Mutual interactions of E. andrei earthworm and pathogens during the process of vermicomposting

  • Radka RoubalováEmail author
  • Petra Procházková
  • Aleš Hanč
  • Jiří Dvořák
  • Martin Bilej
Earthworm and Soil Pollution


Vermicomposting is a process by which earthworms together with microorganisms degrade organic wastes into a humus-like material called vermicompost. This process does not include a thermophilic stage, and therefore, the possible presence of pathogens represents a potential health hazard. To elucidate the effect of earthworms in the selective reduction of pathogens, grape marc substrate was artificially inoculated with Escherichia coli, Enterococcus spp., thermotolerant coliform bacteria (TCB), and Salmonella spp., and their reduction during vermicomposting was monitored. Various defense mechanisms eliminating microorganisms in the earthworm gut were assumed to be involved in the process of pathogen reduction. Therefore, we followed the expression of three pattern recognition receptors (coelomic cytolytic factor (CCF), lipopolysaccharide-binding protein (LBP), and Toll-like receptor (v-TLR)), two antimicrobial molecules (fetidin/lysenins and lysozyme), and heat shock protein HSP70. We detected the significant decrease of some defense molecules (fetidin/lysenins and LBP) in all pathogen-inoculated substrates, and the increase of CCF and LBP in the Salmonella spp.-inoculated substrate. At the same time, the reduction of pathogens during vermicomposting was assessed. We observed the accelerated reduction of E. coli, Enterococcus spp., and TCB in pathogen-inoculated substrates with earthworms compared to that without earthworms. Moreover, the differences between the microbiome of grape marc substrate and earthworm intestines were determined by high throughput sequencing. This analysis revealed that the bacterial composition of grape marc substrate differed from the composition of the content of earthworm intestines, suggesting the elimination of specific bacterial species during food passage through the gut.


Eisenia Grape marc Bacteria Defense molecules Microbiome 



Coelomic cytolytic factor


Colony-forming unit


Lipopolysaccharide-binding protein


Pattern recognition receptor


Thermotolerant coliform bacteria


Toll-like receptor



The authors thank L. Matějů for technical help with selective bacterial cultivation.

Funding sources

This research was supported by the Institutional Research Concept RVO 61388971 and by the Ministry of Agriculture of the Czech Republic under the NAZV project No. QJ1530034 and by CULS Prague under the CIGA project No. 20172018.

Supplementary material

11356_2019_4329_MOESM1_ESM.docx (37 kb)
ESM1 (DOCX 36 kb)


  1. Affar EB, Dufour M, Poirier GG, Nadeau D (1998) Isolation, purification and partial characterization of chloragocytes from the earthworm species Lumbricus terrestris. Mol Cell Biochem 185:123–133CrossRefGoogle Scholar
  2. Aira M, Gomez-Brandon M, Gonzalez-Porto P, Dominguez J (2011) Selective reduction of the pathogenic load of cow manure in an industrial-scale continuous-feeding vermireactor. Bioresour Technol 102:9633–9637CrossRefGoogle Scholar
  3. Aira M, Bybee S, Perez-Losada M, Dominguez J (2015) Feeding on microbiomes: effects of detritivory on the taxonomic and phylogenetic bacterial composition of animal manures. FEMS Microbiol Ecol 91(11):1–10Google Scholar
  4. Beschin A, Bilej M, Hanssens F, Raymakers J, Van Dyck E et al (1998) Identification and cloning of a glucan- and lipopolysaccharide-binding protein from Eisenia foetida earthworm involved in the activation of prophenoloxidase cascade. J Biol Chem 273:24948–24954CrossRefGoogle Scholar
  5. Bilej M, De Baetselier P, Van Dijck E, Stijlemans B, Colige A et al (2001) Distinct carbohydrate recognition domains of an invertebrate defense molecule recognize Gram-negative and Gram-positive bacteria. J Biol Chem 276:45840–45847CrossRefGoogle Scholar
  6. Bodo K, Ernszt D, Nemeth P, Engelmann P (2018) Distinct immune-and defense-related molecular fingerprints in sepatated coelomocyte subsets of Eisenia andrei earthworms. ISJ 15:338–345Google Scholar
  7. Chen Y, Zhang Y, Zhang Q, Xu L, Li R, Luo X, Zhang X, Tong J (2015) Earthworms modify microbial community structure and accelerate maize stover decomposition during vermicomposting. Environ Sci Pollut Res Int 22:17161–17170CrossRefGoogle Scholar
  8. Cole JR, Wang Q, Fish JA, Chai BL, McGarrell DM et al (2014) Ribosomal database project: data and tools for high throughput rRNA analysis. Nucleic Acids Res 42:D633–D642CrossRefGoogle Scholar
  9. Dvorak J, Mancikova V, Pizl V, Elhottova D, Silerova M et al (2013) Microbial environment affects innate immunity in two closely related earthworm species Eisenia andrei and Eisenia fetida. PLoS One 8:e79257CrossRefGoogle Scholar
  10. Dvorak J, Roubalova R, Prochazkova P, Rossmann P, Skanta F et al (2016) Sensing microorganisms in the gut triggers the immune response in Eisenia andrei earthworms. Dev Comp Immunol 57:67–74CrossRefGoogle Scholar
  11. Edwards CA (2010) Human pathogen reduction during vermicomposting. In: Edwards CA, Arancon NQ, Sherman RL (eds) Vermiculture technology: earthworms, organic wastes and environmental management. CRC Press, Boca RatonCrossRefGoogle Scholar
  12. Hanc A, Pliva P (2013) Vermicomposting technology as a tool for nutrient recovery from kitchen bio-waste. J Mater Cycles Waste Manag 15:431–439CrossRefGoogle Scholar
  13. Homa J, Zorska A, Wesolowski D, Chadzinska M (2013) Dermal exposure to immunostimulants induces changes in activity and proliferation of coelomocytes of Eisenia andrei. J Comp Physiol B 183:313–322CrossRefGoogle Scholar
  14. Homa J, Stalmach M, Wilczek G, Kolaczkowska E (2016) Effective activation of antioxidant system by immune-relevant factors reversely correlates with apoptosis of Eisenia andrei coelomocytes. J Comp Physiol B 186:417–430CrossRefGoogle Scholar
  15. Joskova R, Silerova M, Prochazkova P, Bilej M (2009) Identification and cloning of an invertebrate-type lysozyme from Eisenia andrei. Dev Comp Immunol 33:932–938CrossRefGoogle Scholar
  16. Kiernan JA (2008) Histological and histochemical methods: theory and practice, fourth edn. Scion Publishing Ltd, UKGoogle Scholar
  17. Lung AJ, Lin CM, Kim JM, Marshall MR, Nordstedt R et al (2001) Destruction of Escherichia coli O157:H7 and Salmonella enteritidis in cow manure composting. J Food Prot 64:1309–1314CrossRefGoogle Scholar
  18. Monroy FMA, Domínguez J (2008) Changes in density of nematodes, protozoa and total coliforms after transit through the gut of four epigeic earthworms (Oligochaeta). Appl Soil Ecol 39:127–132CrossRefGoogle Scholar
  19. Monroy F, Aira M, Dominguez J (2009) Reduction of total coliform numbers during vermicomposting is caused by short-term direct effects of earthworms on microorganisms and depends on the dose of application of pig slurry. Sci Total Environ 407:5411–5416CrossRefGoogle Scholar
  20. Pachepsky YA, Sadeghi AM, Bradford SA, Shelton DR, Guber AK, Dao T (2006) Transport and fate of manure-borne pathogens: modeling perspective. Agric Water Manag 86:81–92CrossRefGoogle Scholar
  21. Pass DA, Morgan AJ, Read DS, Field D, Weightman AJ, Kille P (2015) The effect of anthropogenic arsenic contamination on the earthworm microbiome. Environ Microbiol 17:1884–1896CrossRefGoogle Scholar
  22. Prochazkova P, Silerova M, Felsberg J, Joskova R, Beschin A et al (2006) Relationship between hemolytic molecules in Eisenia fetida earthworms. Dev Comp Immunol 30:381–392CrossRefGoogle Scholar
  23. Prochazkova P, Hanc A, Dvorak J, Roubalova R, Dreslova M et al (2018) Contribution of Eisenia andrei earthworms in pathogen reduction during vermicomposting. Environ Sci Pollut Res Int 25:26267–26278CrossRefGoogle Scholar
  24. Roubalova R, Dvorak J, Prochazkova P, Elhottova D, Rossmann P et al (2014) The effect of dibenzo-p-dioxin- and dibenzofuran-contaminated soil on the earthworm Eisenia andrei. Environ Pollut 193:22–28CrossRefGoogle Scholar
  25. Roubalova R, Dvorak J, Prochazkova P, Skanta F, Navarro Pacheco NI et al (2018) The role of CuZn- and Mn-superoxide dismutases in earthworm Eisenia andrei kept in two distinct field-contaminated soils. Ecotoxicol Environ Saf 159:363–371CrossRefGoogle Scholar
  26. Segata N, Izard J, Waldron L, Gevers D, Miropolsky L, Garrett WS, Huttenhower C (2011) Metagenomic biomarker discovery and explanation. Genome Biol 12:R60CrossRefGoogle Scholar
  27. Sinha RK, Herat S, Bharambe G, Brahambhatt A (2010) Vermistabilization of sewage sludge (biosolids) by earthworms: converting a potential biohazard destined for landfill disposal into a pathogen-free, nutritive and safe biofertilizer for farms. Waste Manag Res 28:872–881CrossRefGoogle Scholar
  28. Skanta F, Roubalova R, Dvorak J, Prochazkova P, Bilej M (2013) Molecular cloning and expression of TLR in the Eisenia andrei earthworm. Dev Comp Immunol 41:694–702CrossRefGoogle Scholar
  29. Skanta F, Prochazkova P, Roubalova R, Dvorak J, Bilej M (2016) LBP/BPI homologue in Eisenia andrei earthworms. Dev Comp Immunol 54:1–6CrossRefGoogle Scholar
  30. Soobhany N (2018) Preliminary evaluation of pathogenic bacteria loading on organic Municipal Solid Waste compost and vermicompost. J Environ Manag 206:763–767CrossRefGoogle Scholar
  31. Swati A, Hait S (2018) A comprehensive review of the fate of pathogens during vermicomposting of organic wastes. J Environ Qual 47:16–29CrossRefGoogle Scholar
  32. Turner C (2002) The thermal inactivation of E-coli in straw and pig manure. Bioresour Technol 84:57–61Google Scholar
  33. Vetrovsky T, Baldrian P (2013) Analysis of soil fungal communities by amplicon pyrosequencing: current approaches to data analysis and the introduction of the pipeline SEED. Biol Fertil Soils 49:1027–1037CrossRefGoogle Scholar
  34. Yu Y, Lee C, Kim J, Hwang S (2005) Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction. Biotechnol Bioeng 89:670–679CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Radka Roubalová
    • 1
    Email author
  • Petra Procházková
    • 1
  • Aleš Hanč
    • 2
  • Jiří Dvořák
    • 1
  • Martin Bilej
    • 1
  1. 1.Institute of Microbiology of the Czech Academy of SciencesPrague 4Czech Republic
  2. 2.Czech University of Life Sciences PraguePrague 6Czech Republic

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