Microbial Ecology

, Volume 77, Issue 4, pp 913–930 | Cite as

Assessing the Microbiota of Black Soldier Fly Larvae (Hermetia illucens) Reared on Organic Waste Streams on Four Different Locations at Laboratory and Large Scale

  • E. WynantsEmail author
  • L. Frooninckx
  • S. Crauwels
  • C. Verreth
  • J. De Smet
  • C. Sandrock
  • J. Wohlfahrt
  • J. Van Schelt
  • S. Depraetere
  • B. Lievens
  • S. Van Miert
  • J. Claes
  • L. Van Campenhout
Environmental Microbiology


This study aimed to gain insight into the microbial quality, safety and bacterial community composition of black soldier fly larvae (Hermetia illucens) reared at different facilities on a variety of organic waste streams. For seven rearing cycles, both on laboratory-scale and in large-scale facilities at several locations, the microbiota of the larvae was studied. Also samples of the substrate used and the residue (= leftover substrate after rearing, existing of non-consumed substrate, exuviae and faeces) were investigated. Depending on the sample, it was subjected to plate counting, Illumina Miseq sequencing and/or detection of specific food pathogens. The results revealed that the substrates applied at the various locations differed substantially in microbial numbers as well as in the bacterial community composition. Furthermore, little similarity was observed between the microbiota of the substrate and that of the larvae reared on that substrate. Despite substantial differences between the microbiota of larvae reared at several locations, 48 species-level operational taxonomic units (OTUs) were shared by all larvae, among which most belonged to the phyla Firmicutes and Proteobacteria. Although the substrate is assumed to be an important source of bacteria, our results suggest that a variety of supposedly interacting factors-both abiotic and biotic-are likely to affect the microbiota in the larvae. In some larvae and/or residue samples, potential foodborne pathogens such as Salmonella and Bacillus cereus were detected, emphasising that decontamination technologies are required when the larvae are used in feed, just as for other feed ingredients, or eventually in food.


Hermetia illucens Microbiota Laboratory scale Industrial scale High-throughput sequencing Plate counts 



The research that yielded these results was funded by the Belgian Federal Public Service of Health, Food Chain Safety and Environment through the contract RT 15/9 EDINCO.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethical Approval

All applicable international, national and/or institutional guidelines for the care and use of animals were followed.

Supplementary material

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  1. 1.
    Lalander CH, Fidjeland J, Diener S, Eriksson S, Vinnerås B (2015) High waste-to-biomass conversion and efficient Salmonella spp. reduction using black soldier fly for waste recycling. Agron Sustain Dev 35(1):261–271Google Scholar
  2. 2.
    European Commission (2015) Flash Eurobarometer 425. Report: Food waste and date marking. Accessed 10 August 2018
  3. 3.
    Martinez J, Dabert P, Barrington S, Burton C (2009) Livestock waste treatment systems for environmental quality, food safety and sustainability. Bioresour Technol 100:5527–5536Google Scholar
  4. 4.
    Diener S, Zurbrügg C, Tockner K (2009) Conversion of organic material by black soldier fly larvae: establishing optimal feeding rates. Waste Manag Res 27(6):603–610Google Scholar
  5. 5.
    van Huis A (2013) Potential of insects as food and feed in assuring food security. Annu Rev Entomol 58:563–583Google Scholar
  6. 6.
    Salomone R, Saija G, Mondello G, Giannetto A, Fasulo S, Savastano D (2017) Environmental impact of food waste bioconversion by insects: application of life cycle assessment to process using Hermetia illucens. J Clean Prod 140(2):890–905Google Scholar
  7. 7.
    Jeon H, Park S, Choi J, Jeong G, Lee S, Choi Y, Lee S (2011) The intestinal bacterial community in the food waste-reducing larvae of Hermetia illucens. Curr Microbiol 62:1390–1399Google Scholar
  8. 8.
    Nguyen TTX, Tomberlin JK, Vanlaerhoven S (2015) Ability of black soldier fly (diptera: stratiomyidae) larvae to recycle food waste. Environ Entomol 44(2):406–410Google Scholar
  9. 9.
    Sheppard DC, Newton GL, Thompson SA, Savage S (1994) A value added manure management system using the black soldier fly. Bioresour Technol 50(3):275–279Google Scholar
  10. 10.
    Myers HM, Tomberlin JK, Lambert B, Kattes D (2008) Development of black soldier fly (diptera: stratiomyidae) larvae fed dairy manure. Environ Entomol 37(1):11–15Google Scholar
  11. 11.
    Li Q, Zheng L, Cai H, Garza E, Yu Z, Zhou S (2011) From organic waste to biodiesel: black soldier fly, Hermetia illucens, makes it feasible. Fuel 90:1545–1548Google Scholar
  12. 12.
    Lalander C, Diener S, Magri ME, Zurbrügg C, Lindström A, Vinnerås B (2013) Faecal sludge management with the larvae of the black soldier fly (Hermetia illucens)—from a hygiene aspect. Sci Total Environ 45:312–318Google Scholar
  13. 13.
    Banks IJ, Gibson WT, Cameron MM (2014) Growth rates of black soldier fly larvae fed on fresh human faeces and their implication for improving sanitation. Tropical Med Int Health 19(1):14–22Google Scholar
  14. 14.
    Stadtlander T, Stamer A, Buser A, Wohlfahrt J, Leiber F, Sandrock C (2017) Hermetia illucens meal as fish meal replacement for rainbow trout on farm. J Insects Food Feed 3(3):165–175Google Scholar
  15. 15.
    Henry M, Gasco L, Piccolo G, Fountoulaki E (2015) Review on the use of insects in the diet of farmed fish: past and future. Anim Feed Sci Technol 203:1–22Google Scholar
  16. 16.
    Maurer V, Holinger M, Amsler Z, Früh B, Wohlfahrt J, Stamer A, Leiber F (2015) Replacement of soybean cake by Hermetia illucens meal in diets for layers. J Insects Food Feed 1:1–8Google Scholar
  17. 17.
    Wang YS, Shelomi M (2017) Review of black soldier fly (Hermetia illucens) as animal feed and human food. Foods 6(10):91Google Scholar
  18. 18.
    Zheng L, Hou Y, Li W, Yang S, Li Q, Yu Z (2012) Biodiesel production from rice straw and restaurant waste employing black soldier fly assisted by microbes. Energy 47:225–229Google Scholar
  19. 19.
    Elieh-Ali-Komi D, Hamblin MR (2016) Chitin and chitosan: production and application of versatile 653 biomedical nanomaterials. Int J Adv Res 4:411–427Google Scholar
  20. 20.
    Dillon RJ, Dillon VM (2004) The gut bacteria of insects: nonpathogenic interactions. Annu Rev Entomol 49(1):71–92Google Scholar
  21. 21.
    Engel P, Moran NA (2013) The gut microbiota of insects–diversity in structure and function. FEMS Microbiol Rev 37(5):699–735Google Scholar
  22. 22.
    EFSA Scientific Committee. (2015). Risk profile related to production and consumption of insects as food and feed. EFSA Journal 2015, 13(10), 60 pp.
  23. 23.
    Boccazzi IV, Ottoboni M, Martin E, Comandatore F, Vallone L, Spranghers T, Eeckhout M, Mereghetti V, Pinotti L, Epis S (2017) A survey of the mycobiota associated with larvae of the black soldier fly (Hermetia illucens) reared for feed production. PLoS One 12(8):e0182533Google Scholar
  24. 24.
    Erickson MC, Islam M, Sheppard C, Liao J, Doyle MP (2004) Reduction of Escherichia coli O157: H7 and Salmonella enterica serovar enteritidis in chicken manure by larvae of the black soldier fly. J Food Prot 67(4):685–690Google Scholar
  25. 25.
    Čičková H, Newton GL, Lacy RC, Kozánek M (2015) The use of fly larvae for organic waste treatment. Waste Manag 35:68–80Google Scholar
  26. 26.
    Liu Q, Tomberlin JK, Brady JA, Sanford MR, Yu Z (2008) Black soldier fly (Diptera: Stratiomyidae) larvae reduce Escherichia coli in dairy manure. Environ Entomol 37(6):1525–1530Google Scholar
  27. 27.
    Xiao X, Mazza L, Yu Y, Cai M, Zheng L, Tomberlin JK, Yu J, van Huis A, Yu Z, Fasulo S, Zhang J (2018) Efficient co-conversion process of chicken manure into protein feed and organic fertilizer by Hermetia illucens L.(Diptera: Stratiomyidae) larvae and functional bacteria. J Environ Manag 217:668–676Google Scholar
  28. 28.
    De Smet J, Wynants E, Cos P, Van Campenhout L (2018) Microbial community dynamics during rearing of black soldier fly larvae (Hermetia illucens) and impact on exploitation potential. Appl Environ Microbiol 84(9):e02722–e02717Google Scholar
  29. 29.
    Cheng JY, Chiu SL, Lo IM (2017) Effects of moisture content of food waste on residue separation, larval growth and larval survival in black soldier fly bioconversion. Waste Manag 67:315–323Google Scholar
  30. 30.
    Stoops J, Crauwels S, Waud M, Claes J, Lievens B, Van Campenhout L (2016) Microbial community assessment of mealworm larvae (Tenebrio molitor) and grasshoppers (Locusta migratoria migratorioides) sold for human consumption. Food Microbiol 53:122–127Google Scholar
  31. 31.
    Dijk, R., van den Berg, D., Beumer, R., de Boer, E., Dijkstra, A., Mout, L., … in ’t Veld, S. (2015). Microbiologie van voedingsmiddelen: Methoden, principes en criteria. (R. Dijk, D. van den Berg, R. Beumer, E. de Boer, A. Dijkstra, L. Mout, … S. in ’t Veld, Eds.) (5th ed.). Capelle aan den Ijssel, The Netherlands: MYbusinessmediaGoogle Scholar
  32. 32.
    Wynants E, Crauwels S, Verreth C, Gianotten N, Lievens B, Claes J, Van Campenhout L (2018) Microbial dynamics during production of lesser mealworms (Alphitobius diaperinus) for human consumption at industrial scale. Food Microbiol 70:181–191Google Scholar
  33. 33.
    Chao A (1984) Nonparametric estimation of the number of classes in a population. Scand J Stat 11:265–270Google Scholar
  34. 34.
    Pielou EC (1966) The measurement of diversity in different types of biological collections. J Theor Biol 13:131–144Google Scholar
  35. 35.
    Shannon CE (1948) A mathematical theory of communication. Bell Syst Tech J 27:379–423 & 623–656Google Scholar
  36. 36.
    Development Core Team R (2013) R: a language and environment for statistical computing. R Foundation for Statistical Computing, ViennaGoogle Scholar
  37. 37.
    Adams MR, Moss MO (2008) Food microbiology3d edn. RSC Publishing, CambridgeGoogle Scholar
  38. 38.
    Park SI, Kim JW, Yoe SM (2015) Purification and characterization of a novel antibacterial peptide from black soldier fly (Hermetia illucens) larvae. Dev Comp Immunol 52(1):98–106Google Scholar
  39. 39.
    Zdybicka-Barabas A, Bulak P, Polakowski C, Bieganowski A, Waśko A (2017) Immune response in the larvae of the black soldier fly Hermetia illucens. ISJ 14:9–17Google Scholar
  40. 40.
    Liu X, Chen X, Wang H, Yang Q, ur Rehman K, Li W, Cai M, Li Q, Mazza L, Zhang J, Yu Z, Zheng L (2017) Dynamic changes of nutrient composition throughout the entire life cycle of black soldier fly. PLoS One 12(8):e0182601Google Scholar
  41. 41.
    Pimentel AC, Montali A, Bruno D, Tettamanti G (2017) Metabolic adjustment of the larval fat body in Hermetia illucens to dietary conditions. J Asia Pac Entomol 20(4):1307–1313Google Scholar
  42. 42.
    Vogel H, Müller A, Heckel DG, Gutzeit H, Vilcinskas A (2018) Nutritional immunology: diversification and diet-dependent expression of antimicrobial peptides in the black soldier fly Hermetia illucens. Dev Comp Immunol 78:141–148Google Scholar
  43. 43.
    Vandeweyer D, Crauwels S, Lievens B, Van Campenhout L (2017) Microbial counts of mealworm larvae (Tenebrio molitor) and crickets (Acheta domesticus and Gryllodes sigillatus) from different rearing companies and different production batches. Int J Food Microbiol 242:13–18Google Scholar
  44. 44.
    Vandeweyer D, Crauwels S, Lievens B, Van Campenhout L (2017) Metagenetic analysis of the bacterial communities of edible insects from diverse production cycles at industrial rearing companies. Int J Food Microbiol 261:11–18Google Scholar
  45. 45.
    Zheng L, Crippen TL, Singh B, Tarone AM, Dowd S, Yu Z, Wood TK, Tomberlin JK (2013) A survey of bacterial diversity from successive life stages of black soldier fly (Diptera: Stratiomyidae) by using 16S rDNA pyrosequencing. J Med Entomol 50(3):647–658Google Scholar
  46. 46.
    Filippidou S, Junier T, Wunderlin T, Lo CC, Li PE, Chain PS, Junier P (2015) Under-detection of endospore-forming Firmicutes in metagenomic data. Comput Struct Biotechnol J 13:299–306Google Scholar
  47. 47.
    Astudillo-García C, Bell JJ, Webster NS, Glasl B, Jompa J, Montoya JM, Taylor MW (2017) Evaluating the core microbiota in complex communities: a systematic investigation. Environ Microbiol 19(4):1450–1462Google Scholar
  48. 48.
    Yu G, Cheng P, Chen Y, Li Y, Yang Z, Chen Y, Tomberlin JK (2011) Inoculating poultry manure with companion bacteria influences growth and development of black soldier fly (Diptera: Stratiomyidae) larvae. Environ Entomol 40(1):30–35Google Scholar
  49. 49.
    Zheng L, Crippen TL, Holmes L, Singh B, Pimsler ML, Benbow ME, Tarone AM, Dowd S, Yu Z, Vanlaerhoven SL, Wood TK, Tomberlin JK (2013) Bacteria mediate oviposition by the black soldier fly, Hermetia illucens (L.) (Diptera: Stratiomyidae). Sci Rep 3:2563Google Scholar
  50. 50.
    Osimani A, Milanović V, Cardinali F, Garofalo C, Clementi F, Pasquini M, Riolo P, Ruschioni S, Isidoro N, Loreto N, Franciosi E, Tuohy K, Petruzzelli A, Foglini M, Gabucci C, Tonucci F, Aquilanti L (2018) The bacterial biota of laboratory-reared edible mealworms (T. molitor L.): from feed to frass. Int J Food Microbiol 272:49–60Google Scholar
  51. 51.
    Early AM, Shanmugarajah N, Buchon N, Clark AG (2017) Drosophila genotype influences commensal bacterial levels. PLoS One 12(1):e0170332Google Scholar
  52. 52.
    Dobson AJ, Chaston JM, Newell PD, Donahue L, Hermann SL, Sannino DR, Westmiller S, Wong CAN, Clark AG, Lazzaro BP, Douglas AE (2015) Host genetic determinants of microbiota-dependent nutrition revealed by genome-wide analysis of Drosophila melanogaster. Nat Commun 6:6312Google Scholar
  53. 53.
    Näpflin K, Schmid-Hempel P (2018) Host effects on microbiota community assembly. J Anim Ecol 87(2):331–340Google Scholar
  54. 54.
    Vorburger C, Perlman SJ (2018) The role of defensive symbionts in host–parasite coevolution. Biol Rev 93:1747–1764Google Scholar
  55. 55.
    Bickford D, Lohman DJ, Sodhi NS, Ng PKL, Meier R, Winker K, Ingram KK, Das I (2007) Cryptic species as a window on diversity and conservation. Trends Ecol Evol 22(3):148–155Google Scholar
  56. 56.
    Popa R, Green TR (2012) Using black soldier fly larvae for processing organic leachates. J Econ Entomol 105(2):374–378Google Scholar
  57. 57.
    Ma J, Lei Y, Rehman KU, Yu Z, Zhang J, Li W, Li Q, Tomberlin JK, Zheng L (2018) Dynamic effects of initial pH of substrate on biological growth and metamorphosis of black soldier fly (Diptera: Stratiomyidae). Environ Entomol 47(1):159–165Google Scholar
  58. 58.
    Schneider JC (2009) Principles and procedures for rearing high quality insects. Mississippi State University, MississippiGoogle Scholar
  59. 59.
    Choi WH, Yun JH, Chu JP, Chu KB (2012) Antibacterial effect of extracts of Hermetia illucens (Diptera: Stratiomyidae) larvae against gram-negative bacteria. Entomol Res 42(5):219–226Google Scholar
  60. 60.
    Spranghers T, Michiels J, Vrancx J, Ovyn A, Eeckhout M, De Clercq P, De Smet S (2018) Gut antimicrobial effects and nutritional value of black soldier fly (Hermetia illucens L.) prepupae for weaned piglets. Anim Feed Sci Technol 235:33–42Google Scholar
  61. 61.
    Stenfors Arnesen LP, Fagerlund A, Granum PE (2008) From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiol Rev 32(4):579–606Google Scholar
  62. 62.
    Fasolato L, Cardazzo B, Carraro L, Fontana F, Novelli E, Balzan S (2018) Edible processed insects from e-commerce: food safety with a focus on the Bacillus cereus group. Food Microbiol 76:296–303Google Scholar
  63. 63.
    Grabowski NT, Klein G (2017) Microbiology of processed edible insect products—results of a preliminary survey. Int J Food Microbiol 243:103–107Google Scholar
  64. 64.
    Wells-Bennik MHJ, Eijlander RT, Den Besten HMW, Berendsen EM, Warda AK, Krawczyk AO, Nierop Groot MN, Xiao Y, Zwietering MH, Kuipers OP, Abee T (2016) Bacterial spores in food: survival, emergence, and outgrowth. Annu Rev Food Sci Technol 7:457–482Google Scholar
  65. 65.
    van der Voort M, Abee T (2013) Sporulation environment of emetic toxin-producing Bacillus cereus strains determines spore size, heat resistance and germination capacity. J Appl Microbiol 114(4):1201–1210Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • E. Wynants
    • 1
    Email author
  • L. Frooninckx
    • 2
  • S. Crauwels
    • 3
  • C. Verreth
    • 3
  • J. De Smet
    • 1
  • C. Sandrock
    • 4
  • J. Wohlfahrt
    • 4
  • J. Van Schelt
    • 5
  • S. Depraetere
    • 6
  • B. Lievens
    • 3
  • S. Van Miert
    • 2
  • J. Claes
    • 1
  • L. Van Campenhout
    • 1
  1. 1.Department of Microbial and Molecular Systems (M2S), Lab4FoodKU LeuvenGeelBelgium
  2. 2.Thomas More University of Applied Sciences, RADIUSGeelBelgium
  3. 3.Department of Microbial and Molecular Systems (M2S), Laboratory for Process Microbial Ecology and Bioinspirational Management (PME&BIM)KU LeuvenSint-Katelijne-WaverBelgium
  4. 4.Research Institute of Organic Agriculture (FiBL)FrickSwitzerland
  5. 5.Koppert Biological SystemsBerkel en RodenrijsThe Netherlands
  6. 6.MillibeterTurnhoutBelgium

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