Use of different dry materials to control the moisture in a black soldier fly (Hermetia illucens) rearing substrate

Introduction

The black soldier fly (BSF), Hermetia illucens (L.) (Diptera: Stratiomyidae), has gained in popularity recently as a biological waste recycler, converting the waste into larval biomass that can then serve as a protein source for diet supplementation in poultry and livestock rearing (da Silva & Hesselberg, 2020).

There have been no reports of BSF larvae (BSFL) having a negative effect on the growth performance or meat quality in pigs or poultry, although a lower feed conversion rate has been reported in pigs (Lu et al., 2022). The high amount of fat obtained from the defatting BSFL can be used to produce high-quality biodiesel because it contains high levels of lauric, palmitic, and myristic acids (Kim et al., 2021). In addition, the fat from BSFL has a high potential for use in cosmetics (Franco et al., 2022). The BSF frass, a byproduct from the larval decomposition process, is composed of 37% C, 3% N, 1–5% P, and 0.5–4.1% K by wet weight (w/w) and so can be utilized as a biofertilizer (Lopes, Yong & Lalander, 2022). As a result of these potential benefits from BSF rearing, insect farming is being practiced worldwide in an effort to create a sustainable circular bioeconomy by extracting value from biowaste management (Kim et al., 2021; Liu et al., 2022; Lopes, Yong & Lalander, 2022).

When it comes to BSF farming, there are several substrate factors that can affect the ability of BSFL to efficiently convert waste materials. These factors include the nutrient content, fiber content, particle size, substrate ventilation, pH, structure, and moisture content (Barrett et al., 2023; Cheng, Chiu & Lo, 2017; Joly & Nikiema, 2019; Ma et al., 2018; Yakti et al., 2023; Zhang et al., 2021). The nutrient content of the substrate plays a significant role in the growth, development, and body composition of the BSFL (Barragán-Fonseca et al., 2018; Eggink et al., 2022; Meneguz et al., 2018). While BSFL can survive on substrates with a wide range of pH levels, from highly acidic to highly basic (pH 2.0–10.0), the optimum initial pH for favorable gut microorganisms and higher biomass production ranges from pH 6.0 to 8.0 (Ma et al., 2018).

Substrate ventilation is crucial to allow oxygen diffusion for consumption by the BSFL. These insects prefer feeding on the top 2–4 cm of the substrate, avoiding the anaerobic zone towards the bottom (Barrett et al., 2023). Furthermore, the moisture content of the substrate influences the vertical distribution behavior of the BSFL. Indeed, BSFL are found at different substrate depths depending on the moisture content, with higher moisture contents leading to higher mortality rates (MRs) (Bekker et al., 2021). The moisture content significantly affects the BSF larval abilities and performances, with negative effects on feed reduction and wet weight and positive effects on developmental time. One study indicated that BSFL reared on a substrate with a 60% moisture content exhibited the highest growth performance, while the growth was lowest at an 80% moisture content (Dzepe et al., 2020). In contrast, a moisture content below 80% was found to have no effect on larval survival rates, while above 90% it reduced the survival rates (Dzepe et al., 2020; Khairuddin, Ghafar & Hassan, 2022; Lalander et al., 2020. However, the larvae were unable to develop on a 40% moisture content (Cammack & Tomberlin, 2017). In fact, the moisture contents did not influence BSFL performance directly but were directly associated with the activities of substrate microbiota (Bekker et al., 2021). In addition, high-moisture substrates can present challenges during residue separation at harvest time (Cheng, Chiu & Lo, 2017). By considering and optimizing these substrate factors, BSF farmers can enhance the overall efficiency and success of their farming practices while ensuring the well-being and productivity of the BSFL.

Many organic wastes, such as food waste, fruits and vegetables, have a high water content. Due to the importance of substrate moisture on biomass production, several techniques have been evaluated to reduce high moisture levels and increase larval yield. One approach is the design of an automated incubation system that utilizes pressure sensors to control the temperature and maintain a substrate moisture level of 60% through water evaporation (Erbland, Alyokhin & Peterson, 2021). Another method involves increasing the aeration rate of the larval rearing containers, which can help control the air temperature and relative humidity, ultimately reducing the substrate moisture (Abduh et al., 2022; Cheng, Chiu & Lo, 2017). Additionally, excess moisture can be minimized by adding dry substrates, such as coconut coir dust (CC), palm kernel meal, or milk powder, on top of the substrate to adjust the water content to below 80% (Dortmans, Diener & Verstappen, 2017). Despite the use of some dry substrates, limited information is available regarding the optimum quantity of each type of dry substrate to be added.

The objective of this study was to evaluate the efficiency of various types of dry substrates when added to a minced mixture of vegetables with high moisture content. Each dry substrate, with its distinct physical properties, may exhibit a different efficiency in controlling excessive moisture and impact the BSFL performance differently. Therefore, six different dry substrates derived from agricultural byproducts were selected, and their quantities were varied to formulate suitable diets with optimal substrate moisture levels, in terms of enhancing the BSFL rearing process. These dry substrates offer a potential low-cost technique that can benefit BSF farming, especially for small-scale production where automated machinery may be lacking.

Materials & Methods

Results

Larval survival

In contrast to the CON group (6.7 ± 4.1% MR), a higher MR was observed in larval stages when high quantities (up to 25%) of dry substrates were present. Specifically, substrates containing, all % (w/w), 25–50% RB, 50% SCG, and 50% RHA showed elevated MRs (17.8 ± 10.0%, 25.3 ± 5.8%, 22.7 ± 7.1%, and 19.2 ± 5.7%, respectively; F = 8.362, df = 12, P = 0.000) (Fig. 2A). However, when 25% (w/w) dry substrates were used, the MR was low, except for with RB due to the absence of moisture in this substrate.

In Trial 2, high MRs were observed when the larval food was mixed with all % (w/w), 15% RHA, 10–15% SCG, and 5–10% CC (12.8 ± 6.1%, 18.3 ± 8.3%, 13.2 ± 8.3%, 19.8 ± 6.8% and 15.8 ± 5.7%, respectively; F = 4.723, df = 18, P = 0.000). Among the low dry substrate addition mixtures, only the CC and SCG treatments exhibited a high MR.

Growth performance

Due to the consistent feed rate supply, mixing with higher quantities of dry substrates resulted in a lower substrate nutrition level and reduced biomass in the BSF larvae prepupae and pupae (Fig. 3). Compared to the control substrate, a wet weight loss of over 40% was observed in the larvae, prepupae, and pupae when cultured with media containing 50% (w/w) of any of the dry substrate mixtures except for the 50% RH (F = 109.213, df = 12, P = 0.000; F = 72.886, df = 12, P = 0.000 and F = 139.152, df = 12, P = 0.000, for the larvae, prepupae, and pupae, respectively). Interestingly, BSF reared on substrate mixtures containing 25–50% (w/w) RH displayed the highest weight compared to those with the same quantity of the other dry substrates. In contrast, 25–50% (w/w) RHA resulted in the smallest BSFL biomass, although the addition of a lower quantity of 10–15% (w/w) RHA showed improved results. Similarly, CC treatments showed the smallest biomass at the lower levels of added dry substrates (5–15% (w/w)), with a weight loss of 50–61% in the larval stage (F = 97.158, df = 18, P = 0.000 in larvae; F = 140.912, df = 18, P = 0.000 in prepupae, and F = 82.832, df = 12, P = 0.000 in pupae).

Sex ratio

Overall, the proportion of female offspring produced ranged from 48% to 51%. The sex ratio of the offspring was not significantly affected by the addition of a low level of each dry substrate. However, high-level mixtures resulted in a reduction in the proportion of female offspring, particularly in the case of 50% RHA (29.1 ± 6.2%) and 50% RSD (35.4 ± 3.0%; F = 6.416, df = 12, P = 0.000) (Fig. 4).

Scale-up tests for medium-scale production

Four treatments (5% RHA, 10% RH, 5% RB, and 15% SCG) were selected from the small-scale tests for evaluation in medium-scale production using the same quantities of added dry substrate. With the exception of 15% SCG, the M of the substrates was reduced compared to that in the small-scale tests (Table 2). Consequently, the substrates did not dry out during development of the prepupae and larger larvae and pupae were produced. Figure 5 reveals that substrate mixtures containing 15% SCG and 5% RHA resulted in a significantly increased weight of the larvae and prepupae (F = 63.220, df = 4, P = 0.000 and F = 137.642, df = 4, P = 0.000, respectively).

Table 2:

Moisture range (%) and the percentage of moisture removal (M) (%) of media with various dry substrate additions in small- and medium-scale experiments.

Treatment (% (w/w))	Small-scale		Medium-scale		Difference
	Moisture range	M (%)	Moisture range	M (%)
CON	60.53–63.47	–	62.03–63.63	–	–
5% RHA	60.08–62.93	2.49	61.80–63.23	1.47	decrease
10% RH	59.05–62.03	4.16	59.93–63.13	1.84	decrease
5% RB	59.55–61.85	3.34	43.87–63.40	2.64	decrease
15% SCG	59.00–61.58	4.24	61.30–63.47	4.81	increase

DOI: 10.7717/peerj.17129/table-2

Discussion

Globally, fruits and vegetables are common organic wastes that have the potential to be used as BSFL substrates (Jucker et al., 2017) with a high waste-to-biomass conversion level (Bekker et al., 2021). However, their high moisture content presents a challenge in BSF production. Studies have shown that adjusting the moisture levels can both improve the survival rates and increase the obtained BSFL biomass when using certain substrates, such as apples and spinach (Ribeiro, Costa & Ameixa, 2022). The lower nutritional value of fruits and vegetables, in terms of their low protein concentration (around 13%) and high a carbohydrate-to-protein ratio, can lead to a decreased larval growth performance (Bonelli et al., 2020; Jucker et al., 2017; Kinasih et al., 2018; Lalander et al., 2019). As a generalist feeder, BSFL have demonstrated their ability to adapt their digestion process and optimize larval performance (Bonelli et al., 2020).

Optimizing the feed composition for BSFL rearing through the incorporation of vegetables as the main ingredient can significantly improve the waste-to-biomass conversion efficiency. The combination of vegetable canteen waste and mill byproducts was previously demonstrated to augment the BSFL biomass production compared to poultry feed despite the latter’s higher protein content (Gold et al., 2020). Apart from the enhanced food quality, this substrate combination can also improve the moisture content. Fruits like apples, bananas, or a blend of both have a low nutrient density and a high moisture content, rendering them suitable substrates for BSFL when integrated with spent grains at a 1:1 (w/w) ratio (Scala et al., 2020). Wheat bran has been used to regulate the substrate moisture (Yang & Tomberlin, 2020).

In this investigation, RB emerged as a potentially effective dry substrate; however, numerous studies have highlighted its limitations. Previous examination of the proportion of RB mixed with coconut pulp residue revealed that BSFL did not thrive when the mixture contained over 50% (w/w) RB due to the resulting high substrate temperatures (Muchdar et al., 2021). Similarly, and consistent with this study, RB could sustain BSF larval growth and rapid development when used at less than 25% (w/w) in the substrate due to its elevated protein content. However, BSFL exhibited a higher MR when exposed to 25% and 50% (w/w) RB substrates since these resulted in high substrate temperatures and substrate drying out. This is consistent with the recommendation for the control of high moisture substrates of placing the RB on the substrate’s outer edge (Barrett et al., 2023), where the small quantity of RB on the surface of the media prevents excessive substrate heating.

The utilization of toxic dry substrates deserves considerable attention. For instance, CC has an abundant tannin content with the potential to release these compounds into the BSFL substrate as they are water soluble substances. These can be toxic to the digestive tissues of insects, including BSFL, and they also possesses antimicrobial properties that can potentially adversely alter the insect’s gut microbiome as well as substrate fermentation (Barbehenn & Peter Constabel, 2011). Despite CC exhibiting a high water-holding capacity (Evans, Konduru & Stamps, 1996) and its potential to effectively manage a substrate with a high moisture content (Table 1), its utilization appears to result in a high BSFL mortality and low BSF biomass. Nevertheless, this study did not specifically examine tannin or other toxic substances within the substrates. The removal of tannin through leaching by washing with 60 °C water (Tejano, 1985) or microwave treatment (Adeel et al., 2020) before adding the CC to the larval substrate for rearing BSFL could potentially enhance its beneficial properties.

Likewise, the presence of toxic phenolic compounds in SCG could impact the larval growth (Permana & Ramadhani Eka Putra, 2018; Sideris et al., 2021). However, fermented SCG, derived from cofermentation with pineapples at levels of less than 60% (w/w) in the substrate mixture gave larger BSFL (Khaekratoke, Laksanawimol & Thancharoen, 2022). Although the SCG used in this study underwent self-fermentation, the BSFL exhibited similar trends. Additionally, the use of SCG has been recommended for its ability to minimize the odor from substrate decomposition.

The particle sizes of dry substrates are related to the level of oxygen diffusion within the media. In conditions of high moisture, the fine particle sizes of dry substrates, like SCG, can lead to a compact texture. This can limit the level of oxygen diffusion, particularly at the lower regions of the substrates and so prevent utilization of this substrate region (Barrett et al., 2023). Fortunately, the substrate depth in the medium-scale study fell within a range of approximately 5–7 cm, allowing the larvae to exhibit their typical feeding behavior. Furthermore, the tested substrates received active ventilation once a day. However, the observed differences in the BSFL growth performances between the small-scale and medium-scale studies are likely to be attributed to the differences in substrate depth.

Both RH and RHA possess the ability to absorb heavy metals (Kumar et al., 2013; Lata & Samadder, 2014; Syuhadah & Rohasliney, 2012). Previously, RH biochar was recommended for use when contaminated organic waste substrates were used for rearing BSFL due to its capacity to absorb up to 97% of the cadmium (Mutanekelwa, Okumu & Andika, 2022). However, that study did not evaluate the efficiency with live BSFL, nor did it investigate the effects of different proportions of RH biochar on the larval mortality or growth. Our data demonstrate that substrate mixtures containing RHA correspondingly reduced the pH of the substrates depending on the added quantity (pH = 6.2 for 50% RHA), whereas the pH of the RH substrate ranged from 7.0–7.1. Ma et al. (2018) discovered that substrates with a pH level between 6.0 and 8.0 were optimal for substantial BSFL production, in contrast to levels below pH 4.0 that were detrimental. Therefore, in this study, the observed high MRs of BSFL in substrates with low quantities of dry substrates is not likely to have been caused solely by a low pH. With respect to the %M, it is evident that substrate mixtures with low quantities of RHA (5–15%) exhibited a minimal %M, resulting in a high moisture content that led to an increased MR during the prepupal stage. In contrast, substrates containing 5–10% RHA could support a larger BSFL mass compared to those with higher quantities of RHA.

Typically, RH has a higher %M and larger particle sizes than RHA. Yakti et al. (2023) demonstrated that fibers with larger particle sizes possess a greater water-holding capacity and facilitate an improved aeration of larval substrates, consequently leading to larger larval sizes and a lower MR. This is consistent with the findings of the present study, where RH demonstrated the capacity to effectively support BSFL performance, even when present in significant quantities. Substrates containing 25–50% (w/w) RH could promote shorter BSFL development and yield a larger BSFL biomass compared to the control, but without a significant difference in the MR compared to the control group.

Comparison of the BSFL growth performance between the small-scale and medium-scale experiments revealed distinct outcomes. Evidently, in the medium-scale trials, substrates with dry substrate mixtures yielded a greater BSFL biomass, particularly in the cases of 5% RB, 15% SCG, and 5% RHA. Notably, factors such as the container volume, substrate surface area, and substrate thickness likely contributed significantly to these differences. Nevertheless, the %M of most substrates was decreased in the medium-scale trials, making them suboptimal for the pupation of prepupae. As a result, some prepupae exhibited a climbing behavior to escape from the damp substrate. In light of this, we propose considering the incorporation of higher quantities of the dry substrate when working with larger-scale setups.

Based on the findings of this investigation, the selection of dry substrates for controlling the substrate moisture level requires careful consideration of diverse properties, including the water-holding capacity, nutritional content, presence of toxic compounds, particle sizes, and substrate cost. We have detailed the pros and cons of each dry substrate for moisture control in Table 3, where RB and RH emerge as promising dry substrates for use in BSF rearing. A quantity of RB less than 15% effectively controls substrate moisture content and promotes BSFL growth with a shorter harvest time. BSFL grown in substrates with less than 50% RH exhibit low MR and large larval growth. However, it is worth mentioning that a combination of different dry substrates can be employed to strike a balance in substrate moisture and enhance the efficiency of vegetable-fruit substrates for successful BSFL rearing.

Conclusions

Several low-cost agricultural byproducts exhibit the potential to serve as supplements for BSFL rearing substrates to effectively manage the substrate moisture level. Based on their moisture control efficacy and impact on BSFL performance, RB and RH are potential dry substrates for BSF rearing. However, care in the use of RB is required to prevent its excessive usage (>15%), which could lead to increased temperatures or substrate desiccation. In contrast, CC stands out as an ideal option for moisture control, yet a prerequisite for its application involves the removal of tannins. The combination of substrates can be performed to enhance the efficiency and cost-effectiveness.

Supplemental Information