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OPEN

Inoculation with black soldier
fly larvae alters the microbiome
and volatile organic compound
profile of decomposing food waste
Rena Michishita 1,2, Masami Shimoda

3, Seiichi Furukawa

2& Takuya Uehara

1*

The black soldier fly (BSF; Hermetia illucens) is used in sustainable processing of many types oforganic waste. However, organic waste being decomposed by BSF produces strong odors, hindering
more widespread application. The odor components and how they are produced have yet to be
characterized. We found that digestion of food waste by BSF significantly alters the microbial
flora, based on metagenomic analyses, and the odor components generated, as shown by thermal
desorption gas chromatography mass spectrometry analysis. Inoculation with BSF significantly
decreased production of volatile organic sulfur compounds (dimethyl disulfide and dimethyl trisulfide),which are known to be released during methionine and cysteine metabolism by Lactobacillus andEnterococcus bacteria. BSF inoculation significantly changed the abundance of Lactobacillus andEnterococcus and decreased microbial diversity overall. These findings may help in optimizing use of
BSF for deodorization of composting food waste.
As the global population continues to grow, the amount of organic waste generated also continues to increase
and must be managed. For example, global average food waste is estimated at 121 kg/capita/year, according to
the report on organic food waste by the United Nations Environment P­ rogramme1, and Sustainable Developmental Goals (SDG) 12.3 aims to reduce this by half by 2030. Organic waste generated in cities is processed inthree major ways: incineration, landfill, and ­composting2. However, these waste disposal methods can cause
secondary pollution, such as generation of greenhouse gasses, odors, and contamination of groundwater. Use of
insects (crickets, litter beetle, black soldier fly, etc.) has recently shown promise for improved decomposition offood ­waste3. In particular, the black soldier fly (BSF, Hermetia illucens, Fig. 1a) can feed on many types of waste,
including food residues and livestock manure, and the adult flies can be used as an alternative source of protein
in livestock f­ eed4–7. Use of BSF also contributes to reduction of greenhouse gas emissions, such as N
­ H3 and­CH4, from livestock m­ anure8. Many aspects of BSF biology are being investigated, such as chromosome-scale
genome ­sequencing9, CRISPR-Cas9 genome ­editing10, low-cost ­rearing11, bioconversion ­rate12, and potential
application to circular ­bioeconomy13,the results of these studies may reveal ways to improve the suitability of
BSF for industrial-scale applications.
In most large-scale BSF breeding facilities, the waste containers and other facilities are open, and emit strong
odors. On the other hand, when the flies are reared in closed, small-scale facilities, the odors decrease withina few days even if the flies are fed partially decomposed food scraps. In other words, BSF can decompose foodwaste without generating strong odors. Here, changes in the composition of volatile organic compounds (VOCs),
especially those that generate odors, generated during processing of food scraps were identified by thermal desorption gas chromatography mass spectrometry (TDU-GC/MS) analysis. In addition, since the strong odors may
be generated by microbial decomposition of food waste, we investigated how addition of BSF to decomposing
food waste altered the microbial community by using metagenomic analysis.

1Division of Insect Sciences, Institute of Agrobiological Sciences, NARO, Tsukuba, Ibaraki 305‑8634,
Japan. 2Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305‑8572,
Japan. 3Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113‑8657, Japan. *email:tue@affrc.go.jp
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Figure 1.  Black soldier fly (BSF) and methods for inoculating the larvae to the artificial food waste. (a)Developmental stages of BSF. (b) Schematic diagram of the method for preparing artificial food waste. (c)Artificial food waste one week after BSF inoculation. (d) Larval weights before and after BSF inoculation.
Weights of larvae in the 3- and 10-larva vials were compared by using Student’s t-test. *Significant difference(P < 0.05).

Results

Digestion of organic waste by BSF larvae. The vials with and without BSF larvae differed in appearance. In no-larvae vials, only the upper surface of the food waste exposed to air was darker, and the food waste
contained pockets of gas generated during decomposition (Fig. 1c). In vials with larvae, the food waste was uniformly black from exposure to the air, as the larvae mixed the scraps by moving through the vial while feeding.
The vials with larvae did not contain pockets of gas.
The average weight of individual larva at inoculation was 26.96 ± 3.79 mg. After 7 days, the average weightof individual larva in 3- and 10-larva vials was 154.86 ± 5.46 mg and 129.85 ± 4.12 mg, respectively, increasing
about 4–6 times in 7 days (Fig. 1d). The weight of larva reared in 3-larva vials was significantly higher than thatof those reared in 10-larva vials.
Alteration of VOCs by inoculation of BSF larvae. The number of odor components detected in the 0-,3-, and 10-larva vials was 232.6 ± 27.1, 178.2 ± 6.2, and 182.0 ± 8.4, respectively (Fig. 2b,c). The three treatments
did not differ significantly in the number of components, but the no-larvae treatment had greater variation in thenumber of components. In all vials, the most prominent peak was limonene, likely from fruit peels. In the principal component analysis of VOCs, each treatment clustered tightly in the principal component space (Fig. 2d).
To investigate which odor components were altered by introduction of the BSF larvae, we performed a volcano
plot analysis of components that differed between the 0- and 10-larva vials (Fig. 3a). Levels of 64 components
were lower in the 10-larva vials than in the no-larvae vials (Supplementary Data 1). Aliphatic esters and alcohols,
as well as terpenes and sulfides were increased or decreased between treatments. Some of these components are
known to have foul odors, such as dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS) and trimethylamine.
In particular, comparison of DMDS and DMTS levels between the 0-, 3-, and 10-larva vials showed that theabundance of both components decreased with the addition of the BSF larvae (Fig. 3b).
To clarify the deodorizing effect of BSF larvae themselves, we added standard odor components to the artificial
diet and compared odor profiles after 7 days. The amount of odor components was not significantly altered by
BSF (Supplementary Fig. 1).
Changes in the microbiome in food waste following inoculation of BSF larvae. We carried out
metagenomic analysis of the microbes present in each treatment to assess how adding BSF to food waste changed
the microbiome (Fig. 4a). The 16S rRNA gene PCR and MinION sequencing and analysis showed that mostreads were assigned to 173 species and 34 genera (Supplementary Data 2, 3). To investigate the variation andqualitative differences in each treatment, we performed a non-metric multidimensional scaling (NMDS) analysis of species and number of reads. Data points for each of the three treatments clustered tightly in the NMDS
space (Fig. 4b). Comparing alpha diversity in each treatment, microbial diversity decreased as the number of
BSF larvae increased (Fig. 4c).
Abundances of genera in each treatment were compared by heatmap analysis (Fig. 4d). Bacillus was the mostabundant genus in every treatment. Companilactobacillus, Levilactobacillus, Lactobacillus, and Acetobacter were
found only in vials with larvae. Additionally, abundances of Bacillus, Companilactobacillus, and Levilactobacillus increased as the number of larvae increased. On the other hand, Enterococcus and Lacticaseibacillus were
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Figure 2.  Overview of thermal desorption gas chromatography mass spectrometry (TDU-GC/MS) analysis
of food waste. (a) Adsorptive medium and its use for adsorption of odor components. (b) Typical total ion
chromatograms. (c) Mean number of volatile organic compounds. Mean number of components did not differ
significantly (N.S.) between treatments (ANOVA). (d) Principal component (PC) analysis of odor components
by treatment.

Figure 3.  Odor components differentially detected between treatments. (a) Volcano plot analysis of odor
components detected in 0- and 10-larva vials. Blue and red squares show components detected with higher
abundance in 0- and 10-larva vials, respectively. Green lines show thresholds of fold change and P value. (b)Abundance of DMDS (upper) and DMTS (lower) in each treatment.
observed only in the no-larvae vials. Most of the microbes identified are anaerobic, and there was no evidence
that introduction of BSF favored the growth of either aerobic or anaerobic microorganisms.

Discussion

Our TDU-GC/MS analysis showed that inoculating food waste with BSF larvae caused qualitative and quantitative changes in VOC composition, which may explain the differences in the odors produced when BSF larvae areused for decomposition of food waste. Metagenomic analysis revealed that introduction of BSF also significantly
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Figure 4.  Overview of metagenomic analysis of microbes present in food waste. (a) Schematic diagram ofthe methods for metagenomic analysis. (b) Non-metric multidimensional scaling (NMDS) analysis of species
detected by 16 s amplicon sequencing. (c) Comparison of alpha diversity (Shannon Index) of species detectedin each treatment. (d) Heatmap analysis of genera detected in each treatment (no-larvae: BC01–BC05, 3-larva:
BC06–BC10, 10-larva: BC11–BC15).
changed the composition of the microbial community in food waste. These results suggest that introductionof BSF larvae into food waste causes physical and biochemical changes in the degradation process, altering the
microbial community and the odor composition.

Physicochemical and biological effects on microorganisms. Most previous studies focused on thegut microbial flora of BSF l­arvae10,14–16, and research focusing on the surrounding microbiological environmentis extremely limited. Studies on the BSF gut microbial flora identified Morganella and Dysgonomonas as genera
that are typically ­present10,14,17; however, we did not find these bacteria in our analyses. We found that introduction of BSF larvae to organic waste resulted in decreased alpha diversity, or species richness, of the microbial
flora; microbial diversity decreased as the number of larvae increased. Wu et al.18 also reported lower microbial
species diversity in larval frass, which is consistent with our results. The observed changes in microbial flora
seem to be due to physical and biochemical changes caused by the larvae, and to depend on the number of larvae
introduced, as described below.
The primary effect of introducing BSF larvae into food waste is physical agitation of the waste as the larvae
move through it by peristaltic action. We found that waste with and without larvae showed distinct physicaldifferences (Fig. 1c). In no-larvae vials, the gas produced by decomposition created pockets in the food waste.
Vials with larvae lacked these gas pockets because the agitation of the food waste by the larvae allowed the gas toescape to the vial headspace. Food waste in vials with larvae turned black, likely due to oxidation of polyphenols
contained in the fruit peels. In addition, the larvae were able to digest and metabolize available nutrients in thewaste and to grow to maturity. Although the growth rate of larvae varied with the age, larval weight increased by afactor of approximately 4 or more in 7 days in our experiments. Bioconversion rates and physicochemical changes
in organic wastes following introduction of BSF larvae have been reported in several s­ tudies15,19,20. In general,digestion by BSF larvae tends to make the pH of waste more basic (pH 8.0–9.0)21 and to decrease the nutrient­content15. The BSF would also have reduced microbial diversity by directly consuming the microbes present in
the substrate. These changes are likely to have a significant impact on the microbial community in the food waste.
BSF larvae can grow in environments with high levels of pathogenic microorganisms, such as livestock
manure, and are thought to have a highly developed immune ­system22. The BSF genome contains 50 genes that
encode antimicrobial peptides, one of the largest numbers so far identified in i­ nsects10. Several peptides, such as
defencin, diptericin, and stomoxyn, have been cloned from BSF and confirmed to show antimicrobial activity
in in vitro e­ xperiments23–25. A transgenic silkworm expressing these three genes showed an increased resistanceto both gram-positive and gram-negative entomopathogenic ­bacteria25. These findings suggest that not only
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physical but also biochemical effects, such as production of immunity-related compounds, are involved in thealteration of the microbial flora.

Effects on odor composition. Inoculation with BSF larvae changed the composition of odor components. This finding has also been reported following odor analysis of BSF fed on spent grain and fruit ­peels26.
We hypothesize that these changes reflect changes in the microbiota and the odor compounds they produce.
Although information on the relationship between VOC emission and microbial flora is extremely limited, several bacteria are reported to affect emission of VOCs. For example, volatile organic sulfur compounds (VOSCs)such as DMDS and DMTS are positively correlated with the presence of microorganisms including Lactobacillus and Leuconostoc27. Another study showed that Bacillus and Actinomycetes might produce VOSCs during­decomposition28. VOSCs are known as key components from meat and vegetable ­wastes27. During catabolism of
sulfur-containing amino acids such as methionine and cysteine, microorganisms including lactic acid bacteria
and Bacillus produce methanethiol, which can easily be converted to DMDS and ­DMTS29,30. Conversion of theseamino acids is catalyzed by microbial enzymes, and the enzymatic activity differs among ­strains29. We foundthat the abundance of both VOSCs and Lactobacillus, Leuconostoc, Lacticaseibacillus, and Enterococcus differedsignificantly between BSF-inoculated and control vials, which is consistent with these reports.
Although BSF larvae appear to reduce VOSC emission via alteration of the microbial flora, BSF larvae themselves seem to have less ability to reduce these components directly (Supplementary Fig. 1). For example, there
was no difference in levels of limonene, which is contained in fruit peels, between BSF-inoculated and control
vials. In other words, the lower the initial odor of the organic waste, the higher the deodorizing effect.
Potential use of BSF to control odors in organic waste recycling. In general, organic waste processing with BSF is known to produce strong odors. Our results, however, showed that the introduction of
BSF can reduce certain odor components. BSF processing is also likely to be effective for reducing the odor of
livestock ­manure31, and we believe that our results can be generalized not only to food waste but also to otherorganic waste. Below we present our recommendations for eliminating odors during processing of organic wastewith BSF. Our results show that the deodorizing effect of the BSF itself is small. Therefore, it is unlikely that BSF
can reduce the odor of organic waste that already has a strong odor by feeding on it. On the other hand, if theodor of the organic waste is caused by microbial decomposition, introduction of BSF at the early stage can behighly effective in eliminating odors. The insect’s ability to dramatically alter the surrounding microbiological environment would strongly inhibit the generation of odorous components such as DMDS and DMTS. To
maximize production of protein in the form of adult flies, it is important to have a high waste to larvae ratio.
However, to optimize deodorization, a lower waste to larvae ratio is preferable to maximize the biochemical andphysical effects.

Methods

Insects. Successive generations of lab-reared flies were used in this study. Flies were originally collected in
Tsukuba, Japan in 2013 and reared under a 16L:8D photoperiod, at 25 °C11. Eggs collected from colonies of adultflies were placed on a Petri dish and incubated for 3 days for hatching. Larvae were transferred to plastic containers (12 cm diameter, 10 cm height, Mineron-Kasei Co., Ltd., Osaka, Japan) and fed an artificial diet for thevinegar fly until inoculation to food waste.
Artificial food waste. We used an artificial food waste in the lab (Fig. 1b), based on the food found in a
typical Japanese home, using food from the following five categories: vegetables, fruits, carbohydrates, meat, andfish (Supplementary Table 1; Food Waste Suitable for Treatment Using Black Soldier Fly L­ arvae32). To create
decomposed waste prior to addition of larvae, freshly prepared waste was kept for 3 days in the same conditions
used for rearing the BSF. Decomposing food waste (10 g) was dispensed into a glass vial (2.12 cm diameter,7.55 cm height, Gerstel GmbH and Co., KG, Germany). Then 3, 5, or 10 individuals of 10-day old larvae (5thinstar larvae) were washed with distilled water, wiped with a paper towel and transferred to the vial. Vial openings were covered with a plastic mesh to prevent escape of larvae and to permit aeration. Vials without larvae
were used as controls. We collected samples 7 days after larval inoculation, because at this point microbial proliferation is high, making it ideal for sampling both VOC production and microbial diversity.
VOC sampling. Volatile components in the vial headspace were collected by passive adsorption onto a
Monotrap (Fig. 2a, RGC18 TD, GL-Science, Tokyo, Japan) for 3 h at room temperature. Following adsorption,
samples were transferred to a 1.5-ml vial and stored at − 20 °C until analysis.

TDU‑GC/MS analysis. Headspace volatiles collected using the Monotrap were analyzed by gas chromatography-mass spectrometry (GC–MS, GC: Agilent 7890A/MS5977B MSD, Agilent Technologies, CA, USA) withan HP-5MS UI capillary column (30 m, 0.25-mm ID, 0.25-μm film thickness; Agilent Technologies) equippedwith a thermo-desorption system, cooled injection, and cold trap (Gerstel GmbH and Co.). The GC was maintained at 40 °C for 3 min, increased to 150 °C at a rate of 10 °C/min, then to 280 °C at 20 °C/min, and held atthat temperature for 5 min. Helium was the carrier gas at a constant flow of 1.1 ml/min. The compounds weretentatively identified using data contained in the NIST Mass Spectral Library, 2017 release.
Data analysis. GC/MS data were deconvoluted using Unknowns Analysis software (ver. B.09.00, Agilent
Technologies Inc.) and aligned using Mass Profinder Professional (ver. 14.9, Agilent Technologies Inc.). Peaks

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with amplitudes of less than 1% of the maximum peak height were ignored. Only entities present in at least 60%
of replicates from one condition were included in subsequent analyses. To determine changes in components ineach condition, the data were subjected to principal component analysis by using the statistical software R (version 4.1.0)33. To determine which components varied between conditions, we performed volcano plot analysis
on the 0- and 10-individual vials (P value 0.05, fold change 2) in the Mass Profinder Professional software. Compound identification was performed by comparing mass spectra to the NIST 2017 Library.

Metagenomic analysis. DNA was extracted from the rotten food waste with NucleoSpin DNA Stool kits

(Macherey–Nagel GmbH & Co. KG, Düren, Germany) and quality checked by using a NanoDrop (Thermo
Fisher Scientific Inc., MA, USA) and Qubit 4 Fluorometer (Thermo Fisher Scientific Inc.). Sequencing libraries
were prepared with a 16S Barcoding Kit (SQK-16S024, Oxford Nanopore Technologies, UK) and sequenced byusing MinION (Oxford Nanopore Technologies). The data were demultiplexed by using the adapter trimming
tool ­Porechop34, and E­ mu35 was used to estimate sequence relative abundance, with default settings. NMDS
analysis was performed by using the vegan package in ­R36.

Data availability

All raw reads have been uploaded to the DNA Data Bank of Japan under BioProjectID PRJDB14354 and SRA
under DRA014852. Supplementary materials have been uploaded to the following data repository: https://​doi.​
org/​10.​6084/​m9.​figsh​are.​21534​735.
Received: 7 November 2022; Accepted: 10 March 2023

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Acknowledgements

We thank Drs. Tetsuya Kobayashi and Liu Chia-Ming for comments on the manuscript, and members of theInsect Design Technology Group for maintenance of the BSF colony. This work was supported by the Cabinet
Office, Government of Japan, Cross-ministerial Moonshot Agriculture, Forestry and Fisheries Research andDevelopment Program, “Technologies for Smart Bio-industry and Agriculture” (funding agency: Bio-oriented
Technology Research Advancement Institution) [JPJ009237]. This research was in partial fulfillment of an MSc
degree (RM) from the University of Tsukuba. The MinION image in Fig. 4a was provided by DBCLS Togo Picture
Gallery (©2016 DBCLS TogoTV; https://​togotv.​dbcls.​jp/​pics.​html).

Author contributions

R.M., M.S., and T.U. conceived the project and designed and interpreted all of the experiments. R.M. performed
the experiments shown in Figs. 1, 2, 3 and 4. M.S. helped to design and perform insect conditioning and artificial
food waste preparation. T.U. analyzed data for all figures and helped to perform TDU-GC/MS and 16s ampliconanalyses. R.M., M.S., and T.U. wrote the paper with help from S.F.

Competing interests

The authors declare no competing interests.

Additional information

Supplementary Information The online version contains supplementary material available at https://​doi.​org/​
10.​1038/​s41598-​023-​31388-z.
Correspondence and requests for materials should be addressed to T.U.
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Scientific Reports |(2023) 13:4297 |https://doi.org/10.1038/s41598-023-31388-z

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