Optimize using black soldier fly larvae reared on chicken manure as an alternative feed in the insect farming industry

doi:10.21203/rs.3.rs-5004505/v1

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Optimize using black soldier fly larvae reared on chicken manure as an alternative feed in the insect farming industry

https://doi.org/10.21203/rs.3.rs-5004505/v1

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In April 2022, the State Information Service (SIS) released statistics indicating that Egypt produced over 1.4 billion birds overall, including 320 million chickens. The high price of chicken feed presents Egypt with a significant challenge. As many research suggested the use of chicken manure (CM) as a substitute feed, Egypt may transform it to a reasonably priced and readily available for chicken feed by using black soldier fly larvae (BSFL). BSFL fed on pure CM don’t gain significant weight and often died, this research aims to investigate the effects of adding different ratios of bread waste (BW) to CM to increase BSF larval development. The best results were obtained when BW was added to CM at a 1:1 ratio. Four bacterial isolates were isolated from BSF eggs, while one bacterial isolate, Morganella morganii, was isolated from the larval gut. On adding the bacterial isolates to different substrates, M. morganii showed the highest effect on larval weight and conversion rate, which may highlight its potential as a beneficial bacterial isolate for BSF cultivation. Further research is needed to explore these mechanisms, assess the potential of these bacterial isolates, and optimize the use of bacterial supplementation in insect farming systems.

Biological sciences/Ecology

Biological sciences/Microbiology

Earth and environmental sciences/Ecology

Earth and environmental sciences/Environmental sciences

Physical sciences/Chemistry

Chicken manure

black soldier fly

larval gut bacteria

Morganella

digestive enzymes

16S rRNA gene sequencing.

Hermetia illucens (Linnaeus) (Diptera: Stratiomyidae), better known as the black soldier fly (BSF), is one of the most promising insect species for commercial production. High protein and fat contents support its potential uses in chicken feed composition. The protein content and amino acid profile of H. illucens larvae are comparable to those of various high-protein feedstuffs, including fish and soybean meal. According to Makkar, et al. ^1,2, there could be a scarcity of soymeal, fishmeal, and other protein sources, which would raise the demand for cattle and poultry to eat alternate foods.

By converting organic waste into useful nutrients, BSF has the potential to contribute to the reduction of environmental pollution. Insect feeding may yield lipids, proteins, and chitin². Developed nations may find it advantageous to extract resources in a more sustainable manner when transforming organic wastes through BSF into a more useful alternative. Additionally, employing black soldier fly may offset the growing costs of ecological raw materials and animal feed^3,4.

Incorporating insects as a sustainable protein source in the diet of chickens could potentially contribute to achieving food security and addressing related issues. Feeding insects that have been raised on organic waste to chickens may offer a viable means of maintaining the economic sustainability of the poultry industry⁵.

Benefits of BSF include optimizing fertilizer use and turning it into safe waste; creating biofuel; and using biomass as an alternative protein source for chicken feed that takes cheap prices into account rather than soybeans. Chitin, which is extracted from the cuticle of insects and cocoons to form chitosan, has potential use in the fields of cosmetics, medicines, and insecticides^6,7,8. Using BLF larvae to break down organic waste is more efficient than using traditional methods when it comes to improving fertilizing properties within a short time^9,10.

Larvae of BSF, in addition to the related bacteria, decompose organic matter such as chicken manure (CM)¹¹. Poly-bacterial components of BSF increase the efficiency of manure conversion into nutrients accumulation in larvae, by reducing the amount of CM and enhancing larval biomass. Additionally, it is possible to lower the financial costs associated with the removal of chicken manure¹².

Numerous related symbiotic bacteria coexist with insects like Hermetia illucens¹³. According to Chen, et al. ¹⁴, host-associated microorganisms may play a role in the development of insect physiological functions, such as the immune system. According to recent research, symbiotic bacteria are essential for the growth of insects. The environment has an impact on the development of the gut microbiota and the generation of digestive enzymes throughout the life cycle ¹⁵. BSF-associated microorganisms help in sustaining insect performance¹⁶. According to documented studies, larvae fed chicken manure supplemented with isolated Bacillus increased in weight and developed faster¹⁷.

According to Prosdocimi, et al.¹⁸, adding certain isolate probiotics to the host food can effectively increase larval weight, growth rate, and adult survival. The gut microbiota's microbial composition, which includes bacteria and fungi, is greatly affected when used as a feeding substrate¹⁹. The microbiota harbored by insects contributes to complex processes such as improving the gastrointestinal tract, modulating the immune system²⁰, the digestion of plant polymers ²¹, synthesizing essential amino acids and vitamins ²², pheromone and kairomone synthesis for inter- and intraspecific communication, and additionally, the defense against pathogen and parasite colonization ¹³.Generally, the ability to grow on media with a high microbial content, such as feces, is related to a highly adaptable immune response.

The impact of symbiotic bacteria on nutrient production activity and their capacity to increase the rate at which BSF converts waste into insect biomass have not been well studied. In order to increase the larval biomass, this study was designed to investigate the biological characteristics of the black soldier fly when fed on chicken manure that has been treated with isolated bacteria.

Hermatia illucens

Study area

Laboratory experiments were carried out during 2023–2024 at the Environmental Studies Lab, Plant Protection Department, Faculty of Agriculture, Ain Shams University.

Insect culture

The black soldier fly larvae were obtained from a farm in Obour City, Ahmed Orabi Neighborhood, Egypt. It was reared in lab more than one generation. The larvae were reared on bread waste, while adults were reared in wooden cages covered with muslin cloth to allow ventilation and avoid insect escape. The cage was provided with substrate from food waste. Rolled cardboard paper was placed on top of the substrate for oviposition. Laids eggs were collected continuously and incubated to hatch.

Experimental design

The collected larvae in the experiments were six days old; they were similar in size and activity. Three experiments were designed as follows: feeding larvae on chicken manure only (CM), feeding larvae on chicken manure and bread waste with a ratio of (3 CM: 1 BW), and (1 CM: 1 BW). To investigate these experiments, three bacterial isolates were used. They are Microbacterium paraoxidans (EN2), Bacillus subtilis (EO6), and Morganella morganii (GL). Three replicates (100 larvae) were repeated for each. Eighteen grams of each substrate were added three times into a plastic plate with dimensions (5 cm h.×15 cm d.) when substrate humidity was 50%. Temperature and relative humidity were 25°C and 60–65%, respectively. At the end of the fifth larval stage, fifty larvae for each replicate were washed with water, dried with cotton cloth, and weighed. The other fifty larvae were allowed to pupate. An electric oven (Nüve En 500) was used to dry larvae and pupa for 48 hours at 65°C. Dried larvae and pupae weights were recorded using a digital balance (KERN ADJ 200–4)

Biochemical analysis of different substrates and BSFL biomass

Fresh chicken manure was collected from a poultry farm located in the Faculty of Agriculture at Ain Shams University, Egypt, and sun dried for one month before the experiment.

Proximate chemical analysis of chicken manure, bread waste, and BSF larvae in triplicates per each determination was performed for dry matter (DM), crude protein (CP), and ether extracts (EE), crude fiber (CF), and ash content according to A.O.A.C.²³. The nitrogen-free extract (NFE) was calculated by subtracting the summation percentages of CP, EE, CF, and ash from one hundred, as shown in Table 1.

Table 1

Chemical composition of different black solider fly substrates
	Substrates
	CM	BW	3CM:1BW	1CM:1BW
Chemical composition (%)
Dry matter (DM)	96.17	95.12	95.9	95.6
Crude Protein (CP)	18.65	9.49	16.3	14.07
Ether Extracts (EE)	22.28	5.93	18.19	14.1
Nitrogen-Free Extract (NFE)	25.20	75.30	37.72	50.25
Crude Fiber (CF)	6.70	8.24	8.08	7.47
Ash	27.18	1.03	20.64	14.1
*Ether Extracts (EE) = Fats, Nitrogen-Free Extract (NFE) = Carbohydrates, CM: Chicken manure, BW: Bread waste.

Media used in isolation and maintaining microorganisms

Luria-Bertani (LB) agar medium¹² was used to isolate, maintain, and preserve microorganisms. Its components were prepared as follows: (g/L): tryptone, 10.0; yeast extract, 5.0; NaCl, 10.0; agar, 18.0; and pH was adjusted to 7.0. Skimmed milk (SM) agar medium²⁴ was used for the protease-producing bacteria. It consists of (g/L): skimmed milk, 28.0; peptone, 5.0; yeast extract, 25.0; glucose, 1.0; CaCl₂.2H₂O 0.1; KH₂PO₄ 0.5; agar, 20.0; and pH was adjusted to 7.0. Urea-agar medium was used for the urea-producing bacteria.

Bacterial isolation

Isolation from eggs of BSF

Bacteria used in this study were isolated from the egg surface of BSF within 6 hours of oviposition. For the isolation of bacterial isolates from the egg surface, 0.5 g of BSF eggs were homogenized with 2 mL of distilled water. The mixture was constantly stirred and incubated for 5 minutes at 28 ºC. Following this, the suspension was diluted it by the serial dilution method²⁵ and used to isolate bacteria, and plates were incubated at 28–30°C for 5 days. Following the incubation period, colonies were selected and purified using the streak plate technique²⁶. The pure bacterial colonies were grown on LB agar slant, then kept in a refrigerator at 5 ºC and subcultured at monthly intervals for continued study.

Isolation from the larval gut of BSF

To isolate bacteria from larvae, the guts of 4th instar larvae were separated. The larval surface was sterilized with 75% ethanol for 3 minutes and then rinsed three times in sterile phosphate buffered saline (PBS) at pH 7.4. The whole gut (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 method²⁵. The diluted cell suspensions were plated on solid LB medium and incubated for 5 days at 28–30°C. To ensure the purity and stability of bacterial species depending on morphological features, they were subcultured three times. Bacterial isolates were inoculated into LB broth medium and agitated in a rotary shaker for one hour at 150 rpm for 48 h at 28–30°C to obtain a final concentration of 10⁸ CFU/mL.

Morphological characteristics and gram staining for the bacterial isolates

The selected bacterial isolates (EN2, EO3, EO4, EO6, and GL) were identified based on the cultural shape and color of the colony. Microscopic shape, Gram staining, and confirmed identification were based on molecular characterization (16S rRNA sequencing). The bacterial isolates were grown on sterile Petri plates containing autoclaved LB agar medium and incubated for 2 days at 28–30°C.

Culture was extracted at 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 fungal 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 in 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 mixture²⁷. 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.

Screening of enzymes produced by bacterial isolates

Urease Assay

The selective medium used for the urease test was urea agar medium with a phenol red indicator. Inoculating bacteria was carried out at 30°C for 48 hours in urea medium. Furthermore, identify ureases by looking for the red color.

Protease Assay

The protease assay used a petri dish that had been filled with protease medium and frozen. All isolates were incubated at 30°C for 48 hours. The presence of a clear zone refers to the protease enzymes produced by isolating bacteria. It could be expressed as the presence of a clear zone as (+) and the absence of a clear zone as (-)²⁴.

Effect of tested bacterial isolates on the chemical components of BSFL

This used 250 ml plugged Erlenmeyer flasks, each containing 100 ml of LB broth medium, inoculated with 5 ml of the selected bacterial culture, and incubated on a rotary shaker at 150 rpm and at 28–30°C for 2 days. At the end of the incubation period, the culture medium was added to the substrate.

Co-conversion of BSF larvae and individual bacterial isolates

The three BSF-related bacterial isolates, namely Microbacterium paraoxidans (EN2), Bacillus subtilis (EO6), and Morganella morganii (GL), were individually inoculated (30 mL) as supplements into 80 g, where each substrate was a proportion of 1% (v/w). One hundred, 6-day-old BSFL were inoculated into each substrate-bacterial mixture, as well as into 80 g of each substrate without bacterial supplementation, serving as the control. Each treatment was set up in three replicates and kept in the climatic chamber for approximately three weeks at 25°C with 60–70% RH. After that, the weight and conversion rate of BSFL were recorded using the previously described methodology²⁸ as follows:

BSFL conversion rate = total larval biomass (g) / feed added (g) X 100%

Statistical analysis

To show differences between treatments, analysis of variance (ANOVA) was employed, and Tukey's HSD post-hoc comparison was performed at a significance level of P < 0.05. Data analyses were performed with SPSS 16.0.²⁹.

Influence of different substrates on black solider fly larval biomass

The fresh and dry weight of black solider fly larvae biomass showed highly significant differences between four substrates (CM, 3CM:1BW, 1CM:1BW, and BW). The lowest weight was recorded when larvae were treated with CM substrate. The fresh larval weight was increased by 7.2%, 46.1%, and 49.9% when treated with BW, 3CM:1BW, and 1CM:1BW substrates, respectively. The dry larval weight was increased by 68.2%, 70.69%, and 99.5% when treated with BW, 3CM:1BW, and 1CM:1BW substrates, respectively (Table 2).

Influence of different substrates on BSF pupal weight

Statistical analysis showed highly significant differences between pupal dry weights of BSF when treated with four substrates (CM, 3CM:1BW, 1CM:1BW, and BW). The lowest weight was observed when treated with CM substrate. The dry pupal weight was increased by 41.7%, 51.3%, and 105.9% at 3CM:1BW, BW, and 1CM:1BW substrates, respectively (Table 2).

The co-conversion efficiency of BSFL

As shown in Table 2, the conversion rate of the different substrates recorded was highly significant. The lowest was achieved at CM and BW substrates. The conversion rate was increased for 3CM:1BW and 1CM:1BW substrates, by 46.3% and 50.5%, respectively. But, as with waste conversion, CM treatment lowered BSFL weight gain as compared with BW treatment.

Table 2

Larval, pupal weight and percentage conversion rate of black solider fly on four substrates.
Substrates	FW/50 Larvae ± se¹	DW/50 Larvae ± se¹	DW/50Pupae ± se¹	Conversion rate
CM	8.67 ± 0.33^b	2.17 ± 0.05^c	1.87 ± 0.05^c	10.8^b
BW	9.3 ± 0.33^b	3.65 ± 0.16^b	2.83 ± 0.01^b	11.62^b
3CM :1BW	12.67 ± 0.33^a	3.71 ± 0.01^b	2.65 ± 0.09^b	15.8^a
1CM:1BW	13 ± 0.58^a	4.33 ± 0.05^a	3.85 ± .18^a	16.25^a
F value²	29.944***	83.422***	60.511***	29.944***
H.S.D.³	1.33g	0.32g	0.34g	1.66%
¹se = standard error. ²F values determined after ANOVA (SPSS date), *** signifies p < 0.001. ³Means followed by different letters are significantly different at P < 0.05 level; Tukey’s HSD test. CM: Chicken manure, BW: Bread waste, FW: Fresh weight, DW: Dry weight

Essential chemical composition of larval biomass

Chemical compositions of larval biomass were analyzed to investigate the effect of larval feeding on the previous substrates. Table 3 shows the variation in contents of protein, fat, carbohydrate, crude fiber, and ash depending on treatments. Crude protein recorded the lowest value (25.2%) at CM treatment. CP content was increased by 3%, 3.4%, and 26.5% at 3CM:1BW, 1CM:1BW, and BW substrates, respectively, compared to CM treatment.

Fat content recorded the lowest value at CM treatment, where it recorded 12.77%. The percentage of fat increased to be 115.2%, 128.7%, and 282.8% compared to CM treatment when treated with 3CM:1BW, 1CM:1BW, and BW substrates, respectively.

Carbohydrates recorded the highest value at CM treatment (13%). Carbohydrate content decreased by 32%, 46.2%, and 48.6% in 3CM:1BW, BW, and 1CM:1BW substrates, respectively, compared to CM treatment.

Crude fiber (CF) contents recorded the lowest value at CM treatment (5.7%). CF content decreased by 10.3%, 11.2%, and 42.4% at 3CM:1BW, 1CM:1BW, and BW substrates, respectively, compared to CM treatment.

When ash contents were analyzed, they achieved the highest value at CM treatment (43.33%). Ash content decreased by 7.4%, 26.8%, 27.5%, and 90.53% at 1CM:1BW, 3CM:1BW, and BW substrates, respectively, compared to CM treatment.

Table 3

Chemical composition of BSFL reared on different substrates.
	Chemical component ± se¹
	%CP²	%Fat²	%Carbohydrate²	%CF²	%Ash²
Substrates
CM	25.2 ± 1.4^b	12.77 ± 0.69^c	13 ± 0.92^a	5.7 ± 0.17^b	43.33 ± 2.37^a
BW	31.89 ± 1.8^a	48.89 ± 2.3^a	6.99 ± 0.54^b	8.12 ± 0.57^a	4.1 ± 0.23^c
3CM :1BW	25.97 ± 1.39^ab	27.48 ± 1.73^b	8.83 ± 0.64^b	6.29 ± 0.49^ab	31.42 ± 1.04^b
1CM:1BW	26.06 ± 1.25^ab	29.2 ± 1.65^b	6.68 ± 0.83^b	6.34 ± 0.38^ab	31.72 ± .98^b
F. value³	4.430**	76.384***	15.118***	5.876**	144.689***
H.S.D.⁴	4.79%	5.53%	2.4%	1.4%	4.5%
¹se = standard error. ²The chemical composition of larvae. See Material and methods. ³F values determined after ANOVA (SPSS date), *** signifies p < 0.001. ⁴Means followed by different letters are significantly different at P < 0.05 level; Tukey’s HSD test. CM: Chicken manure, BW: Bread waste, CP: Crude protein, CF: Crude fiber.

The obtained data investigated the effect of different treatments on larval, pupal weight, conversion efficiency, and nutritional accumulation of BSFL. Data analysis proved that chicken manure treatment (CM) recorded the lowest values of weight and nutritional accumulation of BSF, while bread waste (BW) recorded higher weight and protein values when compared with CM treatment, but it recorded the opposite result with fat and cost. In addition, it is notable that the 3CM:1BW and 1CM:1BW demonstrated the highest values in weight and nutritional accumulation of BSFL. 1CM:1BW treatment is 14% larger in weight than 3CM:1BW treatment, and at the same time, it is 14% more expensive.

Among these results, the 1CM:1BW treatment may be the best one. It was greater in weight and conversion efficiency compared to other treatments. The nutritional accumulation of BSFL was balanced. Adding 50% of the bread waste enhances the properties of the chicken manure and promotes its benefits in terms of aeration and the accumulation of harmful bacteria.

The best chemical composition substrates of BSFL were recorded when adding of 1CM:1BW substrate. This remarkable result in BSFL weight could be attributed to the change of chemical composition ingredient that achieved after mixing chicken manure (CM) with bread waste (BW). Regarding to Table 1 CM show high percentage of protein (18.65%), fat (22.28%), and ash (27.18%) compared to BW which represents percentages 9.49%, 5.93%, and 1.03%, respectively. However, after mixing CM with BW, we recorded different values of all chemical composition. in this respect, the mixing 1CM:1BW show more efficiency larval weight (4.33 g/50 larvae) compared to 3CM:1BW (3.71 g/50 larvae) and individual substrate either CM (2.17 g/50 larvae) or BW (3.65 g/50 larvae). The 1CM:1BW substrate percentage was modified as follow: protein (14.07%), Carbs (50.25%), fats (14.1%), crude fiber (7.47%), and ash (14.1%). Regarding to previous percentage, on one side could be revealed that this adding of CM with BW led to elevate protein value from 9.49 to14.07%, fats from 5.93 to14.1%, and ash from 1.03 to 14.1%, on the other side, decreasing carbs from 75.30 to 50.25%.

This modulation in substrate composition percentage revealed that mixing substrates resulted in best balanced ingredient substrate for BSFL to produce the best weight of BSFL (4.33 g/50 larvae). This result could be interpreted that CM contains high percent of protein and fat along with lower percent in carbs which either palatable or efficient for neediness for larvae to gain best weight, not only that but also the BW gives the same result (3.65 g/50 larvae).

This increase in BW compared to CM could be attributed to high percentage of carbs and lower fat in this substrate which more digestible to larvae, and supply larvae with fast energy where, in contrary lower weight of larvae in CM could be form lower carbs of CM 25% which reflect the neediness of larvae in this age phase to more bioenergetics power

BSFL organic waste treatment varied in reliability and efficiency³⁰. Recent studies recorded various types of organic waste in BSF treatment as a substrate, including kitchen waste³¹, poultry waste³², chicken manure¹², dairy manure³³, and cereals, fruits, and vegetables³⁴. In contrast, Msang, et al.³⁵ demonstrated the effects of three organic waste substrates: Irish potato peels, kale remains, and bovine ruminal content, on larval weight gain, prepupal yield, and bioconversion rate of BSFL. Kale remains recorded outperformed Irish potato peels and bovine ruminal contents, suggesting that they can be enriched to provide an alternative feed for BSFL instead of chick mash. Nguyen, et al.³¹ investigated the consumption and reduction of five different organic waste types by black solider fly: rendered fish, pig liver, pig manure, kitchen waste, fruits and vegetables, and control poultry feed. BSFL reduced all types of waste that were examined. They recorded the heaviest black soldier flies that fed on kitchen waste, which also had the highest mean rate of reduction per day. Although some waste generated larvae with a higher nutritional content, larvae grown on liver, manure, fruits and vegetables, and fish were almost the same length and weight as larvae fed the control feed.

When analyzing the CP, EE, NFE, CF, and ash for different treatments, the nutrient accumulation of BSFL raised on various organic waste substrates has been the subject of some recent studies^31,36,12.

The performance of the process is significantly impacted by the macronutrients found in organic waste, including protein, carbohydrates, fats, and fibers³⁷. Protein has such a good impact on larval development; it is a necessary nutrient in larval feeding substrates³⁸. BSFL raised on organic wastes with greater protein contents has larger larval weights, higher protein levels, higher feed conversion rates, shorter growth times, and lower lipid contents, according to research by Nguyen, et al. ³¹ and Oonincx, et al.³⁹.

Jalil, et al.⁴⁰ proved that larvae reared in protein food sources are larger than those feeding on a carbohydrate food source. Jucker, et al.⁴¹ found that larvae feeding on substrates with low protein contents have a longer development time, are smaller, and have higher lipid contents if the carbohydrate content is high. The performance of the BSFL is determined by the nutritional contents of the organic wastes. For example, animal manure and municipal organic solid waste have higher protein contents than fruits and vegetables, which have higher carbohydrate contents; city municipal organic solid wastes have the largest lipid contents. The median fiber and ash content is higher in animal manures and fruit and vegetable wastes³⁰. Consequently, BSFL biomass values could be enhanced by mixing nutrient-rich substrates during the pre-treatment step of the BSFL feeding.

In comparison to traditional composting, black soldier fly larvae (BSFL) lower the original weight of organic waste by approximately 50% in a shorter period of time. They do this by feeding voraciously on a variety of organic waste, including food wastes, agro-industrial by-products, and poultry and dairy manure. The nutrients in the wastes are converted by BSFL into proteins and lipids that are valuable animal feed. The residue left over from this process can be applied as organic fertilizer⁴².

Identification of the bacterial isolates

Bacteria were isolated from the BSF egg-associated and gut-companion bacteria and reared on BW substrate to obtain isolates with properties potentially relevant for host nutrition. Upon the appearance of colonies on the plates, the isolates were selected according to their morphology and then purified, establishing a collection of pure cultures (Fig. 1(A-E)).

The isolated selected bacteria were identified based on their microscopic shape and Gram staining (Fig. 1(F-I)). Bacteria were identified based on molecular characterization (16S rRNA sequencing). The bacterial isolate was grown on sterile Petri plates containing autoclaved LB agar medium and incubated for two days at 30 ºC. For DNA extraction, the cultures were sent to the Molecular Biology Research Unit at Assiut University. Intron Biotechnology Company, Korea, supplied the Patho-gene-spin DNA/RNA extraction kit. After that, the fungal DNA product was shipped to SolGent Company in Daejeon, South Korea, for rRNA gene sequencing and polymerase chain reaction (PCR). Two universal primers, 27F (5'-AGAGTTTGATCC TGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTA CGACTT-3'), were used in the PCR process. By electrophoresis on 1% agarose gel, the purified PCR products (amplicons) were verified using a size nucleotide marker (100 base pairs). Dodeoxynucleotides (dd NTPs) were added to the reaction mixture and 27F and 1492R primers were used to sequence the purified amplicons in both the sense and antisense directions²⁷. Sequences were further analyzed using Basic Local Alignment Search Tool (BLAST) from the National Center of Biotechnology Information (NCBI) website. Phylogenetic analysis of sequences was done using MegAlign (DNA Star) software version 5.05.

The five bacterial isolates were classified into three phylums; Proteobacteria (1 isolate), Firmicutes (3 isolates), and Actinobacteria (1 isolate) by 16S rRNA gene partial sequencing. The phylum Proteobacteria, with one isolate belonging to the Morganellaceae family, was assigned to the genus Morganella. Within the phylum Firmicutes, there are three isolates belonging to the Bacillaceae family (all assigned to the genus Bacillus). Within the phylum Actinobacteria, one isolate belonging to the Microbacteriaceae family was assigned to the genus Microbacterium⁴³ (Fig. 2).

Proteases and ureases enzyme activity

At the end of the incubation period, urea degradation ability was present among the isolates: three out of a total of five isolates were able to degrade urea (Fig. 3 (A–E)). All five isolates were screened to characterize the potential bacteria for nitrogen uptake. At the end of the incubation period of skimmed milk agar medium, we found that three out of a total of five isolates were able to degrade protein (Fig. 3 (F–I)).

Effect of bacterial isolates supplementation on larval development

Four bacterial isolates were isolated from BSF-associated eggs, namely: Microbacterium paraoxidans (EN2), Bacillus proteolyticus (EO3), Bacillus megaterium (EO4), and Bacillus subtilis (EO6). One bacterial strain, Morganella morganii (GL), was isolated from the larval gut. These bacterial isolates were individually inoculated to a sterile medium. After the end of the incubation period, supplement the substrate individually.

The bacterial isolates achieved positive effect on the development and growth parameters of BSFL. In CM treatment, M. morganii (GL) isolated from the larval gut, was significantly higher than that of the control by 12.44% in dry larval weight. M. paraoxidans (EN2), isolated from the egg, recorded 5.1% higher than control. In 3CM:1BW treatment, M. morganii was significantly higher than control by 9.7%, followed by M. paraoxidans and B. subtilis, which recorded 5.7 and 5.4%, respectively. In 1CM:1BW treatment, there were non- significant differences between treated with bacteria isolated and control (Fig. 4-A).

The conversion rate of CM treated with bacteria into BSFL biomass was significantly greater in the M. morganii treatment by 11.11% compared to control. In 3CM:1BW treated, the conversion rate was non-significantly greater by 3.16% in both M. morganii and M. paraoxidans compared to control. The conversion rate of 1CM:1BW treated with bacteria into BSFL biomass was significantly greater in both M. morganii and B. subtilis treatments by 4.9% compared to control (Fig. 4-B).

The influence of companion bacteria on chemical components of BSFL biomass:

Chemical components of BSFL after co-conversion were analyzed, such as the CP, EE, NFE, CF, and ash for the isolates (Bacillus subtilis, Microbacterium paraoxidans, Morganella morganii, and control) at the treatments (CM, 3CM:1BW, and 1CM:1BW substrates). Biochemical components varied depending on treatments. In the CM treatment, M. morganii was significantly higher than the control in all contents but carbohydrates. The carbohydrate content of BSFL resulting from M. morganii was significantly lower than control (Fig. 5).

In the 3CM:1BW treatment, there were non-significant differences between the four treatments in CP and ash contents. M. morganii was significantly higher than the control in carbohydrate content and lower in fat and CF contents. (Fig. 5)

In the 1CM:1BW treatment, there were non-significant differences between the four treatments in chemical components except carbohydrate. M. morganii was significantly higher than the control in carbohydrate contents (Fig. 5).

Feed production contributes to 25.5% of the CO2 emissions produced in the chicken meat and egg supply chains, amounting to 606 million tons of CO2 per year. The transportation of soy products across the Atlantic increases the carbon footprint. Recent research has revealed that 5% of the European carbon footprint associated with imported Brazilian soybean is a result of maritime transport, further diminishing the sustainability of egg production. The deforestation and land use change in the soybean supply chain contribute to over 50% of the EU's carbon footprint, which highlights a significant opportunity to decrease greenhouse gas emissions in poultry production by utilizing locally produced larvae fed with organic waste as a protein alternative⁴⁴.

The present study investigated the effects of BSF egg-associated and gut-companion bacteria on dry larval weight gain, conversion efficiency, and nutritional accumulation of BSFL reared on chicken manure and bread waste with different ratios. Callegari, et al.⁴⁵; Zhang, et al.⁴⁶; Mannaa, et al.⁴⁷ found that the administration of BSF-associated bacteria contributed to enhanced insect growth, development, and involvement in organic waste bioconversion processes. Isolated companion bacteria from BSFL and inoculated with chicken manure could promote larval growth and development, according to Mazza, et al.¹². Our results proved that the companion bacteria from BSF eggs and gut could improve weight, conversion efficiency, and nutrient accumulation of BSFL, especially BSF-CL (Morganella morganii), which contributed positively in increasing BSFL biomass, with a significant increase of 12.44% compared to the control in the CM treatments and a 9.7% increase in the 3CM:1BW treatment. In the 1CM:BW treatment, BSFL biomass increased significantly by 4.6%. The conversion rate of 1CM:1BW treated with bacteria into BSFL biomass was significantly greater in both M. morganii and B. subtilis treatments by 4.9% compared to control. This finding is similar to Rehman, et al.⁴⁸; Lalander, et al.⁴⁹;Vandeweyer, et al.⁵⁰ who studied how gut microbiota composition varies depending on rearing substrates, via a mechanism that might support the recruitment of microorganisms to facilitate digestion of a specific substrate. Our results were also in agreement with Meneguz, et al.³⁶ as they indicated that the evaluation of an appropriate feeding system and the initial pH value of the substrate are important parameters to reduce the time and increase the weight in the production of larvae. BSFL weight was increased by increasing the pH value of the substrates.

Makkar, et al.¹ discussed that if an increased demand for cattle and poultry to alternate foods may arise if there is a scarcity of soymeal, fishmeal, and other protein sources; H. illucens larvae are comparable to high-protein feedstuffs according to their protein content and amino acid profiles. Also, egg mass was positively affected by the insect meal diets, as was the lay percentage, although only at the lowest inclusion level. Overall, a protein replacement of 25% with an insect meal from Hermetia illucens larvae in the diet of laying hens seems to be more suitable and closer to the optimal level⁴⁴. Bovera, et al.⁵¹ studied how egg weight, feed intake, and feed conversion rate were not affected by the soybean meal substitution at both inclusion levels of insect meal (25% and 50%). Based on the current findings indicating a 25% decrease in concentrate consumption among the 20% hens, it can be inferred that for this diet to be more cost-effective than the conventional one, 1 kg of live BSF larvae would need to be priced at less than 40% of the cost of 1 kg of concentrate if the 20% concentrate is retailed at the same price as the conventional concentrate⁴⁴.

As many research suggested the use of chicken manure (CM) as a substitute feed, Egypt can make use of the available chicken manure (CM) and transform it to a reasonably priced and readily available alternative source for chicken feed using black soldier fly larvae. The black soldier fly (BSF), Hermetia illucens, is one of the most important insects in commercial production. And as BSF larvae fed on pure CM don’t gain significant weight and often died, this research aims to investigate the effects of adding different ratios of bread waste (BW) to CM as a source of carbohydrates to enhance its properties and increase the larval development. The results showed that the larvae's dry weight increased by 99.5% when BW was added to CM at a 1:1 ratio. Four bacterial isolates were isolated from Black Soldier Fly (BSF) eggs, namely Microbacterium paraoxidans, Bacillus proteolyticus, Bacillus megaterium, and Bacillus subtilis, while one strain, Morganella morganii, was isolated from BSF larval guts. These bacterial isolates were evaluated for their ability to enhance BSF larval development and growth when supplemented with the rearing substrate. In the chicken manure treatment, M. morganii exhibited the greatest increase in larval dry weight, while in the 3:1 chicken manure-to-bread waste treatment, both M. morganii and M. paraoxidans showed significant improvements in larval weight. The presence of M. morganii also significantly improved the conversion efficiency of chicken manure into BSF larval biomass. And in the 1:1 chicken manure-to-bread waste treatment, M. morganii and B. subtilis had higher larval conversion rates compared to the control. The choice of substrate significantly affected BSF larval biomass and nutritional composition. Overall, the bacterial isolates isolated from BSF eggs and guts demonstrated positive effects on larval growth and development, with M. morganii showing the greatest impact. The supplementation of these bacterial isolates positively influenced positively the growth parameters of BSFL, with significant increases in dry larval weight and conversion rate observed in specific treatments. These findings contribute understanding microbial interactions in BSF production and rearing techniques. Further research is needed to elucidate the underlying mechanisms and explore the potential of these bacterial isolates on a larger scale. Insects used to produce animal feed, might decrease the need for soybean production as well as the loss of natural resources and deforestation. BSF larvae, in particular, are a promising choice for large-scale production due to their high growth performance, low mortality rate, superior disease resistance, and protein quality. As such, BSF larvae meal holds promise as a potential substitute for soybean meal and could lower the need for growing soybeans and help in reducing deforestation, and so BSF has the potential to contribute to the reduction of environmental pollution which is a recommended a topic for further studies.

Acknowledgements

The authors express their deep thanks to Prof. Dr. Safwat H. Ali, Dept. of Chemistry, Fac. of Agric., Ain Shams Univ. for helping in writing.

Author contributions

All authors shared the aim and design of the work; E.M.A. and S.A.A. contributed to the study's conception and design. Material preparation, data collection and analysis were performed by E.M.A., S.A.A. and S.A.H. Statistical analyses were performed by E.M.A. S.A.A and E.M.A. wrote the first draft of the manuscript. All authors commented on previous versions of the manuscript. W.E. and R.S.R. revised the manuscript. All authors read and approved the final version of the manuscript.

Competing interests

The authors claim to have no conflicts of interest.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Supplementary information

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No competing interests reported.

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Optimize using black soldier fly larvae reared on chicken manure as an alternative feed in the insect farming industry

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