 409, 1425, 1421, and 1413 bp for EN2, EO3, EO4, EO6, and GL, respectively. These sequences were aligned using BLAST and RDP against NCBI’s database. The isolates showed 100% identity with Microbacterium paraoxydans, Bacillus proteolyticus, Priestia megaterium, Bacillus subtilis, and Morganella morganii, respectively. Phylogenetic trees were constructed using MEGA11 (Fig. 3A-E). Taxonomically, they belonged to Actinomycetota (1 isolate), Bacillota (3 isolates), and Pseudomonadota (1 isolate). The respective sequences were deposited in under accession numbers: PQ763439, PQ763441, PQ763491, PQ763537, and PQ763412, respectively. (Table 5).

Table 4. Phenotypic identification (colony morphology and microscopic characteristics) of the selected bacterial isolates.CharacteristicsCode of bacterial isolatesEN2 isolateEO3 isolateEO4 isolateEO6 isolateGL isolateColony morphologyShapeSmallMediumMediumMediumSmallRoundRoundRoundRoundRoundSmoothSmoothSmoothSmoothSmoothYellow-pigmented coloniesWhite-pigmented coloniesWhite-pigmented coloniesWhite-pigmented coloniesWhite-pigmented coloniesDiffusible pigments-----Microscopic charactersShapeCocci in clusterLong bacilli in chainsLong bacilli in chainsLong bacilli in chainsSingle, short rodGram stain react+ve+ve+ve+ve-veEndospores form++++-Motility-+++-Genus Microbacterium

Bacillus

Bacillus

Bacillus

Morganella

Fig. 2. Different microbial colonies from BSF eggs-associated and larval guts on LB agar medium plates (A), Growth of the bacterial isolates (EO4, EO6, EO3 and GL isolates) on LB agar medium (B-E), and Gram stain and cell morphology of some of the bacterial isolates EN2, EO4, EO6 and GL (F-I).

Fig. 3. Neighbor-joining tree based on 16 S rRNA sequences of the genus bacterial isolate codes EN2 (A), EO3 (B), EO4 (C), EO6 (D) and GL (E) obtained from BLAST search showing the position of isolate and related strains.

Table 5. Strains were designated as Microbacterium paraoxydans, Bacillus proteolyticus, Priestia megaterium, Bacillus subtilis, and Morganella morganii.KingdomPhylumClassOrderFamilyGenusScientific NameAccession NumberBacteriaActinomycetotaActinomycetesMicroccalesMicrobacteriaceae Microbacterium Microbacterium paraoxydans strain SR PQ763439 BacillotaBacilliBacillalesBcillaceae Bacillus Bacillus proteolyticus strain SR PQ763441 Priestia megaterium strain SR PQ763491 Bacillus subtilis strain SR PQ763537 PseudomonadotaGammaproteobacteriaEnterobacterialesMorganellaceae Morganella Morganella morganii srtain SR PQ763412

Effects of bacterial inoculation on BSFL performance

This study investigated how BSF egg- and gut-associated bacteria influence larval dry weight gain, bioconversion efficiency, and nutrient accumulation when BSFL are reared on CM and BW at different ratios. Four isolates (M. paraoxydans, B. proteolyticus, P. megaterium, and B. subtilis) were obtained from eggs, while M. morganii was isolated from the larval gut. Each strain was separately inoculated into sterile substrates, which were then applied to CM, 1CM:1BW, and 3CM:1BW. The results (Table 6) showed that M. morganii supplementation significantly increased larval dry weight by 12.44% in CM and 9.7% in 3CM:1BW, followed by M. paraoxydans and B. subtilis, while no significant effects were observed in 1CM:1BW. Similarly, bioconversion efficiency improved in CM and 3CM:1BW treated with M. morganii (11.7% and 5.6% increases, respectively), whereas changes in 1CM:1BW were not statistically significant. Feed conversion ratio (FCR) data further revealed a 14.47% decrease in CM treated with M. morganii, indicating more efficient feed-to-biomass conversion. These findings are consistent with previous reports highlighting the positive role of BSF-associated bacteria in larval development and organic waste bioconversion^24,60–62^.

The larval gut hosts a diverse microbiota, including Enterococcus, Providencia, Morganella, and Lactobacillus, which contribute to nutrient digestion and host health^63^. Gut microbial composition was strongly shaped by the rearing substrate. For instance, larvae fed fermented sericulture waste harbored higher abundances of beneficial genera (Sedimentibacter, Clostridium, Enterococcus, Bacteroides, and Bacillus), whereas unfermented waste led to higher levels of potentially pathogenic genera (Providencia, Klebsiella, and Escherichia)^64^. These shifts correlated with improved metabolic activity, immune response, survival, and substrate conversion efficiency, emphasizing the critical interaction between substrate and microbiota. Supporting this, Gorrens et al.^65^ identified 172 dominant aerobic bacterial isolates from BSFL reared on chicken feed or fiber-rich substrates, with major contributions from Proteobacteria (66.3%) and Firmicutes (30.2%), and the most abundant genera being Enterococcus (29.1%), Escherichia (22.1%), Klebsiella (19.8%), Providencia (11.6%), Enterobacter (7.6%), and Morganella (4.1%). Among these, the role of Morganella is particularly noteworthy in our study. The functional role of M. morganii in the black soldier fly gut is central to understanding variations in larval performance. Our findings suggest that M. morganii may play an important role in protein and amino acid metabolism, nitrogen recycling, and substrate degradation. Collectively, these microbial functions are likely to enhance nutrient solubilization and availability, thereby facilitating nutrient assimilation by the larvae and potentially improving both feed conversion ratio and larval biomass production. The relatively high abundance of M. morganii detected in the hindgut in the present study is consistent with the observations of Vandeweyer et al.^66^, who reported that nitrogenous waste products are released at the onset of the hindgut region. This anatomical localization supports the interpretation of M. morganii as a stable and functionally relevant member of the BSF gut microbiota. Under the controlled rearing conditions applied in this study, M. morganii appears to contribute to digestive efficiency through its metabolic activity and interactions within the gut microbial community, rather than exerting pathogenic effects. Accordingly, its presence may be considered beneficial in this context, supporting a symbiotic system that promotes efficient nutrient utilization and robust larval growth. Overall, these results demonstrate that both substrate composition and associated microbial communities are key determinants of gut microbiota structure and functionality. Strategic manipulation of rearing substrates, combined with supplementation of beneficial microbes, can improve larval growth, optimize waste bioconversion, and enhance the nutritional quality of insect-derived protein, thereby supporting sustainable BSFL production and advancing circular bioeconomy applications.

Table 6. Dry weight, feed conversion ratio and bioconversion efficiency of BSFL reared on three different diets; CM, 3CM:1BW, and 1CM:1BW treated with Bacillus subtilis,* Microbacterium paraoxidans*, Morganella morganii, and control (without bacteria).DietsParametersControlBacterial treatmentsp value Bacillus subtilis

Microbacterium paraoxidans

Morganella morganii F valueCMDW (mg)43.5 ± 1.85^b^44.2 ± 2.25^b^45.66 ± 0.61^ab^48.86 ± 0.75^a^7.130.01FCR7.46 ± 0.55^bc^10.5 ± 1.0^a^8.1 ± 0.0^b^6.38 ± 0.0^c^28.30.0001**BCE%4.94 ± 0.23^b^5.025 ± 0.28^b^5.208 ± 0.07^ab^5.6 ± 0.09^a^7.130.013CM: 1BWDW(mg)74.2 ± 0.41^b^78.2 ± 4.4^ab^81.5 ± 1.74^a^78.4 ± 0.50^ab^4.60.03FCR4.97 ± 0.24^a^5.4 ± 0.28^a^4.85 ± 0.39^a^4.85 ± 0.39^a^1.90.2 nsBCE%8.78 ± 0.05^b^9.28 ± 0.55^ab^9.69 ± 0.21^a^9.31 ± 0.06^ab^4.60.031CM: 1BWDW(mg)90.6 ± 3.14^a^86.2 ± 2.61^a^84.6 ± 7.97^a^84.4 ± 1.85^a^1.170.3 nsFCR4.85 ± 0.39^a^4.59 ± 0.2^a^4.85 ± 0.39^a^4.59 ± 0.2^a^0.690.5 nsBCE%10.825 ± 0.39^a^10.28 ± 0.32^a^10.075 ± 0.99^a^10.05 ± 0.23^a^1.170.3 nsValues are presented as mean ± standard deviation (n = 3). Data were analyzed using a one way ANOVA. Means within the same row followed by different letters (a, b) are significantly different at P < 0.05 level according to Tukey’s HSD test. ( P < 0.05, *** P < 0.001, ns, not significant P ˃ 0.05)Abbreviations: CM, Chicken manure; BW, Bread waste, DW, Dry weight (mg/larvae), FCR, feed conversion ratio, BCE, bioconversion efficiency.

The chemical components of BSFL biomass

The biochemical composition of BSFL reared on CM, 3CM:1BW, and 1CM:1BW diets supplemented with different bacterial strains is summarized in Table 7. Both substrate type and bacterial supplementation significantly influenced proximate composition. Under the CM diet, M. morganii significantly increased ash (46.54 ± 3.22%) compared to the control (43.33 ± 4.1%) and B. subtilis (33.99 ± 1.99%) (F = 7.68, p = 0.009). Fat content was also higher with M. morganii (19.08 ± 1.8%; p < 0.001), whereas carbohydrates were strongly reduced (3.52 ± 0.3%) compared to the control (13.0 ± 1.6%) (F = 43.4, p < 0.001). Fiber was likewise higher (7.1 ± 0.16% vs. 5.7 ± 0.3%; p < 0.001), while crude protein (CP) did not vary significantly (p > 0.05). These results indicate that M. morganii shifted metabolism toward lipid and fiber accumulation at the expense of carbohydrates. In the 3CM:1BW diet, the effect of bacterial strains was more moderate. EE was significantly lower in M. paraoxydans (19.92 ± 1.93%) than in the control (27.48 ± 3.0%) (F = 5.9, p = 0.01), while carbohydrates were markedly higher (15.42 ± 1.99% vs. 8.83 ± 1.1%; F = 5.3, p = 0.02). Fiber was lowest under M. paraoxydans (3.48 ± 0.21% vs. 6.29 ± 0.85%; p < 0.001). CP and ash showed no significant variation (p > 0.05). This pattern suggests that M. paraoxydans enhanced carbohydrate retention and reduced fiber, potentially improving digestibility and energy availability. For the 1CM:1BW diet, differences among bacterial treatments were mostly non-significant (p > 0.05). However, carbohydrates varied significantly (F = 7.78, p = 0.009), with M. morganii reaching the highest value (9.69 ± 1.2% vs. 6.68 ± 1.43% in the control). Although EE and CP were not statistically different, M. paraoxydans produced the highest absolute values (30.14 ± 3.1% EE; 26.62 ± 1.79% CP). Overall, bacterial supplementation substantially modulated BSFL biochemical composition. M. morganii under CM promoted lipid and fiber accumulation while depleting carbohydrates. In contrast, M. paraoxydans under 3CM:1BW increased carbohydrates and protein simultaneously, a favorable balance for larval growth since combined protein and carbohydrate (P + C) are key determinants of biomass quality and energy supply^67^. These findings align with earlier studies (Nguyen et al.^68^ and Mazza, L. et al.^24^) showing that microbial supplementation enhances fat and protein but in strain- and substrate-specific ways. From an application perspective, the CM-only diet was the most responsive to bacterial supplementation, but the 3CM:1BW + M. paraoxydans treatment provided the best compromise between cost, nutritional balance, and production efficiency. Given its higher proportion of low-cost manure and improved P + C profile, this combination was selected for further investigation.

Table 7. Chemical composition of BSFL reared on three diets; CM, 3CM:1BW, and 1CM:1BW treated with Bacillus subtilis,* Microbacterium paraoxidans*,* Morganella Morganii* and control (without bacteria).DietsChemical compositionBacterial treatmentsp valueControl Bacillus subtilis

Microbacterium paraoxidans

Morganella morganii F valueCMAsh43.33 ± 4.1^a^33.99 ± 1.99^b^40.55 ± 3.65^ab^46.54 ± 3.22^a^7.680.009Fat12.77 ± 1.2^b^14.48 ± 1.1^b^12.66 ± 0.98^b^19.08 ± 1.8^a^15.80.001CP25.2 ± 2.43^a^23.21 ± 2.3^a^25.32 ± 2.8^a^23.76 ± 2.55^a^0.510.68 nsCF5.7 ± 0.3^b^4.6 ± 0.4^c^5.28 ± 0.28^bc^7.1 ± 0.16^a^37.70.000TC13 ± 1.6^b^23.72 ± 3.1^a^16.19 ± 2.66^b^3.52 ± 0.3^c^43.40.0003CM: 1BWAsh31.42 ± 1.8^a^30.98 ± 3.15^a^32.32 ± 2.98^a^34.19 ± 2.1^a^0.910.47 nsEE27.48 ± 3.0^a^25.11 ± 2.2^ab^19.92 ± 1.93^b^21.15 ± 2.7^ab^5.90.01CP25.97 ± 2.41^a^27.49 ± 2.39^a^28.87 ± 3.21^a^28.04 ± 2.04^a^0.680.58 nsCF6.29 ± 0.85^a^5.17 ± 0.17^ab^3.48 ± 0.21^c^4.69 ± 0.28^bc^18.60.0006TC8.83 ± 1.1^b^11.25 ± 2.62^ab^15.42 ± 1.99^a^11.96 ± 2.14^ab^5.30.021CM: 1BWAsh31.72 ± 1.7^a^31.91 ± 3.14^a^32.05 ± 4.0^a^31.46 ± 4.0^a^0.010.99 nsEE29.2 ± 2.85^a^28.47 ± 2.9^a^30.14 ± 3.1^a^26.84 ± 2.0^a^0.770.54 nsCP26.06 ± 2.16^a^26.97 ± 2.7^a^26.62 ± 1.79^a^25.98 ± 3.4^a^0.090.95 nsCF6.34 ± 0.65^a^6.84 ± 1.0^a^5.71 ± 0.71^a^6.02 ± 0.89^a^1.020.43 nsTC6.68 ± 1.43^b^5.81 ± 0.81^b^6.6 ± 0.6^b^9.69 ± 1.2^a^7.780.009Values are presented as mean ± standard deviation (n = 3). Data were analyzed using a one way ANOVA. Means within the same row followed by different letters (a, b) are significantly different at P < 0.05 level according to Tukey’s HSD test. (* P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant P ˃ 0.05)Abbreviations: CM, Chicken manure; BW, Bread waste, CP, Crude protein, CF, Crude fiber, TC, Total carbohydrate.

Amino acids composition analysis

The amino acid profile of BSFL was significantly improved by supplementation with M. paraoxidans compared to the control (Table 8; Fig. 4). EAAs increased from 141.79 to 213.36 mg/g (~ 50%), and NEAAs from 191.18 to 302.34 mg/g (~ 58%), with statistically significant differences (p < 0.05). Valine, Leucine, Tyrosine, Isoleucine, Phenylalanine, Lysine, and Threonine showed marked increases, while Histidine and Methionine remained unchanged. Among NEAAs, Alanine, Glutamic acid, Aspartic acid, Glycine, Serine, and Arginine increased significantly, whereas Proline showed no significant change. These improvements can be attributed to the proteolytic activity of M. paraoxidans, enhancing protein bioavailability by hydrolyzing dietary proteins into free amino acids. The increase in EAAs is particularly relevant for poultry and aquaculture nutrition, as BSFL are often limited in Lysine and Methionine^67,69^. Elevated levels of Valine, Leucine, and Lysine indicate improved protein quality, enhancing the suitability of BSFL meal as a sustainable alternative to conventional protein sources such as fishmeal and soybean meal. Likewise, the increase in NEAAs, including Glycine, Alanine, and Glutamic acid, supports energy metabolism, growth performance, and feed palatability. These findings align with previous reports that gut-associated microbes can synthesize or enhance the bioavailability of amino acids in insects^70,71^. Beyond amino acid composition, substrate-associated microbial communities influenced larval performance and metabolic efficiency. Spranghers et al.^67^ and Sealey et al.^69^ highlighted that optimal protein and carbohydrate balance is a key determinant of larval biomass quality and energy supply. Memon et al.^53^ demonstrated that BSFL reared on fermented sericulture waste had higher abundances of beneficial genera (Sedimentibacter, Clostridium, Enterococcus, Bacteroides, Bacillus), enhancing metabolic activity, immune responses, and substrate conversion, whereas unfermented waste increased potentially pathogenic genera (Providencia, Klebsiella, Escherichia).

Collectively, these results indicate that M. paraoxidans supplementation not only improves crude protein and amino acid profiles but also enhances the overall nutritional value and functional quality of BSFL as animal feed. Furthermore, BSFL-based bioconversion of organic waste offers ecological benefits, producing high-value protein and fat while reducing environmental impact, including greenhouse gas emissions, compared to conventional protein sources^72,73^. The frass by-product additionally has potential as a biofertilizer, contributing to a circular bioeconomy.

Fig. 4. Retention times (min) of the amino acids analysis for each numbered peak using high-performance amino acid analyzer system. (a) After feeding on 3CM:1BW alone as a control; (b) After feeding on 3CM:1BW in combination with M. paraoxidans strain SR.

Table 8. Effect of dietary supplementation with Microbacterium paraoxidans strain SR on the amino acid composition of BSF larvae.Essential Amino AcidsCystineHistidineIsoleucineLeucineLysineMethioninePhenylalanineThreonineTyrosineValineSumTreated (mg/g)0.7 ± 0.04^a^17.44 ± 1.9^a^21.05 ± 1.68^a^49.96 ± 4.6^a^15.88 ± 1.88^a^2.7 ± 0.2^a^12.92 ± 1.22^a^15.41 ± 1.95^a^23.04 ± 2.41^a^54.26 ± 3.1^a^213.36Control (mg/g)0.48 ± 0.09^b^24.43 ± 4.14^a^12.94 ± 2.05^b^28.82 ± 2.9^b^10.99 ± 1.5^b^2.61 ± 0.2^a^8.09 ± 0.68^b^11.07 ± 1.5^b^15.65 ± 2.1^b^26.71 ± 1.6^b^141.79F value14.97.062845.312.40.335.89.316.03187.1H.S.D0.15-4.28.73.8-2.233.025.125.59P value0.010.05 ns0.0060.0020.020.6 ns0.0030.030.010.0002* Non-Essential Amino Acids

Alanine

Arginine

Aspartic

Glutamic

Glycine

Serine

Proline

Sum Treated (mg/g)124.59 ± 12^a^13.82 ± 2.1^a^41.56 ± 3.7^a^45.85 ± 3.85^a^40.23 ± 2.23^a^17.91 ± 0.91^a^18.38 ± 1.88^a^302.34Control (mg/g)58.23 ± 6.23^b^8.41 ± 0.65^b^31.15 ± 2.75^b^37.9 ± 2.4^b^27.64 ± 1.64^b^12.08 ± 0.9^b^15.77 ± 1.64^a^191.18F value72.218.115.29.262.0562.23.2H.S.D21.63.57.37.24.42.05-P value0.0010.010.010.03*0.0010.0010.14 nsValues are presented as mean ± standard deviation (n = 3). Data were analyzed using a one way ANOVA. Means within the same column followed by different letters (a, b) are significantly different at P < 0.05 level according to Tukey’s HSD test. ( P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant P ≥ 0.05)Treated: BSFL feeding on 3CM:1BW diet supplemented with Microbacterium paraoxidans strain SR.Control: BSFL feeding on 3CM:1BW diet without bacterial supplementation.Abbreviations: BSF, Black soldier fly; CM, Chicken manure; BW, Bread waste.

Conclusion

This study was conducted in two experimental phases. In the first experiment, co-processing CM with BW significantly improved the growth performance and nutritional quality of BSFL. The 1:1 BW: CM ratio produced the highest larval biomass, feed conversion efficiency, and nutrient accumulation compared to other substrate ratios, highlighting the benefit of balancing nitrogen-rich CM with carbohydrate-rich BW. In the second experiment, the addition of beneficial bacteria isolated from BSFL eggs and larvae B. subtilis,* M. paraoxydans* and M. morganii further enhanced bioconversion outcomes. A key novel finding is the substantial improvement in amino acid composition following microbial inoculation. BSFL reared on 3CM:1BW diets supplemented with M. paraoxydans exhibited a 50.5% increase in EAA (from 141.79 to 213.36 mg/g dry matter) and a 58.1% increase in NEAA (from 191.18 to 302.34 mg/g dry matter) compared to the control. Importantly, this enrichment included feed-limiting amino acids such as lysine, methionine, valine, and leucine, which are often deficient in plant-based feed ingredients. Together, these findings demonstrate that (i) co-utilizing CM and BW provides a balanced substrate that maximizes BSFL performance, and (ii) microbial inoculation plays a vital role in enhancing protein quality by increasing the synthesis and availability of both EAA and NEAA. The combined approach supports sustainable organic waste management, reduces dependency on fishmeal and soybean meal, and contributes to a circular bio-economy. Furthermore, BSFL frass obtained as a by-product has potential application as an organic bio-fertilizer. Future research should focus on scaling up this integrated system, evaluating the digestibility and safety of the enriched BSFL protein in animal feeding trials, and developing targeted microbial consortia to further optimize amino acid synthesis and overall larval performance.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1