Diverting organic waste from landfills via insect biomanufacturing using engineered black soldier flies (Hermetia illucens)

A major roadblock towards the realisation of a circular economy are the lack of high-value products that can be generated from waste. Black soldier flies (BSF; Hermetia illucens) are gaining traction for their ability to rapidly consume large quantities of organic wastes. However, these are primarily used to produce a small variety of products, such as animal feed ingredients and fertiliser. Using synthetic biology, BSF could be developed into a novel sustainable biomanufacturing platform to valorise a broader variety of organic waste feedstocks into enhanced animal feeds, a large variety of high-value biomolecules including industrial enzymes and lipids, and improved fertiliser.

Management of organic waste is a major global challenge.Currently, ~40-70% of organic waste is disposed of in landfills [1][2][3][4] , where it is tightly compacted to economise space.This leads to the anaerobic microbial decomposition of organic waste into methane, a greenhouse gas (GHG) that is 28 times more potent than carbon dioxide 5 .Consequently, the solid waste sector is estimated to be responsible for 5% of global annual CO 2 -equivalent emissions, with the majority of this due to the methane generated from landfilling organic waste 6 .Continued rising GHG emissions have led to the Intergovernmental Panel on Climate Change reporting that we are facing an imminent climate disaster as we expect to surpass the 1.5 °C critical global warming threshold in 2031 7 .Urgent intervention from policy makers, bolstered by innovative technologies to handle organic waste, is imperative to achieve net-zero annual GHG emissions.
Adding to the problem are municipal biosolids, another significant source of organic waste.Municipal biosolids are nutrient-rich organic byproducts from treating sewage at municipal wastewater treatment plants.Despite their high nutrient content and utility as an alternative to synthetic fertiliser, there is an increasing body of research demonstrating that biosolids are contaminated with hazardous chemicals [8][9][10] .Notable examples include heavy metals, microplastics, per-and poly-fluoroalkylated substances (PFAS), pharmaceuticals and 35 chemicals on the EPA priority pollutant list 10,11 .Regulations are increasingly restricting using biosolids as fertiliser 12 , and alternative management practices are required for their disposal.
Challenges to sustainable waste management are particularly severe in developing countries where public services for waste management can be limited or non-existent.Organic waste is often openly dumped, which contributes to the proliferation of pathogens and pests, contaminates water used for drinking and irrigation and results in habitat destruction 6 .Additionally, a large portion of biomass from food crops is non-edible and is frequently managed by open burning that results in unsafe air quality 6,13 .
Many developed nations are implementing organic waste landfill bans to hasten the adoption of waste management strategies that align with the public good.Yet scalable and cost-effective strategies are lacking as the products that can be generated from waste, such as compost, biofuels and animal feed have limited value and there are significant upfront and maintenance costs required to develop the infrastructure to collect, sort, and fully utilise organic waste.
The circular economy is an economic system that aims to minimise waste and maximise resource efficiency by promoting the reuse, recycling and regeneration of materials in continuous cycles, and offers a promising framework to address these challenges.Technological innovations that increase the variety and value of products that can be regenerated from organic wastes will incentivise greater organic waste circularisation and provide an opportunity for entrepreneurs to valorise organic waste.
Synthetic biology has the potential to provide many of the needed technological innovations that could address climate change and sustainability 14 .Though underdeveloped in comparison to microbial synthetic biology, insect synthetic biology could be used to clean up and valorise organic waste by engineering insects that are already adept at processing organic wastes as a platform for biomanufacturing and bioremediation.This type of insect biomanufacturing could convert organic wastes into higher 1 Applied BioSciences, Macquarie University, Sydney, NSW, Australia. 2ARC Centre of Excellence in Synthetic Biology, Macquarie University, Sydney, NSW, value products such as enhanced feed, industrial biomolecules and highquality fertiliser.Globally, it is estimated that the animal feed market is worth USD $500 billion 15 .The global industrial enzyme and lipid market is worth USD $6.95 billion 16 and USD $13.63 billion 17 , respectively.The global fertiliser market is worth USD $207 billion 18 .
In this perspective we describe why insects are a very promising synthetic biology platform to improve sustainable biomanufacturing.We describe the opportunity, advantages and disadvantages and how to mature insect biomanufacturing with a focus on black soldier flies.

Modern biomanufacturing
Biomanufacturing is the use of biological systems and their cellular machinery to generate valuable biomolecules with utility in industries such as agriculture, food, pharmaceutical, material and energy 19 .Typically, mono-cultures of microbes, fungi and cell lines are used for commercial biomanufacturing, predominantly via large scale bioreactor-based fermentation.To a lesser extent, plant and algal systems are also used for biomanufacturing.Products include small molecule primary metabolites such as acetone, butanol, ethanol, amino acids, and organic acids 20 ; and complex secondary metabolites such as antibiotics, flavourings, colourings and fragrances [21][22][23] .Genetically engineered organisms are used to produce many larger high-value proteins including insulin 24 , growth hormones 25 , erythropoietin 26 , industrial enzymes 27 , enzymes for molecular biology 19 , monoclonal antibodies 28 and vaccines 29 .Microbial fermentation systems and cell lines have been particularly invaluable to commercial biomanufacturing due to the relatively simple rearing conditions, fast growth rates, ease of storage, established genetic manipulation methodologies and diverse metabolic potentials.
The United Nation's Sustainable Development Goals (SDGs) 30 have catalysed a shift in focus to develop technologies that reduce emissions and pollution, and to replace processes based on fossil fuels with sustainable biobased approaches.This includes changing approaches to the production of high-value biomolecules to utilise waste as feedstocks.Current industrial biomanufacturing strains are resource and energy intensive as they typically require sophisticated infrastructure, large quantities of water, feedstocks predominantly sourced from food crops and feedstock preparation that is resource and energy intensive 31 .Plant molecular farming is less energy and resource intensive than bioreactor-based biomanufacturing as it can directly fix carbon dioxide to produce valuable biomolecules, and could be used as a carbon capture technology for emissions reductions 32 .However, it has a large land-use footprint and requires large quantities of water and fertiliser that could otherwise be used for growing food.It could impact food security as well as deforestation and biodiversity as more land is cleared to grow these plants.Furthermore, plants grow more efficiently in the field and to commercialise any one transgenic plant for growth outside of physically contained greenhouses requires long and expensive regulatory hurdles 33 .Effective transgene biocontainment strategies are limited and transgenic plants growing as volunteer crops have contaminated food supply chains; leading to expensive recalls, food wastage and damages to public trust 34,35 .Diversification of biomanufacturing technologies for greater sustainability is therefore required.The UN's SDGs will be described in more detail in the 'Addressing the SDGs' section.

Insect biomanufacturing
Beekeeping for honey, beeswax and pollination has existed since the dawn of agriculture, with evidence of humans using beeswax found across Neolithic Europe, the Near East, and North Africa 36 .Silk production from the cocoon of the silkworm (Bombyx mori) originated in China in the 3rd millennium BC and is a high-value product to this day.Recently silkworms have been used as a platform for synthetic biology and nanotechnology to generate products that harness the properties of silk fibre.It has been used to generate high-value protein therapeutics, spider silk, antimicrobial bandages, drug delivery systems, dissolvable and biocompatible medical implants, scaffolding for tissue engineering, precision agriculture for the controlled delivery of bioactives to plants, among other applications [37][38][39][40][41][42] .Extracts from the Coccoidea superfamily of scale insects are used for colourful dyes and resins 43 .Products include kermes, Polish cochineal, Mexican cochineal, lac dye and shellac.These have important historical, cultural and economic value as the low production output and low supply of these eye-catching products were sought after by nobility.
Insect cell lines and insect larvae/pupae are often used in molecular biology research and commercial biomanufacturing to transiently overexpress proteins of interest 44 .They are less likely to become contaminated by human pathogens than mammalian cell lines and they provide many of the post-translational modifications required for eukaryotic protein stability and function.Approximately 1% of FDA/EMA approved protein therapeutics are biomanufactured by insect cell lines 44 .Examples include: vaccines for COVID-19 (Novavax) 45 , influenza (Flublok) 46 and cervical cancer (Ceravix) 47 ; cellular immunotherapies for prostate cancer (Provenge) 48,49 ; and adenovirus production for familial lipoprotein lipase deficiency gene therapy (Glybera) 50 .Cocoon Biosciences, a spin out company of Algenex, is using cabbage looper (Trichoplusia ni) pupae biomanufacturing process to generate growth factors for the cultivated meat industry and enzymes for industrial biotechnology 51 .
Insects as a source of food is a delicacy in many cuisines across the world and will likely benefit from economies of scale to make protein that is more affordable than more conventional livestock 52 .Though insects are not regarded as a delicacy in the West, they are gaining interest as a sustainable source of protein due to their high feed conversion efficiency of preconsumer food wastes 53 .Insects are commonly utilised as nutritious feed for pets and livestock 54,55 .Insect meal is set to become less expensive than other protein feed products such as fishmeal 56,57 .Though it is still more expensive than soy protein, it is considered a more sustainable option 58 .
Various industrial biomanufacturing applications of cell line and microbial systems and whole insects are shown in Tables 1 and 2, respectively.

BSF biomanufacturing for the valorisation of organic waste
Insect pupae biomanufacturing platforms, including the cabbage looper (T.ni), are already used to commercially biomanufacture biomolecules 51,59 .However, these insects cannot be reared on organic waste and their relatively narrow dietary requirements 60 can hinder scalability.Adoption of insect species with an appetite for a variety of organic wastes have the greatest potential for contributing to sustainable biomanufacturing on a global scale.
Many insect species are adept at decomposing organic waste and perform an important ecosystem service to recycle nutrients.Black soldier flies, (BSF; Hermetia illucens), house fly maggots (Musca domestica), crickets and grasshoppers (order Orthoptera), superworm beetle larvae (Zophobas morio), meal worms (Tenebrio molitor) and wax worms (Galleria mellonella) have all been adopted commercially for their strengths in processing organic waste into insect protein (Table 2).
BSF especially have gained interest as a rapid means to convert large quantities of organic wastes into a variety of products (Table 2), as many aspects of their biology and lifecycle readily support large-scale rearing practices in commercial settings.They are not considered a pest species and they are globally distributed, except for Antarctica 61 .Furthermore, they are not a carrier for human pathogens as they do not feed on waste as mobile adults and do not bite 62 .BSF larvae (BSFL) can consume up to 500 mg of organic matter per day per larvae 55 .Optimal rearing conditions for BSF can be applied within rearing facilities and is largely influenced by temperature, humidity, substrate composition, among other conditions 63 ; it is an active area of BSF research 64 .BSFL thrive on a wide variety of organic wastes including kitchen waste, fruits and vegetables, meat and fish, livestock manure and even human excrement 64 .The insects are easily harvested from the processed organic waste with a sieve or when the larvae migrate out of the organic waste to pupate 65 .Typically, on a dry weight basis, the pupae contain 40-44% protein and 15-50% lipid 55 .The composition of BSFL, particularly the lipid fraction, can vary considerably depending on the lipid composition of the substrate mixtures they consume as larvae 66 .The main products made from BSFL/pupae are pet food and livestock feed 54,67 .
Though BSFL/pupae have also been used to make other value-added products including biofuels 68 , bioplastics 69 , lubricant additives 70 , antimicrobial compounds (AMPs) 71 , chitin and chitosan 72 , sustainable protein for human consumption 73 , among other applications 74 .
BSF is used in waste management to reduce the quantity of organic waste, with waste reduction efficiencies between 65.5% and 78.9% under conditions that rival standard composting methods and on a timescale of weeks compared to months 67 .The processed organic waste, insect exuviae and associated microbes, are referred to as frass which is used as an effective soil amendment to recycle nutrients, such as nitrogen and phosphorous, thereby promoting plant growth 75 .

Engineering BSF for biomanufacturing
Utilising transgenic BSF to support a sustainable circular economy involves considering both waste streams and products.Waste-streams may be broadly divided into two categories: those that are suitable to produce food/ feed and those that are not.The former high-grade wastes includes horticultural, food manufacturing, fisheries and pre-consumer food waste 76 .The latter includes low-grade wastes such as restaurant and post-consumer household organic waste, slaughterhouse waste, livestock manure, municipal biosolids and organic waste contaminated with hazardous compounds.The suitability of a particular waste stream to be used as feed will depend on safety, public perception and regulatory factors 76 .

Perspective
A major limitation of using wild-type BSF to generate predominantly animal feeds from waste are regulations which preclude using low-grade organic wastes to rear BSF for feed 76 .BSF could, however, valorise both highand low-grade wastes by engineering BSF as a platform for biomanufacturing and bioremediation (Fig. 1).BSF could convert greater varieties of low-grade organic wastes into insect biomass engineered to biomanufacture a large variety of high-value biomolecules such as industrial enzymes and lipids, that are not subject to food/feed regulations.Engineered BSF could also convert high-grade organic wastes into enhanced animal feeds.Furthermore, there is opportunity to generate enhanced fertiliser from both high-and low-grade organic wastes.

Valorising high-grade organic wastes
As previously discussed, BSF are becoming widely used to produce animal feed and animal feed ingredients 54,55,67 .The value of these ingredients could be increased by engineering BSF to express enzymes that are commonly added into feed ingredients.Amylases, pectinases, lipases, proteases and phytases are currently made by microbial fermentation and incorporated into animal feeds to boost digestion of other ingredients 77 .These enzymes enable the use of less expensive feed ingredients, increase growth rates and in the case of phytases-also reduce phosphorous waste in manure.BSF engineered to express these enzymes may additionally improve their own FCR on otherwise difficult to digest organic waste feedstocks.Altering the lipid profile may be desirable by, for example, engineering BSF to produce marine-derived omega-3 fatty acids 78 which would improve the fatty acid profile of aquaculture animals reared using BSF.BSF engineered to improve feed for these purposes is not likely to encounter significant regulatory hurdles as many genetically engineered products are already approved for use as feed 77 .For example, food crops genetically engineered to improve productivity in the field are inexpensive sources of animal feed 79 and enzyme supplements used to improve digestibility are sourced from genetically engineered microbes 77 .
It may also be possible to engineer BSF to produce oral veterinary medicines and vaccines for livestock 80 .BSF is already palatable for livestock rearing which may enable these veterinary medicines to be administered easily at scale 73 .Moreover, they act as a pre-biotic to promote a healthy and diverse microbiome 81,82 .BSF could improve feed and food safety further by engineering BSF to prevent the growth of harmful microbes 83 and to remediate chemical pollutants found in pre-consumer waste such as heavy metals 84 , biocides 85 and mycotoxins 86 .Currently, therapeutic vaccines for rabbit haemorrhagic disease are already produced by genetically engineered cabbage looper pupae 87 , which have marketing authorisation in the European Union and United Kingdom 88 .Expanding to BSF is therefore likely to be possible from both technical and regulatory perspectives.

Valorising low-grade organic wastes
Transgenic BSF should also be capable of valorising waste streams not suitable for feed due to hygiene or chemical contamination regulations.These could be used as feedstocks to produce high-value industrial biomolecules, which are not subject to hygiene considerations required for feed or food consumption 76 .This includes enzymes such as laccases, amylases, hemicellulases, cellulases, proteases, peroxidases and lipases which have applications in textile processing, feedstock pre-treatment for biofuels, paper manufacturing and as ingredients in detergents 89 .Enzymes could also be biomanufactured by BSF to clean up industrial wastewater.These enzymes may be produced from BSF in crude or purified form, lyophilised for ease of storage and transport and sold to industry.

Perspective
For biofuels, extracellular carbohydrase and ligninolytic enzymes can be initially extracted in aqueous buffers to provide an economical enzymatic pre-treatment method for processing complex lignocellulose feedstocks into fermentable sugars for industrial microbes.The BSF lipid fraction could then be extracted from the insoluble fraction and processed into biodiesel 68 .The protein in the remaining solid phase fraction could be fed to industrial microbes as their nitrogen source for biofuel production.
Beyond valorising wastes that BSF can currently consume, BSF could be engineered to express enzymes that can increase the scope of wastes they are able to process.This could include agricultural lignocellulosic wastes 90 , fats/grease 88 , untreated sewage 91 and chemically contaminated wastes [8][9][10]92 . Soied mixed plastic waste could also be used as a feedstock, though its mineralisation will generate net-positive CO 2 emissions.This could potentially be offset if the BSF generate other biomolecules with high carbon content or traditionally produced via fossil fuels e.g. for generating bioplastics 69 , industrial lipids 93 , or carbon polymers for carbon credits.Alternatively, BSF could generate lyophilised enzymes for the plastic recycling industry, to regenerate the starting monomers from plastic waste 94,95 .
BSF could be engineered for organic waste bioremediation to clean up and redeem value to organic wastes that are chemically contaminated.This could include 25% of all food wastes contaminated by mycotoxins 86 and chemical pollutants typically found in biosolids such as heavy metals and PFAS 8,10,96 .Contaminated soil could additionally be mixed into organic waste in optimised ratios as a potential ex vivo bioremediation service 97 .BSF may be engineered to achieve bioremediation via enzymatic catabolism or potentially via engineering the larvae to hyperaccumulate contaminants and leave behind clean frass for fertiliser.
We demonstrated a proof of concept for engineering insects for biomanufacturing and bioremediation by engineering the model insect Drosophila melanogaster to produce and secrete a functional fungal laccase from Trametes trogii 98 .Fungal laccases are powerful enzymes with a broad substrate range and they have demonstrated utility for a variety of applications.For example, lyophilised laccase could be made from BSF pupae for their use in the textile 99 , paper and pulp 100 , food 101 , pharmaceutical 102 , chemical synthesis 103 and forestry 104 industries.Furthermore, they can biodegrade lignin in lignocellulose and certain plastics 105,106 and may allow these organic wastes to now be processed by the BSFL.They can also be used for bioremediation to clean up contaminants in organic waste and make clean frass; for example, pollutants such as bisphenols 107 , PFAS 108,109 , mycotoxins 110 , microplastics 105 , biocides 111 , pharmaceuticals 112 , among others.We demonstrated that engineered D. melanogaster was capable of degrading laccase substrates within their diet, including bisphenol A (BPA), while a lyophilised powder made from adult flies could decolourise the dye indigo carmine for industrial wastewater treatment 98 .Approximately 200,000 tons of dye is lost to textile industry effluents each year 113 .In waterways, dyes hinder light penetration, which affects photosynthesis and oxygen availability 113 .These effluents could potentially be economically treated by lyophilised laccase generated by BSF.
We additionally demonstrated that D. melanogaster could be engineered to express the microbial enzymes, organomercurial lyase (MerB) and mercuric reductase (MerA) from E. coli, to bioremediate methylmercury 114 .

Perspective
The engineered insects exposed to methylmercury in their diet were able to demethylate methylmercury and remove it from their biomass as atmospheric elemental mercury.Currently, a quarter of the mercury inventory that is released into the environment is from municipal sewage 115 .BSFL could be engineered to express MerA and MerB to clean up and restore value to organic wastes contaminated with mercury, such as municipal 115 or fisheries 92 organic wastes.The atmospheric elemental mercury can be trapped 116 in physically contained facilities and safely stored away from the biosphere.Meanwhile, the clean insect biomass could be used to biomanufacture high-value biomolecules or animal feed and the clean frass can be maintained as fertiliser for crop growth.

Improved fertiliser
Increased BSF production and generation of frass could reduce our reliance on synthetic fertilisers.Synthetic fertiliser manufacturing accounts for 2.5% of global CO 2 -equivalent of annual GHG emissions 117 .This statistic does not account for the quantity of N 2 O volatilisation once it is applied to the soil, which is a GHG that is 300-times more potent than CO 2 117 .
From a soil science perspective, frass provides several additional advantages over synthetic fertilisers.The insect exuviae found in frass stimulate plant defences and may attract beneficials to promote plant health 75,118 .Synthetic fertilisers notoriously cause nutrient runoff and waterway eutrophication.Frass can retain phosphorous and potassium while maintaining absorption by plants and may reduce eutrophication from runoff 75,119,120 .Meanwhile, frass can improve healthy microbial diversity in the soil 121 .Microbes present in organic compost temporarily incorporate nitrogen from the soil into the amino acids of their proteins, preventing runoff of soluble nitrogen species 122 .As the microbes exhaust the nutrients in the fertiliser and microbial populations decline, this provides plants with slow-release nitrogen that promotes root growth.
BSF could be engineered to improve the quality of frass for crop outcomes.BSF could be engineered to improve arable soil; for example, through secreting enzymes or binding molecules into the frass that can remediate a contaminant or improve other biotic or abiotic conditions in the target site 123 .Soil is also increasingly recognised as a powerful sink for carbon capture 124 and with economic policies that reward carbon capture, BSF could be engineered as a commercial carbon capture option.

Advantages and disadvantages of BSF biomanufacturing
BSF have several advantages over industrial bioreactor-based biomanufacturing platforms traditionally used to produce high-value biomolecules.Industrial bioreactor-based biomanufacturing requires feedstocks that provide carbon and nitrogen (e.g.sugar and amino acids), enzyme cofactors, growth factors (e.g.foetal bovine serum for mammalian/insect cell culture) and other mineral and trace nutrients 31 .These can be sourced from a variety of resources ranging from petrochemicals and food crops to waste by-products 31 and a full life-cycle assessment is necessary to evaluate the relative sustainability of each.Regardless of the source, these ingredients must be processed via resource and energy intensive mechanisms.Currently, carbon in large scale biomanufacturing is largely derived from food crops, which is typically processed by energy intensive milling and grinding and juice extraction and heating during clarification.Additional chemical, thermal and enzymatic pre-treatments may be required for waste feedstocks such as lignocellulose 125 and certain microbial strains are only able to metabolise hexose or pentose sugars for fermentation 126 .Nitrogen is typically sourced from inorganic ammonia made in the fossil fuel intensive Haber-Bosch process 31 .Notable trace elements like phosphorous is unsustainably mined using energy-intensive practices 31 .Moreover, sterilisation in some applications may be necessary to remove competing microorganisms that may reduce the product yield.
BSF are able to derive carbon, nitrogen and trace elements like phosphorous from the wide variety of organic waste they are able to consume.Minimal organic waste feedstock pre-processing is required as animal digestion is highly evolved to extract and absorb nutrients: food is physically broken into smaller pieces by mastication, the digestive tract consists of several compartments with acidic and alkaline regions and various digestive enzymes process biopolymers into components that can be absorbed along with other essential nutrients 127 .Furthermore, additional chemical energy from complex variable dietary fibre from a diversity of plants can be broken down by the diverse microbiome in the insect hind-gut to generate short chain fatty acids that can be absorbed and utilised by the insect host 128 .
BSF do not require thermally treated or sterilised inputs as they have evolved excellent tolerance to their fellow organic composters such as bacteria and yeast 128 .These organisms may digest feedstocks that are difficult to process, which can in turn be consumed by BSFL 128 .However, BSF are susceptible to entomopathogenic bacteria, viruses, fungi, nematodes, protozoa, parasitoids, or mites [129][130][131] .Unexplained colony collapse has been reported for BSF colonies (unpublished) and further research is required to determine whether this is due to abiotic and/or biotic causes.
Microbial biomanufacturing requires sophisticated infrastructure and highly skilled personnel which are significant barriers to adoption by lowincome countries.This includes oversight by scientists and engineers and infrastructure for feedstock extraction and quality assurance; equipment sterilisation; fermentation; significant water purification facilities; waste management; heating and cooling; control and monitoring; and downstream genetically modified organism (GMO) processing, product extraction, purification and quality assurance.
Though industrial BSF farming can be automated with sophisticated infrastructure 132 , it is not required and can be adopted and maintained readily worldwide 133 .BSF facilities that house genetically engineered strains however, will need to design/upgrade their facilities and train personnel to comply with biocontainment standards relevant to the country.An overview of a potential BSF biomanufacturing facility is shown in Fig. 1.The benefit of housing engineered strains in physically contained facilities is that it will avoid onerous regulatory hurdles required for intentional release of transgenic organisms into the environment, such as what is required for transgenic plants 34,35,134 .Similar to microbial biomanufacturing, the products made by transgenic BSF can be removed from the contained facility after the insects are treated with a 'physical or chemical process which removes, kills or renders non-viable the GMOs used' 134 .For example, freezing, microwaving, baking, boiling, blanching, desiccation, grinding, asphyxiation, or high hydrostatic pressure can be used to kill black soldier flies 135 .Genetic biocontainment strategies could be stacked for additional security.Genetic biocontainment has been demonstrated in insects to prevent interbreeding with their wild counterparts 136 , as well as strains that cannot fly 137 and would incur a significant fitness cost in the wild but not within the facility.Genetic biocontainment strategies will be discussed further in the 'Biocontainment' section below.
BSF is likely to be regulated similarly to other GMO biomanufacturing platforms, with the exception that products intended to make feed or food products would need to be regulated for feed or food production.Numerous modern products are available on the market in Europe, Asia, Australia, Africa and the Americas that are derived from GMOs.These range from food and feed derived from engineered crops 79 to vitamins [138][139][140] , enzymes 77 , polymers 141 and pharmaceuticals 24,26,142 produced by microbial fermentation.Moreover, BSF is approved for feed or food production using highgrade organic wastes in the European Union, Asia, Africa, Australia and the Americas 76 .
Though single cell organisms have a fast turnaround for generating results rapidly for research and development, scaling microbial industrial biomanufacturing continues to be challenging, particularly at the fermentation stage 143 .BSF farming is already scaling considerably, with BSF facilities growing in number across the globe.One of the largest industrial BSF facilities, AgriProtein in South Africa, processed 250 tonnes of organic wastes per day and generates 7 tonnes of larvae meal, 3 tonnes of oil and 20 tonnes of fertiliser daily 144 .Enterra in Canada processes 100 tonnes of preconsumer organic waste per day and produces 7 tonnes of protein and oil feed ingredients and 8 tonnes of fertiliser per day 144 .Engineering BSF as a biomanufacturing platform for high-value biomolecules could therefore be https://doi.org/10.1038/s42003-024-06516-8 Perspective readily applied at scale, building on this existing infrastructure.Though not directly comparable, according to Capacitor data, globally there are 20 microbial bioreactor facilities in the range of 20,000-99,999 L capacity and 9 bioreactor facilities with greater than 100,000 L capacity 145 .Large companies such as Genentech use bioreactor-based fermentation to make 10 metric tonnes of biologics annually 146 .
A major bottleneck for BSF biomanufacturing is that the genetic toolkit for BSF is yet to be as developed as other industrial biomanufacturing platforms.The first BSF genome was published in 2015 147 and has since been sequenced by other groups 137,148 .There are currently four reports in the literature describing heritable BSF genetic manipulation: one involving Cas9 gene knock-outs 137 and our research group and two other research groups have demonstrated piggyBac transposase mediated random chromosomal integration [149][150][151] .Though commercially valuable products could currently be generated using piggyBac transposase-mediated integration for one or two transgenes, random chromosomal integration complicates strain development and strain performance.It results in variable transgene expression levels due to positional effects and frequently results in the integration of multiple transgene copies.Uncertainty over the location and copy number of a transgene creates challenges for establishing homozygous strains and some genetic manipulations benefit from integration in sexchromosomes.Further research will be required to demonstrate heritable locus-specific genome integration methods to address these challenges, which will be discussed in more detail in the 'Research and Genetic Tools' section.Further, into the future, more complex metabolic engineering will be required.Design considerations for genetic and metabolic engineering from other organisms will speed up its implementation in BSF.

Addressing the sustainable development goals (SDGs)
The SDGs are a set of 17 objectives established by the United Nations (UN) to address the world's most pressing challenges before 2030 30 .Broadly, these goals provide a framework to address issues from poverty and inequality, to environmental degradation and peace.BSF biomanufacturing as showcased here could address multiple SDGs (Table 3) for a more sustainable and equitable future.

Establishing BSF biomanufacturing
The critical next steps for realising BSF manufacturing will involve first developing the research and genetic tools for BSF as a synthetic biology and biomanufacturing platform (Fig. 2).It will also involve identifying functional candidates for high-value biomolecules that are appropriate to the strengths of insect biomanufacturing and developing BSF strains that can process a greater scope of organic wastes.These could be licensed to BSF facilities as stable germline (heritable) transgenic strains.BSF facilities will need to ensure their facilities and staff comply with physical containment standards for housing the genetically engineered insects.BSF facilities will also be required to obtain regulatory approvals.BSF biomanufacturers can then begin to scale up organic waste collection appropriate to the type of biomanufacturing.It will be critical for both BSF strain developers and BSF facilities to establish relationships with downstream industries to ensure BSF products will ultimately be used.

Research and genetic tools
Substantial effort is required for BSF to catch-up to more established research strains and biomanufacturing platforms.Scientific research into BSF synthetic biology as well as basic research into their biology (for example investigating colony collapse, improving fertility, optimising the proportion of protein or lipids, investigating overwintering mechanisms, studying microbiome interactions, etc.) is required to maximise their utility as a biomanufacturing platform.This will require establishing lab-scale research tools for BSF husbandry to enable high-throughput scientific testing for multiple test conditions and simplifying maintenance for multiple strains (Fig. 2a).This will include scaling down rearing containers, establishing simplified low-odour media formulations and developing tools for long-term storage.
It will be critical to establish robust and efficient genetic engineering tools in BSF to establish heritable transgenic lines (Fig. 2b).Genetic engineering, particularly for multicellular eukaryotic organisms, is complex and requires a highly specialised skillset and equipment 152 .While genetic engineering has been described in BSF using random chromosomal integration methods 149 , further research is required to develop efficient locus specific chromosomal integration methods.This will facilitate the characterisation of copy number and positional effects and allow for the easier generation of homozygous lines to maintain stable transgenic BSF strains.Such advancements can be achieved with technologies such as CRISPR-Cas9 or PhiC31 integrase-mediated transgene integration 152 .A PASTE system was also described recently that uses prime-editing to add a bacteriophage AttP landing site into a target locus and simultaneously integrate a target genetic construct 153 .These tools could be used in BSF to insert constructs into chromosomal regions known to be in euchromatin for the desired expression patterning, or to disrupt genes that are required for fitness in the wild, but not within a biomanufacturing facility 137 .
Generating strains with well-characterised PhiC31 AttP landing sites that are broadly available proved very useful for D. melanogaster research, where they are frequently used by commercial transgenesis services 154 .These services have allowed new frontiers in D. melanogaster research to rapidly progress as the complex transgenesis procedures can be outsourced by wellequipped professionals 154 .Similar commercial providers for BSF will allow widespread adoption of research into BSF synthetic biology.
Further research is required to characterise regulatory elements (promoters, untranslated regions (UTRs), and terminators) that can be used to express transgenes at different strengths ubiquitously; inducibly; in specific tissues such as the salivary gland, midgut, hindgut, haemolymph, fat body, or casings; or at specific developmental stages such as during larvae development, pupation, or adulthood (Fig. 2b).Prior research from other insects/organisms or investigating BSF entomopathogenic viruses may reveal constitutive (e.g. from endogenous housekeeping genes, or strong viral promoters) and inducible promoters (e.g.nutrient, chemical, thermal, or light sensing promoters).Inducible promoters have been established in insects for utilising antibiotic responsive promoters 155 or plant hormones, such as auxin 156 , which are inexpensive to make at industrial scales.Prior transcriptomics studies in BSF may guide tissue-and developmental-stage 157 specific promoter discovery before validating these promoters by using RT-qPCR and/or colorimetric/fluorometric reporter fusion constructs.
Biodegradative enzymes that will be required for enhancing waste processing could be expressed specifically during the larval life-stage from a larval promoter.Meanwhile, enzymes required for bioremediation could be expressed from pollutant sensing inducible promoters or at all life-stages.Target high-value biomolecules could begin to be overexpressed in all cells specifically during pupation, when the insects are separated and collected, to maximise target biomolecule yield while minimising a fitness cost to the larvae during waste processing (Fig. 1).

Prototyping insect biomanufacturing
Heterologous expression of microbial, algal, fungal, and plant enzymes in a distantly related insect host can be difficult to achieve as enzymes adapt to function in the conditions of their native host.An enzyme may not be functional in a new host due to differences in cellular chemistry or cellular machinery.This could include issues with cellular redox potential, pH, oxygen sensitivity, as well as problems with protein solubility, misfolding, post-translational modifications, localisation, pre-pro-peptide processing and the requirement for chaperones.These issues can be difficult and time consuming to troubleshoot.Moreover, an enzyme may be toxic to the animal host, which would also preclude their suitability.As there is a vast library of useful enzymes and proteins characterised to date, libraries of enzymes and proteins can be screened and it is likely at least some will be active and will not cause harm.
BSF cell lines (Fig. 2c) or the insect genetic model, D. melanogaster (Fig. 2d), could serve as initial test platforms to rapidly screen libraries of https://doi.org/10.1038/s42003-024-06516-8 Perspective enzymes and proteins.BSF cell lines could be developed to rapidly screen for enzyme function and then many hundreds of variants can be generated for directed evolution to optimise enzyme activity in the conditions of the intended downstream application (for example, in the BSF digestive tract, in the digestive tract of livestock/aquaculture, in the downstream industrial setting, etc.) D. melanogaster could be used to optimise an enzyme or protein's spatial and temporal expression patterning.D. melanogaster is closely related to its dipteran cousin, BSF and enzymes and proteins that are active in D. melanogaster are likely to also be active in BSF.D. melanogaster will provide a rapid turnaround of results as it has a short generation time of 10 days at 25 °C, high fecundity where one female can produce >75 eggs per day 158 and it is simple and inexpensive to maintain.Its genome is thoroughly characterised and its genetic engineering can be reliably outsourced commercially for multiple genomic loci with known positional effects 154 .Well characterised balancer chromosomes also enable the rapid generation of stable homozygous transgenic stocks 159 .Experimental designs can be rapidly adjusted in BSF cell lines and D. melanogaster, and these validated experimental designs can be readily applied to functional assays in BSF.SDG #13: Take urgent action to combat climate change and its impacts • The waste sector is estimated to be responsible for 5% of global annual greenhouse gases 6 , mainly due to landfilling organic wastes.BSF biomanufacturing will reduce organic waste landfilling.• Synthetic fertilisers are estimated to be responsible for 2.5% of annual greenhouse gas emissions 117 and this does not include the volatilisation of the potent greenhouse gas, N 2 O, from synthetic fertiliser applied to soils.Greater adoption of BSF biomanufacturing and production of frass could reduce our reliance on synthetic fertilisers.• Agriculture and its associated land use are estimated to be responsible for 17% of total annual greenhouse gas emissions 117 .Improved animal feeds will improve livestock productivity.BSF engineered to improve frass could improve horticulture productivity.• BSF biomanufacturing does not require feedstocks from food crops and will reduce land clearing and preserve forests as a large carbon sink.• BSF could be engineered as a platform for soil carbon capture for carbon credits.SDG #14: Conserve and sustainably use the oceans, seas and marine resources for sustainable development • BSF engineered to synthesise marine omega-3 fatty acids could reduce the requirement for wild-caught fish feed to support adequate aquaculture nutrition.• BSF engineered as a platform for biomanufacturing and bioremediation will reduce pollutants and organic waste eutrophication in the environment and improve species conservation.• BSF engineered to process soiled mixed plastic wastes will allow plastics to be processed locally and reduce plastic pollution in the ocean.
SDG #15: Protect, restore and promote sustainable use of terrestrial ecosystems, sustainably manage forests, combat desertification and halt and reverse land degradation and halt biodiversity loss • BSF biomanufacturing could reduce land clearing for crops and thereby preserve forests, habitats and biodiversity: 1) Compared to other biomanufacturing methods, BSF biomanufacturing does not require feedstocks derived from food crops.2) BSF frass could improve soil quality and improve crop productivity per unit of land.
• BSF biomanufacturing could halt biodiversity loss: 1) Climate change and pollution are major threats to species conservation 171 and BSF engineered as a platform for biomanufacturing and bioremediation could address significant sources of emissions, as well as reduce pollution in the environment. https://doi.org/10.1038/s42003-024-06516-8

Perspective
Optimised enzyme variants with optimal expression patterning could then be engineered into insects, such as BSF, that are well suited for biomanufacturing, but might not be optimal as a research tool.
Recently, ref. 160 reviewed how D. melanogaster could be an excellent bioreactor for plastic degradation 160 .They list microbial, fungal, insect and algal enzymes that are able to degrade different types of plastic and describe what spatial and temporal expression patterning could be used to express these enzymes in D. melanogaster for plastic degradation.They also describe how the microbiome in the insect hind-gut could be altered with plastic degrading microbes or microbial communities to synergistically break down plastic.
For the purpose of BSF biomanufacturing, the technology readiness to alter an animal's microbiome is not as established as genetically engineering an animal.To date, it remains very difficult to stably alter an animal's microbiome, as these microbes face fierce competition from the native microbiome 161 , which are well adapted to survive in the intestinal mucosa and derive energy from indigestible nutrients commonly available in the lower gastrointestinal tract 128,162 .It would therefore require constant selection, and effective transgene biocontainment strategies for the microbiome are not yet established 163,164 .It would potentially also require expensive sterilisation of the organic waste feedstocks.A diverse microbiome performs a service to the host to degrade indigestible fibre from a diversity of plants, as well as prevent colonisation of harmful bacteria 162 .Significantly altering BSF's native microbiome may impact BSF's ability to process a wide variety of organic waste feedstocks 128 .Though it is challenging to alter an animal's microbiome, it may be possible to develop microbial pre-treatments to improve BSF digestibility of challenging substrates 90 .Research has shown that pre-treatment of lignocellulose feedstocks with microbes and fungi can improve the bioconversion of lignocellulose by BSF 90 .Nevertheless, implementing pre-treatments is challenging at industrial scales 90 and directly engineering BSF to express the required degradative enzymes would mitigate the necessity for pretreatments.

Biocontainment
The risk landscape for this technology is not yet fully understood and biocontainment strategies and transparent risk assessments are required to reveal how this technology can be used to maximise benefit and minimise harm.Though unanticipated risks are by definition not predictable, unintended risks could include escape of transgenic BSF, that are now able to exploit a broader scope of food sources, disrupting ecosystems or leading to the evolution of new pest species.Fungal laccases are able to degrade pesticides 111 and escapees engineered to express these enzymes may be difficult to eradicate.It is notable however that no genetically engineered microbial strain from a physically contained facility has escaped or caused known harm 165 .
The main biocontainment strategy to prevent transgenic BSF escape will be to rear BSF in physically contained facilities 134 .This strategy is widely used to date without issue for a variety of transgenic organisms from microbes to animals in both research and industrial settings 165,166 .Additional genetic biocontainment strategies could also be stacked for greater security.For example, by developing strains that cannot reproduce with their wild counterparts 136 and disrupting genes essential for survival in the wild but not in the contained composting facilities (e.g.flight, sight, eclosion etc.).Silkworms are an example of domesticated insects that cannot survive in the wild as they can only subsist on mulberry leaves and have now lost the ability to fly.Flightless BSF has been demonstrated by using CRISPR-Cas9 knock outs 137 .Moreover, survival could be regulated by synthetic auxotrophies to make strains dependent on compounds not available out of the contained facilities 165 .These tools have been demonstrated in various single cell organisms 165 .This could be achieved by engineering BSF with a repressible biomanufacturing genetic circuit.In the absence of the repressor, the biomanufacturing genetic circuit switches global gene expression from the prepupae life-stage onwards to favour only genes required for high-yield target biomolecule overexpression.All other genes are not expressed and the host is thereby rendered non-viable (Fig. 1).The repressor for the biomanufacturing genetic circuit could be supplied in the contained facilities for the maintenance of breeding colonies.

Conclusion
BSF is a promising synthetic biology platform to improve sustainability in biomanufacturing and allow greater valorisation of organic wastes.To turn this idea into a reality, further research is required to genetically characterise BSF as a synthetic biology platform and to develop BSF biomanufacturing strains that can produce high-value biomolecules on a broader scope of organic wastes.Entrepreneurs and BSF facilities will require regulatory approvals and facility upgrades to license these strains and begin turning waste into valuable products.Research about industry and public perceptions will be required to ensure products will be used.Organic waste is an abundant resource worldwide, and worldwide development of this technology may be influenced by access to funding for businesses and infrastructure; government regulatory processes; and whether scientists and BSF facilities can make products that will be adopted by downstream industries.Ultimately, BSF biomanufacturing may reduce landfilling, open dumping and burning of organic wastes and clean up biosolids to safely grow crops and close nutrient cycles.

Fig. 1 |
Fig. 1 | Engineering BSF for industrial biomanufacturing and bioremediation.BSF biomanufacturing facilities could valorise a broader variety of organic wastes into high-value products such as improved feed, oral therapeutics, industrial

Fig. 2 |
Fig. 2 | Developing the BSF synthetic biology platform.a Optimising lab-scale rearing protocols to minimise space requirements, simplified media formulations and tools for long-term storage.b Streamlining transgenesis protocols and characterising genetic parts such as tissue specific and small molecule regulated promoters.c Establishment of high-throughput enzyme testing and directed evolution capabilities in BSF cell culture.d Establishing the optimal expression patterning in the insect genetic model, D. melanogaster.Figure created using Adobe Illustrator, Adobe Stock Images under an education license and Biorender.com.

Table 2 |
Performance characteristics of example whole insects commonly used for commercial purposes

Table 3 |
73F Biomanufacturing and the SDGs SDG(s)Addressing the SDGs with BSF Biomanufacturing SDG #1: End poverty in all its forms everywhere • BSF biomanufacturing will offer economic opportunities and could be adopted worldwide to valorise widely available and underutilised organic wastes.SDG #2: End hunger, achieve food security and improved nutrition and promote sustainable agriculture• Compared to other biomanufacturing methods, BSF biomanufacturing does not require feedstocks derived from food crops and will improve food security.•BSFbiomanufacturingfor generating value-added animal feeds will improve nutrition and livestock productivity.BSF biomanufacturing for the generation of high-value products would incentivise the development of infrastructure to collect organic waste, reducing open dumping and the consequent proliferation of pests and contamination of waterways.•BSFbiomanufacturingcould generate products that can inexpensively clean up industrial wastewater.•BSFfarmingrequires significantly less water than livestock and horticulture73.SDG #8: Promote sustained, inclusive and sustainable economic growth, full and productive employment and decent work for all• BSF biomanufacturing provides exciting job opportunities for researchers and entrepreneurs that will help to solve some of the world's most pressing problems.