1. Introduction
The continuous growth in the global population and the inevitable increase in demand for natural resources have had a negative impact on all life on Earth. Annual global waste generation has reached approximately 17 billion tons and is expected to rise to 27 billion tons by 2050. Currently, waste production and improper management is responsible for the production of 1.6 billion tons of CO
2 emissions [
1]. The depletion of natural resources, the increasing use of valuable land surface for human activities, and the generation of waste are detrimental to our planet. More sustainable alternatives to guarantee a sufficient supply of food, feed, and biomaterials are needed.
In the last decades, an evolution toward organic, sustainable, and more environmental friendly systems has emerged. This necessary evolution is reflected by the Sustainable Development Goals (SDG) of the United Nations [
2]. Additionally, the European Green Deal, which aims to implement the United Nation’s SDG, provides objectives and an action plan to move to a clean, circular economy that restores biodiversity and cuts pollution [
3]. In line with this, EC Directive No. 2008/98 establishes the order of priority in the choice of by-product treatments ranging from prevention, preparing for re-use, recycling and other recovery, down to disposal [
4]. Many initiatives in diverse domains have been undertaken with the aim to implement sustainable processes for (a) generating food and feed alternatives; (b) energy; (c) reducing waste production; (d) recycling materials; (e) implementing renewable feedstock; and (f) generating biomaterials. Many people consider plants as sustainable and environmental-friendly sources for food, feed, and biomaterials. However, this perception is not entirely correct as plants require massive amounts of land, which results in land-use for agriculture, leading to deforestation and a loss of biodiversity [
2]. Additionally, in the case of plant products derived from tropical regions, transport to EU countries also imposes an environmental impact. The use of plant-based biomaterials for feed and technical applications also counters opposition, especially when there is competition to implement it in food products [
5].
One of the alternatives that may hold an interesting position in a circular economy is the implementation of insects [
6]. As insects have proven to be able to efficiently convert low-value biomass into their own biomass consisting of high-quality components (i.e., proteins, fats and chitin [
7]), they have the potential to help tackle the societal challenges [
8]. Using insects for food, feed, biomaterial production, and to valorize side-streams is a strategy that has gained increased interest. Insects are believed to have a lower ecological impact than current livestock. Moreover, research has shown that insects produce less greenhouse gases and emit significantly less ammonia [
9]. In addition, insects convert feed into biomass more efficiently than conventional livestock [
8] because they are ectothermic and therefore use little energy to maintain their body temperature [
10]. Furthermore, insects can be produced in vertical farming systems, making them more productive per m
2 [
11] and they generally require less water [
12]. Therefore, insects might be able to play a key role in the circular economy, meeting the requirements for a more sustainable society.
In particular,
Hermetia illucens (Diptera: Stratiomyidae), better known as the black soldier fly (BSF), is a very interesting insect species that is intensively investigated to implement as a waste-converter [
13,
14]. It is a non-pest insect species that can convert diverse (non-value) organic waste streams into biomass that can be used for the production of feed [
15], biomaterials for biodiesel production [
16], technical applications such as surfactants [
17], and protein-based bioplastics [
18]. BSF larvae (BSFL) can play an important role in organic waste reduction and renewable biomaterial production, providing a useful addition to a circular economy, and can contribute to the EU Green Deal strategy of reducing food waste by 50% by 2030 [
3]. However, several aspects to improve the sustainable production and economic viability of BSFL and their derived biomaterials need to be investigated [
19]. LCA analyses indicate that the production of BSFL can be sustainable and economically viable if they can be reared on low quality biomass such as manure or mixed organic wastes [
20].
BSFL have been shown to thrive on a wide variety of substrates including food waste, agri-industry co-products, animal waste, and meat [
19,
21]. However, there are differences in the growth, survival, and bioconversion efficiency of BSFL grown on different substrates. One of the conceivable factors that influence this variation is their nutrient content. Indeed, varying macronutrient content (i.e., the protein, fat, or carbohydrate content) has a significant influence on the performance and composition of BSFL [
22,
23]. Several studies have been published in which BSF is reared on waste streams of which the macronutrient content is also investigated [
22,
24,
25,
26,
27,
28,
29,
30,
31,
32]. These studies show that the macronutrient content influences BSFL growth performance and composition, however, diets with similar macronutrient composition show differences that may be due to other factors not analyzed such as texture or nutrient quality, vitamins, and minerals [
22]. Often, streams classified as the same type of side-stream vary greatly in nutritional content as demonstrated by Gold and coworkers [
14]. These differences emphasize the need to evaluate the potential of local side-streams.
Here, we evaluate the potential of BSFL to process a selection of low-value side-streams available in Flanders, Belgium, in order to evaluate the larval performance on each of these. By analyzing the macronutrient content (proteins, lipids, non-fiber carbohydrates), fibers (cellulose, hemicellulose, and lignin), and the mineral content of the side-streams, we investigated their influence on larval performance and bioconversion.
4. Discussion
We evaluated the survival and growth of black soldier fly larvae reared on a set of 12 low-value organic side-streams (mono-streams) and two standard diets. Black soldier fly larvae have been shown to thrive on a wide variety of substrates including food waste, agri-industry co-products, animal waste, and meat, with a minimal need for pre-treatment. This confirms that BSFL are able to upcycle low-value side-streams into valuable biomass [
19,
21]. For this research, side-streams were selected that are mainly used for biogas production. Some of these side-streams are not valorized as feed due to legal restrictions. Moreover, some of the side-streams tested (household food waste, industrial food waste, and chicken manure) are currently also not allowed to be used as feeding substrates for BSF larvae [
19]. Other side-streams are not used because they are of low interest as feed due to low quality or overproduction.
Several parameters influence the survival and growth of BSF larvae, making it difficult to compare different studies in the literature. There are differences between laboratories in breeding conditions (light, temperature, humidity, larval density, time of harvesting, …), substrate compositions (that differ between laboratories or between batches within the same laboratory), and physico-chemical properties of substrates may be different or the used BSF strain may be different. Otherwise, differences in study setup and in analytical techniques used to determine the nutritional composition of substrates might also cause variation. In addition, there are inconsistencies regarding the calculation of bioconversion and waste reduction (fresh weight, dry weight bases, and combinations thereof) and other parameters. A recent paper by Bosch et al. also focused on the possible sources of the variability observed among BSF feeding experiments and argues for the development of procedures to improve harmonization and reproducibility among studies [
19]. For these reasons, an international working group was established during the EAAP 2019 conference, aiming at the standardization of methods, parameters, and terminology in future insect research [
42].
As expected, relatively high survival rates of 97.2% and 96.3%, respectively, were obtained for larvae reared on control diets. Similar high survival rates (>95%) on standard substrates have been observed in the literature [
24,
43,
44,
45]. The average weight of larvae reared on CSM was 148.4 mg, which is significantly higher than larvae reared on GVD (
p < 0.001). This is possibly due to the higher gross energy as well as lower lignin and cellulose content present in CSM compared to GVD. Larvae reared on apple pulp had a relatively high survival rate of 95.5%, however, the average larval weight was only 38.3 mg. This is remarkably low, especially since the gross energy content of apple pulp is higher than GVD. Possibly the low protein content of 3.4% was the reason for the limited larval growth. Liland et al. [
46] also observed that BSF larvae grown on seaweed substrates with low protein content had similar survival rates but lower weights compared to the control substrate. They hypothesize that this was also due to limiting amounts of proteins in the substrates and that a 7% protein content in rearing substrate is advisable for proper growth of BSF [
46]. Another factor might be the high crude fiber content of 25.7% or the high cellulose content of 21.5% present in apple pulp, impairing the digestive processes. In addition, it must be noted that the apple pulp was remarkably acidic, with a pH of 3.7 (data not shown). This could potentially also have been a factor hindering growth. Similar to apple pulp, larvae grown on fruit puree had a very high survival rate of 99.0%. However, the average larval weight was 80.1 mg. Fat contents in this stream were comparable to CSM and the protein content was also rather low (10.0%), however, a relatively high non-fiber carbohydrate content of 39.9% was present. A possible explanation for the lower average larval weight is the relatively high crude fiber content of 17.6%, a lignin content of 3.5%, and a cellulose content of 20.7%. These fiber contents might result in this stream being more difficult to digest. The adverse effect or negative correlation (especially of lignin) on larval growth could also be observed by studying principal component analysis (
Figure 2b), which was conducted using the data acquired in this study. With the exception of Scala et al. [
47], who observed higher larval weight (150 mg) in larvae grown on apple substrate, several studies have been published that also indicate lower larval weight of BSF larvae reared on fruit substrates [
29,
48,
49]. Scala et al. indicate that the rearing of BSF larvae at the industrial scale, in contrast to lab scale rearing, might have positively influenced their development performance [
47].
Larvae reared on beer draff had a survival rate of 95.5% and an average larval weight of 130.9 mg. The protein and fat contents were comparable to CSM. The non-fiber carbohydrate content was only 20.0%, which is remarkably low and the crude fiber content was 18.4%. This, however, did not halt their growth, as the average larval weight was only 11.8% lower than those reared on CSM.
Larvae reared on industrial food waste had a survival rate of 88.8% and a maximal mean larval weight of 176.4 mg, which was the highest mean larval weight measured in this study. The high larval weight can be explained by the very high gross energy amount of 386 kcal/100 g DM due to a very high fat content of 11.9%, a protein content of 19.4%, and non-fiber carbohydrate content of 50.6%. Even though the survival rate was only 88.8%, statistical data analysis showed that this was not significantly different from that measured in control substrates. Interestingly, high sodium content was present in this stream (1.30%). Little information is known about insect sodium requirements and limitations. For other farm animals such as poultry, more information is available. Poultry feed requires sodium contents ranging from 0.10 to 0.25% [
50]. Sodium contents of 0.40% lead to significantly higher water intake in broilers, while sodium contents of 0.9 to 1.2% lead to high mortality rates [
51]. Both industrial and household food waste contain high sodium contents of 1.30 and 0.55 g/100 g DM, respectively. However, larvae still managed to grow well on these sodium-rich substrates without a significant negative effect on larval survival rates. For household food waste, similar results to industrial food waste were expected, since the nutritional composition is relatively similar. Even though a survival rate of only 83.2% was achieved for larvae reared on this stream, the survival rate was not significantly lower from the control substrates. The average maximal larval weight, however, was significantly lower, being only 65.3 mg. This low average weight was unexpected, since a gross energy content of 377 kcal/100 g DM was present. The stream had a fat content of 14.6%, which is relatively high, as this was even 33.9% higher than the industrial food waste, being the stream with thee second-highest fat content. However, research has shown that canteen waste or poultry slaughterhouse waste with a fat content of 34.9% and 42.9%, respectively, are still suitable as a substrate for rearing black soldier fly larvae [
24], indicating that the high fat content is probably not the reason for the poor larval growth and low survival rates. The protein and non-fiber carbohydrate contents of household food waste were comparable to CSM. The fiber contents of household food waste were relatively low. Furthermore, no outliers in mineral contents were found for this stream. Since it is household food waste, the possible presence of other harmful substances cannot be excluded, which could be a reason for the larvae performing poorly on this nutrient-rich side-stream.
Larvae reared on chicken manure did remarkably well, as 97.7% of all larvae survived and a maximal mean larval weight of 134.9 mg was measured, which is a little bit higher than, but comparable to beer draff. The gross energy content of this stream was slightly lower than GVD and comparable protein, fat, and non-fiber carbohydrate contents were measured. This stream contained the highest zinc concentration (0.041 mg/100 g DM). High zinc contents were expected, as the ‘Poultry NRC’ recommends 35 mg of zinc per kg of feed in the diets of laying hens as it is an essential trace mineral in poultry diets, which is required to regulate bone resorption and DNA replication [
52,
53]. The high substrate pH of 8.2 (data not shown) did not have a visible adverse effect on larval growth. The results were similar to Rehman et al. [
54], who also observed high survival of larvae on chicken manure, but lower larval weights of 97 mg were observed in their study.
Larvae reared on corn meal had a survival rate of 91.8% and an average maximal larval weight of 110.4 mg, which was higher than the larvae reared on GVD, but lower than the larvae fed with CSM. A possible reason of the mean maximal larval weight being lower than larvae reared on CSM is the relatively low protein content of 9.5%, which might be the growth-limiting nutrient in this side-stream.
Forced chicory roots was a stream with a gross energy content of 224 kcal/100 g DM, however, a very low fat content of 0.9% as well as a very low protein content of 4.6% was present. Most of the gross energy was due to a non-fiber carbohydrate content of 49.3%. Larvae reared on this stream had a survival rate of 98.0%, however, larvae had stunted growth as the maximal average larval weight was only 46.7 mg. A similarly low protein content also led to a low maximal average larval weight in apple pulp. In this forced chicory root stream, a remarkably high iron content of 0.567 ± 0.024 g/100 g DM was present, but it is not known whether this might have had an adverse effect on larval growth.
Larvae reared on grain middlings 1 had a survival rate of only 50.0%, which is significantly lower than all other tested side-streams (with the exception of tomato leaves). Out of six feeding trials respectively 74, 122, 243, 344, 387, and 328 of 500 larvae survived. The fat content of 4.5% was comparable to CSM, a protein content of 14.1% was comparable to GVD and a non-fiber carbohydrate content of 53.1% was also relatively high, resulting in a gross energy content of 295 kcal/100 g DM, which was similar to CSM. No extreme values in fiber contents were found. A relatively high calcium content of 2.29 g/100 g DM was measured. The effect of high calcium contents on larval survival is still unknown.
Grain middlings 2 contained a significantly higher amount of fibers and the gross energy content was only 144 kcal/100 g DM, which was only half compared to grain middlings 1. Only 1.4% fat content was present, a protein content of 9.6% was present, and the non-fiber carbohydrate content was only 23.4%. However, a survival rate of 97.5% was measured with an average maximal larval weight of 78.0 mg, which is not significantly different from larvae reared on GVD.
Tomato leaves were not suitable as a substrate for black soldier fly larvae, as all larvae died quickly and no notable growth was observed. The protein content, however, was 12.1%, the fat content was 1.2%, and the non-fiber carbohydrate content was 36.5%. This resulted in a gross energy content of 205 kcal/100 g DM. This makes the stream comparable to GVD, on which larvae did manage to grow well. Tomato leaves may contain insecticidal compounds such as glycoalkaloids, as already described in recent research, where tomato leaf extract is used as an insecticide against aphids [
55]. Additionally, during the determination of the mineral contents, the addition of nitric acid to the ashes leads to H
2S production, which implies high sulfur contents present in the stream. This might be due to the use of a sulfur evaporator, which is commonly used in tomato growing to prevent mildew and other fungal diseases [
56]. The high amounts of sulfur may have had a toxic effect on the larvae. Moreover, a calcium content of 8.14 g/100 g DM was measured for tomato leaves, which was remarkably high compared the other substrates. However, as already described, the effects of high calcium content on larval growth is still unknown.
The final substrate tested for the growth of black soldier fly larvae was vegetable overproduction from auctions. The larvae survived well (98.0% survival rate) and the growth was not significantly different from larvae grown on GVD (mean maximal average weight of 88.1 mg). This stream had a relatively low protein content of 10.5%, a relatively low fat content of 2.0%, and a non-fiber carbohydrate content of 42.0%, resulting in a gross energy content of 228 kcal/100 g DM, which is similar to GVD.
The aim of this study was to evaluate whether the growth of larvae was possible on each of the selected side-streams, in order to evaluate the potential of different side-streams for black soldier fly rearing. However, by also conducting a chemical analysis to determine nutritional composition, fiber contents, and micro/macro-element contents of each of the side-streams, we attempted to find out ‘why’ some side-streams would work and some would not. However, this study showed that side-streams are complex matrices and not all relevant parameters could be determined within this study. Compounds such as insecticidal residues, amino/fatty acid profiles, water-retention properties of substrates, microbial loads, hemicellulose profiles, and the exact non-fiber carbohydrate composition, etc. were not included in this study. However, these might have played an important role in modeling larval growth. In this study, for example, we were not able to specifically declare why tomato leaves resulted in a 0% survival rate or why only 50% of all larvae grown on grain middlings 1 survived. Additionally, when comparing the nutritional composition of industrial food waste and household food waste, the result is that those compositions were relatively similar. Both side-streams had high protein, fat, and NFC contents. However, the maximal larval weight of larvae grown on household food waste is significantly lower compared to industrial food waste. Even though most fiber contents were higher in household food waste than in industrial food waste, these fiber contents were not higher than, for example, chicken manure. However, even though chicken manure is a nutritionally poorer side-stream, the maximum larval weight of larvae grown on chicken manure was significantly higher. This highlights an issue when working with side-streams in order to build a prediction model, as it is extremely difficult to control every single parameter. Another interesting parameter is substrate dry matter content. In this study, we brought each substrate to a dry matter content of 30%, however, the dry matter content of the residues had a lot of variation when the experiment started. This means that over the course of the experiment, the gradual change in substrate dry matter content was not constant for each side-stream. Therefore (even though the dry matter contents at the start of the experiment were equal), the amount of accessible moisture during the course of the experiment might have had an influence on larval growth. Moreover, there is room for discussion whether substrate dry matter content is the right parameter to standardize, as depending on the matrix, 30% DM can result in either a soaking wet, a saturated, or a dry substrate. Therefore, subsequent research is required to determine whether it is better to standardize substrate moisture content based, for example, on dry matter content or on maximal water binding capacity. The rate in which water is leaving the substrate (for example by evaporation) should potentially also be described, as this turned out not to be constant for all substrates. Exploring methods to monitor loss of water and to maintain adequate moisture content throughout the entire experiment should be explored. Even though these issues are present, this experiment might still provide potentially interesting correlations. A standard least square regression model was built describing both the main effects and interaction effects of substrate protein, fat, and NFC contents on the maximal larval weight. As main parameters, substrate protein, fat, and NFC content were used to describe the model as these contribute to the gross energy content of the substrate. It was decided to exclude side-streams with survival rates being significantly lower compared to the control substrates. Therefore, data from grain middlings 1 and tomato leaves were excluded from the model. A proximate component analysis was run (
Figure 2a) using the remaining substrates, which showed that no correlation between survival rate and maximal larval weight was present. This may indicate that larval density during this experiment was low enough and that the larvae did not hinder each other’s growth. In
Figure 2b, it can also be observed that larval survival rate is positively correlated with substrate fibers. This is potentially due to the substrate structure or water retention properties of the substrate fibers [
57].
The model predicted maximal larval weight when protein content was maximized (between the borders of protein concentrations tested in this study) and when the substrate fat content was low (0.9%). Previous studies also show that final weight of larvae, bioconversion rate, and feed conversion rate positively correlated with the amount of protein in the rearing substrates [
31,
58]. For larvae grown on apple pulp and chicory roots, it was observed that low protein contents led to poor larval growth. Not only the protein content itself, but also the amino acid composition and/or digestibility of proteins is important. If essential amino acids are limited or digestibility is low, the potential for larval growth is also limited [
14].
The model also includes the influence of NFC (%) on maximal larval weight, however, leverage of the predicted influence of substrate NFC content was way lower compared to the substrate protein and fat content. The low influence of substrate NFC content is remarkable, however, it can be verified by comparing the nutritional composition of beer draff to chicken start mash. The protein contents were similar (19.4% vs. 20.4%), as were the ether extracts (5.5% vs. 4.6%), however, the NFC content of beer draff was much lower compared to CSM (20.0% vs. 48.2%). However, both larval survival and maximal larval growth did not significantly differ. This comparison supports the findings described by the model above.
Another possibly important set of factors playing a role in larval growth is substrate fiber. As observed in the principal component analysis (
Figure 2b), a negative correlation is potentially present between mainly ADL (lignin), ADF, and cellulose contents. Not only may fibers make the side-stream harder to digest, but high fiber contents also leave less room for proteins, fats, and carbohydrates, thus lowering the gross energy content of the side-stream.
The bioconversion efficiency, feed conversion ratio, and waste reduction of the substrates are important parameters when evaluating BSF larvae as potential bioconversion technology. Several studies have evaluated these parameters and a lot of variability is observed, which is influenced by BSF strain, rearing circumstances including larval density, feeding rates and amounts, nutritional composition of the substrates, pretreatment of the substrates, [
19,
24]. Diener et al., for example, showed that waste reduction is highly dependent on the amount of diet that is provided [
54]. For chickenfeed, this ranged between 39.7% and 26.2% when providing 12.5 mg or 200 mg chicken feed per larva and day. As discussed previously, calculations of bioconversion efficiency, feed conversion ratio, and waste reduction also differed between studies. In this study, all conversion efficiencies were calculated on a DM basis as recommended by Bosch et al. [
59].
Larvae reared on CSM had a mean bioconversion efficiency of 17.56%. This is in line with the bioconversion efficiencies of other studies that are also using chicken feed as a control diet, having a bioconversion efficiency between 12% and 21% [
24,
44]. The waste reduction for CSM was 49.9%. Similar studies calculating waste reduction for chicken feed had report numbers ranging between 30.9% and 80.4% [
25,
28,
47]. Larval density and the amount of diet provided had a negative influence on waste reduction [
60,
61]. The variation in density and provision of diet between these studies does indeed vary and hampers comparison. The second control diet GVD is less often used as a control diet although it may differ less between studies as it is prepared in a similar manner between labs (GVD is composed of 50% wheat bran, 20% maize, and 30% alfalfa) and could overcome differences that are currently seen between commercial chickenfeed coming from different providers. [
33]. Bioconversion efficiency and waste reduction were respectively 7.72% and 45.9% for GVD, which is similar to previous studies [
62]. For side-streams in this study, bioconversion efficiencies between 3.59% and 20.71% were calculated and waste reductions were measured in the range between 17.0% and 53.9%, which illustrates the larval ability to both efficiently convert organic waste into larval biomass and to significantly reduce the waste amounts. As indicated from the PCA analysis, maximal larval weight and bioconversion efficiency were closely correlated, which is to be expected when having a similar survival rate. These values are similar to those obtained by other authors for a diversity of organic streams [
19,
24,
44].