Application of Black Soldier Fly Frass, Hermetia illucens (Diptera: Stratiomyidae) as Sustainable Organic Fertilizer for Lettuce, Lactuca sativa Production

In recent years, black soldier fly (BSF) has been shown to efficiently convert organic waste into nutrient-rich larval biomass while generating frass as the main by-product. This work aims to investigate the potential of BSF frass (BSFF) as an organic fertilizer for agricultural production. BSFF was produced by recycling household waste using BSF larvae, and a portion was taken to the lab for physicochemical and microbial analyses before the field trial on lettuce growth and health. The field trial consisted of two doses of BSFF (15 t∙ha −1 and 30 t∙ha −1 ) and one dose of urea (0.214 t∙ha −1 ). An unfertilized plot was prepared and used as a control, and the effects on lettuce growth were assessed using agronomic parameters while the health effects were assessed using parameters such as: the number of leaves affected per plant, the incidence of affection, the severity of the affection and the microbial analysis. BSFF exhibited acceptable physicochemical properties as an organic fertilizer. Its application improved the growth parameters of lettuce plants compared to urea and control. The application rates of 15 t∙ha −1 and 30 t∙ha −1 did not reveal any significant difference (p > 0.05). Further studies are therefore needed to determine the minimum applicable dose. The health parameters of the lettuce plants were slightly altered regardless of the treatment and the microbial analysis of the affected leaves revealed pathogenic micro-organisms. We therefore recommend that decontamination methods be considered when producing BSFF as an organic fertilizer.


Introduction
The depletion of raw material sources and soils leads to hunger and increased food insecurity. The situation is more noticeable in urban areas with high rates of urbanization and scarcity of agricultural land [1]. To cope with such a situation, there is a need for strategies that can match future food demands with increasing population growth while conserving the soil resources. Most African countries rely on agriculture for livelihood, which is a better way to increase food production. However, the use of agricultural land without a conservation method leads to a rapid decline in its fertility which is a major cause of the decline in production yields. Organic fertilizers offer a promising way to sustainably improve soils and crop productivity [2]. They are a good source of nitrogen, which is one of the most limiting soil nutrients for agricultural production [3].
Organic fertilizers improve soil microbial activities and provide macronutrients necessary for the growth of crops [4]. The use of organic fertilizers, such as compost and manure, constitutes a direct input of carbon (C) to the soil, which can be stabilized through physical, chemical, and biochemical mechanisms [5], contributing to long-term storage of C in soils [6] [7] [8]. In addition, organic fertilizers supply secondary nutrients (Calcium, Magnesium, and Sulfate) and micronutrients (zinc, boron, and manganese), which play a key role in the uptake and utilization of macronutrients such as nitrogen, phosphorous, and potassium [9] [10].
Despite its many advantages, the use of organic fertilizers in agriculture is still largely limited because of their low availability. Most organic manures are used to feed animals and leave little or no organic resources for use in agricultural production [11] [12]. Therefore, improving soil productivity using organic resources requires a venture into new organic fertilizer sources. In recent years, black soldier fly (BSF) has been shown to efficiently convert organic wastes into high quality essential nutrients [13] [14]. Globally, this has attracted interest in the face of rising prices of animal feedstuff and accumulating amounts of waste.
The main by-product of this bioconversion technology is BSF Frass (BSFF), which can be used as an organic fertilizer in agriculture [15].
Frass refers to the excrement of insects and will often contain other components such as exoskeleton sheds, death insects and insect food residue. The frass of commercially produced insect larvae, including BSF have great potential as a sustainable fertilizer source with many applications, since commercially produced insects are a way of utilizing pre-consumer food by-products that may otherwise create waste streams [16]. Additionally, the use of insect frass can reduce the need for chemical fertilizers [17]. Insect frass is rich in chitin from the shed exoskeleton, which is known to have several beneficial effects on plant growth and health [18] [19]. It functions as a nematicide and fungicide, and promotes mycorrhization, but in some cases appears to be phytotoxic [20]. Also, BSFF is reported to have insecticidal and insect-deterring properties [21].
In natural conditions, it is well known that frass deposition to soil has a great  [23]. Therefore, several insect companies have already decided to sell it as organic fertilizer [24], and even some farmers are already reporting its beneficial effects on crops [25]. However, there is still very limited information on its ability to improve soil fertility [26]. It still needs further research before a major scaling up in the industry. For example, its effects on soil characteristics and plant growth, its nutrient composition and influencing factors on variation, and the ecological aspects of its application. To contribute to these knowledge gaps, this work aimed at investigating some physicochemical and microbiological characteristics of BSFF and its fertilizing potential on lettuce growth and health.

Black Soldier Fly Frass
The BSFF was produced at the BSF rearing unit of the Agri-Business Vocational Training Center in Dschang, Cameroon, following the bioconversion process of organic waste by BSF larvae (BSFL) as described by Dzepe et al. [27]. Figure 1 summarizes the engineering of the production system, which integrates the BSF life cycle and the recovery process of bio-waste by the BSFL. The bio-waste used in this study was household waste from the students' guesthouse. It consisted mainly of food waste and was collected on a weekly basis and subjected to the BSFL from a pre-established BSF colony. The BSF colony was established using young BSFL from a wild-trapped BSF population reared since 2016 at the International Institute of Tropical Agriculture (IITA) in Yaounde, Cameroon.
Upon receipt, the young larvae were fed in a plastic container with chicken feed until mature, and then they were taken to a new container with pupation substrate (wood shavings) to undergo metamorphosis. The maturity of BSFL was marked by a change in skin color from white to dark-brown. At this stage, they are called pre-pupae and have taken a few days to become pupae, characterized by complete immobility and a hardening of the exoskeleton. The pupae were transferred to an untreated mosquito net cage where they completed their metamorphosis and emerged as adult flies. As the adult BSF do not feed on solid food but can take in liquid substances, they were provided with water twice a day to ensure their survival, and an egg-laying medium was also provided to them in the cage to collect the eggs which were incubated to obtain young larvae used to renew the cycle. Water was sprayed directly onto the cage, while egg-laying medium was adapted from Dortmans et al. [28].
Every two days, clusters of eggs from the established BSF colony were collected and incubated following the procedure described by Dzepe et al. [29]. After hatching, the neonate larvae were fed chicken feed for four days, before being transferred into the household waste from the students' guesthouse. The treatment took place in plastic containers (60 × 40 × 12 cm), at a rate of 100 mg of waste per larva per day as recommended by Dortmans et al. [28] and lasted 15 days, which is the normal time required by BSFL to effectively process organic  waste. At the end of the treatment, BSFLs were separated manually, and the remaining residue (frass) was collected and stored at room temperature for further use as organic fertilizer. A small fraction was sampled for physicochemical and microbiological analyses.

Soil Preparation and Experimental Design
The fertilizing potential of the BSFF was evaluated on a single piece of land at plot was considered a replicate for a given treatment.

Physicochemical and Microbiological Analyses
Soil and BSFF samples were transferred to the laboratory of soil sciences and en- NH ions which saturate the complex and release the cations (Ca 2+ , Mg 2+ , K + , Na + ) which will be then determined.
The concentrations of Ca and Mg were determined by complexometry with EDTA (Ethylene diamine tetraacetic acid), while those of K and Na were determined by flame spectrophotometry. The soil texture was also determined using the texture triangle method based on the USDA grain size classification.
Only BSFF was considered for microbiological analysis. A suspension of 0.1 g of sample in 10 ml of sterile water was prepared, and 0.1 ml of the suspension was then diluted serially (ten-fold) and used in the estimation of bacterial and fungal populations by the standard spread-plate dilution method described by Seeley and VanDemark [32]. Nutrient agar containing 0.015% (w/v) nystatin (to inhibit fungi growth) was used for the isolation of bacteria and incubation took place at 35˚C for 05 days. While potato dextrose agar mixed with 0.05% (w/v) chloramphenicol (to inhibit bacteria growth) was used for fungal isolation, and the incubation took place at 28˚C for 07 days. Pure isolates of representative communities were maintained on agar slant at 4˚C, and identification was based on cultural, microscopic, and biochemical characteristics with reference to Bergey's manual of determinative bacteriology for bacteria, and fungal identification kit of Botton et al. [33] for fungi.

Black Soldier Frass and Lettuce Growth
The agronomic potential of BSFF was investigated using young lettuce plants from a pre-established lettuce nursery. The nursery was carried out on a 3.6 m 2 (3 × 1.2 m) plot of unfertilized soil using lettuce seeds of the Batavia variety purchased from a local market. Using a string and poles, seeding lines spaced 20 cm apart from each other were materialized on the previously plowed plot, and the lettuce seeds were sown at a rate of 2 g•m −2 . The nursery was watered twice a day, and germination was observed from day 4 to day 10 before transplanting b) Plant height was measured from collar to apex using a graduated ruler.
c) Crown diameter was measured for each plant using a digital caliper.
d) The length of the largest leaf was measured from the leaf blade using a graduated ruler e) The width of the largest leaf was also measured using a graduated ruler.
f) The leaf area was calculated using the length of the largest leaf and the width of the largest leaf according to the following formula: Leaf area = (πLl)/4; where π = 3.14, L = largest leaf length and l = largest leaf width.
At the end of the experiment, the lettuce plants were harvested by treatment, weighed using an electronic scale, and the production yield was determined using the following formula: Y (t•h −1 ) = W (t) × S (ha) −1 ; Where Y (t•h −1 ) is the production yield in tonnes per hectare, W (t) the weight of lettuce harvested in tonnes, and S (ha) the area harvested in hectares.

Black Soldier Frass and Plant Health
The influence of BSFF on the health of lettuce plants was evaluated in the dif- c) The severity of affection was determined by field observation using the severity scale of Horsfall and Barrat [34].
For each treatment, all affected leaves were collected and transferred to the laboratory for microbiological analysis. The analysis consisted in identifying the possible micro-organisms responsible for the affections observed in the field and were carried out according to the same procedure as those of the BSFF previously described.

Statistical Analyses
The effects of BSFF on lettuce growth and health were statistically tested using one-way analysis of variance (ANOVA). In case of significant difference, the Tukey's HSD (honestly significant difference) post hoc test was performed.
Analyses were performed using the SPSS v. 22.0 Software, and the tests were considered significant at P < 0.05. (loamy soil with a moderately coarse texture). With a low C:N ratio (18 < 25), the experimental soil presented a poor quality of organic matter. The concentration of major elements N and K was high, while that of trace elements P and Ca was very low. According to the NFU44 051 standard for organic fertilizers, the BSFF produced in this study from household waste was rich in major elements N, P, K and trace elements Ca and Mg (Table 1). It had an organic matter content of 90% and a high C/N ratio (85 > 25). It also had a relatively alkaline pH (7.8).

Influence of Black Soldier Fly Frass on Lettuce Growth
As shown in Figure 2, the average number of leaves per lettuce plant was significantly influenced from the 2 nd to the 4 th week (p < 0.05       Lettuce yield was significantly influenced by the different treatments applied during the study (p < 0.05). It ranged from 31.17 ± 5.77 to 63.28 ± 7.34 t•ha −1 and the BSFF 30 t•ha −1 treatment had the highest influence, followed by the BSFF 15 t•ha −1 treatment (55.57 ± 6.89 t•ha −1 ). The lowest yield was recorded in the Control, no fertilizer, while the Urea 0.214 t•ha −1 had an intermediate value ( Figure 6).

Influence of Black Soldier Fly Frass on Lettuce Health
The application of BSFF influenced the health parameters of lettuce plants during the experiment. Figure 7 presents the average number of leaves per plant that showed signs of pathology during the study. It appears that until day 21 Open Journal of Applied Sciences    after transplanting, less than one leaf per plant was affected whatever the treatment and no significant difference was observed (p > 0.05). On the other hand, from week 4, the plants of the BSFF 15 t•ha −1 treatment were significantly more affected (1.21 ± 0.14 leaves per plant), followed by the plants of the Control, no fertilizer (1. 34 ± 0.85 leaves per plant) at week 5. At week 6, plants from the Urea 0.214 t•ha −1 were more affected (2.40 ± 1.03 leaves per plant), followed by plants from the BSFF 15 t•ha −1 treatment (1.84 ± 0.67 leaves per plant). Figure 8 presents the affection dynamics of lettuce plants according to the different treatments during the study. The incidence of the affection increased with time whatever the treatment and reached more than 50% of the plants before the harvest period (day 42). At the end of week 5, the affection incidence recorded with the 1st Control, no fertilizer was significantly higher compared to the other treatments. Similarly, at the end of week 6, the value recorded with the BSFF 30 t•ha −1 treatment was significantly lowers (p < 0.05).
A similar trend was recorded with the severity of the affection. Disease incidence was generally low (below 20%) on day 7 after transplanting and had increased to about 60% on day 42 for most of the treatments (Figure 9). Lettuce plants in the control, no fertilizer and the Urea 0.214 t•ha −1 were more susceptible to the disease compared to plants in the BSFF 15 t•ha −1 and BSFF 30 t•ha −1 treatments. Throughout the study period, the plants treated with BSFF 30 t•ha −1 showed less signs of disease than the rest of the treatments.
The microbiological analysis of the affected leaves collected in the fields revealed the presence of two genera of fungi (Cercospora and Aspergillus) and three genera of bacteria (Actinomycetes, Bacillus and Clostridium), whatever the treatment. The genus Cercospora is a pathogenic fungus and has also been found in the BSFF, along with all the other genera previously listed.

Discussion
As previously depicted in Figure 1, the application of BSF technology generates two main products: a nutrient-rich larval biomass and a waste residue commonly     [36] report that the N content of a good organic fertilizer is between 0.5% and 6% depending on its basic elements, and that its application in sufficient quantity makes it possible to improve the properties of the soil. According to the NFU44 051 standard for organic fertilizers, the following parameters must be taken into consideration when producing organic fertilizers: MO ≥ 20%, total N < 3%, total P 2 O 5 < 3%, total K 2 O < 3%, C:N ratio > 8, and the sum of nutrients (total N + total P 2 O 5 + total K 2 O) < 7%. For the present study, all these parameters were respected, which testifies to the quality of BSFF. The C:N ratio was higher compared to that obtained by Gajalakshmi [37] with compost from solid urban Open Journal of Applied Sciences waste. Indeed, Ajaweed [38] characterized compostable urban waste and reported that the nature of the latter could influence the C:N ratio, as well as other nutrient elements in the compost. The BSFF mineral element concentrations obtained in this study were different from those obtained by Anyega et al. [38] with larvae fed on brewers' spent grain, and Gärttling et al. [15] with larvae fed with a mixture of urban waste. However, all values were within the suitable range for organic fertilizers. These observations confirm the hypothesis that the physicochemical characteristics of BSFF vary widely depending on the type of substrate used to rear the BSF larvae. Poveda et al. [26] also found that this feeding substrate can affect the microbial composition of frass as well as its nutrient content.
In the present study, the pH of BSFF was slightly alkaline (7.84) like that of most organic fertilizers [39]. It therefore provides favorable conditions for improving soil biological properties and cation availability. It could also be used to increase the pH of acidic soils.  [40] who reports that organic compost provides the plant with the nitrogen necessary for its aerial growth and improves its stem/leaf ratio. Rosmiati et al. [41] also report good growth performance in lettuce plants with BSFF produced from Coffee Husk. The same observation was made with the production yield where the values obtained with the BSFF treatments were significantly higher compared to those of the control treatments. Varying the BSFF application rate had no significant effect on lettuce growth and production, although the yield recorded with the BSFF 30 t•ha −1 treatment was relatively higher. Although urea provides enough nitrogen to the plant compared to BSFF, the latter also provides other nutrients such as phosphorus, potassium, calcium, and magnesium essential for plant growth. Also, with a C:N ratio > 25, the application of BSFF can lead to nitrogen mineralization in the soil and release accessible forms for plants. These preliminary results confirm the fertilizing potential of BSFF which can be used as a sustainable alternative to chemical fertilizers. With the increasing importance of the insect production sector, BSFF will soon be a highly available organic fertilizer. Current insect producers are mostly small or medium-sized enterprises, of which about 80% raise BSF [13]. Present production sums up to a few thousand tonnes per year but will expand substantially in the coming years and frass will emerge toward a considerable by-product. Frass represents about 70% to 90% of the total output in a BSF production system [42] and must be valued for the system to be profitable.
Microbial analyzes revealed the presence of a pathogenic fungus (Cercospora sp) both in the lettuce plants and in the BSFF. This fungus has been shown to be the main cause of affection in lettuce leaves, and we hypothesize that Cercospora Open Journal of Applied Sciences sp is transmitted from BSFF to plants, and plants in treatments without BSFF would have been accidentally contaminated by wind or during handling. Another fungus of the genus Aspergillus has also been identified. Although, it is not pathogenic to lettuce plants, this fungus (Aspergillus) can cause allergies or respiratory illnesses in humans been. Fuchs and Larbi [43], report that not all organic fertilizers have the potential to protect plants against infections. Some can constitute sources of contaminants if they are improperly treated. Bacteria found in BSFF (Actinomycetes sp, Bacillus sp and Clostridium sp), were also found on the affected lettuce leaves. This could be explained by the anatomy of lettuce plants, where leaves are found very close to the ground, thus facilitating the migration of bacteria from the soil to the plants.
The results found in this study suggest that BSFF can be used as an organic fertilizer and improve crop growth and production yield. The variation of two doses of BSFF (BSFF 15 t•ha −1 and BSFF 30 t•ha −1 ) did not reveal any significant difference in the parameters studied. Further studies are therefore needed to determine the minimum applicable dose, in order to better advice farmers for sustainable use. The microbial analysis revealed the presence of pathogenic microorganisms capable of affecting plants, as well as humans. It is therefore necessary that decontamination methods be considered when producing BSFF for soil fertilization. This can be done by composting the BSFF for a few more weeks after production.