Removal of Pathogens during Vermi-Stabilization
Earthworm promises to provide cheaper solutions to several social, economic and environmental problems plaguing the human society. Their body work as bio-filter and they can purify and also disinfect and detoxify the solid wastes. They are both protective and productive for environment and society. The microbiomics of gut worm (Eisenia foetida Savigny) and their association with vermistabilization was studied to determine the quantification of microbial groups in reactors. Worms were reared in three reactors viz. rice, straw, sugarcane trash, kitchen waste along with cow dung in 2:1 ratio for seventy days. The microbial load (Pseudomonas sp., Streptosporangium sp., Salmonella sp., Shigella sp. Flexibacter sp. and Escherichia sp.) in gut and cast, total nitrogen, potassium, organic carbon in reactors was determined, periodically. The microbial population in gut and reactors were determined using pour plate method The microbiomics of gut and casting revealed the removal of Salmonella (12-17x103±0.02 to 0-4x103±0.05 cfu g-1), Shigella (3-5x103±0.04 to 0.00±0.08 cfu g-1), Flexibacter (6-10x104±0.05 to 00.0±0.05 cfu g-1), Escherichia (8x102±0.02 to 0.00-1x101±0.05 cfu g-1) during vermistabilization, indicating the selective nature of feeding of earthworms which helps in the removal of pathogens as they enter in food chain of worms. However, increase in the number of Pseudomonas (13-16x104±0.05 to 18-22x105±1.0 cfu g-1), Streptosporangium (12-14x104±0.05 to 18-20x105±1.0 cfu g-1) seems to be responsible for reduction of pathogens and mineralization of other organics during the waste degradation process.
Received: April 25, 2011;
Accepted: May 26, 2011;
Published: July 19, 2011
A revolution is unfolding in vermiculture studies for multiple uses in environmental
protection and sustainable development. Earthworms have over 600 million years
of experience as environmental managers in the ecosystem. Vermiculture
scientists all over the world knew about the role of earthworms as waste
managers, as soil managers and fertility improvers and plant
growth promoters for long time (Sinha et al.,
2010). But some new discoveries about their role in removal
of pathogen may revolutionized the vermiculture industry.
Soil, the major reservoir of microbes, meets the food requirement of earthworms
and this has necessitated the establishment of different kinds of relationships
viz., a part of food for earthworm, proliferated in the gut and reactors. The
microbes and earthworm together mineralize, humifies organic matter and also
facilitates chelation of some metal ions (Canellas et
al., 2002; Rizl and Novokova, 2003; Maboeta
and von Rensburg, 2003). Earthworms have the capacity to utilize soil microbes
as their food (Parthasarathi and Ranganathan, 1999;
Verma and Shweta, 2011).
Selective proliferation of microorganisms in the gut of different kinds of
earthworms, due to the environment and food material available for growth of
microorganisms have been reported earlier viz., fungi in Perionyx millardi
(Chhotaray et al., 2011), Lampito mauritii
(Ansari, 2011), Onychochaeta borincana (Barik
and Gulati, 2009), bacteria in Amynthas caliginosa (Omrani
et al., 2005), Lampito mauritii (Idowu
et al., 2006), L. terrestris (Ansari,
2011) and actinomycetes in L. terrestris, A. longa and A.
caliginosa (Chhotaray et al., 2011), L.
rubellus and A. caliginosa (Ansari, 2011).
Higher microbial population and the activity in the casts of earthworms compared
to surrounding soils have been demonstrated by Tangjang
et al. (2009). The available information on solid waste management
suggests that solid wastes should be biocomposted before applying to soil in
order to achieve biological transformation of the organic matter and to avoid
potential risk of pathogens (Masciandaro et al.,
2000; Ansari and Saywack, 2011). Biocomposting of
solid wastes brings about stabilization of the organic matter and effectively
reduces pathogen concentrations (Kumar and Shweta, 2011).
However, absolute removal of pathogens difficult to achieve and many pathogens
survive the composting process (Sinha et al., 2009b).
Incorporation of earthworm in biocomposting process has been considered to be
an appropriate technology for bio waste management for producing nutrient enriched
vermicompost. Various investigators have, therefore, established the viability
of the technology using earthworms as a treatment system for different wastes
(Ansari and Sitaram, 2011; Elviror
et al., 1997).
Much of the research on vermistabilization has been focused on changes in the
chemical parameters. The information available on the microbiota which determine
the rate of degradation is however, very little (Edwards and
Bohlen, 1996a; Lavelle et al., 1998). Hence,
the present investigations were under taken and cover the dynamics and quantification
of microbial groups during vermistabilization process, changes in composition
of microbes in the gut, casting and their association with stabilization.
MATERIALS AND METHODS
The experiment was carried out in 2009-2010.Worms (Eisenia foetida Savigny) were reared in three reactors viz., rice straw and cow dung (RC) 2:1 ratio, sugarcane trash and cow dung (SC) 2:1 ratio and vegetable waste and cow dung (VC) 2:1 ratio. The reactors were mixed well and moistened with water up to 40% moisture contents. These treatment groups were investigated in two replicates; the reactor were placed in fiber bins and kept in natural environment to maintain favorable temperature for worms, as they are highly sensitive to temperature fluctuations. Two hundred mature earthworms were introduced into the bins containing 20 kg reactors each.
The treatment groups of different composition were allowed to stabilize for 70 days. Samples from the entire units were taken out, monthly, to measure the biomass of individuals. The worms were washed with distilled water and were weighed. After weighing, they were immediately placed in bins to prevent their desiccation.
During stabilization process of the reactors, five earthworms were collected
and washed with sterile water to remove the surface microbial flora from its
outer skin. The earthworms were then sacrificed by freezing and their whole
body was dissected. The gut region approximately (3-5 cm) was taken for analysis
for the reason the higher glutamic activity favours the microbial growth as
reported by Edwards (1998) and Lavelle
et al. (1998). Microbial analysis was carried out immediately following
Lavelle et al. (1998).
For microbial analysis of castings, worms were collected from all the experimental units and rinsed thoroughly in sterile water. The worms were then placed in petriplates containing tissue paper for 5 min. The fresh casts deposited were analyzed for different microbial count.
The castings were analyzed at interval of 30 days. The total nitrogen, total
potassium and total organic carbon measured by following Shaw
and Beadle (1949), Pearson (1952) and Walkey
and Black (1934), respectively.
Statistical analysis: Results are the means of three replicates. One way Analysis of Variance (ANOVA) was done using the INDOSTAT programme. The objective was to determine any significant differences among the parameters analyzed in different reactors during the stabilization process.
General: The average body weight (g) of earthworm was monitored on 30th
and 60th days of vermistabilization (Table 1). No mortality
was observed throughout experiment. The biomass (weight) increased from 0.64±0.02
to 1.2±0.02 in RC and SC and 0.64±0.02 to 0.84±0.02 in
VC reactor. The readily available nutrients in RC and SC might have enhanced
the feeding activity of worms (Kumar and Shweta, 2011)
in present study.
Total organic carbon: During the process of stabilization process worms
bio-transform waste constituents into a more useful fertilizer by grinding and
digesting organic wastes with the help of aerobic and anaerobic micro flora.
The percentage of organic carbon was remarkably decreased from 34.42±0.01
to 32.93±0.01 in RC; 36.39±0.05 to 32.82±0.02 in SC; 36.41±0.05
to 32.39±0.04 in VC at 30th day and remains ±30% at 60th day in
all reactor. The total organic carbon was metabolized into CO2 by
various micro-floras present in gut of worm and in reactor, involved in the
vermistabilization (Table 2).
|| Biomass (weight) of worms during stabilization process of
|All values are mean and standard deviation of the replicates.
|| Chemical analysis of reactors during vermistabilization
|All values are mean and standard deviation of the replicates.
*Significant (p<0.01), RC: Rice straw+cow dung, SC: sugarcane trash+cow
dung, VC: Vegetable waste+cow dung
Total kjeldahl nitrogen (TKN): The total nitrogen content of the vermicompost
increased with time due to rapid mineralization of organic nitrogenous compounds
(Table 2). However, the nitrogen content of the final produce
depends on the initial nitrogen concentration of the reactor (Kumar
and Shweta, 2011). The percentage of nitrogen significantly increased in
all reactors viz. 0.73±0.02 to 1.00±0.02 in RC and from 0.76-0.86±0.02
to 1.44±0.02 in SC and VC at the end of vermistabilization. Their enhanced
decomposition resulted in lowering of C:N ratio (Talashilkar
et al., 1999).
Total potassium: Potassium is an essential micronutrient for both animals
and plants. It plays a significant role in food metabolism and maintenance of
cellular organization. It activates the enzymes in protein and carbohydrate
metabolism. The percentage of potassium were found maximum in VC (0.85±0.003)
followed by SC (0.83±0.03) and a slight decrease in RC (0.65±0.03)
treatment (Table 2) at 60th day (Table 2).
Microbiomics of gut of worms during stabilization: At the initial stage
of stabilization process various pathogens viz. Salmonella, Shigella,
Escherichia and Flexibacter were found to be dominant in the gut
of worm but during the subsequent days their level decreased significantly (Table
3). However, Pseudomonas and Streptosporangium count increased
from 12-16x104±0.05 to 18-22x105±10.0 cfu
g-1. The microbial population of Salmonella decreased from
12x103±0.05 to 4x103±0.05 cfu g-1
in RC, 13x103±0.02 to 0.00±0.02 cfu g-1
in SC, 17x103±0.02 to 0.00±0.05 cfu g-1
in VC reactor at 60th day of vermistabilization.
|| Microbiomics of gut of worm during stabilization process
|All values are mean and standard deviation of the replicates
and expressed in cfu g-1. *Significant (p<0.01)
||Microbiomics in castings of worm during stabilization of reactors
|All values are mean and standard deviation of the replicates
and expressed in cfu g-1. *Significant (p<0.01)
Similarly, Escherichia count decreases from 8x102±0.02
to 1x101±0.05 cfu g-1 in RC, 8x102±0.03
to 0.00±0.05 cfu g-1 in SC and VC reactor at the 60th day.
The reduction in Shigella and Flexibacter recorded at earlier
(30th day of stabilization) stage. Shigella decreased from 3-5x102±0.04
to 0.00-1x101±0.05 cfu g-1 in all reactors at 30th
day and completely removed at 60th day. Flexibacter sp. observed only
in RC reactor (8x102±0.05 cfu g-1) at 30th day
of stabilization while, in others completely removed at the same point of time.
Population of Pseudomonas gradually increased from 14x104±0.05
to 22x105±1.0 cfu g-1 in RC, 16x104±0.05
to 22x105±1.0 cfu g-1 in SC, 13x104±0.05
to 18x105±1.0 cfu g-1 in VC reactor. The microbial
population of Streptosporangium also significantly increased from 12x104±0.05
to 20x105±1.0 cfu g-1 in SC, 12x104±0.05
to 18x105±1.0 cfu g-1 in VC and 14x104±0.05
to 15x105±1.0 cfu g-1 RC reactor.
Microbiomics of cast during stabilization: The casting analysis did not show much difference with microbial count of gut. However, Shigella and Flexibacter were not recorded in any reactor at 30th day of vermistabilization. Salmonella sp. was completely removed out in all reactors except in RC (2x103±0.02 cfu g-1) at 60th day. Similarly, Escherichia was also not recorded in any reactor at same point of time. The significant increase in Pseudomonas (16x104±0.05 to 28x105±1.0 cfu g-1 in RC, 16x104±0.05 to 28x105±1.0 cfu g-1 in SC, 13x104±0.05 to 20x105±1.0 cfu g-1 in VC) and Streptosprangium (15x104±0.05 to 17x105±1.0 cfu g-1 in RC, 14x104±0.05 to 20x105±1.0 cfu g-1 in SC, 14x104±0.05 to 18x105±1.0 cfu g-1 in VC were recorded in castings of all reactors (Table 4).
Earthworms routinely devour on the protozoa, bacteria and fungus as food in
any substrate where they inhabit (Sinha et al., 2010).
They seem to realize instinctively those anaerobic bacteria and fungi are undesirable
and so feed upon them preferentially, thus arresting their proliferation. More
recently, Sinha et al. (2010) has found in her
research that worms living in pathogen-rich materials (e.g., sewage and sludge),
when dissected show no evidence of pathogens beyond 5 mm of their gut (Sinha
et al., 2009a). The experimental results of microbiomics of gut and
casting in present study proved the reduction/removal of Salmonella,
Shigella, Escherichia and Flexibacter in all reactors.
These results corroborate with the findings of Edwards and Bohlen
(1996a), Bohlen and Edwards (1995) who have reported
that earthworms food source include microorganism and can digest them
selectively. Kumar and Sekaran (2005) also reported
removal of Salmonella and Escherichia in 35 days in municipal
solid wastes by Lampito mauritii.
Bajsa et al. (2003) reported the removal of
faecal Coliform, Salmonella, enteric viruses; helminths ova from
sewage and sludge appear to be much more rapid when they are processed by E.
foetida. In another study Bajsa et al. (2004)
reported the pathogen die-off in vermicomposting of sewage-sludge stipends with
E. coli and E. faecalis at the 1.6-5.4x106 cfu g-1
and 3- 4x104 cfu g-1, respectively and achieved
a safe product in 4-5 months of vermicomposting and the product remained the
same quality without much reappearance of pathogens after in the remaining month
of the test, confirming the observation of present study. Other studies (Singleton
et al., 2003; Safawat et al., 2002)
also confirmed significant human pathogen reduction in biosolids, vermicomposted
Increase in population of Streptosporangium and Pseudomonas in
present study corroborates the findings of Sidhu et al.
(2001) who described the role of Streptosporangium in the suppression
of pathogenic bacterial population in earthworm. Kumar and
Sekaran (2005), Masciandaro et al. (2000)
and Sidhu et al. (2001) also proved that the
indigenous micro-flora play a significant role in the suppression of pathogenic
bacterial population growth in composted sludge. This confirms the role of Streptosporangium
and Pseudomonas in suppression of pathogens in present study. However,
there are conflicting observations relative to the proliferation of specific
bacterial group in earthworm gut micro flora; indeed passage through the gut
may selectively decrease the number of certain microbes such as Shigella
and Escherichia (Edwards and Bohlen, 1996b).
Vinceslas-Apka and Loquet (1997) and Lavelle
et al. (1998) have reported that organic carbon is metabolized into
CO2 and the various microflora present in the gut of worm and in
reactor are involved in the degradation of organic carbon. The total nitrogen
content of the reactors increased with time, due to rapid mineralization of
organic nitrogen compounds. Present results have supported the findings of Bansal
and Kapoor (2000) which have shown increase in the nitrogen content as a
result of carbon loss in vermicomposting of crop residues and cattle dung. In
the present study C:N ratio of reactors lowered because of enhanced decomposition,
agreeing to the earlier observations of Talashilkar et
Nevertheless, the removal of pathogens may be due to release of antibiotics
by earthworms, responsible to kill pathogenic organisms inhabit and render it
virtually sterile (Sinha et al., 2009a). The
worms also release the coelomic fluids that have anti-bacterial properties which
may destroy all pathogen in reactors (Pierre et al.,
1982). Further, there is some thing inside the worms which destroys the
pathogens and makes their excreta pathogen free needed to be explored. However,
absolute removal of pathogens is difficult to achieve, some may survives even
The study suggests that the worm selectively inhibits the growth of pathogens in reactors. The presence of pathogenic organisms Salmonella, Shigella, Escherichia and Flexibacter was observed at initial stage of stabilization process but after vermistabilization these were reduced to zero concentration. This amply demonstrates that these pathogens have been eliminated as they entered in food chain of worms. And for this, Pseudomonas, Streptosporangium seems to be responsible for reduction of pathogens and metabolization of reactors. However, it may not be possible to remove pathogens completely but at least worms change the microbial make-up of reactors to make it harmless to the soil and enable its use as a nutritive organic fertilizer.
The authors are thankful to the Department of Biotechnology, Ministry of Science and Technology, Government of India, New Delhi, for financial assistance.
Ansari, A.A. and K. Sitaram, 2011.
An investigation into the anti-microbial and anti-fungal properties of earthworm powder obtained from Eisenia fetida
. Am. J. Food Technol., 6: 329-335.CrossRef | Direct Link |
Ansari, A.A. and P. Saywack, 2011.
Identification and classification of earthworm species in Guyana. Int. J. Zool. Res., 7: 93-99.CrossRef | Direct Link |
Ansari, A.A., 2011.
Worm powered environmental biotechnology in organic waste management. Int. J. Soil Sci., 6: 25-30.CrossRef | Direct Link |
Bajsa, O., O.J. Nair, K. Mathew and G.E. Ho, 2004.
Pathogen die-off in vermicomposting process. Proceedings of the 6th International Conference on Small Water and Wastewater Treatment Systems, Feb. 11-13. Australia, pp: 1-8
Bajsa, O., J. Nair, K. Mathew and G.E. Ho, 2003.
Vermiculture as a tool for domestic wastewater management. Water Sci. Technol., 48: 125-132.PubMed | Direct Link |
Bansal, S. and K.K. Kapoor, 2000.
Vermicomposting of crop residues and cattle dung with Eisenia foetida
. Bioresour. Technol., 73: 95-98.CrossRef | Direct Link |
Barik, T. and J.M.L. Gulati, 2009.
Use of vermicompost in field crops and cropping systems: A review. Agric. Rev., 30: 48-55.
Bohlen, P.J. and C.A. Edwards, 1995.
Earthworm effects on N dynamics and soil respiration in microcosms receiving organic and inorganic nutrients. Soil Biol. Biochem., 27: 341-348.CrossRef |
Canellas, L.P., F.L. Olivares, A.L. Okorokova-Facanha and A.R. Facanha, 2002.
Humic acids isolated from earthworm compost enhance root elongation, lateral root emergence and plasma membrane H+
-ATPase activity in maize roots. Plant Physiol., 130: 1951-1957.CrossRef | Direct Link |
Chhotaray, D., C.S.K. Mishra and P.K. Mohapatra, 2011.
Diversity of bacteria and fungi in the gut and cast of the tropical earthworm Glyphodrillus tuberosus
isolated from conventional and organic rice fields. J. Pharmacol. Toxicol., 6: 303-311.CrossRef | Direct Link |
Edwards, C.A., 1998.
The Use of Earthworms in the Breakdown and Management of Organic Wastes. In: Earthworm Ecology, Edwards, C.A. (Ed.). CRC Press LLC, Boca Raton, Fl., ISBN: 084931819X, pp: 327-354
Edwards, C.A. and P.J. Bohlen, 1996.
Biology and Ecology of Earthworms. 3rd Edn., Chapman and Hall, London, UK., ISBN-13: 9780412561603, Pages: 426
Edwards, C.A. and P.J. Bohlen, 1996.
Biology and Ecology of Earthworms. 1st Edn., Chapman and Hall, London, UK
Elviror, C., L. Sampedro, J. Dominguez and S. Mato, 1997.
Vermicomposting of wastewater sludge from paper pulp industry with nitrogen rich materials. Soil Biol. Biochem., 29: 759-762.CrossRef |
Kumar, R. and Shweta, 2011.
Enhancement of wood waste decomposition by microbial inoculum prior to vermicomposting. Bioresour. Technol., 102: 1475-1481.CrossRef | PubMed |
Kumar, A.G. and G. Sekaran, 2005.
Enteric pathogen modification by anaecic earthworm Lampito mauritii
. J. Applied Sci. Environ., 9: 15-17.Direct Link |
Idowu, A.B., M.O. Edema and A.O. Adeyi, 2006.
Distribution of bacteria and fungi in the earthworm Libyodrillus violaceous
(Annelida: Oligochaeta), a native earthworm from Nigeria. Rev. Biol. Trop., 54: 49-58.PubMed |
Lavelle, P., B. Pashanasi, F. Charpentier, C. Gilot and J.P. Rossi et al
Large-Scale Effects of Earthworms on Soil Organic Matter and Nutrient Dynamics. In: Earthworm Ecology, C.A. Edwards (Ed.). Boca Raton, Lucie Press. 103-122
Maboeta, M.S. and L. von Rensburg, 2003.
Vermicomposting of industrially produced woodchips and sewage sludge utilizing Eisenia fetida
. Ecotoxicol. Environ. Safety, 56: 265-270.Direct Link |
Masciandaro, G., B. Ceccanti and C. Garcia, 2000. In situ
vermicomposting of biological sludges and impacts on soil quality. Soil Biol. Biochem., 32: 1015-1024.CrossRef |
Omrani, G.A., M. Zamanzadeh, A. Maleki and Y. Ashori, 2005.
Earthworm ecology in the Northern part of Iran: With an emphasis on compost worm Eisenia fetida
. J. Applied Sci., 5: 1434-1437.CrossRef | Direct Link |
Parthasarathi, K. and L.S. Ranganathan, 1999.
Longevity of microbial and enzyme activity and their influence on NPK content in pressmud vermicasts. Eur. J. Soil Biol., 35: 107-113.CrossRef |
Pearson, R.W., 1952.
Potassium supplying power of eight alabama soils. Soil Sci., 74: 301-310.Direct Link |
Pierre, V., R. Phillip, L. Margnerite and C. Pierrette, 1982.
Anti-bacterial activity of the haemolytic system from the earthworms Elsinia foetida
andrei. J. Invertebr. Pathol., 40: 21-27.CrossRef |
Rizl, V. and A. Novokova, 2003.
Interactions between micro-fungi and Eisenia andrei
(Oligochaeta) during cattle manure vermicomposting the 7th international symposium on earthworm ecology Cardiff Wales. Pedobiologia, 47: 895-899.CrossRef |
Shaw, J. and L.C. Beadle, 1949.
A simplified Ultra micro
kjeldahl method for estimation of protein and total nitrogen in Fluid samples of less than 1.0 ll. J. Exp. Biol., 26: 15-23.
Sidhu, J., R.A. Gibbs, G.E. Ho and I. Unkovich, 2001.
The role of indigenous microorganisms in suppression of Salmonella
regrowth in composted biosolids. Water Res., 35: 913-920.CrossRef |
Singleton, D.R., P.F. Hendrix, D.C. Coleman and W.B. Whitman, 2003.
Identification of uncultured bacteria tightly associated with the intestine of the earthworm Lumbricus rubellus
(Lumbricidae: Oligochaeta). Soil Biol. Biochem., 35: 1547-1555.CrossRef |
Sinha, R.K., K. Chauhan, D. Valani, V. Chandran, B.K. Soni and V. Patel, 2010.
Earthworms: Charles Darwin's unheralded soldiers of mankind: Protective and productive for man and environment. J. Environ. Protect., 1: 251-260.CrossRef | Direct Link |
Sinha, R.K., S. Herat, D. Valani and K. Chauhan, 2009.
Vermiculture and sustainable agriculture. J. Agric. Environ. Sci., 5: 1-55.
Sinha, R.K., S. Heart, G. Bharambe and A. Brahambhatt, 2009.
Vermistabilization of sewage sludge (biosolids) by earthworms converting a potential biohazard destined for landfill disposal into a pathogen free nutritive and save biofertilizer for farms. Waste Manage. Res., 28: 872-881.CrossRef | Direct Link |
Talashilkar, S.C., P.P. Bhangarath and V.B. Metha, 1999.
Changes in chemical properties during composting of organic residues as influenced by earthworm activity. J. Indian Soc. Soil Sci., 47: 50-53.
Tangjang, S., K. Arunachalam, A. Arunachalam and A.K. Shukla, 2009.
Microbial population dynamics of soil under traditional agroforestry systems in Northeast India. Res. J. Soil Biol., 1: 1-7.CrossRef | Direct Link |
Verma, D. and Shweta, 2011.
Earthworm resources of western himalayan region, India. Int. J. Soil Sci., 6: 124-133.CrossRef |
Vinceslas-Apka, M. and M. Loquet, 1997.
Organic matter transformations in lignocellulosic waste products composted or vermicomposted (Eisenia fetida andrei
): Chemical analysis and 13C CPMAS NMR spectroscopy. Soil Biol. Biochem., 29: 751-758.
Walkey, J.A. and J.A. Black, 1934.
Estimation of organic carbon by the chromic acid titration method. Soil Sci., 37: 29-31.
Shakir, S.H.S. and R.W. Weaver, 2002.
Earthworm survival in oil contaminated soil. Plant Soil, 240: 127-132.CrossRef |