Black Soldier Fly Cultivation: Conditions and a Proposed Methods

Iowa State University Capstones, Theses and Creative Components Dissertations

Spring 2021

Black soldier fly cultivation: conditions and a proposed methods

Julius Au

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Breeding and developing the BSFL

Julius Au Weng Lee

A non-thesis submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

Major: Industrial and Agricultural Technology

Program of Study Committee: Dr. Thomas Brumm, Major Professor Dr. Erin Bowers Dr. Joseph Morris

The student author, whose presentation of the scholarship herein was approved by the program of study committee, is solely responsible for the content of this non-thesis. The Graduate College will ensure non-thesis is globally accessible and will not permit alterations after a degree is conferred.

Iowa State University

Ames, Iowa

2020

Table of Contents LIST OF FIGURES ...... i LIST OF TABLES ...... ii NOMENCLATURE ...... iii ABSTRACT ...... iv CHAPTER 1: INTRODUCTION ...... 1 CHAPTER 2: EXPLANATION OF THE BSF LIFECYCLE ...... 4 CHAPTER 3: FISHMEAL SUBSTITUTION WITH THE BSFL ...... 6 CHAPTER 4: THE BSFL PROTEIN AND LIPID CONTENT ...... 9 CHAPTER 5: MYCOTOXIN ACCUMULATION ...... 12 CHAPTER 6: HEAVY METAL ACCUMULATION ...... 15 CHAPTER 7: CONSIDERATION AND PREVENTION FOR ENTOMOPATHOGENS ...... 18 CHAPTER 8: THE PROPOSED BSFL REARING SYSTEM ...... 20 CHAPTER 8.1: ABIOTIC FACTORS ...... 21 8.1.1: TEMPERATURE ...... 21 8.1.2: RELATIVE HUMIDITY ...... 24 8.2: THE BREEDING UNIT ...... 26 8.2.1: ARTIFICIAL LIGHTING ...... 28 8.2.2: OVIPOSITION ATTRACTANT ...... 31 8.3: THE BREEDING SITE ...... 32 CHAPTER 8.3.1: OVIPOSITION SITE PREFERENCE ...... 34 CHAPTER 8.4: RESETTING THE BREEDING UNIT ...... 36 CHAPTER 8.5: NURSERY FEEDING CONTAINER ...... 37 CHAPTER 8.6: GROW OUT FEEDING CONTAINER ...... 39 CHAPTER 8.6.1: WASTE MATERIAL ON DEVELOPMENT AND SURVIVABILITY ...... 40 CHAPTER 8.6.2: FEED MOISTURE ON DEVELOPMENT AND SURVIVABILITY ...... 48 CHAPTER 8.6.3: FEEDING RATE ON WASTE REDUCTION AND PREPUPAL WEIGHT ...... 52 CHAPTER 8.7: PREPARING THE WASTE FEED ...... 60 CHAPTER 8.8: LARVAE FOR REPOPULATING THE BREEDING UNIT ...... 61 CHAPTER 8.9: THE PUPATION CONTAINER ...... 62 CHAPTER 8.9.1: PUPATION MATERIAL ON SURVIVABILITY.EMERGENCE TIME ...... 62 CHAPTER 9: APPROXIMATE COST OF THE PROPOSED SYSTEM ...... 67 CHAPTER 10: CONCLUSION ...... 68 REFERENCE ...... 70

LIST OF FIGURES

Figure 1: Life cycle of the black soldier fly sourced from De Smet et al. (2018) ...... 4 Figure 2: The survivability of the BSF from eggs to its prepupal stage sourced from Chia et al. (2018)...... 23 Figure 3: Illustration of the breeding cage construct without the plastic screen mesh...... 27 Figure 4: The oviposition response using varying oviposition material from Nyakeri et al. (2017) ...... 32 Figure 5: Wooden stack arrangement used in the breeding cage without rubber bands...... 34 Figure 6: Illustration of information recorded on clipboard...... 34 Figure 7: Amount of egg masses deposited and number of masses deposited onto different oviposition materials from Boaru et al. (2019)...... 36 Figure 8: Tiered rack for nursery/feeding...... 38 Figure 9: Example of nursery/feeding container...... 38 Figure 10: Illustration of figure 10 in the nursery/grow out container...... 38 Figure 11: Placement of the wooden stacks onto supporting metal stand...... 38 Figure 12: The performance of the black soldier fly’s larvae survivability, prepupal wet weight and waste reduction based on differing feed moisture concentrations from Lalander et al. (2019)...... 51 Figure 13: The results of Diener dry matter waste reduction percent in relation to the prepupal mass. The waste material was chicken feed with a moisture content of 60%...... 56 Figure 14: The results of Manurung dry matter waste reduction percent in relation to the prepupal weight. The waste material was human fecal sludge. Manurung used a mixture of 30 gram of straw with 60.ml water...... 56 Figure 15: The results of Nyakeri dry matter waste reduction percent in relation to the prepupal weight. Nyakeri used fresh human fecal...... 57 Figure 16: The results from Myers et al. (2013) dry matter waste reduction percent in relation to prepupal weight. The waste material was fresh dairy manure with a moisture content of 70%...... 57 Figure 17: The results from Bonso (2013) dry matter waste reduction percent in relation to the larvae conversion rate. The waste material was a mixture of cooked and uncooked food with a moisture content of 90%...... 58 Figure 18: The dry matter waste reduction results from Mutafela (2015) using lump and daily feeding with 3 different waste material...... 58 Figure 19: Proposed BSFL production process flow chart...... 66

LIST OF TABLES

Table 1: Mean value of crude protein % (non-chitin corrected) and lipid % (ether extract) dry matter of BSF prepupae on different diets...... 10

Table 2: Mean mycotoxin concentration in feeding substrate and bioaccumulation in BSFL...... 13

Table 3: Initial heavy metal concentration in feedstock comparison with BSFL pre-pupal and residue ...... 16

Table 4: The effect of relative humidity on egg development and successful hatching of BSFL. . 26

Table 5: The mean period required to reach the pre-pupal stage with varying protein and carbohydrate content...... 42

Table 6: The mean survivability of pre-pupae and adult fly when fed various substrate...... 45

Table 7: Prepupal biomass yield of different systems ...... 47

Table 8: Feeding rates and resulting mean dry weight (mg) of prepupae fed varying waste...... 53

Table 9: The reduction in waste from different authors using common feeding rates with differing waste and WRI...... 54

Table 10: Effects of pupation material on development and survivability of post-feeding larvae.

...... 64

Table 11: Literature-based operational conditions for growing and breeding BSF...... 65

Table 12: The approximate cost for the proposed system...... 67

iii

NOMENCLATURE

LSHTM London School of Hygiene and Tropical Medicine BSF Black Soldier Fly

BSFL Black soldier fly larvae

WW Wet Weight

DW Dry Weight

MOW Municipal Organic Waste

N/A Not Available

AMP Anti-Microbial Peptides

PUFA Poly Unsaturated Fatty Acids

MCFA Medium Chain Fatty Acids

LCFA Long Chain Fatty Acids

FCR Food Conversion Ratio

SGR Specific Growth Rate

WRI Waste Reduction Index

WR Waste reduction

LOQ Limit of Quantification

ND Not Detected

MC Moisture Content

ABSTRACT

With the global population expected to increase to 9.8 billion in 2050, we have to look to more waste management due to the inherent increase in organic was comprising fecal matter and food waste (an issue we are currently facing). To sustain the increased population, we are projected to require a 60% increase in protein production. However, in order to accomplish this, we will have to redirect more resources to the feed industry. In regard to the feed industry, they are facing pressure to use more sustainable feed. The BSF prepupae has been shown to consume a wide variety of waste products including animal manure, human fecal matter, rotting fruits and vegetables and converting themselves into high protein and lipid feed stuff. The cultivation of the fly requires specific conditions to established and maintain a fly colony effectively. This paper focuses onto the culmination of information and conditions needed to breed and cultivate the fly from egg to adult, provides a proposed physical pilot system to breed fly as well as expected observations during the production process.

CHAPTER 1: INTRODUCTION

Within the context of the projected population growth, the earth will be experiencing a two billion increase in population. This inherently means the volume and intensity of waste management will have to be increase for the additional two billion waste units produced.

Moreover, to sustain the increased population the FAO (2011) estimates that we will require a

60% increase in protein production. However, in order to successfully meet this need a tremendous amount of resources has to be allocated to produce the said amount with feed being the most challenging. According to Ray et al (2013) by 2050, although wheat, rice and maze are projected to increase by 38%, 42% and 67% respectively they will insufficient to keep up with population demand. This brings about the competition for human food or animal feed (Rehman et al., 2017) such as for beef cattle which uses maize as an energy source. Additionally, we are also aware that the livestock feed sector is under pressure to use more environmentally sustainable animal food ingredients (FAO, 2011). A recourse to bridge the 60% protein gap, satisfy the challenge of sustainability and manage waste may lie in aquaculture by complete or partial substitution of fishmeal with the BSFL as the protein source.

Among livestock, fish has the comparative advantage of having a lower food conversion ratio and is expected to contribute significantly to the increasing need for fishery products (FAO,

2014). In cultivating fish, the feed cost accounts for approximately 60-80% of its operational cost and of that, protein constitute 50% making it the costliest ingredient in intensive aquaculture

(Nguyen et al., 2009). From 1990 to 2017 the global aquaculture production has increased by

527% (The State of World Fisheries and Aquaculture, 2020). Following the increase in 2 production, the percentage of stocks fished within biologically sustainable levels decreased by

24.4% from 1990 to 2017 and the price of fishmeal increased by 340% (The State of World

Fisheries and Aquaculture, 2020). The BSFL has been approved by AAFCO for salmon feedstuff

(AAFCO 2016) but consumer acceptance of the fly still needs to be further researched although from Belgium, two thirds of participants from a survey were willing to include insect meal into livestock feed highlighting fish and poultry (Verbeke et al., 2015). A possible reason whereby the BSFL may not be accepted could be a societal issue or a palatability issues stemming from the unknown. Consuming insects may be seen as a “poor man’s” food in certain mindsets but with more understanding of the insect, more adventurous pallets and coupled with its inclusion in high-end cuisine there is a hope that that the fear-based reasons will subside.

Fish-derived commercial feed is regarded as the optimal protein source for fish feed however the limitation of wild caught marine stock has been reached drawing up the question of sustainability of the harvested fish from an ecological sustainability standpoint (Renna et al.

2017; Elwert et al. 2010). In fishmeal production forage fish and lower trophic fish (anchovies and sardines) are harvested by the masses. The literature on plant-based protein has been evaluated which has the comparative price advantage and nutritional consistency to fishmeal but are often lower in essential amino acids and fatty acids (Cummins et al., 2017; Kroeckel et al.,

2012). They also represent the highest potential for hosting mycotoxins (Marijani et al., 2019).

As an alternative, insect meal has been considered promising because it can be sustainability sourced for lipid and protein not to mention carnivorous freshwater fish consume insects in their natural ecosystem (Renna et al., 2017). The urgency to find an alternate protein source for animal 3 feed is even more urgent in some European Union countries because fishmeal is either banned or expensive (Elwert et al., 2010).

Sustainability in this context is defined as the responsible use of resources to produce safe feed ingredients to maximize protein production demands for human population expansion while ameliorating long-term environmental and health impacts. The larvae of the black soldier fly have been demonstrated to ingest animal manure (Sheppard et al., 1994; Newton et al., 2005), human feces (Banks, 2014; Lalander et al., 2019), vegetable waste (Sprangher et al., 2017), kitchen waste (Shumo et al., 2019) and fish offal (St. Hilaire et al., 2007). They have also been demonstrated to consume mycotoxin-contaminated waste without showing toxic presence within their body (Purschke et al., 2017; Bosch et al., 2017; Gülsünoğlu et al., 2019). However, studies have shown that the larvae do have a higher tendency to accumulate cadmium compared to other heavy metals (van der Fels-Klerx, 2016; Diener et al., 2015b). The larvae are able to utilize these excretions and waste containing half of the nutrients from feed (Steinfeld, 2012) by assimilation and converting themselves into high protein and lipid feedstuff for animals (Oonincx et al.,

2015). They also have a well-balanced essential amino acid profile making it possible to be used as a replacement for fishmeal (Kroeckel et al., 2012) and has also received industrial interest as food and animal feed as a source of protein (Meijer et al. 2019).

Due to their saprophytic nature and the imbalance of available feed substrate, a range of by-products will be evaluated on the performance of the fly’s development. From a safety aspect, since a range of inedible substrates are a potential food source the bioaccumulation levels of mycotoxins and heavy metals will be evaluated to avoid fish illness and unintended carry-over to 4 humans. As a result of substituting fishmeal to an alternative feed source, a small section of the performance of the larvae will also be evaluated using the available literature including Nile

Tilapia (Oreochromis niloticus) and other aquatic life as the study of BSF is in its infancy (Huis,

2020). The motive of this paper is to lay down guidance for breeding and developing of the

BSFL and propose a pilot scale production system. The system is not intended to provide enough larvae for intense aquaculture production. For breeding the larvae, its survivability and egg yield are dependent upon abiotic factors such as temperature and relative humidity but also required three other conditions to produce the eggs. Along with temperature, feed moisture and its quality are influential factors in the development and survivability of the larvae to adulthood.

Consideration and prevention methods for entomopathogens will also be provided as a safety measure.

CHAPTER 2: EXPLANATION OF THE BSF LIFECYCLE

Figure 1: Life cycle of the black soldier fly sourced from De Smet et al. (2018)

The black soldier fly (Hermetia illucens) is from the Diptera order and of the

Stratiomyidae family is indigenous to the subtropical and warmer parts of America (Makkar et al., 2014). An adult black soldier fly has a black wasp-like appearance and can measure up to

20mm in length. When full grown the BSF does not have a sponge-like feeding structure like the common house fly making the BSF unattracted to potentially hazardous waste; therefore, having a decreased risk probability of transmitting zoonotic diseases. The fly maintains its bodily functions and survives by primarily depleting their fat reserves accumulated during their larval feeding stages making its feeding stage crucial to its longevity. The BSF is considered a non-pest fly and spends most of its time on leaves having a 5 to 12-day life expectancy without water

(Mwaniki, 2019; Makkar et al., 2014; Nakamura et al., 2015) and may live for 14-21 days with water (Tomberlin et al. 2002; Nakamura et al., 2015).

After 2 days from pupal emergence the female and male begin their mating ritual with a courtship flight up to a 1.5m vertical height and lands on surfaces to mate (Caruso et al., 2013) when conditions allow and compel them to (Mwaniki, 2019; Makkar et al., 2014).The gestation period followed by oviposition takes place 2 days after mating and not earlier (Bertinetti et al.,

2019; Tomberlin & Sheppard, 2002). Each female fly will lay between 400-900 eggs (Dortmans et al., 2017; Sripontan et al., 2017; Tomberlin & Sheppard, 2002a; Booth et al., 1984). When the eggs have hatched after 3-4 days (Diener et al. 2011b) the larvae begin feeding and undergoes five larval stages before reaching the 6th pre-pupal stage. At this stage, the BSFL ceases feeding and excretes its gut content. The growth and survivability are also dependent on the insect’s nutrient bioavailability but unfortunately not much is known about it (Lalander et al., 2019). As the larvae progressed, its mouthpart undergoes morphological changes which suggests a 6 development in feeding habits whereby more feed is consumed from stage 1- 4 and slows down as it reaches the prepupal stage (Gligorescu et al., 2018) indicating non-linear consumption.

An attractive feature of this species is that during its 6th stage they innately migrate to protected and dry areas (Park et al., 2016) and have been seen to climb up vertical surfaces provided moist enough to retain water tension (Banks, LSHTM, personal observation). This migratory behavior is termed “self-harvesting” (Wang & Shelomi, 2017). The pre-pupae’s physical appearance is distinguished by the sclerotized pigmentation of the exoskeleton while larvae remain white (Zhang et al., 2010). The difference between the pre-pupal and pupal stage is that the pupae is completely immobile and inelastic at the last abdominal segment (Dzepe et al.,

2020; Holmes et al., 2013). The pre-pupae will take 7-9 days to reach the pupal stage and another

6-9 days to complete metamorphosis and holometabolism (Diener et al. 2011b; Holmes et al.,

2013).

CHAPTER 3: FISHMEAL SUBSTITUTION WITH THE BSFL

Aquaculture production is the cultivation of aquatic life under controlled inputs ranging from backyard hobbyist to intensely managed systems using advanced technologies but all often depend on using the ocean’s resources for feed ingredients. Nevertheless, the central purpose of any business is to reduce resource inputs and optimize profits by setting an efficient diet conforming with the fish’s growth stage and maintaining a consistent predictable growth rate to meet market demands with the sustainability of the feed ingredients becoming increasingly more important. One criterion for an ingredient to be considered a suitable alternative protein source is the ability to provide adequate and appropriate amino acids ratios for growth and health (Muin et 7 al., 2017) Protein is the main constituent of the fish body thus sufficient dietary supply is needed for optimum growth (Ahmad, 2004). Numerous studies have been conducted on the dietary protein content and growth response of a variety of fish that have a wide variety of dietary needs. Tilapia is the commonly cultured species that has robust tolerance towards abiotic factors and can grow well on low protein diets. Tilapia is expected to be one of the fastest supply growths with an expectation of a two-fold increase from 2010 to 2030 with China accounting for

37% of food fish production (FAO, 2011).

A study conducted by (Muin et al., 2017) prepared five distinct BSFL diets with

41.74±1.09% crude protein and 28.74±1.44% lipid at inclusion rates of 25% increments from 0% for fingerling Nile tilapia, Oreochromis niloticus. An interesting fact about this experiment is that the pellets were highly accepted by the fingerling because usually when fishmeal is replaced by an alternate protein source the palatability decreases (Rodríguez‐Serna, 1996) as is the case for vegetable oil (Wang & Shelomi, 2017). Muin et al. (2017) study concluded that half of the feed could be replaced with BSFL while achieving a 2.91 food conversion ratio (FCR) being the lowest among the test diets. The pellet with 50% of BSFL meal produced the highest specific growth rate (SGR) and PER value but also stated that as the inclusion rate increased the FCR became less efficient beyond 50%. The FCR is defined as the consumed feed to average weight gain ratio where a lower value is signifying higher feed utilization for tissue synthesis. This increase in FCR can be explained by Marono et al. (2015) which elaborated that chitin present in the insect’s exoskeleton negatively affected protein digestibility of the BSFL by protein-chitin linking.

The data on chitin fractions on the experiment for Muin et al. (2017) was not provided.

Muin et al. (2017) concluded that, at best, a 50% replacement by the BSFL meal into fishmeal is 8 possible without affecting its carcass quality, growth and health and the same conclusion was reached in Teye-Gaga (2017). It is worth noting that as a fish increases in size, they required less protein. This is because smaller fish use protein for protein synthesis when adequate lipids are present but if they are not adequate protein will be used as an energy source (Craig & Helfrich,

2002). For warm water omnivorous fish, it is recommended that the dietary lipid content be low

(El-Sayed, 2006) varying from 80-120g/kg (Jauncey, 1998). Nevertheless, since the larvae are able to reach lipid content of between 250-350g/kg, defatting the larvae would allow a higher inclusion as pointed by Fasakin et al. (2003).

With another study on juvenile carp (Cyprinus carpiovar. Jian) using the BSFL oil and soybean oil, the growth rate was not affected by the substitution with BSFL oil but lipid deposition decreased with higher BSFL oil (Senlin et al., 2016). With a study on the European seabass (Dicentrarchus labrax) fingerlings, Magalhães et al. (2017) drew an inference that without adversely affecting the growth performance, feed utilization or digestibility a partial replacement of up to 45% of FM can be substituted with meal containing 19.5% BSFL. With crustacean, the lipid and protein of the pacific white shrimp (Litopenaeus vannamei) decreased with more than 7% BSFL inclusion but noted that improvement is possible through supplementation and balancing the essential and non-essential amino acid ratios (Cummin et al.,

2017). The replacement of fishmeal with the black soldier fly meal differs in amount between aquatic species without adversely affecting its growth.

CHAPTER 4: THE BSFL PROTEIN AND LIPID CONTENT

Compared to carnivorous terrestrial animals, fish require a higher crude protein content because sources of carbohydrates in their natural ecosystem is scarce therefore, through adaptation, they developed an efficiency in metabolizing protein as an energy source. As a fish culturist this is unwanted as we would like protein to be used for the fish’s own protein synthesis for growth and not be used as an alternative energy source for metabolic activities. This makes the lipid content in a pellet important as it will serve as an energy source. The black soldier fly’s larvae are able to attain a lipid concentration of 25-35% and a crude protein of up to 45% live. In regard to crude protein, fish do not depend on the crude protein level per say but they depend on the amino acid profile (Mjoun et al., 2010). For proper application in least-cost formulation software, it is of paramount importance to understand the factors that contribute to the variation in nutritional value (Spranghers et al., 2017).

The nutritional composition of the black soldier fly prepupae is highly dependable on the feeding substrate provided (Wang & Shelomi, 2017; Julia et al., 2017) and could crucially replace fish meal in animal nutrition (Tschirner & Simon, 2015). When compared to fishmeal

(FM), soybean meal (SBM) has lower essential amino acid concentrations, particularly methionine, lysine, and threonine as well as a lack of essential n-3 fatty acids; eicosapentaenoic, docosahexaenoic acids (Cummin et al., 2017). Because of this conflict, animal nutritionists are eager to find novel protein sources for feeding non-ruminants with high protein quality and digestibility (Tschirner & Simon, 2015. In Thomas Spranghers 2017 study three waste stream substrates were used in rearing black soldier fly larvae. The analysis concluded that the feeding substrate had no substantial influence over the amino acid profile. Lalander et al. (2019) data 10 using 11 feeding material also displayed the same conclusion drawn by Spranghers et al. (2017) although the individual essential amino acids quantity was higher in Lalander et al. (2019) but both had similar crude protein concentrations.

Table 1: Mean value of crude protein % (non-chitin corrected) and lipid % (ether extract) dry matter of BSF prepupae on different diets.

Diet Protein, % Crude fat, % Source

Substrate Prepupae Substrate Prepupae

Total 77 Chicken Spranghers et al. 17.5 41.2 0.53 33.6 feed (2017)

Spranghers et al. Vegetable waste 0.86 39.9 0.21 37.1 (2017)

Spranghers et al. Restaurant waste 15.7 43.1 13.9 38.6 (2017)

Spranghers et al. Bio digestate waste 24.6 42.2 0.62 21.8 (2017)

70% Vegetable 11.99 31.29 2.6 26.28 Meneguz et al. (2017) 30% fruit

Fruit 4.6 22.97 2.78 40.7 Meneguz et al. (2017)

Brewery by-product 20 39.57 8.6 29.87 Meneguz et al. (2017)

Winery by-product 11.7 25.73 7.9 32.22 Meneguz et al. (2017)

Cow manure - - - 21.42 St. Hilaire et al. (2007)

50% cow manure - - - 30.44 St. Hilaire et al. (2007) 50% fish offal 11

Swine manure - - - 33.1 St. Hilaire et al. (2007)

Mixture of 22 37.2 5.9 30.8 Tschirner et al. (2015) middling

Dried DDGS 31.2 44.6 8.4 38.6 Tschirner et al. (2015)

Dried sugar beet 8.5 52.3 1.1 34.0 Tschirner et al. (2015) pulp

Municipal organic - 39.8 - 30.1 Mutafela (2015) waste

Horse manure - 40.9 - 12.9 Mutafela (2015)

Fresh fruit waste - 37.8 - 41.7 Mutafela (2015)

Swine manure - 43.2 - 28 Newton et al. (2005)

Poultry manure - 42.1 - 34.8 Newton et al. (2005)

Wild (West Kenya, - 40 - 20 Nyakeri et al. (2017) Bondo area)

Chicken manure 15.3 41.1 2.7 30.1 Shumo et al. (2019)

Brewers-spent 12.2 41.3 7.2 31 Shumo et al. (2019) waste

Kitchen waste 20 33 7.2 34.3 Shumo et al. (2019)

Spranghers et al. (2017) showed that the fat content and conventional protein used in feed formulation can be replaced by insects without adverse effect on the product’s quality. Research has also shown that when reared with fish fecal matter, insect crude protein increased to 42.5%

(Schmitt et al., 2019) which may prove useful to produce a micro circular economy. The research 12 also indicates that substrate type has an effect on the lipid content but only to a limited degree which agrees with Spranghers et al. (2017). The n-3 PUFA levels were shown to be between relatively low (9-23mg kg-1) compared to n-6 PUFA concentration (46-120mg kg-1) and characterized by high levels lauric acid (436.5-608.9mg kg-1) in Spranghers et al. (2017). St.

Hilaire et al. (2007) demonstrated that when the BSF larvae were fed with fish offal the n-3

PUFA concentration went from negligible to approximately 3% of the lipid. The same author also proposed that by including small amounts of fish waste into the larvae’s diet 24 hours before pupation the same levels can be attained in the pre-pupae. One particular reason why the soldier fly is of interest is because during its last larval stage their lipid content is higher than other larvae (Cičková et al., 2015). This makes them interesting for biodiesel production (Surendra et al. 2016) but the economic feasibility of using the larvae for waste reduction depends on the nature of the input material (Sheppard et al., 1994; Newton et al., 2005; Diener et al., 2011a;

Lalander et al., 2019; Banks, 2014).

CHAPTER 5: MYCOTOXIN ACCUMULATION

There is a vast knowledge involving the interaction between the biological activities of mycotoxins with terrestrial animals especially in the livestock industry and may represent a problem. Mycotoxins are the secondary metabolite of molds that can have significant effect on animal and human health (Zain, 2011). The outcomes of ingesting mycotoxin-contaminated feed by fish are similar to that of terrestrial animals commonly inducing stunted growth, compromising the immune system and in severe intoxication, death (Marijani et al., 2019). The most common chemical compound in grain is aflatoxin which is produced by the fungi 13

Aspergillus flavus. Food waste can be used as feed material for the BSFL, but precautionary measures have to be taken to safeguard from mycotoxicosis. As an illustration, if aflatoxin were consumed by dairy cattle the milk harvested will contaminate chemical compounds which will transfer into humans. Similarly, toxic residues can be carried down the line through fish meat

(Marijani et al., 2019).

Table 2: Mean mycotoxin concentration in feeding substrate and bioaccumulation in BSFL.

Feeding substrate Residual Mycotoxin Larvae Source concentration substrate

Deoxynivalenol Purschke et al. 697.7 µg kg-1 ND 1135.7 µg kg-1 (DON) (2017)

Purschke et al. Aflatoxin B1 13.3 µg kg-1 ND 10.9 µg kg-1 (2017)

< LOQ of 4 Purschke et al. Aflatoxin B2 2.6 µg kg-1 ND µg kg-1 (2017)

< LOQ of 16 Purschke et al. Aflatoxin G2 7 µg kg-1 ND µg kg-1 (2017)

< LOQ of 20 Purschke et al. Ochratoxin A 39.4 µg kg-1 ND µg kg-1 (2017)

103.9 Purschke et al. Zearalenone 130.4 µg kg-1 ND µg kg-1 (2017)

Aflatoxin B1 7478 mg kg-1 <0.1 µg kg-1 1270 mg kg-1 Bosch et al. (2017)

Gülsünoğlu et al (2019) evaluated the deoxynivalenol (DON) accumulation and nutrient recovery using the black soldier fly and concluded that the toxin did not accumulate in the larvae but rather passed through it while assimilating the nutrient into its body but the exact nature of this was not explained. An added benefit from a disease transmission standpoint is that when the post-feeding stage is reached the larvae excretes its intestinal contents which reduces its hazard potential (Spranghers et al., 2017). However, the defecation period after the pre-pupal stage initiation requires more research as harvesting the pre-pupae too early may cause an uptake of hazards by animal but has been indicated to require 1-3 days (Diener et al., 2015b). Research by

Purschke et al. (2017) also showed that for the various mycotoxins, the concentrations was below the detection limit but considerably levels of deoxynivalenol was detected in the residual substrate while zearalenone showed no major difference post and pre-larvae.

The higher levels in the residue can also be attributed by continual fungal growth during the bioprocessing period of the larvae. Another study by Bosch et al. (2017) concurs with

Purschke et al. (2017) where the BSFL had a high tolerance for aflatoxin B1 and M1 measuring below 0.10µg/kg and did not affect the larvae’s growth which was also reported by Camenzuli

(2018). The BSFL larvae was also shown to metabolize and excrete aflatoxin B1, (DON), ochratoxin A, zearalenone and a mixture of mycotoxins in Camenzuli et al (2018) but cautioned that additional research and toxicology test need to be conducted to understand the limits of contamination possible in feed. To my knowledge, unfortunately the study of mycotoxin accumulation in the larvae’s gut has not been well documented as the earliest study on this was in 2017 (Purschke et al., 2017; Bosch et al., 2017). The existing studies concerning the mycotoxin bioaccumulation in the pr-pupae is promising as studies display the same conclusions. 15

CHAPTER 6: HEAVY METAL ACCUMULATION

In 2014, approximately 587 billion tons of animal manure was produced (Afazeli et al.

2014) which contain heavy metals such as copper, lead, zinc, chromium and cadmium (Moral et al. 2008). Animal manure can be digested by the BSF larvae but there are safety issues which have to be addressed because of the nature of manufactured animal feed (Qiao et al., 2017).

Production of BSFL is an opportunity for farmers to generate extra income directly from the farms but high traces of heavy metals have been detected in animal manure and some have exceeded some country’s heavy metal thresholds in feed such as China and the European Union

(EU) (Qiao et al., 2017). For example, the typical Cadmium concentration in swine manure in northeast China is 15.1mg/kg dry weight (Qiao et al., 2017). This concentration far exceeded

China’s 0.1mg/kg limit and is 7.5 times more than the EU’s. For Chromium, the highest concentration in poultry manure was 2402.95mg/kg and this exceeds China’s limit by 12-fold.

These levels are an issue because cadmium and chromium are generally poisonous to animal and humans even at low levels (Saha et al., 2011). There have been studies which indicate that certain heavy metals have negative developmental and survivability effects on the BSF such as elevated zinc concentration in Diener et al. (2011a) and Diener et al. (2011b) where strong fluctuations in daily prepupal harvest was experienced. Zinc contaminated food has been shown to severely affect the survivability of larvae (40%) and pupae (70%) of the common house fly attributed by the decrease in hemocyte density which is a fitness indicator of the fly’s immune system. (Borowska et al., 2004).

Table 3: Initial heavy metal concentration in feedstock comparison with BSFL pre-pupal and residue

Chemical Feed concentration, Pre-pupae, Residue, BAF Source element mg kg-1 mg kg-1 mg kg-1

15.2 3.4 19.9 0.22 Purschke et al. (2017) Chromium 300 19.525 N/A 0.07 Qiao et al. (2017)

Nickle 15.2 4.2 19.7 0.28 Purschke et al. (2017)

3 2.8 3.8 0.93 Purschke et al. (2017)

Arsenic van der Fels-Klerx 1-4 0.58-1.96 N/A 0.58-0.49 (2016)

1.5 13.7 1.8 9.13 Purschke et al. (2017)

5 19 N/A 4.20 Qiao et al. (2017)

2.7 7.9 2.9 2.93 Diener et al. (2015b)

Cadmium 13.3 36.2 16 2.72 Diener et al. (2015b)

61.5 142.9 89.8 2.32 Diener et al. (2015b)

van der Fels-Klerx 0.25-1.0 2.4-6.9 N/A 9.5-6.9 (2016)

Mercury 0.2 0.1 0.3 0.50 Purschke et al. (2017)

15.2 35.6 19.8 2.34 Purschke et al. (2017)

5.9 1.5 7.8 0.25 Diener et al. (2015b) Lead 34.3 25.3 53.2 0.74 Diener et al. (2015b)

142.9 40.1 267.9 0.28 Diener et al. (2015b) 17

van der Fels-Klerx 2.5-10.0 3-14 N/A 1.2-1.4 (2016)

177.4 N/A N/A N/A Diener et al. (2015b)

Zinc 616 596 1196 0.97 Diener et al. (2015b)

2044 866 3313 0.42 Diener et al. (2015b)

All respective chemical elements displayed consistent bioaccumulation trends across listed studies except for Pb. Diener et al. (2015b) tested for low, medium, high concentration. The data for the lowest zinc concentration was contaminated and could not be provided. All values for feed concentration were spiked.

It is understood that accumulation of heavy metals does occur in the body of the prepupae (table 3). However, there has not been many that consider the accumulation into the pupal stage for reproductive purposes. In an interesting study where Qiao et al. (2017) tested the presence of cadmium and chromium at different life stages and in different parts of the body, it was found that Cadmium concentration in the larvae and pre-pupae were significantly more concentrated compared to the diet but Chromium produced results in the opposite direction where it was lower. In the larval and pre-pupal stage, most of the two metals were found in the body, not in the integument. However, in the pupa inverse results were seen with higher amounts in the puparium. The findings show that the different concentration of both these heavy metals at different life stages in different body parts may be a strategy of the BSF to tolerate and discard heavy metals stress through molting.

In regard to the effects of heavy metals on the larvae progression, cadmium and chromium had no effect on eclosion rate or larval survival. Cadmium does not significantly affect the accumulated number of pupae while chromium does (Qiao et al. 2017). A possible 18 reason for the lower pupation rate is that the high chromium concentration may have disrupted the metabolic activity of protein, carbohydrate, lipid causing a deficit in energy for life stage progression (Tylko et al. 2005). Another possible explanation for the high accumulation of cadmium is that Cd2+ is similar to Ca2+ which allows absorption into cells through calcium channels (Diener et al., 2015b). Despite the 18-fold increase in Cadmium concentration in Diener et al. (2015b), which should have cause toxicity, the development time from eclosed egg to prepupae only varied by a mean of 1.3 days. The low effect on the development time could be explained by a protein belonging to the HSP70-family found by Braeckman et al. (1999). This protein is induced by high cadmium levels in the cellular environment of Aedes albopictus

(Diptera: Culicidae) and acts as a defense mechanism against the effects of Cadmium

(Braeckman et al., 1999). On another note, heavy metals also encourage reduced lipid accumulation in BSFL as explained by Ortel (1995). A paper by Purschke et al. (2017) agrees with the findings of Ortel (1995) by lower post-larval mass.

CHAPTER 7: CONSIDERATION AND PREVENTION FOR ENTOMOPATHOGENS

In a dense population, the transmission of pathogens from one animal to the other weighs heavily on the product yield and the financial side of production. As of 2020 there has not been any documented pathogenic outbreaks (Joosten et al., 2020) but it is a good practice to understand the methods of preventing them especially when other insects being mass produced has experienced pathogenic outbreaks such as the yellow meal worm, Tenebrio molitor (Eilenberg et al., 2018). The BSF facility is basically a fort that attempts to keep the pathogens out which makes it important to understand how the pathogen may get in. 19

In designing a production facility, there are two choices. The first would be a closed facility and the other would be a semi-open facility. In a closed facility there is the advantage of a physical barrier which prevents other insects from invading. It also protects the fly colony from escapees which return infected by outside sources or by a foreign infected fly. In addition to this, the climate can also be controlled but only in a well-designed building. A close facility enables the climate to be controlled inside for efficient production but this may also create pockets of exceptionally desirable regions for pathogens to multiple when poor airflow is provided (Joosten et al., 2020). Unfortunately, to my understanding there is no literature which addresses this but since no outbreak has been reported it can be assumed that current settings are sufficient and the facility design is determined mainly by regional weather. In tropical locations, a semi-open facility would be a better choice (Eilenberg et al., 2018) although it is stressed that the physical barrier separating the colony from the outside world should be secure (Joosten et al., 2020).

Aside from escapees introducing the pathogens, another risk and source would be larvae themselves. Stressed animals may reduce their immunity towards opportunistic pathogens when provided with insufficient food (Joosten et al., 2020). When the larvae do they succumb to death, their carcasses serve as an energy source spreading the disease and increasing the infection pressure (Joosten et al., 2020). Stressed larvae may very well turn cannibalistic and spread the infection as shown by Maciel-Vergara et al., (2018) with Zophobas morio Fabricius with pathogen Pseudomonas aeruginosa. One prevention method described by Eilenberg et al. (2018) is to practice good hygiene within the facility.

CHAPTER 8: THE PROPOSED BSFL REARING SYSTEM

The proposed system will rely on several conditions: a. A steady and reliable supply of waste materials with consistent nutritional composition. b. A rough approximation of number of eggs from a new generation. c. Proper sanitation procedures throughout the production process. d. Larvae fed with high quality feed for maintaining a high adult fly count. e. All spray bottles must be properly labeled and kept in the appropriate location. e. Adequate air ventilation throughout the breeding facility. f. No food allowed into the production area.

Through personal communication with Sio Chun Jia, co-founder of Life Origin, he stated that reading the material to understand breeding and growing the soldier fly is not as difficult but performing the task and gaining technical wisdom is much tougher because of fluctuating conditions such as the feed’s moisture especially when not done in laboratory settings. Because of this, Sio suggested using measuring instrumentation to monitor the temperature and water content of the feed and air. An Arduino Uno can be used with a MAX31865 breakout board and a PT100 RTD to monitor temperature and a STEMMA Soil Sensor - I2C Capacitive Moisture

Sensor can be purchased on www.adafruit.com. The code is also provided and assembly is also provided on the website. He also noted that his initial fly colony was ravaged by a virus obtained from another breeder. As such, the new flies should be isolated in their own system to avoid production meltdown. Getting the feeding rate right is crucial because if there are leftover when the larvae are done feeding the residue will turn pungent because of the warm temperature and water activity. Providing an excessive amount of feed to the larvae will cause odorous 21 anaerobic smell but gain higher mass. However, if provided with lesser feed waste reduction increases and the odorous smell reduces but the pre-pupal mass will be the trade-off. Therefore, it is important to find the right feeding rate where the larvae feed just enough to gain mass and leave minimal material to rot. One solution is to provide aeration through a soaker drip line but this increases the operating cost. Although the waste’s odorous smell turns less pungent or earthy, they larvae may not feed fast enough to keep up with the microbial anaerobic digestion with 60-70% MC and at a warm 25-27℃ temperature but can mitigated with forced aeration through a soaker hose buried in the feed. An artificial rearing system involves maintaining an adult fly colony in an enclosure to produce an uninterrupted flow of eggs to be used as bioconversion agents for waste (Nyakeri et al., 2017)

CHAPTER 8.1: ABIOTIC FACTORS

8.1.1: TEMPERATURE

In addition to one other condition the rearing temperature is a key parameter in the larvae’s development and mortality. In particular, the newly hatched eggs are especially sensitive to environmental changes (Caruso et al. 2013). At 27℃ the eggs hatch in 4 days (Booth &

Sheppard 1984). If artificial heating should fail, the lowest threshold temperature for egg hatching was recorded at 10˚C (Chia et al., 2018) but Holmes et al., (2016) recorded it at 12℃.

The lowest thermal limit for eggs to hatch was proven when eggs incubated at 10℃ completely collapsed but hatched at 15℃ (Chia et al. 2018).

The larvae typically bury themselves 2-3 cm below the surface of the substrate but they emerge when conditions are unfavorable indicating discomfort (Nyakeri et al. 2017) and look for 22 cooler conditions to feed (Joly & Nikiema, 2019). Chia et al. (2018) determined that the development time from larva to fly was at 15℃ was 183 days. Under cool conditions, the larvae feed less when conditions are not conducive and triggers metabolic reduction in order to survive

(Dortmans et al., 2017). When compared to 30℃ the development time was drastically reduced to 30 days (Chia et al., 2018). The ideal temperature to culture the larvae has been recommended to be between 24 - 33℃ (Alvarez 2012) but also has been reported to be around 35℃ by Newby

(1997). However, maintenance at 35˚C is risky because at 36˚C none of the larvae survived in

Tomberlin et al. (2009). Newby (1997) also reported that at 47℃ the larval mortality rose dramatically also but the difference could be attributed to genetic variation (Zhou et al., 2013).

Possibly also due to a genetic variation, at 16℃ the eggs managed to hatch but the larvae died after 3 days (Holmes et al 2016) which differs from Chia et al. (2018) 15˚C results. As the maggot consumed food through the medium, they also generate heat and for this reason Alvarez

(2012) suggested that lower temperatures would be more suitable for the immatures. Chia et al.

(2018) recorded its highest egg eclosion count at 30℃ and 35℃ with 80% and 75% respectively and also significantly a longer time for the eggs to hatch when lower temperatures were used.

Egg, larvae, prepupae survivability over temperature, % 100 90 80 70 60 50 40

Survivability, % 30 20 10 0 10 15 20 25 30 35 40 45 Temperature, ˚C

Egg survivability, % larval survival pre-pupae survival

Figure 2: The survivability of the BSF from eggs to its prepupal stage sourced from Chia et al. (2018).

Besides modulating the growth rate of the larvae, the rearing temperature also plays a role in the lifespan and weight of the resulting fly. Tomberlin et al. (2009) reported that flies needed less time to develop at higher temperatures agreeing with previous studies because this factor is among one of the most influential for development, survival and to progress the colony

(Chia et al. 2018). However, a trade-off was also observed when the temperature increased from

27℃ to 30℃. The larvae grew at a faster rate but it also weighed less and additionally decreased the fly’s lifespan. Gligorescu et al. (2018) also produced similar weight results from 20-27℃. On one hand Tomberlin et al. (2009) reported a decreased prepupal weigh when temperature increased and on the other hand in Harnden & Tomberlin (2016), it was observed that the larval final weight was on average 30% lower at 24.9℃ when compared to 27.6℃ and 32.2℃.

This would suggest that below ~27℃ the larval weight trend is reversed (Joly &

Nikiema, 2019). This suggestion favors the range deduced by Alvarez (2012) of 24-33℃ for 24 larval rearing temperature. The longevity of the adult soldier fly was found to be significantly affected by temperature and performs best at an intermediate temperature following a quadratic model between 20-25℃ although this range compromises the maximum mean number of eggs produced by a female being at 30℃ (Chia et al. 2018). The same author reported that there was no significant difference in pre-oviposition time between 25-30℃.

The adult fly can be supplemented with additional energy (sugar) therefore allowing a higher temperature of 30℃ rather than 20-25℃ but no oocytes were found in female flies after 3 days of oviposition (Tomberlin et al., 2002a) making sugar supplementation unnecessary and water more sensible. The temperature showed a significant interaction with larval weight by producing heavier weight in the instar larvae, pre-pupae, pupae and adult stages at higher temperatures (Chia et al. 2018). Fly eclosion takes between 10-14 days in a 27-30℃ environments (Sheppard et al. 2002). To conclude, the optimal larval rearing temperature is at

27˚C with respect to survivability and development (Tomberlin et al., 2009; Chia et al., 2018).

8.1.2: RELATIVE HUMIDITY

Apart from the temperature contributing to the survivability and development of the fly, relative humidity also has a part which works in tandem with temperature. Referring to a psychometric chart, the maximum amount of water which the air is able to contain increases with temperature which moistens the outer layer of the eggs. The eggs and pupal of the soldier fly are dependent upon the amount of water vapor in the air to hatch. At temperatures of >26℃ and >60 elative humidity (RH) approximately 80% of the eggs can be expected to hatch (Tomberlin &

Sheppard 2002a). Particularly during the eggs stage low RH is associated with high egg 25 mortality (Schausberger, 1998) which could impede year-round production and waste management in cold locations. In a low relative humidity surrounding, the water loss experienced by the egg and pupal membrane can result in deleterious effects by desiccation.

Unlike their mature stages which are able to regulate their water loss as a trade-off from respiration, the eggs are unable to control water during respiration (Wigglesworth 1984; Zrubek and Woods 2006) nor are they able to defend themselves against predatory insects but these predators can be built out of the system on the premise that a safety system is built in during the facility design process.

In determining the effect of humidity on egg and pupal eclosion Holmes et al. (2012) reported conclusive results of higher eclosion success at 70% RH (86.22%) for eggs and 70%

RH (93%) for adults when compared to lower values. At 70% RH, the post-feeding and pupal survivorship was above 95%. The higher relative humidity also produced a longer adult longevity of 7.93 days which is typical when not provided with water (Nakamura et al 2015).

Holmes at al. (2012) also reported decreasing development time as the humidity rose. Egg hatching occurred 3 days after laying at 93% RH at 24℃ (Diener et al. 2011a). Egg hatching took 4-5 days at 60% RH at 28˚C (Nyakeri et al. 2019). The desiccation of the eggs was observed on the outer layer of the egg clusters while eggs shielded inside the cluster successfully emerged as larvae (Holmes et al, 2012). When oviposition occurs within a crevice the eggs cluster takes on the three-dimensional form of the crevice. The preference of the fly to lay into crevices may be a strategy for egg survival but more studies are needed to test this hypothesis with different humidity levels (Holmes et al., 2012). From table 4, the post-feeding stages appear to be more resilient towards low humidity compared to the eggs. 26

Table 4: The effect of relative humidity on egg development and successful hatching of BSFL.

Relative Successful adult Development time, days Survivability/eclosion, % Humidity emergence, %

, % Egg Prepupae Pupae Egg Prepupae Pupae

25 5.2 10.36 8.92 8 38 35 16 40 3.8 9.72 8.97 19.86 74 77 59 50 3.7 - - 38 - - - 60 3 - - 72.74 - - - 70 3.3 9.48 8.41 86.22 97 98 93 Data sourced from Holmes et al. (2012) using growth chamber held at 27℃

8.2: THE BREEDING UNIT The black soldier fly has been recorded to mate in large enclosure sizes ranging from

0.39x0.28x0.28m to 3x3x6m (Julita et al. 2018; Zhang et al. 2010; Sheppard et al. 2002;

Heussler et al., 2018). Nakamura et al. (2015) also showed that even in a small cage of

0.27x0.27x0.27m the flies were able to produce eggs with a high fly density. Sørensen &

Loeschcke (2001) also supported the high-density claim but with the reason to prevent inbreeding depression however in Sio’s system, there were no signs of depression after 10 generations and will considered.

The breeding cage will be constructed using a wooden frame with a plastic mesh screen measuring at a vertical height of 1.5m to accommodate the fly’s flight mating ritual and a base of

1m2 to allow a higher number of required materials into the unit. A small zipper door will be 27 used to replace material during the production process. Eventually the adult flies will expire therefore, a sleeve will be built into the cage providing an entry route for the following fly generation. An illustration of the breeding cage is provided in figure 3. As we may experience predation by insectoids, the unit will be rested on a plastic table (or stainless steel) to discourage oviposition on the base and each leg will be placed into a container with soap solution to prevent invasions and unwanted larval breeding from other insects. Succeeding the flight, artificial plants will be provided for the fly to mate on. The plants will also serve by providing a shady oviposition site (layered wooden stacks) by placing the plants in close proximity oviposition site.

A container with water and a cloth on the water’s surface will also be placed in the unit. The position of the unit relative to the room will be placed facing the morning sunlight if natural lighting is dependable but if it is not, 100W LED will be hung above. No other material other than the wooden stacks, water container, artificial plants and lure container should be present in the unit. This may be time-counterproductive from laying eggs other than in the stacks. If eggs are laid on other surfaces carefully use a toothless knife to remove them and place onto the supporting stand.

Figure 3: Illustration of the breeding cage construct without the plastic screen mesh.

The breeding unit for maintaining a black soldier waste processing unit is key as producing enough young and healthy larvae is needed and is the most delicate step as highlighted by Diener et al (2015) and mentioned by Lohri et al (2017). Most information regarding the black soldier fly larvae is associated with their capacity to be used as a waste management tool because they are efficient organic waste converters into valuable fertilizers (Park et al., 2016).

Other studies have been focused on using the larvae as a protein replacement source particularly in the aquaculture industry. However, to bring about these solutions a more important piece of the puzzle is the constant exposure to abiotic factors which influence the production of the eggs.

In an artificial breeding system, current studies indicate the egg yield is affected by the type light source used, the lure used to attract the fly and the type of material used to a laying site.

8.2.1: ARTIFICIAL LIGHTING

In a natural setting sunlight is used as the sole source of light which functions as a stimulant for mating (Tomberlin et al., 2002) but in locations where winter occurs the exposure to sunlight is reduced which hampers production. Few mating was observed during cloudy days by Tomberlin et al. (2002) and agrees with a recent study (Heussler et al., 2019). This also tells us in a dark environment egg production can be controlled. With sunlight as the source, mating count positively regressed with light intensity but oviposition was not influenced (Tomberlin et al., 2002, Sripontan et al., 2017). The regression was also observed by Park et al. (2016) using sunlight but the most egg clutch count was observed with sunny-shady side and not direct sunlight. Artificial light is a constant source and tend to produce more eggs when light is stable 29

(Sripontan et al., 2017). With sunlight, mating was observed in the morning (Heussler et al.,

2019) and oviposition was observed only in the afternoon (Sripontan et al., 2017). As this is the observation, in an artificial rearing system the intensity of the light source can be regulated and gradually reduced when mating has taken effect. Another option is to place artificial plants above the oviposition site to create shade. To resolve the lack of sunlight, artificial lighting can be used to reduce the dependency of natural light but the effects on larval production and development has to be considered as they may not be similar with natural lighting.

Zhang et al. (2010) performed a study where they concluded elicitation to reproduce was comparatively effective when a 500W quartz-iodine lamp (QIL) with a 135 μmol/m2s1 light intensity was used. The QIL resulted in a 61% mating rate of sunlight most probably because its light spectrum is similar to sunlight. However, mating was non-existent when rare earth lamps were used with wavelengths 350-450nm which lead Tomberlin & Sheppard (2002) to speculate that the fly only responds to specific wavelength cues. No mating or oviposition took place when a 40W Sylvania Gro Lux or 430W Pro Light System was used (Tomberlin et al. 2002). Using sunlight, a high 85% of the breeding occurred in the morning where light intensity was 110

μmol/m2s1 but decreased when light intensity increased (Zhang et al 2010). Tomberlin and

Sheppard (2002) recorded the highest mating of over 200 μmol/m2s1 under sunlight but no mating took place with light intensity below 63 μmol/m2s1. The difference in results could be, other than light intensity, mating has also been shown to be influenced by time of day. Oonincx et al. (2016) concluded that the BSFL’s visual photoreceptors were sensitive between 332-

535nm and influenced mating behavior which did produce mating. However, Schneider (2019) reported a narrower 440-540nm finding using 100W LED at 431W/m2 and produced 93% 30 mating under 7 hours. The range by Schneider (2019) is supported by Briscoe & Chittika (2001) where 12 of 16 examined Diptera species had receptors for the 480-530nm range and color vision allows the insect to react to the intensity of the stimulus. Schneider (2019) results made sense since insects typically cannot see beyond the visible light spectrum (Briscoe & Chittika,

2001).

Further literature showed successful oviposition between halogen, LED and fluorescent lamps as light sources with no significant difference but did not record the mating frequencies

(Heussler et al., 2019). The studies provide proof warranting the claim that sunlight dependency for BSFL production is reducible using artificial lighting emitting specific wavelength ranges but sunlight produced greater results for mating the soldier fly (Tomberlin and Sheppard 2002;

Zhang et al. 2010).

Nakamura et al. (2015) reported an oviposition period of 7.6-9.4 days with a peak on the

6th and 7th day depending on the light source. Heussler et al. (2018) reported a longer oviposition period of 8-13 days. It should be note that using the halogen bulbs increase the breeding cage’s temperature by 2℃ in Heussler et al. (2018) which effectively decreased the adult’s longevity by 4-7 days. In conclusion halogen light bulbs, LED and fluorescent lamps can be used to stimulate and produce the black soldier fly eggs but Heussler et al. (2018) recommended using LED for their outstanding efficiency. Light intensity and wavelength effects mating. Light intensity does not affect the oviposition but light stability does by favoring a shady site (Sripontan et al., 2019).

8.2.2: OVIPOSITION ATTRACTANT

As mentioned, one of the three criteria for gravid females to oviposit is the nearby decaying organic material. It is well known that putrescent odor induces females to lay eggs but there exists a scarcity of information about the baiting attractant’s effect on the amount of eggs laid (Nyakeri et al., 2017). Although it is generally agreed that most decomposing materials are able to attract the female flies, for the purpose of mass rearing, it is necessary to determine the most egg-producing lure. Results from Nyakeri et al. (2017) using five varying attractants, cow manure produced the highest amount of eggs but decreased as the experiment progressed compared to the rest (fruit mix, commercial sweet-smelling liquid, rotten fish and frass tea). This could be attributed to microbes feeding on the manure which reduced its pungent odor. Manure drying is also a reason for decreased egg yield (Nyakeri et al. 2017). Fish also displayed the same decreasing pattern. It should be noted that in Nyakeri 2017 study, the light source was not stated but as the experiment was conducted in Kenya it can be assumed that they used sunlight.

In Bonso’s (2013) work among the other baits used (fish, chicken feed) human feces and rat meat scored a high rating on the selection matrix.

It is important to note that in Nyakeri et al. (2017) and Bonso (2013) organic decomposing material specifically fresh manure was effective in attracting the female flies but decreased as the freshness decreased. However, the adult fly may search for a specific food source for its larvae. In Sripontan et al. (2017) reported that fruit mix (papaya, banana, pineapple) was the only attractant in which the flies lay their eggs when dairy, pig and chicken manure was present in a 2-month study. They postulated that BSF selected it attractant based on prior exposure. The fly chooses the feed that it fed on during its larval stages. To conclude, it is 32 best to suit the oviposition attractant which is local to the region and perform tests to determine if the attractant is suitable and not depend solely on literature.

The oviposition response of the BSF with different baiting attractants over 8 days 9 8 7 6 5 4 3 Egg mass/day 2 1 0 0 1 2 3 4 5 6 7 8 9 Days of experiment

Dairy manure Commercial Fruit mixture Frass tea Rotting fish sweet scent

Figure 4: The oviposition response using varying oviposition material from Nyakeri et al. (2017)

8.3: THE BREEDING SITE

In providing a suitable site for the females to lay their eggs, cardboard and wood have shown the most promise in egg laying performance. However, using cardboard may distort calculations for determining the number of eggs deposited because it absorbs moisture; absorbing moisture by cardboard will create a higher variability when determining the egg count.

To minimize the error, wooden planks measuring 15x5cm will be used in constructing the oviposition structure for more consistency. This consistency of the number will be useful in maintaining the adult fly population and quantity of the resulting product. Each construct will 33 use four wooden planks stacked atop each other separated by two ice-cream sticks on each opposing side of each plank (see figure 5). They are to be secured together using rubber bands.

The wood’s dimension is adjustable with the core understanding that excessive volume between the planks is a waste.

As the flies are stimulated by mating and laying their eggs close to foul smelling odors, the wooden stacks will be rested on the attractant container’s rim. To ensure that enough containers are present for their own purpose, the lure and nursery/feeding container will be color coded. The lure container will contain a mixture of waste material and dead flies at an 90% MC which will serve a single purpose because using aerobic organic waste material even a few days old has shown to produce slower growth rates when consumed by larvae (Beard & Sands 1973).

The lure will also not be reused. It will be stored away and mixed with the bio-processed waste.

The wooden stacks will be collected and replaced every 3 days until the current batch of flies expire starting from the 4th day the adult flies are introduced to the breeding cage. When the adults have expired collect 100 dead flies from each enclosure to be used in the oviposition attractant (Dortmans et al., 2017). As organization is the cornerstone of a successful process keeping track of each larval batch is important. Every time a wooden stack batch is reintroduced to a new generation of adults, they will be provided an identification number written on a writable tape attached to the nursery/grow out container. The batch number, introduction date and other particulars will be recorded onto a clipboard and will follow the specific stacks until it is reused for breeding.

34 Batch number: Introduction date from breeding unit: Harvest date: Mass of feed added: Approximate feed consumed: Water addition: Date # pumps Initial feed Remarks added moisture

Figure 5 : Wooden stack arrangement used in the Figure 6: Illustration of information recorded on clipboard. breeding cage without rubber bands.

CHAPTER 8.3.1: OVIPOSITION SITE PREFERENCE

In choosing a suitable oviposition site, the female fly uses the tip of its abdomen to scan the material for suitability (Joly & Nikiema, 2019). The tip contains sensors which inform the female on the presence of larvae, other BSF eggs, pathogens on the material and available nutrients (Tomberlin 2017). When the female chooses the suitable oviposition site, they leave chemical markers for the other female flagging it as an egg laying site (Alvarez 2012). In a recent study using layered structures, evidence was presented where varying material used for oviposition yields varying eggs onto said material. Boaru et al. (2019) used 421-452 adults for each lure and results show that when corrugated cardboard and wood were used the number of eggs masses accumulated onto them (72 and 61) were much higher when compared to the bio- balls (8) and glass (34) and it seems that the medium used is the most important factor for oviposition (Park et al., 2016).

As previously noted, synthetic material such as the bio-balls produced the least desirable results. This is because they might be at an inherent disadvantage because the fly lays the eggs in crevice and the bio-balls had a much lower crevice surface area compared to wood and cardboard. As additional proof that the fly preference over natural material is of higher influence compared to the sites structure. Heussler et al. (2018) tested plastic material similar to the shape of corrugated cardboard was used. The plastic material was proven to be ineffective when only up 22% laid their egg onto it as compared to 93% with natural cardboard. The transparency of the plastic cardboard was not provided and may have had a role because it is possible that the adult was also looking for a dark area to lay her eggs. Dark regions may have signified protected and safe.

Although the glass and corrugated cardboard produced approximately equal amounts of egg masses, they were distributed heterogeneously which is unfavorable for mass production

(Pastor et al., 2015). The wooden material showed the best orderly egg mass distribution that is ideal from a management point of view. The eggs would have to be manually removed if they were laid on other surfaces other than the prepared site. This would represent an unwise choice for resource allocation. In conclusion, wood would be the most suitable material to be used in a layered structure and plastic material should be avoided.

Figure 7: Amount of egg masses deposited and number of masses deposited onto different oviposition materials from Boaru et al. (2019).

CHAPTER 8.4: RESETTING THE BREEDING UNIT

The breeding cage will be reset on the same day the current adults expire to maintain work environment hygiene and avoid lack of breeding cages when it is time to repopulate them.

Although the BSF produces AMP in response to high bacterial load (Vogel et al. 2018) the fly’s surrounding is still vulnerable to hazardous microbial growth which could pose a threat to humans. To minimize or eliminate transferring any pathogens, when handling waste material all personnel are required to wear disposable rubber gloves, face masks and eye protection. To sanitize the lure’s container, remove the container from the breeding cage and dispose of the larval frass into a designated container. When the container appears to be free of waste scrub the container thoroughly with water and dish detergent with a squeegee. When satisfied, continue by 37 applying a layer of at least 70% alcohol solution delivered by a spray bottle making sure that all post-waste contact surfaces are coated. Store the container at the allocated location preferrable where the lure is added to the attractant container. The grow out container/nursery container follows the same sanitation procedure and are stored at the same location in a separate stack.

CHAPTER 8.5: NURSERY FEEDING CONTAINER

Once the eggs are collected it is important that they not be removed from the stacks and provided with minimal light exposure. To avoid any contact between the feed and wood a metal stand (see figure 10) is placed into the container first and the wood planks rests on the stand. We also do not intend the eggs to be in direct contact with the feeding material because they will then be prone to fungal infections during the hatching period. Once figure 11 has been completed, the composite is placed into a tiered rack. The container will follow the placement sequence of top-right to bottom-left with their corresponding batch number. It is important that the stacks do not cross outside the container’s border as they will perish. The container with eggs shall not be placed onto a level where the top is exposed to direct light due to photosensitivity.

The eggs will be harvested every 3 days and provided a 70-75% MC chick starter feed based on the minimal time eggs take to hatch.

Since the feeding material’s moisture content has been shown to adversely affect the larvae’s growth and diminishes throughout the feeding period, fresh water will be added through a spray bottle to achieve a consistency similar to the feeding material when first treated. Water supplementation should not be used if the larvae are expected to reach the pre-pupal stage within 38

3-5 days. A visual cue which indicates that the waste material has been digested and excreted is

the color change to a black slurry (Oonincx et al. 2015) and will also appear to be dryer. The

larvae are maintained in the nursery container and used as the grow out container together with

the frass. It is possible to remove the frass through sieving but runs the risk of losing larvae.

Winnowing is possible but could cause a thermal shock but the effects have not been

documented.

Figure 9: Example of nursery/feeding container. Figure 8: Tiered rack for nursery/feeding.

Figure 11: Placement of the wooden stacks onto Figure 10: Illustration of figure 10 in the nursery/grow supporting metal stand. out container.

CHAPTER 8.6: GROW OUT FEEDING CONTAINER

After 6 days of feeding in the nursery container ~1% of the larvae will be removed to repopulate the breeding cage. The remaining 99% will be left in the feeding container where waste material is added. The lump feeding regime will be used at a rate of 100mg larvae-1 day-1.

At the system’s initial stages data will need to be generated for use in equation 3 to approximate the egg counts. This is done to estimate the amount of waste needed before the eggs are produced in the next cycle. The larvae will feed for approximately 19-24 days depending on the waste material(s) used.

!"#$% '()*ℎ# ", 6 − /$0 − "%/ %$12$( ,1"3 4$#5ℎ # ≈ ", %$12$( !"#$% '()*ℎ# ", (**7 ,1"3 4$#5ℎ #

Equation 3: Estimation of the number of eggs produced on wood

Although interesting and convenient, the larvae’s natural migration will not be used in this system because we would then have to wait for the larvae to crawl in waves representing a bottleneck therefore slowing down the harvesting process and sequentially production. The likeliness that all pre-pupae self-harvesting is also unlikely (Banks 2014). Using a batch production method, once majority of the larvae are seen to have reach their pre-pupal stage they will be left for 3 days for intestinal evacuation and other larvae to reach the pre-pupal stage.

They will be transferred to a 3mmx3mm chicken wire sieve placed onto an off-the-shelf container. To separate the pre-pupae from the digested feed, vibrate the mesh and allow the waste material to fall into a collecting container. The larvae’s sensitivity to light can also be used 40 by allowing them to burrow, pass and drop into the container but the frass may also follow. The mesh size was selected to be smaller than the pre-pupae width based on measurements reported by Julita et al. (2018). The processing step forward depends on the need of the customer but for sanitation purposes, the larvae will be submerged into boiling water to deactivated them

(Dortmans et al., 2017). This method also forces the larvae to excrete its gut content if it was not excreted. If the larvae are not used directly after harvest it is recommended that they be sun dried or oven dried to 10% MC after deactivation (Dortmans et al., 2017). Oven drying was used for data collection in Dortmans et al., (2017) and is assumed to be sufficient to achieve 10% MC;

105˚F for 24 hours.

CHAPTER 8.6.1: WASTE MATERIAL ON DEVELOPMENT AND SURVIVABILITY

The time to develop from initial instar to the prepupae will determine the degree of power consumption needed for lighting and heating in colder conditions with delayed development costing higher operational cost and rapid costing lower. It may take fourteen days under favorable conditions or a stretched 120 days if food is deficient (Furman et al., 1959; Myers et al., 2008). Although the BSFL are portrayed as ferocious feeders of organic material (Mutafela

2015) they do not feed continuously (Alvarez 2012) but do intake a wide range of substrates with adverse chemical composition which significantly affect the fly’s development (Julita et al.,

2018). The consumed material during the larval stages will also determine the population’s survival (Roper et al., 1996) and should be considered when mass rearing is intended. When producing a biological product for sale from a range of waste, the development time of the product will vary with heterogeneous compositions and should be examined to predict the development time, mass and mortality rate. 41

Similar to other animals, insects use carbohydrates, protein and fats as energy reserves for maintenance, growth and reproductive needs (Behmer, 2008). When the larvae were fed diets rich in protein but low in carbohydrates it was observed that the larvae experienced decreased growth size limit (Sripontan et al., 2020). The low carbohydrate content was likely the reason because the larvae may have been forced to metabolize protein as an alternative energy source in the presence of low carbohydrate (Sripontan et al., 2020). Protein requires a longer nutrient assimilation time and the minimum pupation weight (Gobbi et al., 2013) has been recorded at 2 –

4mg dry weight (Manurung et al. 2016). The range of weight described by Manuring et al.

(2016) is without a doubt inferior to previous studies with feedstock containing a higher carbohydrate percent. This is therefore the lowest harvesting weight. Vice-versa diets rich in carbohydrates and poor in protein are suspected to be the reason for slower growth rate for the larvae (Sripontan et al., 2020) which aligns with Gobbi et al. (2013).

A positive response to diets increased the average growth rate and shortens the larval stage’s duration (Sripontan et al. 2020). This is supported by Nguyen et al. (2013) where slower growth rates and lower final larval mass were observed when pig manure containing 2.38% protein and 5% carbohydrates were used compared to kitchen waste containing 5.86% protein and 16.3% carbohydrate. Slow growth was also observed by using rice straws as feeding material mainly caused by straw’s low energy content (Manurung et al. 2016). Although the black soldier fly larvae’s gut contains microbes producing cellulase (Kim et al, 2011) energy is still needed to convert lignocellulose to simple sugars which rice straw is already lacking. The use of more energy to convert lignocellulose saps the energy away from growth. Nguyen et al. (2013) results from using pig manure agrees with the suggestion (Sripontan et al., 2020) that a diet with higher 42 and balanced protein and carbohydrate improved mass and larvae development time. Cammack

& Tomberlin (2017) achieved their fastest development with least amount of feed at protein: carbohydrate of 21:21 and 35:7 at about approximately 33 days but 35:7 weighed significantly more by roughly 14mg (from graph estimation) both at 70% MC.

In general, the performance of manure on the development time and prepupal mass was poor compared to a wide variety of substrates. It would then be possible to assume that pretreatment of waste material would elicit a better response to supplement deficiencies by mixing materials especially protein and carbohydrates (Nijhout, 2013). In fact, improved development time and prepupal mass was shown by Rehman et al. (2017) using soybean curd with dairy manure. Vegetable waste also showed improvement in Julita et al. (2018) study by supplementing horse and sheep manure with vegetable waste. Fats as energy source is an alternative and have been shown to decrease mean development time to prepupae when using high protein high fat feed compared to high protein low fat by 12 days (Oonincx et al., 2013).

Table 5: The mean period required to reach the pre-pupal stage with varying protein and carbohydrate content.

Organic Substrate protein, Carbohydrate, Development Source material % % time, d

Horse manure 10.13 51.97 42 Julita et al. (2018)

Sheep manure 13.34 40.17 42.8 Julita et al. (2018)

Pig manure 2.38 5 25 Nguyen et al. (2013)

Dairy manure N/A N/A 25.26-31.49 Myers et al. (2008)

Kitchen waste 5.86 16.3 20.17 Nguyen et al. (2013) 43

Vegetable + 0.9 3.05 21.67 Nguyen et al. (2013) fruit waste

50% sheep

manure+ 16.43 41.61 30.8 Julita et al. (2018) 50% vegetable

waste

50% horse

manure + 13.89 37.4 28.6 Julita et al. (2018) 50% vegetable

waste

5:00 N/A N/A 24.3 Rehman et al. (2017)

4:01 N/A N/A 22.5 Rehman et al. (2017)

3:02 N/A N/A 20.6 Rehman et al. (2017)

2:03 N/A N/A 21.1 Rehman et al. (2017)

1:04 N/A N/A 21.1 Rehman et al. (2017)

0:05 N/A N/A 19.4 Rehman et al. (2017)

Pig liver 19.41 1.2 19.17 Nguyen et al. (2013)

Fish rendering 9.05 N/A 19.83 Nguyen et al. (2013)

Saturated 14-22 N/A 18 Zhang et al. (2010) grain diet

Rehman et al (2017) data represents dairy manure: soybean curd. N/A. Myers et al (2008) used 27-70g daily feeding rate. Nguyen et al. (2013) data represents the minimum amount of time needed to reach the wandering stage.

The prepupae development would take longer if manure were dried due to heat sensitive vitamins and destruction of microorganisms (Oonincx et al., 2015) indicating microorganisms could aid in speeding up the larval period but bacteria inoculation may be costly depending on the companion. The mean median development time from larvae to prepupae using pig’s manure by Nguyen et al. (2013) was 15.93 days slower than Julita et al. (2018) horse manure showing a low protein and carbohydrate quality as well. The development time with horse manure to prepupae was not significantly different compared to sheep manure. Julita et al. (2018) produced results of lower development time and mortalities for all stages using manure-vegetable mixtures compared to manure only. There is a wide range of factors which can explain the differential growth between feedstocks but the most likely influence is the nutritional content (Nguyen et al.,

2013).

Ideally in a production process with low development time, homogeneous and linear growth is desired but has shown to follow an S-shape curve when using waste material outlined in Sripontan et al. (2020). We cannot assume that any process will produce a 100% success nor can we say with certainty that all waste sources are suitable for the larvae, but we can choose the feeding substrate for the lowest mortality rate. Larvae failing to reach pupation may be the result of lacking certain amino acids involved in cuticle sclerotization such as phenylalanine (Behmer,

2008). When comparing the mortality for prepupae and adult using pig’s liver and manure, liver was higher than pig’s manure by more than 17% for prepupae and significantly higher close to

55% for the adults (Nguyen et al., 2013) although pig’s liver had the lowest development time.

Fish rendering performed considerably poor in all development stages especially adults. For this reason, it is advised not to use fish rendering over pig manure and pig liver. The fish rendering 45 may have contained too much fat for the larvae to metabolize during its metamorphosis making it detrimental but can be used to increase survival rates in suitable amounts (Oonincx et al., 2015;

Ujvari et al., 2009). Individual larvae with heavier weights post-feeding showed higher survivability in reaching pupation and emerging as adults (Georgescu et al., 2020).

Table 6: The mean survivability of pre-pupae and adult fly when fed various substrate.

Survivability, % Feed Source Pre-pupae Pupae

Pig manure 74.33 70.33 Nguyen et al. (2013)

Pig liver 57.22 15.44 Nguyen et al. (2013)

Kitchen waste 46.67 38.78 Nguyen et al. (2013)

Vegetable + 76.67 66.56 Nguyen et al. (2013) fruit waste

Fish rendering 47.22 0.33 Nguyen et al. (2013)

5:0 91.2 N/A Rehman et al. (2017)

4:1 96.6 N/A Rehman et al. (2017)

3:2 98.4 N/A Rehman et al. (2017)

2:3 98.5 N/A Rehman et al. (2017)

1:4 98.8 N/A Rehman et al. (2017)

0:5 99.3 N/A Rehman et al. (2017)

Horse manure 73.6 73.4 Julita et al. (2018) 46

Horse manure + 85.4 84.8 Julita et al. (2018) vegetable waste

Sheep manure 67.6 66 Julita et al. (2018)

Sheep manure + 77.4 77.4 Julita et al. (2018) vegetable waste

Rehman et al (2017) data represents dairy manure: soybean curd. All authors listed did not provide pupation substrate for the pre-pupal and pupal stage.

The consumption before reaching the pre-pupae stage directly affects its survival and hence forward by metabolizing its fat reserves. This means that there is a correlation between the reproductive outcomes of the adult flies and its mass during the prepupae and consecutive stage.

Georgescu et al. (2020) and Boaru et al. (2019) also concluded that the larvae’s weight had a significant positive correlation with the fly’s weight. The female’s larval weight is relevant because it produced a significantly higher clutch weight when heavier and the clutch weight shows a strong positive relationship with number of eggs when a layered oviposition structure was used in Boaru et al. (2019) study. The female clutch weight-egg count relationship has also been theorized by Gobbi et al. (2013) and have provided evidence supporting the hypothesis.

However, according to Georgescu et al. (2020) there is no correlation between clutch weight and the weight of one egg suggesting that eggs size varies between systems. From Zhang et al.

(2010) study it was noted that each egg of the fly was very small weighing between 1-2µg but from other authors the individual egg mass was found to be between 17-25µg (Georgescu et al,

2020; Dortmans et al. 2017; Bertinetti et al, 2019). For this reason, it is important to know the nutritional composition of the feed in order to estimate the number of eggs produced. 47

Table 7: Prepupal biomass yield of different systems

Total feed Total feed Waste Biomass Feed source Source amount consumed reduction yield

Chicken Sheppard et al. 5240kg ww 2620kg ww ~50% ww 196kg ww manure (1994)

Swine Newton et al. 68kg dw 26kg dw ~39% dw ~2.7kg dw manure (2005)

Diener et al. MOW 151kg dw 103kg dw ~68% dw ~17.8kg dw (2011)

Human 480g ww 220g ww ~46% ww ~108g ww Banks (2014) feces

Lalander et al. Food waste 50.7-76g dw 28-42g ww 55.3% 46.7g ww (2019)

Fruit and Lalander et al. 120-180g dw 56-85g dw 46.7% 39.5g ww vegetable (2019)

Abattoir Lalander et al. 45.3-68g dw 21-31.5g dw 46.3% 49.6g ww waste (2019)

Abattoir

waste with Lalander et al. 45.3-68g dw 27.7-31.5g dw 61.1% 48.5g ww fruit and (2019)

vegetable 48

Poultry Lalander et al. 50.7-76g dw 30.4-45.6g dw 60% 30.4g ww manure (2019)

Human Lalander et al. 50.7-76g dw 24.2-36.2g dw 47.7% 45g ww feces (2019)

CHAPTER 8.6.2: FEED MOISTURE ON DEVELOPMENT AND SURVIVABILITY

The moisture content of the feed has also been shown to contribute to the larval development time to the prepupae only and does not affect other stages of development to the adult fly because feed is not consumed in the egg, prepupal, pupae and adult stage. Palma et al.

(2018) reported a 58% increase in harvest weight when the moisture content of their feedstock was 68% compared to 48% using almond by-products (hulls and shells). Cammack & Tomberlin

(2017) compared the moisture of feed and results showed that 70% MC feed performed the best with higher survival to the prepupal stage, grew faster and weighed heavier when compared to

55% MC as was the case for Lalander et al. (2019) from 76% to 90%. From a visual graph estimation in Cammack & Tomberlin (2017), the mean weight difference was highly noticeable when the larvae were fed at 70% MC doubling weight compared to 55% MC. A drawback was that the survivability for both was disappointing and could be caused by liquid build up

(Lalander et al., 2019). From the same study, larvae we’re not able to develop on 40% MC feed. They also noted that the feed’s moisture content has a higher impact compared to carbohydrate: protein which is reasonable because macerating and ingesting the feed will be arduous when not softened by water (Schremmer 1986). Cheng et al. (2017) used feed moisture 49 content of between 70-80% and found that as the moisture increased the larvae reached their maximum weight earlier and did not affect the survivability of the larvae.

These results suggest that the larvae experienced an increase in growth rate with higher moisture content which is desirable because it decreases production time. Banks (2014) also found that 85% MC moisture content produced heavier larvae compared to 65% MC but at the end of the experiment residue in the 65% MC reduced to 10% which is theoretically too dry for the larvae while the 85% MC remained suitable for feeding. The larvae were also able to move around faster in the moister feed compared to the more viscous feed (Banks, 2014) which decreases energy expenditure. Palma et al. (2018) used almond hulls with a moisture range of 58

- 68% and achieved a larval dry weight increase of 33.3%. Banks et al (2014) produced an increase in larval dry weight of 13.4% when the feed’s moisture was increased from 65 - 85%.

But the moisture content of the feed also has to take into account the separation process of the prepupae from the frass.

One of the highlights by Cheng et al. (2017) was to use a feed moisture of between 70-

75% to provide a more efficient sieving process and was backed up Lalander et al. (2019) where

85% MC was not ideal. The initial water content of the feed should not be more than 80% and should be dewatered prior to use (Dortmans et al., 2017) unless ventilation is provided to gradually evaporate the water to a more manageable level for the larvae to consume and survive

(Lalander et al., 2019) while aiding in larval separation when feeding is done. Lalander et al.

(2019) operated under the assumption that 50% dry matter was needed for separation and estimated that 1.42, 1.59 and 1.68 m3 h-1 kg-1 of ventilation was needed to reduce 90, 95 and 50

97.5% feed moisture concentration to 50% respectively using a 2500cm2 container. The same study also noted passive ventilation is insufficient to reach 50% MC when the initial feed’s water concentration was 76, 84 and 88%. As of 2020, to my knowledge, Lalander et al. (2019) study is the only literature which includes ventilation requirements for more efficient separation. To effectively incorporate a ventilation system additional research is required which plays in tandem with the ventilation required for a closed system.

In a comprehensive study on the various effects on the dynamic growth and development of the BSFL a very low and high feed moisture concentrations was used by Padmanabha et al.

(2020). The results show that at very low concentration the feed may have been indigestible causing a slower development and thus a high mortality rate but by gradually increasing the feed moisture the assimilation process improved which bettered growth. Lastly, in a highly saturated feed, high larval mortality together with slower rate was experienced likely from the reduced intake of oxygen. As described in literature, increasing the feed’s water content does show a positive effect on the prepupal mass but it also limits the diffusion of oxygen into the feeding substrate which produced slower growth and lower survivability. The effect of the limiting oxygen creates an anaerobic region as the feeding substrate depth increases (Banks et al., 2014).

This deters and restricts the larvae from burrowing and feeding at greater depths (Palma et al.

2018) making this unideal in maximizing available growing space. The anaerobic regions are created when the biological oxygen demand of the larvae and present microorganisms exceed the natural oxygen diffusion rate from the surface of the feeding surface or from forced aeration making it important to consider the demand of microorganisms (Palma et al. 2018). The anaerobic conditions within the feed is also said to be the cause of high larval mortality in Diener 51 et al (2011). To my knowledge and also supported by Padmanabha et al. (2020), Palma et al.

(2018) paper is the only study which investigated the effects of airflow in feed and its effects on larval growth. The latter’s study was conducted in a closed system where, expectedly, the larval development increased by gradually increasing the aeration rate.

Performance of BSF larvae at feed moisture range of 76-97.5% 400 120

350 100 300 80 250

200 60

150 40 100 Prepupal wet weight, mg 20

50 Survivability/waste reduction , %

0 0 70 75 80 85 90 95 100 Feed moisture concentration, %

Prepupal weight, Survivability, Waste reduction, mg % % wb

Figure 12: The performance of the black soldier fly’s larvae survivability, prepupal wet weight and waste reduction based on differing feed moisture concentrations from Lalander et al. (2019).

With increasing water content, the substrates structure deteriorates and is important to the larvae. The structure is important to the larvae because it allows the larvae to breath while in the substrate. The spiracles which is used for breathing are located at its anterior and posterior section (Barros et al., 2019). As the water content increased and the larval mass increased the larvae is involuntarily partially submerged into the feed with its head into the substrate allowing 52 its posterior to breath by sticking out of the water’s surface. The increase in mortality of the larvae as the feed moisture increase could be a result of drowning from slight disruption to the feeding container (Banks, 2014) by other moving larvae or human intervention. A ventilation rate which is too high may results in the larvae being stuck in the feeding material and drying out

(Lalander et al. 2019). The compromise between the pre-pupal mass and survivability according to Lalander 2019 data is at 84% MC although other studies have concluded the suitable level to be between 70-75% but since frass separation has to be taken into consideration, 70-75% has more merit.

CHAPTER 8.6.3: FEEDING RATE ON WASTE REDUCTION AND PREPUPAL WEIGHT

In providing the feed to the larvae, there are two feeding regimes which can be applied.

The first is a daily feeding regimen. It was verified in Banks (2014) that when 50-200 mg larvae-

1 day-1 was tested, mg larvae-1 day-1 at 85% MC elicited the highest growth rate and also produced the heaviest prepupae (69.6mg dw). Manurung et al (2016) also used the 200/ mg larvae-1 day-1and produced the same conclusion (15.59mg dw) as Banks (2014). Moreover, the latter produced its highest fecal matter reduction of 38.4% with 50mg larvae-1 day-1, 8.9% more than at 200mg larvae-1 day-1. Banks (2014) FMR was much lower than in Lalander et al. (2019) of 47.7%. This could be caused by the lower 40mg larvae-1 day-1 used in Lalander et al. (2019).

An increase in feeding rate from 150mg-300 mg larvae-1 day-1 was also shown to increase the female fly’s weight, number of egg clutches and number of eggs in a clutch (1361-1812 eggs)

(Georgescu et al. 2020). The latter did not provide the carbohydrate content percentage but used a feeding substrate that produces relatively big and healthy larvae (Nguyen et al., 2013). The 53 eggs count/clutch seems to vary vastly from 70-1505 (Bertinetti et al., 2019; Booth & Sheppard.,

1984).

Table 8: Feeding rates and resulting mean dry weight (mg) of prepupae fed varying waste.

Diener et al. Manurung et al. Nyakeri et al. Myers et al. Feeding rate, (2009) (2016) (2019) (2008) mg larvae-1day-1 (chicken fed) (rice straw) (human feces) (Dairy manure)

12.5 33.1 2.5 - -

25 39.1 4.8 - -

27 - - - 33.9

40 - - - 39.2

50 32.7 5.8 - -

54 - - - 46.9

70 - - - 52

100 48 9.2 47.1 -

150 - - 53.2

200 63.3 13.64 66.9 -

600 - - 72.2 -

The moisture content range of prepupae in Spranghers et al. (2017), Diener and Hakim et al. was from 59-66.8 and the calculated average was 62% and was used to approximate the dw in Myers and Nyakeri. Diener used 200 6-day- old larvae fed every 2-3 days. Manurung used 200 6-day-old larvae fed every 3 days. Myer used 300 4-day-old and fed daily. Spranghers et al (2017) used 1000 6-8 day old larvae fed every 3 days. Diener, Manuring, Nyakeri, Myers sourced larvae from Dübendorf (Switzerland), Bandung (Indonesia), Nairobi City (Kenya), Stephenville (Texas, USA)

9:; = =!> (100) Equation (1) "

C = =#$%> (100) Equation (2) #

Where; W = total amount of organic material, g

; R = Residual organic material, g

; D = Material reduction, %

; t = feeding time, days

Table 9: The reduction in waste from different authors using common feeding rates with differing waste and WRI.

Diener et al. Manurung et al. Nyakeri et al. Myers et al.

(2009) (2016) (2019) (2008)

Feeding rate, mg WR, % WRI WR, % WRI WR, % WRI WR, % WRI larvae-1day-1

12.5 39.70 1.1 31.53 0.58 - -

27 ------58.2 1.9

25 37.30 1.3 24.13 0.45 - -

40 ------54.95 2.0

50 43.20 3.1 18.36 0.35 - -

54 ------50.08 1.9

70 ------33.18 1.3

100 41.80 3.8 18.65 0.44 81 4.05 - -

150 - - - - 84 4.67 - -

200 26.20 2.4 9.58 0.24 57 3.41 - -

250 - - - - 54 3.6 - - 55

It appears from Manurung et al. (2016) using rice straw waste and Diener et al. (2009) with moistened chicken feed that 100mg larvae-1day-1 seem to be the optimum trade-off for waste consumption and biomass yield but according to Banks (2014) data it is indicated that the human FMR decreases as higher feeding rate was applied and the same was found for Myers et al. (2008) using dairy manure. The WRI for Myers et al. (2008) however dropped significantly from 1.9 to 1.2 at 54mg larvae-1day-1 to 70 mg larvae-1day-1 The optimal feeding rate based on the three presented data appears to be 100mg larvae-1day-1. The optimal feeding rate prioritizing waste reduction is not able to be determined because of varying feeding material and larval development time. We can only speculate that the intersection points between waste reduction and prepupal mass is the optimal feeding rate for their respective feed because figure 17 displayed irregular curves which restricts the choices to only the data points provided by the authors. The figure also shows that simultaneous maximization of prepupal mass and waste reduction is not possible. The WRI formulas were obtained from Diener et al. (2009, pg. 606) and high WRI values signifies good waste reduction efficiency. The WRI was either the highest or second highest in Diener et al. (2009), Manurung et al. (2016) and Nyakeri et al. (2019) at

100mg larvae-1day-1. From a WRI analysis of table 9 the optimal feeding rate when using 12.5 -

250 mg larvae-1day-1 seem to be 100 mg larvae-1day-1. There is a lack of study on the optimal feeding rate for the BSFL.

Diener et al. (2009) results of Dry weight - WR 70 0.5 1 - 0.45

day 60 1 - 0.4 50 0.35

40 0.3 0.25 30 0.2 20 0.15 0.1 Waste reduction, % 10 0.05 0 0 Prepupal dry weight, mg larvae 0 50 100 150 200 250 Feeding rate, mg larvae-1 day-1

Weight WR

Figure 13: The results of Diener dry matter waste reduction percent in relation to the prepupal mass. The waste material was chicken feed with a moisture content of 60%.

Manurung et al. (2016) results of Dry weight - WR 16 0.5 1 - 0.45

day 14 1 - 0.4 12 0.35 10 0.3 8 0.25

6 0.2 0.15 4 Waste reduction, % 0.1 2 0.05

Prepupal dry weight, mg larvae 0 0 0 50 100 150 200 250 Feeding rate, mg larvae-1 day-1

Weight WR

Figure 14: The results of Manurung dry matter waste reduction percent in relation to the prepupal weight. The waste material was human fecal sludge. Manurung used a mixture of 30 gram of straw with 60.ml water.

Nyakeri et al. (2019) results of Dry weight - WR 140 90 1 - 80 day 120 1 - 70 100 60 80 50

60 40 30 40 20 Waste reduction, % 20 10 0 0

Prepupal dry weight, mg larvae 70 90 110 130 150 170 190 210 230 250 270 Feeding rate, mg larvae-1 day-1

weight WR

Figure 15: The results of Nyakeri dry matter waste reduction percent in relation to the prepupal weight. Nyakeri used fresh human fecal.

Myers et al. (2008) results of Dry weight - WR 1 - 90 70 day 1

- 80 60 70 50 60 50 40

40 30 30 20 20 Waste reduction, % 10 10

Prepupal dry weight, mg larvae 0 0 20 30 40 50 60 70 80 Feeding rate, mg larvae-1 day-1

Weight WR

Figure 16: The results from Myers et al. (2013) dry matter waste reduction percent in relation to prepupal weight. The waste material was fresh dairy manure with a moisture content of 70%.

Bonso (2013) results of dry matter reduction - conversion rates 100 90 80 70 60 50 40 30 20 10 0 Waste reduction/conversion rate, % 0 250 500 750 1000 1250 1500 1750 2000 2250 Feeding rate, mg larvae-1 day-1

Conversion rate, % WR, %

Figure 17: The results from Bonso (2013) dry matter waste reduction percent in relation to the larvae conversion rate. The waste material was a mixture of cooked and uncooked food with a moisture content of 90%.

Difference in dry matter reduction between lump and daily 100

90 83.12 80 74.56

70 61.72 61.66 60

50 37.91 40

30 Dry matter reduction, %

20 16.64

0 Lump Daily Lump Daily Lump Daily Fruit waste Manure Rejects

Figure 18: The dry matter waste reduction results from Mutafela (2015) using lump and daily feeding with 3 different waste material.

The second feeding regime is a batch feeding regimen where feed is added to the grow out container only once throughout the larval development period. A concern with this regime is that as the material ages it loses its nutritional value at the same time the larvae increase in size and its feeding rate increases as well (Banks 2014) until it approaches the 6th stage. This means that the older larvae will have to compensate for nutritional deficiencies by consuming more lower nutritional feed compared (Wright et al. 2003; Mutafela, 2015) to when supplied with fresh feed daily. Lump feeding prolongs the development time by 2-4 days but produces a heavier pre-pupa (Banks, 2014). In regard to whether or not new feed should be mixed or allowed to rest on the surface, no difference was demonstrated with low amounts (1.5kg) but was shown to have additional 5% waste reduction when high amounts (4.6kg) was added unmixed

(Diener et al. 2011a). This should be considered when using a larger system than proposed with higher larval mass.

So far, the larvae’s performance has only been assessed in individual regions and since the BSF management system could be applied globally the role of genetic difference in BSF should be assessed to determine the difference in performance. Zhou et al. (2013) used three different BSF strains. Two strains were from Wuhan (China) and Guangzhou (China) and one from Texas (USA). The result show that although the Guangzhou strain has a significantly lower waste reduction than Texas, its prepupal weight was comparable to Texas and also developed faster than the Texas strain. Hence, the Guangzhou strain was better at assimilating nutrients.

Zhou et al. (2013) study indicated that plasticity among the strains exist with waste reduction and larval development but does not differ much with the protein content when fed swine, chicken and dairy manure. The study on inter and intra-country performance of the BSFL in one paper is 60 scarce and would need more research in order to choose a suitable strain to suit the objective of using the BSFL being waste management or to maximize the products output with minimal input.

Theoretical waste consumption and larval dry mass by proposed system: 700 '00 (250 &'()*' &*+',) . 2 = 175,000 '00, &'()*' &*1 (175,000 '00,)(80% ,89:+:' '00 ;ℎ),') = 140,000 '00 ℎ)>?ℎ'@ (140,000 '00, ℎ)>?ℎ'@)(80% ,89:+:' &''@+A0 ;ℎ),') = 112,000 ,89:+:+A0 *)9:)' 100 (0 &''@ (112,000 *)9:)') . 2 = 11,200,000(0 &''@ *)9:)'

11,200,000 (0 &''@ . 2 (20 @)1,) = 224,000,000(0 &''@ @)1 = 224 B0 &''@ >ℎ9C80ℎ >ℎ' &''@+A0 ;'9+C@

CHAPTER 8.7: PREPARING THE WASTE FEED

As a precaution for handler’s safety rubber gloves, eye protection and face masks should be worn to prevent any form of transmission between the waste and handler. After the pretreatment all equipment must be washed down with water and dishwashing detergent. Before the feeding material is provided to the larvae, they will have to be pre-treated to remove materials hazardous to the larvae including metals such as zinc shards or nuisance materials such as cloth but generally manure and other organic waste material do not contain them. The waste material should be sieved through a 10mm screen mesh to remove any metals which may have been mixed with waste from the supplier’s site. An off-the-shelf broad plastic container can be used as the pre-treatment container. If the waste material is too solid to pass through the sieve use a shovel to push the waste through but if the waste is fluid like it should be dried to approximately 61

70-75% moisture content through evaporative cooling. If the nutritional content of the waste is found to be of low quality; less than 10% protein and carbohydrates mixing the waste with supplemental material can be done using a cement mixer to be run for 5 minutes or until they are judged to be visually acceptably homogenized. Other mixer can be used but should not be coated with any heavy metal to prevent unintentional spiking.

CHAPTER 8.8: LARVAE FOR REPOPULATING THE BREEDING

UNIT

After the larvae have fed for 6 days reaching a more manageable size, 1% of the larvae are removed from each larval batch and placed into another feeding container with 70% MC chick starter feed. They will be allowed to feed ad libitum. Once all the larvae are seen to turn darker, they are separated from the frass by 3mmx3mm sieves and placed into the pupation container filled up to 5cm of wood shavings. The pupation container along with a water container will be placed onto a rack. Since flies pupate on different days, a cloth will be placed onto the water’s surface to help prevent their drowning to extend the fly’s life long enough to be used for producing eggs. The unit will be kept dark to avoid unintended breeding. Similar to the rack used for nursery/grow out, a sleeve will be made to facilitate the flies into the breeding cage and will be secured during pupation. After the flies emerges from the pupal stage, the sleeve will be unsecured and the flies will be lured into the breeding cage with a LED light source.

CHAPTER 8.9: THE PUPATION CONTAINER

Contrary to literature which states that the prepupae will search for a dry location to pupate, the wood shavings will be slightly moistened with a spray bottle to prevent desiccation of the pre-pupae and pupae. In this proposed system it is recommended that the pupation material be wood shavings for faster results and acceptable survivability. The pupation material will only be used for the pre-pupae that will repopulate the breeding cage.

CHAPTER 8.9.1: PUPATION MATERIAL ON SURVIVABILITY.EMERGENCE TIME

In an endeavor to increase Darwinian fitness and further evolve, an organism’s ability to seek suitable living and breeding habitat is crucial (Thorpe, 1945). There are two critical requirements for the fly to deposit eggs and unless they are met gravid flies will not oviposit.

One is a site with small crevices and second is the presence of organic material at the site (Park et al., 2016). In cold climates, for obvious reasons we are unable to breed the fly year-round outside therefore an enclosure which simulates the natural requirements has to be constructed. In a number of studies, the black soldier fly’s oviposition material has been dry corrugated cardboard, but these experiments focused on the mating and oviposition rate paying little to no attention to the preference of pupation material.

Although BSF only consumes waste material during the first five larval stages there is still a need to improve the non-feeding stages to enhance its entire lifecycle (Dzepe et al., 2020).

Previous studies have indicated that the success of the metamorphosis stage is heavily affected by the post-feeding substrate (Dzepe et al., 2020; Holmes et al., 2013). It would be pointless and inefficient to propose an artificial production system when flies fail to emerge for continuous 63 larvae farming because more prepupae will be needed in the repopulation line to compensate for the lower pupal eclosion success. The pupation substrate has to be evaluated for post-feeding and successful adult emergence. The pupation time may vary using various substrates because the ability to pupate may be significantly affected by the burrowing substrate’s compactness (Dimou et al., 2003). The pupation media itself should be porous and loose to allow for easy burrowing of wandering maggots (Dzepe et al. 2020). A medium with these properties should also provide adequate oxygen levels so the pupae can breathe. If the pupation medium is too fine the spiracles, or breathing structures, can become clogged possibly resulting in death (Alvarez,

2012).

Wood shavings and soil has been reported to be in good management with the shortest pupation time (Dzepe et al., 2020; Holmes et al., 2013) and high adult emergence rate (Dzepe et al., 2020). In the experimental setup of Holmes et al (2013), 50ml of water was added to maintain the pupation material’s moisture content which makes sense as the pupae is also susceptible to desiccation. In determining the depth of the pupation medium, Dzepe et al (2020) used pupation mediums of 20mm height and inoculated them with 100 post-feeding larvae.

Unfortunately, the pupation material-dependent development time for pre-pupae and pupae is limited and needs further study but it is clear that the material needs to be porous enough for the larvae to breath. The current knowledge is sufficient for the purpose of pupation for this paper.

Banks et al (2014) theorized in his dissertation that the low pupation was possibly caused by longer initial development from larvae taking longer to settle into the human fecal matter substrate at 85% MC. More larvae were trying to crawl out of the material than into it which stalled feeding and increased competition of feed for each larva. In the absence of pupation 64 material, the development period may have been prolonged because of searching for a substrate to bury into (Holmes et al., 2013). In the absence of a blanket barrier, the post-feeding larvae were observed to use themselves as a pupation substrate, probably a thigmotactic response, by aggregation in an effort to increase their temperature. The larvae can be allowed to undergo metamorphosis in the feeding substrate such as manure but the survivability to adulthood is not as favorable as with wood shavings, topsoil, sand or potting soil.

Table 10: Effects of pupation material on development and survivability of post-feeding larvae.

Mean development period, days Pupation substrate Survivability, % Source Post-feeding Pupal

No substrate 10.28 6.74 87.6 Holmes et al. (2013)

Sand 8.44 8.86 96.8 Holmes et al. (2013)

Topsoil 7.93 6.89 98 Holmes et al. (2013)

Wood shavings 7.71 6.93 95.6 Holmes et al. (2013)

Potting soil 7.56 6.92 97.2 Holmes et al. (2013)

Digested dairy manure 15.87-17.2 61.65-65.78 Myers et al. (2006)

Table 11: Literature-based operational conditions for growing and breeding BSF.

Temperature Feed Relative Photoperiod, System design Source Moisture Humidity h suggestion (light: dark)

Egg 25-30℃ Irrelevant 70% 8:16 Eggs are incubated in a shaded area. Caruso et al. (2013); The wooden stacks are placed above Nyakeri et al. (2017); waste feed. To avoid any possible Holmes et al (2012); laying of the eggs onto the lure, Joly & Nikiema (2019); cover the lure’s container with a Chia et al. (2017); plastic wire mesh. Myers et al. (2008); Tomberlin & Sheppard (2002a)

Larval instar 24-27℃ 70% The literature - Use a spray bottle to maintain the Alvarez et al. (2012); (0-6 days old) focused on the moistures consistency. If necessary, Cheng et al. (2017); feed moisture use an electronic moisture meter. Harnden & Tomberlin content Provide them with chick starter feed (2016); Dzepe et al. (2020); Banks (2014)

Larvae 24-27℃ 70-80% The literature 12:12 Use a spray bottle to maintain the Cammack & Tomberlin focused on the moistures consistency. If necessary, (2017); Alvarez et al. feed moisture use an electronic moisture meter. The (2012); Banks (2014); content feeding rate will be provided at 100 Manurung et al. (2016); mg larvae-1 day-1 using lump feeding Diener et al. (2011b) regime.

Prepupae/pupae 24-27℃ Literature 70% - Provide the pupation site with wood Holmes et al (2012); focuses more shaving and a slightly moist cloth Alvarez et al. (2012); on RH below the shavings aid in preventing desiccation. Pupation unit should be kept dark to avoid untimely breeding.

Adult 25-27℃ Literature 60-70% 7:17 Provide water for the adults with a Chia et al. (2018) focuses more cloth placed onto the water’s surface Sheppard et al. (2002); on RH and to avoid drowning and prolong life for Nakamura et al. (2015); temperature breeding purposes. Breeding cage Sripontan et al. (2017) should face the morning sunlight or Schneider (2019) use LED if sunlight is not dependable. 66

Mating and Oviposition (0-4 days)

Adult flies expired. Reset Hatching breeding cage (3-4 days)

Nursery feeding Prepare HQF Prepare/pre-treat waste (6 days)

1% population Check moisture fed HQF 99% larval population Check moisture level (14-18 days) fed waste (19-24 days) level

1% population fed provided pupation material Deactivation by (~16 days) boiling water

Dried to 10% MC

Figure 19: Proposed BSFL production process flow chart.

CHAPTER 9: APPROXIMATE COST OF THE PROPOSED SYSTEM

Table 12: The approximate cost for the proposed system.

Fixed cost Approximate cost, ~$ Plastic screen mesh (14m2/cage) 15 12 1.5m wood pieces 20 15cm x 5cm wood planks (plywood) Scavenge Pack of rubber bands and ice-cream sticks 6 12 x Industrial containers 80 4 artificial plants 8 4 water-filled buckets with dish detergent 38 1m x 1m flat plastic table 12 Electronic balance 6,000 Oviposition waste material 40 Mixing apparatus; cement mixer/simple stick and bucket 200/10 Metal stand for nursery/feeding container 60 10mm x 10mm metal mesh 10 Writable tape 20 Clip boards 5 2 Snap-on shelves (The Home Depot) 320 Red (alcohol) and green (water) spray bottle 18 5mm x 5mm metal mesh 10 2 x Broad plastic container 15 70% alcohol 30 Forceps 10 Rubber gloves, face mask, eye protection 30 Squeegee 6 Waste container for bio-processed material 15 Electric stove with oven 2,000 Wood shavings 8 Black-out cloth 19 Water container 5 100W LED strips 8 Simple Arduino moisture meter (optional) 30 Total fixed cost 8,848 / 9,038

Variable cost Approximate cost, ~$ Labor/month 7,600 Electrical power/month • Oven (drying at $0.12/hour) 250 • Stove (deactivation at $0.12/hour) 50 HQF (Chick starter feed) 70 Waste material 75 Transportation 120 Total variable cost 8,165

Start-up cost of system 17,013 / 17,203

CHAPTER 10: CONCLUSION

The inclusion of the BSFL into aquatic feed is possible because of the higher protein requirement needed by fish achievable by the larvae up to 45%. Breeding requires attention to several influential factors such as the artificial lighting (which is crucial to mating), the lure in producing higher egg counts laid and the type material provided for the eggs to be laid on. The eggs stage is the most vulnerable stage and producing enough surviving larvae is crucial for managing higher amount of waste. Developing and producing the most larval yield depends heavily on the feed’s carbohydrate and protein content, the temperature of the larvae feeding environment, the surrounding relative humidity, the feed’s water content and the feeding regime type. To ensure that pupation is successful the suitable pupation material should also be used namely a material which provides a blanket barrier and also allows airflow. Unfortunately, more research is need on the effects of a wider mycotoxin variety on the BSFL but recent studies suggest that aflatoxin 69

(BI, B2, G2), ochratoxin A, deoxynivalenol and zearalenone accumulates only in very small amounts. The accumulation of heavy metals is apparent where cadmium the most compared to others in this paper but cadmium and the other heavy metals do not appear to affect the larval growth and fecundity. Zinc however, does decrease in egg count laid by the female fly. The

BSFL are capable consumers of waste from human feces, animal manure, restaurant and vegetable waste and others. Due to the genetic variation of the BSFL found globally, more understanding is needed to access the waste consumption capabilities of the larvae together with its waste-biomass conversion efficiency.

The proposed breeding recommends the conditions needed to produce relatively high amount of eggs compared to a natural setting. The outlined system also provides conditions for the larvae to achieve a heavier weight while also having the highest consumed amount of feed possible. All recommendations were researched from numerous papers involving the breeding and development of the fly. The proposed system also takes into consideration of predatory insects, reptiles and also consideration of preventing entomopathogens. For employee safety, when handing waste material, cleaning the container and handling the pre-pupae all handlers must use protective equipment. The cost of materials used were approximated from various sources and off-the-shelf parts were given priority provided they are suitable to be used. Based on the feed provided, the production period from mating and oviposition, eggs and to the prepupal stage will take between 33-38 days. The system is designed to performed indoor but if outside abiotic factors allow the breeding cage should be placed in the direction where the morning sunlight shines directly onto the cage. If abiotic factors do not allow it, heating has to be supplied and the relative humidity has to be monitored for the egg’s to successfully hatch. 70

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