ZHANG Ji-bin,ZHANG Jia,LI Jia-hui,Jeffery K.TOMERLIN,XIAO Xiao-peng,Kashif ur REHMAN,,CAI Min-min,ZHENG Long-yu,YU Zi-niu

1 State Key Laboratory of Agricultural Microbiology/National Engineering Research Center of Microbial Pesticides/College of Life Science and Technology,Huazhong Agricultural University,Wuhan 430070,P.R.China

2 Department of Entomology,Texas A&M University,TX 77843,USA

3 Livestock and Dairy Development Department,Poultry Research Institute,Rawalpindi 46000,Pakistan

Abstract The number of intensive livestock and poultry farms is expected to increase substantially in future because of consumer demand.Unfortunately,such demand also results in a great deal of manure being generated,which threatens the environment if it is not properly managed.Concurrent developments in biotransformation of these wastes with the black soldier fly (BSF),Hermetia illucens (L.) (Diptera:Stratiomydiae),demonstrates such concerns can be abated,while also producing products(e.g.,protein,chitin,biodiesel,and fertilizer) of value.In this review,we analyzed the factors influencing black soldier fly larvae (BSFL) conversion processes,the role of microorganisms,and the mechanisms used by BSFL when converting livestock and poultry manure into these valuable products.The effects of BSFL conversion technology on reducing the hazards of such materials and their associated pathogens are reviewed,and the economics of livestock and poultry manure conversion by BSF larvae is discussed.

Keywords:black soldier fly,livestock and poultry manure,conversion,mechanism,resource utilization

1.Introduction

Due to its rapid growth,the global human population is predicted to reach 10 billion by 2050 (Finley 2020).This increase in the global human population in conjunction with increased urbanization,and increased income per capita will change nutritional consumption habits (greater protein demands) with demands for meat and milk expected to increase by 70 and 58%,respectively during that time period (Makkaret al.2014;Finley 2020).Therefore,livestock production is expected to increase considerably due to high demand for meat,milk and animal byproducts,resulting in livestock and poultry manure accumulation(Chadwicket al.2011;Awasthiet al.2019).Mass production of livestock results in the production of manure,which is typically left untreated.Presently,only 20 to 30% of manure is treated either by composting,co-composting,or anaerobic digestion for methane production (Awasthiet al.2019).Thus,unprocessed livestock manure could result in environmental contamination,impacting soil,water,and air quality,which in turn poses a number of risks including the spread of pathogens (Konget al.2018;Ravindranet al.2019).It is therefore important to identify effective methods to manage and recycle such wastes.Presently,the treatment of livestock and poultry manure mainly relies on traditional methods,which include using it as organic fertilizer,feed,or the production of energy.However,these processes are time-consuming,offer low value,and are restricted (Fanet al.2019),and have disadvantages such as the spread of pathogens,the requirement for large areas of land,and high investment cost.Consequently,there is pressure to improve management of livestock and poultry manure,while protecting the environment.

A more sustainable,economically viable,and environmentally friendly solution for management of livestock and poultry manure is to convert it into insect biomass(Rehmanet al.2017a,b;Mazzaet al.2020).Insects are the most abundant and diverse group of animals on earth(Allegrettiet al.2018),and are a largely underutilized resource (Roffeiset al.2018).Many insects are able to feed on organic wastes and can effectively convert manure into insect biomass at low environmental cost.Several species,such asHermetiaillucensL.(Diptera:Stratiomyidae)(Rehmanet al.2019;Mazzaet al.2020;Zhanget al.2020),MuscadomesticaL.(Diptera:Muscidae) (Zhanget al.2012),andTenebriomolitorL.(Coleoptera:Tenebrionidae) (Wanget al.2017a;Thévenotet al.2018) are just a few that are being industrialized.Among these species,H.illucens,commonly known as the black soldier fly (BSF),thrives on organic waste (animal manure and food wastes) in nature(Nguyenet al.2015;Rehmanet al.2017a) and has been recognized as a valuable resource insect,which is capable of effectively transforming livestock and poultry manure into larval biomass and compost for producing animal feed and biofertilizer (Lalanderet al.2019).BSF larvae (BSFL)have potential to reduce the accumulation of livestock and poultry manure (Beskinet al.2018),degrade mycotoxin substances (Purschkeet al.2017) and inhibit pathogenic bacteria (Liuet al.2008).BSFL have advantages of being easy to feed,have a low feed cost and are capable of converting manure into higher value-added products than other resource insects,which has resulted in this species being industrialized globally.These features may allow BSFL to be used to reduce environmental contamination caused by livestock and poultry manure.

2.Factors influencing animal manure conversion by BSF larvae

Factors such as larval density (Yang 2010),manure temperature (Harnden and Tomberlin 2016) and pH (Ji 2017;Maet al.2018),type of manure (Mirandaet al.2019),co-digestion manure mixture (Rehmanet al.2017a) and strain ofH.illucens(Zhouet al.2013) may influence manure conversion efficiency.A comparison of different BSFL densities in swine manure (SM) determined that a density of 1 000 larvae kg–1manure resulted in optimal manure reduction (51.0%) and conversion rate (15.1%) (Yang 2010).

Because dairy manure (DM) has low nutrient and high fiber content,mixing DM with a higher nutrient content manure,such as chicken manure (CHM),may be necessary to optimize manure conversion by BSFL.DM and CHM mixed at different ratios results in different levels of biomass conversion.Rehmanet al.(2017a) found that DM and CHM mixed at a ratio of 2:3 resulted in better larval production,waste mass reduction and nutrient reduction than other manure mixtures ratios.In addition,consumption of the cellulose,hemicellulose and lignin by larvae was significantly greater than for dairy-only manure.A DM:CHM ratio of 2:3 resulted in both nutrient-rich larval output and higher organic mass reduction than with dairy manure in other mixtures ratios (Rehmanet al.2017a).Therefore in practical application the ratio of 2:3 could be used in the co-conversion of DM and CHM by BSFL.

Temperature is another important factor to consider in the mass production of BSFL.In manure,BSFL required less time to complete development and had a final weight～30%greater when reared at 24.9 to 32.2°C than when reared at 24.9°C (Harnden and Tomberlin 2016).Black soldier flies required a mean of 4 d longer to complete larval and pupal development at 27°C than at 30°C,whereas at 27 and 30°C,females weighed more than males on average but required longer time to complete larval development(Tomberlinet al.2009).

A study of the effect of initial pH on BSFL growth found that BSFL placed in a strong alkaline (pH 11.0) environment were able to develop into normal pupae,whereas at a substrate pH of 3.0,only 55% of the larvae developed into normal pupae (Ji 2017;Maet al.2018).Furthermore,larval development time in a substrate with an initial pH of 8.0 was approximately 3 d shorter than in substrates with initial pH of 6.0,7.0,or 10.0.BSFL final weight was found to be lowest when reared in substrates with initial pH of 2.0 or 4.0 and the highest when initial pH ranged from 6 to 10(Maet al.2018).

Several studies have shown that manure type significantly influences the development of BSF (Table 1).BSFL fed with CHM have significantly greater weight,require less development time,and have a significantly greater bioconversion rate of manure to BSF biomass than those fed swine manure (SW) and DM (Zhouet al.2013;Oonincxet al.2015;Mirandaet al.2019).Zhouet al.(2013) compared the ability of different BSF strains to convert manure to insect biomass.They found that BSF larvae of Wuhan strain had a higher weight gain and manure dry matter reduction rate than Guangzhou and Texas,USA strains,respectively.

3.Process development of livestock and poultry manure conversion by BSF larvae

Sheppardet al.(1994) built a manure management system using BSFL for caged laying hens where the layer house was modified to support breeding and self-harvesting of BSF larvae and prepupae.A slightly sloping concrete basin was constructed under the cage batteries so that prepupae migrating from the manure basins entered a plastic pipe at the top of the slope through a slot and were directed to holding containers.Females returning to oviposit near manure accumulating in basins under the hen battery cages created the large larval population.The manure management system was able to produce larvae with 42%protein and 35% fat,and reduced manure accumulation by at least 50% (Sheppardet al.1994).Newtonet al.(2005)described a system for on-farm BSF biodegradation of pig waste.In this system waste was collected on a conveyor belt located below the pigs which allowed for separation of manure solids.Manure solids were then conveyed to a larval basin containing larvae of mixed ages.Newly hatched BSF larvae obtained from a laboratory colony were used to maintain sufficient number of larvae in the manure basin.

A new process combining BSFL and microbes to manage chicken manure was developed by Xiaoet al.(2018a).Thirteen days were needed to convert CHM inoculated with functional bacteria to organic fertilizer and harvest BSFL.The moisture and pH of the chicken manure residue were around 52% and 8,respectively.Determination of the germination index of the manure residue is a commonly used procedure to indicate the maturity of the material.The chicken manure residue after co-conversion by BSFL and microbes had a higher maturity (germination index>92%) than residue treated with BSFL only (germination index～86%) (Xiaoet al.2018a).Rehmanet al.(2017a,b) developed a strategy for using BSFL to process DM mixed with CHM or soybean curd residue.This process significantly enhanced BSFL production,waste mass reduction,rate of larval conversion,feed conversion ratio,and fiber utilization.A mixture consisting of 40% DM and 60% CHM had significantly greater cellulose,hemicellulose and lignin consumption (61.2,53.2 and 42.2%,respectively)than DM only (49.89,49.77 and 31.95%,respectively)(Table 2) (Rehmanet al.2017a,b).

Recently,three methods for processing livestock and poultry manure using BSFL have been developed.The first is a manual method that can convert 10 tonnes of poultry manure per day (Xiaoet al.2018b).This model relies on manual labor to pre-treat the manure,feed it to the larvae,and harvest the resulting larvae and fertilizer.The second method is mechanized and is suitable for medium-sized enterprises.The third method is automated and it is mainly operated by mechines and remote control of the manure pre-treatment,manure feeding and residue discharge processes.This method can convert more manure moreefficiently,economically and using less labor than other methods (Zhanget al.2020).

Table 1 Comparison of selected parameters of black soldier fly conversion of different manures

4.The role of microorganisms in livestock and poultry manure conversion by BSF larvae

Microorganisms can be used to enhance BSFL waste digestion and protein production (Rehmanet al.2019;Zhanget al.2020).They have the ability to secrete various digestive enzymes (i.e.,amylase,lipase and protease,cellulases) in the BSFL gut and assist the BSFL in effectively hydrolyzing the fiber which is poorly digested by BSFL alone,and oxidize the fat in manure,and degrade protein and organic matter into smaller molecules for absorption by BSFL and to provide energy for BSFL metabolic activity (Kimet al.2011;De Smetet al.2018;Rehmanet al.2019).The inoculation of specific gut microbes and exogenous bacteria in BSFL may amplify manure decomposition.Microorganisms play a vital role for BSF larval biomass development and waste nutrient utilization.Yuet al.(2011) evaluated four strains ofBacillus subtilisisolated from BSFL for use as a prebiotic for CHM decomposition.It was demonstrated that inoculating CHM with these companion bacteria could enhance larval growth and development (Yuet al.2011).

Researchers have assessed symbiotic bacteria from BSF eggs and larvae gut for use as prebiotics for CHM decomposition.Mixed bacteria and BSFL had greater larvae weight gain and manure reduction rate (28.6 and 52.91%respectively) than BSFL alone.Bacterial treatment not only promoted BSFL growth and development and reduction of manure,but also increased protein and fat accumulation in larvae (Mazzaet al.2020).

Xiaoet al.(2018b) also studied co-conversion of BSFL with its gut bacteriaB.subtilisBSF-CL in CHM decomposition.In the presence of the gut bacteriaB.subtilisBSF-CL,the BSFL conversion rate increased by 12.7%,and CHM reduction rate increased by 13.4% compared to the control (noB.subtilisBSF-CL).The residue inoculated withB.subtilishad higher maturity (germination index >92%)than un-inoculated residue (germination index～86%) (Xiaoet al.2018b).

Historically,only bacteria associated with BSFL have been explored for their potential use as a prebiotic.Recently,Xiaoet al.(2018a) studied CHM degradation using the BSFL Wuhan strain assisted by exogenous bacteria isolated from chicken and pig manure.Their results indicated that compared with the control (no bacterial inoculation),bacterial inoculation increased BSFL survival rate by 10.25%,larval weight by 28.41%,conversion rate by 30.46%,and CHM reduction rate by 7.69%.This study demonstrated that exogenous bacteria could enhance BSFL conversion efficiency (Xiaoet al.2018a) and that the search and selection of microbes for such purposes should not be limited to those associated with BSFL.

Cellulose degradation is a challenge for the BSFL industry as degradation efficiency of such materials is very low.However,Rehmanet al.(2019) developed a strategy to combine cellulose-degrading bacteria with BSFL to break down and recycle cellulose in DM.It was found that cellulose breakdown was highly dependent on the ratio of the bacteria to the manure.BSF larvae assisted byBacillusstrain MRO2(R5),which was isolated and screened from pig manure fermentation at high temperature,had the best outcome with a manure reduction rate of 48.87%,bioconversion rate of 10.8%,food conversion ratio of 4.5,and cellulose reduction rate of 72.96%,hemicellulose reduction rate of 68.52%,lignin reduction rate of 32.86%,and the significant increase in the nutrient utilization (protein,71.20%;fat,67.80%) (Table 2) (Rehmanet al.2019).

5.Mechanism of livestock and poultry manure conversion by BSFL

Increasing evidence has shown that BSFL can be used for efficient management and bioconversion of animal manure (Sheppardet al.2002;Kimet al.2008;Dieneret al.2009).This ability lies within the anatomy of the BSFL gastrointestinal structures,digestive enzymes,and symbiotic systems.

The BSFL can be regarded as a waste reactor with the digestive system serving as the core (Goldet al.2018).The BSFL midgut is a critical section for decomposing the waste,and absorbing associated nutrients.The peculiar features and functions of each midgut region of BSFL have been investigated recently (Bonelliet al.2019).The luminal pH of the anterior,middle,and posterior midgut varies with values progressing from acidic (pH 5.9±0.1),strongly acidic(pH 2.1±0.1) to alkaline (pH 8.3±0.2).In the anterior region,ingested polysaccharides are degraded by amylases,while lipids are hydrolyzed by lipases.In the mid-region of the midgut,active lysozymes in combination with the luminal pH indicates that pathogen suppression could be occurring.The posterior region of the midgut plays a fundamental role in protein digestion by endo-and eso-peptidases,and it is responsible for further digestion of lipids and nutrient absorption (Bonelliet al.2019).

Enzymes are the most important driver for diet decomposition in the BSFL.They hydrolyse macronutrients into smaller molecules that can be absorbed through the gut wall (Chapmanet al.2013).Gut extracts of the BSFL have high amylase,lipase and protease activities,whereas enzyme activities in extracts of the salivary glands were much lower (Kimet al.2011).Higher activities of leucine arylamidase,α-galactosidase,β-galactosidase,α-mannosidase and α-fucosidase were observed in gut extracts of BSFL than in extracts from house fly,Musca domesticaL.(Diptera:Muscidae) larvae,which is another species used to recycle animal waste.The diversity and high activities of digestive enzymes in the gut of the BSFL contribute to their ability to digest large quantities of complex animal waste more quickly and efficiently than any other known species of fly (Kimet al.2011) (Fig.1).

Considering the digestive capacities of BSFL and the presence of the high enzymatic activity in their midgut,gut microbes may play an important role in aiding their host during the digestion of complex manure substrates (De Smetet al.2018).Moreover,studies have shown that a number of metabolic reactions occurring in the insect gut are encoded by the metagenome of its microbiota rather than by its own genome (Parket al.2007;Warneckeet al.2007).Each region of the BSFL midgut possesses its own features and a specific resident microbiota.

The microbiota of the midgut ofH.illucenslarvae has been described (Brunoet al.2019).Brunoet al.(2019)demonstrated that different midgut regions play a role in shaping microbial load and diversity.The microbiota of the anterior part of the midgut showed the highest diversity,which gradually decreased along the midgut,whereas bacterial load had an opposite trend,being the maximal in the posterior region.This shift in microbial diversity is potentially maintained by the shift in pH and the activity of antimicrobial peptides,lysozymes,and digestive enzymes secreted by midgut cells into the anterior and middle midgut(Brunoet al.2019) (Fig.1).

Fig.1 Mechanism of protein and fiber degradation in animal manure black soldier fly larvae (BSFL) conversion.

High abundances ofBacillusand yeasts were identified in the gut of BSFL (De Smetet al.2018;Zhanet al.2020).Bacillusis capable of lignocellulose degradation,while yeasts utilize metabolites and accumulate bacterial proteins and polysaccharides.By balancing nutrition (C/N ratio) and buffering capacity (pH) of the animal wastes,Rehmanet al.(2017a,b) developed an efficient co-digestion strategy for using BSFL to treat DM,which was mixed with an appropriate proportion of CHM or soybean curd.Moreover,adding exogenous probiotics resulted in greater bioconversion efficacy,larvae survival rate,BSFL weight gain,manure reduction rate and fiber content reduction,than using gut microbiota alone.By analyzing the fiber structural and chemical changes with scanning electron microscopy and Fourier-transformed infrared spectroscopy (FT-IR),it was found that the structure and chemical composition of the fiber had been modified.The surface morphology of fibers of DM40:CHM60 when exogenous probiotics were added was relatively compact and rigid before BSFL digestion,but after digestion it showed a porous and more corrugated structure.The effect on the fiber chemical composition was more obvious in the treatment when BSFL were assisted with addition of probiotics (Rehmanet al.2019) (Fig.1).

The BSFL gut microbiome and exogenous bacterial communities can produce and secrete microbial enzymes such as proteases,cellulases,lipases,xylanases and pectinases that degrade organic compounds in the animal manure (De Smetet al.2018).With this toolset,oxidation of fiber and fat can be achieved,resulting in energy release for BSFL metabolic activity.It appears that most protein and other organic matter in animal wastes is degraded by digestive enzymes.These results suggest that microbial richness and diversity in animal wastes influence the ability of BSFL to digest various organic wastes (De Smetet al.2018).

Molecular and genetic research can contribute to the use of the BSF for recycling particular types of waste.Genomic and transcriptomic study using next generation sequencing technology may lay a good foundation for molecular and genetic research in non-model organisms.Recently,a comprehensive omics approach,including genomics,transcriptomics and metagenomics,was utilized to explore the genetic bases underlying the key aspects of BSF biology (Zhanet al.2020).An omics method was used to understand genetic bases of BSF biology.The BSF genome encoded 1 798 duplicated genes,which is species-specific and expressed in late stage of larvae.We speculate these duplicated genes play an important role in organic waste bioconversion of BSFL.Furthermore,intestinal microbiota analysis of BSFL fed with kitchen waste,chicken manure,pig manure and diary manure was investigated.Firmicutes were identified to be the most dominant bacterial phylum in the midgut of BSFL and implied they are important in manure degradation as these microbes produce proteases and pectinases.Bacteroidetes dominated in the midguts of larvae fed with kitchen waste and chicken manure,respectively,and these bacterial phyla play an important role in degrading macromolecule organic wastes (Zhanet al.2020) (Fig.1).

6.Effect of BSFL on the degradation of hazardous substances in livestock and poultry manure

Livestock and poultry manure contain various hazardous materials such as antibiotics,mycotoxins,odorous substances,and heavy metals.In nature,BSFL not only thrive on manure,but also remediate contaminants.As with macronutrients,the decomposition of such contaminants involves a number of complex metabolic processes,mostly taking place in the larval midgut,malpighian tubules and fat bodies (Goldet al.2018).

In terms of antibiotics in animal manure,BSFL could provide an environmentally friendly manure treatment based on their ability to effectively and rapidly degrade tetracycline(TC).Nearly 97% of TC was found to be degraded within 12 days by BSFL,which is up to 1.6-times faster than that achieved by typical composting (Caiet al.2018c).The intestinal microbiota of BSFL play a significant role in the ability of the larvae to effectively and rapidly degrade TC.Caiet al.(2018c) provided novel insights into the metabolic mechanism of biodegradation of antibiotics in the management of organic waste by adjusting the intestinal microbiota of BSFL.Moreover,due to the influence of intestinal microbes,BSFL treatments effectively reduced antibiotic resistance genes (tet A,tet C,tet G,tet M,tet O,tet Q,tet T,tet Wandtet X) and integrin genes (intI 1andintI 2)by 95.0% during the 12-day process,which reduces not only the risk of antibiotics in the environment but also the selection of antibiotic resistant bacteria (Caiet al.2018b).

Suboptimal conditions during storage of feed products may lead to the growth of endophytic and saprophytic fungi that can produce mycotoxins.Such contamination has large financial implications for the feed industry as such adulterated materials cannot be used as feed for livestock and poultry.Recently,Boschet al.(2017) found that BSFL larvae have a high aflatoxin B1 tolerance and that the toxin did not accumulate in harvested larvae.Purschkeet al.(2017) also reached a similar conclusion that mycotoxins had neither been accumulated in the BSFL tissue nor significantly affected the growing conditions.To further understand the metabolic processes,Meijeret al.(2019)investigated the conversion of aflatoxin B1 by S9 extracts of BSFL reared on a contaminated substrate.The S9 enzyme fraction contains both the membrane bound as well as the soluble enzymes.The study showed that approximately 60% of aflatoxin B1 was converted to aflatoxicol and aflatoxin P1.The S9 fraction of BSFL contains cytochrome P450s that metabolically convert aflatoxin B1 into aflatoxin P1,while cytoplasmic reductase is responsible for conversion of aflatoxin B1 into aflatoxicol,for which the toxicity is 18 times lower than that of aflatoxin B1(Meijeret al.2019).

Livestock and poultry manure usually release volatile odorous pollutants.Such odors could be reduced during BSFL conversion of manure.The production of odors,such as 4-methyl phenol,ethyl phenol,3-methyl indole and indole,are reduced by BSFL activity.Short chain fatty acids,such as acetic acid and propionic acid,were found to be completely absent after 48 h of digestion of swine or dairy manure by BSFL,while long-chain fatty acids,such as pentanoic acid and butyric acid,were absent after 72 h(Beskinet al.2018).Chenet al.(2019a) found that BSFL larvae could reduce total greenhouse gas (such as CO2,CH4,N2O and NH3) emissions by 72.63–99.99% compared with conventional composting of pig manure.

Heavy metals,such as copper,zinc or cadmium,are essential for metabolic processes in BSFL.However,high concentrations of such materials in animal wastes could influence the growth and development of BSFL (Purschkeet al.2017;Caiet al.2018a).Wuet al.(2020) examined the influence of copper and cadmium on the growth and gut microbiota of BSFL and the distribution of accumulated heavy metals in the larvae and their feces.It was observed that the contents of both copper and cadmium accumulated in the bodies and feces of BSFL were greatly increased with elevated exposure doses,however the weight gain of BSFL was not significantly inhibited by exposure to Cu (from 100 to 800 mg kg–1) and Cd (from 10 to 80 mg kg–1).In BSFL feces,copper and cadmium mainly existed as residues and in water-soluble states,respectively.Exposure to copper and cadmium remarkably altered the intestinal microbiota of BSFL,particularly in the phyla Proteobacteria,Firmicutes and Bacteroidetes.By investigating the influences of chromium and cadmium on the development of BSF larvae,Gaoet al.(2017) found that chromium and cadmium have effects on larval duration and pupation rate but not on larval eclosion and survival.Although both chromium and cadmium were transferred into larvae,prepupae,and pupae,they were accumulated at low levels in prepupae and pupae.The results suggested that larvae should be harvested at later life stages in order to reduce heavy metal risk.Uptake of cadmium in housefly larvae is mediated through calcium channels in the gut cells.As BSFL have more than ten times the level of calcium than housefly larvae,they may sequester more cadmium than housefly larvae (Wanget al.2016).

7.Mechanism of pathogen inhibition in livestock and poultry manure by BSF larvae

Livestock and poultry manures are ideal reservoirs for many significant foodborne pathogens,includingEscherichia coliO157:H7 andSalmonellaspp.Therefore,it is of key importance to assess how the abundance of these pathogens is affected by the presence of BSFL to ensure they do not accumulate in the larvae or remain in the residue.

Black soldier fly larvae are able to suppress a number of pathogens in animal waste.However,the effectiveness of pathogen reduction may depend on the temperature and the composition of the manure.Ericksonet al.(2004)determined that BSFL could suppressE.coliO157:H7 andS.entericaserovar Enteritidis in CHM.Over a storage period of 3 to 6 days,BSFL accelerated inactivation ofE.coliO157:H7 in CHM at temperatures between 27 and 32°C,but had no effect in CM and enhanced survival in SM.During the initial two-to six-day period after allowing BSFL to feed on contaminated manure,S.entericaserovarEnteritidis populations in CHM were reduced.The ability of BSFL to reduceE.colipopulations in DM was investigated by Liuet al.(2008).They determined BSFL can significantly reduceE.colicounts in DM.However,it should be noted that the greatest level of suppression occurred at 27°C.Interestingly,researchers found a novel,temperateEscherichiabacteriophage designated vB_EcoS_PHB10(PHB10) from the black soldier fly gut,which could lyse two out of 13Escherichiastrains tested (Chenet al.2019).Lalanderet al.(2015) found that BSFL not only inhibited pathogenic bacteria and fungi,but also viruses(i.e.,Adenovirus,Reovirus,and Enterovirus).Elhaget al.(2017) identified seven new gene fragments of three types of antimicrobial peptides from BSFL.Among these types of antimicrobial peptides,stomoxynZH1 was expressed inE.colias a fusion protein with thioredoxin.StomoxynZH1 exhibits diverse inhibitory activity on various pathogens,including Gram-positive bacteriumStaphylococcusaureus,Gram negative bacteriumE.coli,and the fungiRhizoctonia solaniKhün (rice)-10 andSclerotiniasclerotiorum(Lib.) de Bary-14 (Elhaget al.2017).

Immunity of BSFL to pathogens has been investigated at the genetic level.Zhanet al.(2020) reported a highquality genome of 1.1 Gb and a consensus set of 16 770 gene models ofHermetiaillucens,and explored the genetic bases underlying the key aspects of BSF biology.Compared to other dipteran species,the BSF genome has undergone a substantial expansion in functional modules related to adaptation to septic environments,including immune system factors,olfactory receptors,and cytochrome P450s.In the BSF genome,a full set of immune-related genes were annotated and further found to have notable expansions in both recognition and effector molecules.Expansion of recognition components may also facilitate responses to diverse pathogens.Accordingly,the BSF immune system might have adapted to mitigate pathogenic microbes.Furthermore,the genomes of BSF encode more secreted peptidoglycan recognition proteins (PGRPs) and gramnegative binding proteins (GNBPs) compared to those in other dipteran species.The PGRPs regulate signaling pathways during bacterial infection and GNBPs participate in the activation of the Toll pathway.The BSF genome also encodes 50 antimicrobial peptides (AMPs),making it the largest AMP family yet identified in insects.Altogether,these results suggest that expansions of genes involved in the immune response underlie the adaptation of BSF to a pathogen-rich environment such as animal manure and even food waste (Zhanet al.2020).Further research found two BSF immune genesBsfDuoxandBsfTLR3could regulate the key gut bacteriaProvidenciaandDysgonomonashomeostasis to depress zoonotic pathogens (Huanget al.2020).

8.Resource utilization and economic analysis of livestock and poultry manure conversion by BSFL

Countries are producing and accumulating more animal manure with fewer options for its disposal.This situation presents a pressing need for holistic research into strategies and technology for the management and treatment of manure.The treatment of animal manure by BSF larvae is gaining attention (Čičkováet al.2015;Henryet al.2015;Lohriet al.2017;Goldet al.2018;De Smetet al.2018).As previously demonstrated,BSF is now considered to be a resource species for processing animal manure as it is capable of reducing animal waste efficiently,while converting it into insect bio-mass,suppressing pathogenic bacteria,and degrading antibiotics and other contaminants (Tomberlin and van Huis 2020).In addition,BSF has advantages of high fertility and a short life cycle (Liuet al.2019).Sheppardet al.(1994) established an efficient,low-cost poultry manure treatment system using BSF larvae.The system converted half the manure into protein (42%) and grease(35%),and the nitrogen content and the abundance ofE.coliin the manure were significantly reduced (Sheppardet al.1994;Ericksonet al.2004).The nutrient values of the converted organic matter fully met the standard for organic fertilizer (Newtonet al.2005).Additionally,the nitrogen,phosphorus and potassium contents of poultry manure were found to decrease by 55.1,44.1,and 52.8%,respectively,after conversion (Newtonet al.2005).These observations demonstrate that BSFL can offer significant environmental benefits in the conversion of livestock excrement (Liet al.2011a).The Insect–Manure–Digestion treatment system has been adapted to the treatment of waste from animal farming and has achieved satisfactory results (Nguyenet al.2013;Oonincxet al.2015;Nyakeriet al.2017;Rehmanet al.2017b).

Due to the abundance of enzymes and microbes in the intestine of BSFL,they can effectively convert organic waste into BSFL biomass and high-quality fertilizers.Dry matter content of BSF prepupae has been shown to be 33–44%(Sheppardet al.1994;Dieneret al.2009).BSFL biomass contains higher levels of proteins,amino acids,fats,and carbohydrates than other mass-produced insects.BSFL also contain essential amino acids,fatty acids,mineral elements,vitamins and other active substances.The protein derived from BSFL has been used as animal feed(Barraganfonsecaet al.2017;Liuet al.2017;Schiavoneet al.2017) and as a substitute for fish meal (St-Hilaireet al.2007).The grease derived from the BSFL has been used as raw material in the extraction of biodiesel (Nguyenet al.2017;Wanget al.2017b;Rehmanet al.2018).The resulting biodiesel compares favorably with plant-based biodiesels and meets selected criteria of EN14214 (Liet al.2011a,b;Zhenget al.2012;Rehmanet al.2018).Furthermore,BSFL can be used as a raw material for the extraction of biologically active substances (e.g.,antimicrobial peptides) (Parket al.2015;Elhaget al.2017;Park and Yoe 2017;Vogelet al.2018),which may be useful for the pharmaceutical industry.The antimicrobial peptide derived from BSFL has been shown to possess greater thermal stability than antimicrobial peptides derived from other insects and to exhibit good performance after repeated freezing and thawing (Elhaget al.2017).The resource value of BSF products derives not only from their protein,grease,and antimicrobial peptides but also from the properties of their chitin (Wanget al.2020).Chitin derived from crustaceans is a linear biological biopolymer and has been widely used in the textiles industry,papermaking,agriculture and forestry,food,medicine,bio-engineering,and other fields.The changes in physicochemical characteristics of chitin were investigated at every stage of BSF development.Fourier transform infrared spectroscopy,thermogravimetric analysis and X-ray diffraction analysis showed that chitin from different larval and pupae stages of BSF was all α-chitin with similar thermal stability (Bieganowskiet al.2016).The chitin level in BSF was the highest in the puparium and the lowest in the adults (Wanget al.2020).Surface morphology characteristics of chitin was found by scan electron microscopy to vary significantly in the developmental phases(Wanget al.2020).

The management of animal wastes is both costly and a growing environmental concern.However,BSFL are easy to rear and harvest and at higher reproduction rates and low feeding costs.Newtonet al.(2005) suggested that,under select conditions in North America,BSFL used for direct processing of manure in poultry houses could return US$25 000 per house per year with minimal initial investment and maintenance costs.Dieneret al.(2009) established that an 1 100 m2plant processing three tons of organic waste(wet weight) per day yielded an annual revenue of US$55 000.In China,BSF industrialization has grown greatly due to its ability to convert wastes into biomaterials of value safely and effectively.Companies in many Chinese provinces are able to convert livestock manure at a large scale using a variety of models (e.g.,some companies convert 10 tons of poultry manure per day) (Zhanget al.2020).Manure conversion by BSF can be not only operated with manual mode technology in developing nations with minimal capital investment,but also with automated methods to produce consistent,safe,high-quality products.

9.Perspective

The large amount of livestock and poultry manure produced by industrial feeding has a great impact on the environment.On the other hand,the need for more sustainable alternative sources of protein is growing rapidly.A growing body of evidence supports and justifies the sustainable use of insects as a means to produce protein products as feed for livestock,poultry,and aquaculture.One of the most promising species being reared is the black soldier fly(H.illucens).BSFL manure processing is an emerging manure treatment technology which can produce marketable high-value products (organic fertilizer,insect biomass)while consuming livestock and poultry manure.This technology contributes towards sustainable and financially viable resource recovery-based waste management.Factors influencing conversion of manure by BSF,process development,the role of microorganisms in livestock and poultry manure conversion by BSF larvae,and mechanisms and the economic value of livestock and poultry manure conversion by BSFL are reviewed and discussed.Moreover,the effect of BSFL conversion technology on degradation of hazardous substances and inhibition of associated pathogens are also reviewed and discussed.

The promising ability of BSFL to convert animal waste is encouraging,and microbes play an important role in this process.Some microbes could help improve waste conversion efficiency and effectiveness.Bacterial genes encoding enzymes (i.e.,protease,cellulase and lipase)in the BSFL gut can hydrolyze cellulose,proteins and lipids,and can thereby contribute to the decomposition and recycling of biological waste and other accumulated nutrients in insect hosts.However,the mechanisms of BSFL gut microbes in waste conversion are still unclear.Further research should focus on the mechanism of BSFL intestinal microbe interaction with the host and effects on animal waste degradation,BSFL growth and development,and pathogen inhibition in manure.

Industrialization to mass produce BSFL will allow more efficient and economical conversion of livestock and poultry manure.However,there are a number of hurdles related to adult colony and egg production management to overcome.Establishment of standard production techniques appears to be important to ensure that process performance can meet the requirements for large amounts of animal waste conversion.In order to apply BSFL to large-scale livestock and poultry manure treatment,it is necessary to optimize the conversion process,including egg production and hatching,larval survival and adult emergence.BSFL production industrialization can establish a circular economy with minimal wastes remaining after digestion.Quality and safety of conversion products for application should be evaluated and it will be essential to establish a BSFL product and organic fertilizer standard for different animal waste conversions.

Acknowledgements

This work was supported by the National Key R&D Program of China (2018YFD0500203 and 2018YFF0213503) and the National Natural Science Foundation of China (31770136).

Declaration of competing interest

The authors declare that they have no conflict of interest.