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“Earthworms-The intestines of the soil"

– Aristotle.

"The plow is one of the most ancient and most valuable of man's inventions; but

long before he existed, the land was in fact regularly plowed and still continues to be thus

plowed by earthworms. It may be doubted whether there are many other animals which

have played so important a part in the history of the world, as have these lowly organized

creatures. Without the work of this humble creature, who knows, nothing of the benefits he

confers upon mankind, agriculture, as we know it, would be very difficult, if not wholly

impossible"

–Charles Darwin.

These are the words, which reveal the importance of Earthworms. It is well known that

the earthworms have the ability to support the growth of plants and they can increase the fertility

of the soil. There are about 3920 named species of earthworm so far reported worldwide. In

India, so far, 509 species, referable to 67 genera and 10 families, have been reported (Kale,

1991). Earthworms play an important role in agro-ecosystem like enhancing decomposition,

humus formation, nutrient cycling and soil structural development (Kladiviko et al., 1986). The

practice of vermiculture is at least a century old but it is now being revived worldwide with

diverse ecological objectives such as waste management, soil detoxification and regeneration and

sustainable agriculture. Earthworms act in the soil as aerators, grinders, crushers, chemical

degraders and biological stimulators. They secrete enzymes, proteases, lipases, amylases,

cellulases and chitinases which bring about rapid biochemical conversion of the cellulosic and

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the proteinaceous materials in the variety of organic wastes which originate from homes,

gardens, dairies and farms. Recent works have elucidated some of the mechanisms by which

earthworms enhance soil aggregation. Ingested aggregates are broken up in liquid slurry that

mixes soil with organic material and binding agents. The defecated casts become stable after

drying and also earthworms initiate the formation of stable soil aggregates in mining soils.

Ecology of earthworms

Earthworms are burrowing animals and form tunnels by literally eating their way through

the soil. The distribution of earthworms in soil depends on factors like soil moisture, availability

of organic matter and pH of the soil. They occur in diverse habitats specially those which are

dark and moist. Organic materials like humus, cattle dung and kitchen wastes are highly

attractive sites for some species. Earthworms are generally absent or rare in soil with a very

coarse texture, in soil and high clay content, or soil with pH < 4 (Gunathilagraj, 1996).

Earthworms are very sensitive to touch, light and dryness. Water-logging in the soil can cause

them to come to the surface. Worms can tolerate a temperature range between 5ºC to 29ºC. A

temperature of 20ºC to 25ºC and moisture of 50–60 percent is optimum for earthworm function

(Hand, 1988).

Biology of earthworms

Earthworms are long, narrow, cylindrical, bilaterally symmetrical, segmented animals

without bones. The body is dark brown, glistening and covered with delicate cuticle. They weigh

about 700–1400 mg after 10 weeks. They have a muscular gizzard which finely grinds the food

(fresh and decaying plant debris, living or dead larvae and small animals, and bacteria and

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protozoa mixed with earth) to a size of 2–4 microns. The gut of the earthworm is inhabited by

millions of decomposer micro-organisms. They are bisexual animals and cross-fertilization

occurs as a rule. Copulation may last for about an hour, the worms then separate. Later the

clitellum of each worm eject cocoon where sperms enter to fertilize the eggs. Up to 3 cocoons

per worm per week are produced. From each cocoon about 10–12 tiny worms emerge.

Earthworms continue to grow throughout their life and the number of segments continuously

proliferates from a „growing zone‟ just in front of the anus. Earthworms contain 70–80 percent

high quality lysine rich protein on a dry weight basis. They can be useful as animal feed. Usually

the life span of an earthworm is about 3 to 7 years depending upon the type of species and the

ecological situation.

Eudrilus eugeniae

Eudrilus eugeniae is an earthworm species indigenous in Africa but it has been bred

extensively in USA, Canada, Europe and Asia for the fish bait market, where it is commonly

called as the African Night-crawler. E. eugeniae is a large worm appearing brown and red to

dark violet like animal flesh. Their length ranges from 3.2 to 14 cm, and 5 to 8 mm in diameter.

It grows faster and better than other species. Life span of this worm ranges from 1 to 3 years. It

grows rapidly and reasonably prolific. Under optimum conditions it would be ideal for animal

feed protein production. However there has been relatively little work on the biology and

ecology of this species. The African night crawler Eudrilus eugeniae is used extensively in

commercial vermin culture especially in India. Increased attention is also being given to this

species as a possible waste decomposer and as a protein source.

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Coelomic fluid of earthworm

A fluid within the coelom of earthworm is known as the coelomic fluid and this fluid is

collected by stimulating them in different methods like mild electric shock, puncturing of

coelomic cavity and worm water shock method. The coelomic fluid functions as a hydrostatic

skeleton and also serves as the circulatory medium. The fluid contains cytolytic, agglutinating

and/or antibacterial components, which are involved in the immune systems. Presumably the

function of this system is to destroy membranes of foreign cell, a mechanism that causes cell

death by cytosol release, and is attributed to the coelomycetes, which secrete humoral effectors

into the coelomic fluid. Coelomic fluid is also reported for having anticancer activity. The high

concentration of coelomic fluid exhibited toxic effect on HeLa cells, causing the cell lysis and

break down into pieces. Antibacterial activity of coelomic fluid is reported to be selective. The

coelomic fluid from earthworm is known to contain immunoactive cells and molecules involved

in immune defense. Earthworm coelomic fluid is found to contain molecules that bind anti IgA

and anti IgG. Elucidation of the earthworm binding site on anti IgG and anti IgA could make

earthworm coelomic fluid a valuable reagent in immunological, chemical and biological

research.

Vermiculture biotechnology promises to usher in the „Second green revolution‟ by

completely replacing the destructive agro-chemicals which did more harm than good to both the

farmers and their farmland. Earthworms restore and improve soil fertility and significantly boost

crop productivity. Earthworms excreta (vermicast) is a nutritive „organic fertilizer‟ rich in

humus, NPK, micronutrients, beneficial soil microbes - „nitrogen-fixing and phosphate

solubilizing bacteria‟ and „actinomycetes‟ and growth hormones „auxins‟, „gibberellins‟ and

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„cytokinins‟. Both earthworms and its vermicast and body liquid (vermiwash) are scientifically

proving as both „growth promoters and protectors‟ for crop plants. In the experiments with corn

and wheat crops, tomato and egg-plants it displayed excellent growth performances in terms of

height of plants, colour and texture of leaves, appearance of flowers and fruits, seed ears etc, as

compared to chemical fertilizers and the conventional compost. There is also less incidences of

„pest and disease attack‟ and „reduced demand of water‟ for irrigation in plants grown on

vermicompost. Presence of live earthworms in soil also makes significant difference in flower

and fruit formation in vegetable crops. Biomass of earthworms, a byproduct of Vermiculture

Biotechnology (VBT) is rich in „high quality protein‟ and source of nutritive feed materials for

fishery, poultry and dairy industries and also for human consumption (Rajiv et al., 2010).

Vermiculture and environmental management

Vermiculture is practiced for the mass production of earthworms with the multiple

objectives of waste management, soil fertility and detoxification and vermicompost production

for sustainable agriculture. The practice was started in the middle of 20th century and the first

serious experiments were established in Holland in 1970, and subsequently in England, and

Canada. Later vermiculture practices were followed in USA, Italy, Philippines, Thailand, China,

Korea, Japan, Brazil, France, Australia and Israel (Edward, 1988). Collie (1978) and Hartenstein

and Bisesi (1989) have reported on the management of sewage sludge and effluents from

intensively housed livestock by vermiculture in USA. Vermiculture is being practiced and

propagated on a large scale in Australia as a part of the „Urban agriculture development

program‟ which utilizes the urban wastes. Australia‟s „Worm grower association‟ is the largest in

world with more than 1200 members.

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India has yet to appreciate the full importance of vermiculture despite the potential for the

production of 400 million tonnes of vermicompost annually from waste degradation (Sinha,

1996). Senapati (1992) has stressed the importance of vermiculture for the management of all

cellulosic wastes in India. Gunathilagraj and Ramesh (1996) and Gunathilagaraj and

Ravignanam (1996) reported respectively about management of coir and sericultural wastes by

earthworms in India. Kale et al. (1993), Seenappa and Kale (1993) and Seenappa et al. (1995)

have each advocated vermicomposting and management on aspects of sugar factory waste, solid

wastes from the aromatic oil industries, and distillery wastes in India. In 1998, the Government

of India announced exemption from tax liability to all those institutions, organizations, and

individuals in India practicing vermiculture on a commercial scale. Vermicomposting plants are

operating in Pune and Bangalore with 100 tonnes day−1 capacity (Sinha, 1996). Chennai,

Mumbai, Indore, Jaipur and several other Indian cities are also setting up vermiculture farms.

Earthworms in general are highly resistant to many pesticides and have been reported to

concentrate the pesticides and heavy metals in their tissues. They also inhibit the soil borne

pathogens and work as a detoxifying agent for polluted soil (Davis, 1971; Ireland, 1983). These

properties of earthworms can be utilized for effluent treatment and heavy metal and pesticides

removal from industrial and agricultural wastes. Earthworms are important „secondary

decomposers‟ and vermicomposting in nature is an ongoing process if the natural population of

earthworms are undisturbed. Vermiculture engineers the growth of beneficial nitrogen fixing and

decomposer bacteria and actinomycetes fungus in the degraded waste (vermicompost). India has

voracious waste eater tropical species of earthworms. The warm and moist climatic conditions of

India are also favorable for earthworm rapid biodegradation action. An earthworm promotes the

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growth of „beneficial decomposer bacteria‟ in waste biomass and acts as an aerator, grinder,

crusher, chemical degrader and a biological stimulator. Given the optimum conditions of

temperature and moisture, earthworms eat the organic component of the waste biomass, which is

finely ground into small particles in their gizzard and passed on to the intestine for enzymatic

actions.

The worms secrete enzymes; proteases, lipases, amylases, cellulases and chitinases in

their gizzard and intestine which bring about rapid biochemical conversion of the cellulosic and

the proteinaceous materials in the organic wastes (Hand, 1988). The gizzard and the intestine

work as a „bioreactor.‟ Only 5–10 percent of the chemically digested and ingested material is

absorbed into the body and the rest is excreted out in the form of fine mucus coated granular

aggregates called „vermicastings‟ which are rich in nitrates, phosphates and potash. Earthworm

participation enhances natural biodegradation and decomposition of wastes from 60 to 80 percent

(given optimum temperature and moisture) thus significantly reducing the composting time by

several weeks. The process of decomposition is odour-free because earthworms release coelomic

fluids in the decaying waste biomass which have antibacterial properties and kill pathogens

(Pierre et al., 1982). Earthworms also create aerobic conditions in the waste materials, inhibiting

the action of anaerobic micro-organisms which release foul-smelling hydrogen sulfide and

mercaptans. Eisenia fetida, E. andrei, Eudrilus eugeniae, Lumbricus rubellus and Perionyx

excavatus are major waste eater and biodegrader earthworm species. They are used worldwide

for waste degradation and are found to be very successful functionaries for the ecological

management of organic municipal wastes (Edwards, 1988). E. eugeniae and P. excavatus are

believed to be the more versatile waste managers (Graff, 1981; Kale et al., 1982).

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Though earthworms are popular among the farmers to scientists for their plant growth

promoting activities to anticancer activities, there are not enough studies available that has

recorded the molecular basis of these findings. Hence, it was proposed to characterize the

coelomic fluid and its various biological activities such as anti microbial and anti-cancer besides

its obvious plant growth promoting property. Being a soil dweller and a known decomposer, a

study has been conducted to evaluate the impact of textile effluent discharge the population of

the earthworm. Eudrilus eugeniae species of earthworm was chosen for this study and the

objectives were

1. Evaluation of different collection methods for coelomic fluid from Eudrilus eugeniae and

its biochemical characterization.

2. Evaluation of the antimicrobial activity of the coelomic fluid on selected pathogenic

strains.

3. To investigate the plant growth promoting property of the coelomic fluid in plant tissue

culture system.

4. To evaluate the anticancer potential of coelomic fluid of Eudrilus eugeniae in SiHa cell

line

5. To study the impact of textile effluent on the fecundity and population of Eudrilus

eugeniae.

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In 1881, Charles Darwin published his last book "The formation of vegetable mould

through the action of worms with observation on their habits" shortly before his death. The book

drew attention to the great importance of earthworms in the breakdown of dead plant material

and the release of essential nutrients from it. However, only in last few decades, potential of

earthworms for breaking down organic waste has been explored in depth and many large scale

vermicomposting facilities have been developed all over the world with varying success.

Earthworms

The earthworm derives its name from the fact that it burrows and eats its way into the

earth. Earthworms have been on the earth for over 20 million years. There are 3920 species of

earthworms distributed throughout the world. Aquatic worms are called as microdrilli and

terrestrial earthworms are known as megadrilli. In India, there are about 509 species of

earthworms, belonging to 67 genera. Besides these, more than 20 species from other countries

have been introduced into India. These are known as 'peregrines'. Earthworm occur in diverse

habitats, organic materials like manures, litter, compost etc are highly attractive for earthworms

but they are also found in very hydrophilic environment close to both fresh and brackish water,

some species can survive under snow (Sharma et al., 2005).

Classification of earthworm

Kingdom: Animalia, Phylum: Annelida, Class: Oligochaeta, Order: Opisthopora, Family:

Lumbricidae, Genus: A large number of genera have been described in literature, Species: A

large number of species under each genus have been described in literature. Earthworms have

also been classified on the basis of their ecological niche (Bouche, 1977) and feeding behaviour

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(Lee, 1985) [Fig. 2.1]. A brief discussion of different ecological groups of the earthworms is

given below:

Fig. 2.1: Classification of earthworms based on ecological groups and niche

Epigeic species

These species live above the mineral soil surface typically in the litter layers and plant

debris and feed on them. These are phytophagous. Most of the species have insignificant role in

humus formation and are not good for use in field conditions for soil reclamation. They have

high reproductive rate and high cocoon production rate. However, their life span is relatively

short. They show high metabolic activity and hence are particularly useful for vermicomposting.

E. eugeniae

P. excavatus

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Examples are Eisenia fetida, Eisenia andrei, Eudrilus eugeniae, Perionyx excavates and

Drawida modesta.

Endogeic species

These species inhabit mineral soil beneath the top soil surface generally forming

horizontal tunnels to the soil surface. They feed on soil more or less enriched with organic

matter. They are probably important in improvement of soil texture and structure (pedogenesis)

and are not much beneficial in organic matter decomposition and recycling of plant nutrients.

Their reproduction rate is moderate and they have shorter life span. Example is Octochaetona

thurstoni.

Anecic species

These are surface feeding earthworms that construct and live in permanent burrows in the

mineral soil layers but come to the surface to feed on organic matter, mostly plant litter, and pull

it into their burrows. They are important in burying surface litter. They are great help in

incorporation of organic matter into the soil, and distribution and cycling of plant nutrients, and

also in improvement of soil structure and texture (pedogenesis). These species have low cocoon

production rate and limited reproductive capacity, but their life span is longer. Examples are

Lampito mauritti, Lumbricus terrestris and Octochaetona serrata. A summary of characteristics

used by Bouche to distinguish the earthworms on the basis of ecological niche is given in

Table A (Gajalakshmi and Abbasi, 2004a).

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Classification of earthworms based on feeding behavior

According to their feeding habits, earthworms are classified into detritivorous and

geophagus (Lee, 1985). Detritivores feed at or near the soil surface mainly on plant litter or dead

roots and other plant debris in the organic matter rich surface soil or on mammalian dung. These

include epigeic and anecic forms. These are also called as humus formers. Geophagus feed

deeper beneath the surface, ingesting large quantities of organically rich soil. These include

endogeic forms. These are also called as humus feeders. For the purpose of vermicomposting of

different organic wastes, generally epigeic species of earthworms are used widely in India

(Ismail, 2005). It is generally known that the epigeic species Eudrilus eugeniae, Perionyx

excavates and Eisenia fetida have a potential as waste decomposers. In order to utilize these

species successfully in vermicomposting and vermiculture all aspects of their biology and

physical requirements must be known. The life-cycle of each of the three species are now well

documented after intensive studies under controlled conditions. Venter and Reinecke (1988)

presented studies on Eisenia fetida, Reinecke et al. (1992) on Eudrilus eugeniae, and Hallatt et

al., (1990) on Perionyx excavatus. From a comparison of the lifecycle it is evident that all three

species are prolific breeders, maintaining a high reproduction rate under favourable conditions of

temperature, moisture and food availability.

Food and feeding habits of earthworms

Earthworms exhibit a high degree of niche diversity (Table A). Surface dwellers largely

feed upon leaf litter on soil surface. Burrow formers swallow soil and derive nutrition from it.

The quantity and quality of food available in an ecosystem determines population size,

composition and diversity of earthworm community. In general, daily ingestion of feed varies

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from 100 to 300 mg/g of worm body weight. According to one estimate, an earthworm can

consume 8 to 20 g dung/year. So at a population density of 1,20,000 adults/ha, dung

consumption would be 17.20 tones/ha/year (Bhatnagar and Palta, 1996). In a temperate

deciduous forest, annual leaf fall of approximately three tones/ha/year will be consumed just in

threemonths (Satchell, 1983). These estimates thus amply indicate that earthworms are important

in soil biota mixing and incorporating organic matter into soil. Some earthworms are able to

selectively digest certain microorganisms (Dash et al., 1984).

Characteristics Epigeic Endogeics Anecics

Body Size Small Large Moderate

Burrowing habit Reduced Developed Strongly developed

Longitudinal contraction

No Little Developed

Hooked chetae Absent Absent Present

Sensitivity to light Low Strong Moderate

Mobility Rapid Slow Moderate

Skin moistening Developed Feeble Developed

Pigmentation Homochromic Absent Dorsal and Anterior

Fecundity High Low Moderate

Maturation Rapid Slow Moderate

Respiration High Feeble Moderate

Survival under adverse

conditions as cocoons By quiescence True diapause

Table A: Summary of characteristics to classify the earthworms on the basis of ecological niche

(Gajalakshmi and Abbasi, 2004a)

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Biology of earthworms

Earthworms are long, thread-like, elongated cylindrical, soft bodied worms with uniform

ring like structures all along the length of their body. Earthworms vary greatly in size. In India

some peregrine species like Microscotex phosphoreus (Duges) are only 20 mm long while some

endemic geophagous worms such as Drawida grandus (Bourus) may reach up to one meter in

length. The colour of earthworms generally ranges from a brownish- black tinge to purple with

some exceptions. Generally the dorsal side of the worms is darker while ventral side is paler.

Their bodies are segmented which are arranged linearly and outwardly highlighted by circular

grooves which are called annuli.

The number of segments varies from 80-100 or more. The first segment into which the

mouth opens is called as peristomium. On the dorsal surface of the peristomium is a lobe like

structure called prostomium which overhangs the mouth. The last segment is called the anal

segment and it has a perforation for the anus at the hind end. At the sides of the body on the

ventral surface of each segment are four pairs of short, stubby brittles or setae. The setae provide

action for movement and also enable the worms to cling to their burrows when predators try to

pull them out. Earthworms possess both male and female gonads. At maturity, it develops

swollen region behind the anterior which is called as clitellum. It deposits its eggs in a cocoon

without the free larval stage.

Cocoon production starts at the age of 6 weeks and continues till the end of 6 months.

Under favorable conditions, one pair of earthworms can produce 100 cocoons in 6 weeks to 6

months (Ismail, 1997). Cocoon is a translucent, small, spherical protective capsule in which

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earthworms lay their eggs. The shape, size, colour and number of cocoons vary from species to

species. The incubation period of a cocoon is roughly about 3-5 weeks, in temperate worms it

ranges between 3-30 weeks and in tropical worms within 1-8 weeks. Quality of organic waste is

one of the factors determining the onset and rate of reproduction (Garg et al., 2005). Epigeic

earthworms swallow large quantities of decaying animal waste and plant litter. The quantity of

food taken by a worm varies from 100 to 300 mg g-1 body weight day -1 (Edwards and Lofty,

1972). While the worm is feeding, the buccal chamber is everted and the food is drawn into the

mouth by the sucking action of the muscular pharynx. The gizzard serves to break the food into

fine particles which is then sent into the intestine where gastric juices act on the ingested food to

digest proteins, fats and carbohydrates. The excreta is egested through the anus as castings

(Ismail, 2005).

Composting, Vermicomposting and Vermiculture

Composting is bioconversion of organic matter by heterotrophic microorganisms

(bacteria, fungi, actinomycetes and protozoa) into humus-like material called compost. The

process occurs naturally provided the fight organisms, moisture, aerobic conditions, feed

material and nutrients are available for microbial growth. By controlling these factors the

composting process can occur at a much faster rate.

Vermicomposting is the process by which worms are used to convert organic materials

(usually wastes) into a humus-like material known as vermicompost. The goal is to process the

material as quickly and efficiently as possible. Vermiculture is the culture of earthworms. The

goal is to continually increase the number of worms in order to obtain a sustainable harvest. The

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worms are either used to expand a vermicomposting operation or sold to customers who use

them for the same or other purposes. If the goal is to produce vermicompost then we want to

have maximum worm population density all of the time. If the goal is to produce worms then we

keep the population density low enough so that reproductive rates are optimized.

Principle of vermicomposting

Certain species of earthworms can ingest organic waste rapidly and fragment them into

much fine particles by passing them through gizzard. The earthworms maintain aerobic

conditions in the vermicomposting process, ingest solids and convert a portion of it to earthworm

biomass and respiration products and egest peat like material termed as worm castings (Loehr et

al., 1985). Castings are much more fragmented, porous and microbially active than parent

material (Edwards, 1988a; Edwards, 1998b; Edwards and Bohlen, 1996) due to humification and

increased decomposition. The earthworms derive their nourishment from the microorganisms

involved in the waste decomposition; and organic waste to be decomposed. The earthworms and

the microorganisms act symbiotically to accelerate and enhance the decomposition of the organic

waste. The composition of the worm casting depends on the parent material. During this process,

important plant nutrients such as nitrogen, potassium, phosphorus, calcium etc. present in the

waste are converted through microbial action into forms that are much more soluble and

available to plants than those in the parent substrate (Ndegwa and Thompson, 2001). Overall, the

vermicomposting process is a result of the combined action of earthworms and microflora living

in earthworm intestine and in the organic waste (Albanell et al., 1988).

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A revolution is unfolding in vermiculture studies for vermicomposting of diverse organic

wastes by vermiculture technology using waste eater earthworms into a nutritive „organic

fertilizer‟ and using them for production of „safe food‟, both in quantity and quality without

recourse to agro-chemicals. Heavy use of agro-chemicals since the „green-revolution‟ of the

1960‟s boosted food productivity, with the cost of environment and society. It killed the

beneficial soil organisms and destroyed their natural fertility, impaired the power of „biological

resistance‟ in crops to make them more susceptible to pests and diseases. Chemically grown

foods have adversely affected human health. The scientific community all over the world is

desperately looking for an „economically viable, socially safe and environmentally sustainable‟

alternative to the agro-chemicals. Vermiculture biotechnology promises to usher in the „Second

green revolution‟ by completely replacing the destructive agro-chemicals which did more harm

than good to both the farmers and their farmland during the „First green revolution‟ of the 1950 -

60‟s. Earthworms restore and improve soil fertility and boost crop productivity by the use of

their excreta - „vermicast‟. They excrete beneficial soil microbes, and secrete polysaccharides,

proteins and other nitrogenous compounds into the soil. They promote soil fragmentation and

aeration, and bring about „soil turning‟ and dispersion in farmlands.

Worm activity can increase air-soil volume from 8 - 30%. One acre of land can contain

up to 3 million earthworms, the activities of which can bring up to 8 - 10 tons of „top soil‟ to the

surface every year. Presence of worms improves water penetration in compacted soils by 50%

(Kangmin and Peizhen, 2010; Ghabbour, 1996; Bhat and Kambhata, 1994). A study in India

showed that an earthworm population of 0.2 - 1.0 million per hectare of farmlands can be

established within a short period of three months. On an average 12 tons/ hectare/year of soil or

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organic matter is ingested by earthworms, leading to upturning of 18 tons of soil/year, and the

world over at this rate it may mean a 2 inches of fertile humus layer over the globe (Bhawalkar

and Bhawalkar, 1993; White, 1997). Earthworms have over 600 million years of experience in

waste and land management, soil improvement and farm production. No wonder, Charles

Darwin called them as the „unheralded soldiers of mankind and farmer‟s friend working day and

night under the soil‟ (Martin, 1976; Satchell, 1983). Importance of earthworms in growth of

pomegranate fruit plants was indicated by the ancient Indian scientist Surpala in the 10th Century

A.D. in his epic „Vrikshayurveda‟ (Science of tree growing) (Sadhale, 1996).

The concept of sustainable agriculture

It is not enough to produce „sufficient food‟ to feed the civilization but also to produce a

„high quality of nutritive food‟ which should be „safe‟ (chemical free) and also „protective‟ to

human health and to produce it in a sustainable manner to ensure „food security‟ for all, but most

for the poor developing countries in the long term. „Food safety and security‟ is a major issue

everywhere in the world and this urgently needs a change in strategy of farm production. The

new concept of farm production against the destructive „chemical agriculture‟ has been termed as

„sustainable agriculture‟. This is about growing „nutritive and protective foods‟ with the aid of

biological based „organic fertilizers‟ without recourse to agro-chemicals. This is thought to be

the answer for the „food safety and security‟ for the human society in future. The U.S. National

Research Council (1989) defined sustainable agriculture as „those alternative farming systems

and technologies incorporating natural processes, reducing the use of inputs of off-farm sources,

ensuring the long term sustainability of current production levels and conserving soil, water,

energy and farm biodiversity‟. It is a system of food production which avoids or largely excludes

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the use of systematically compounded chemical fertilizers and pesticides and use of

environmentally friendly organic inputs.

A powerful growth promoter and plant protector

Earthworms vermicompost is a highly nutritive organic fertilizer which is rich in humus,

nitrogen (N, 2 - 3%), phosphorus (P, 1.55 - 2.25%), potassium (K, 1.85 - 2.25%), micronutrients,

beneficial soil microbes like „nitrogen-fixing bacteria‟ and mycorrhizal fungi. This organic

fertilizer was scientifically proved as miracle plant growth promoters (Tiwary et al., 1989; Binet

et al., 1998, Chaoui et al., 2003; Guerrero, 2010). Kale and Bano (1986) reports as high as

7.37% of nitrogen and 19.58% of phosphorus as P2O5 in worm‟s vermicast. Furthermore,

Suhane et al. (2007) showed that exchangeable potassium (K) was over 95% higher in

vermicompost compared with conventional compost. There are also over 60% higher amounts of

calcium (Ca) and magnesium (Mg). Vermicompost has very high porosity, aeration, „drainage‟

and water holding capacity. The more important is that it contains plant-available nutrients and

appears to increase and retain the nutrients for longer period of time. Pajon (Undated) rated it as

4 - 7 times more powerful growth promoter than conventional compost. A matter of still greater

agronomic significance is that worms and vermicompost increases biological resistance in plants

(due to actinomyctes) and protect them against pest and diseases either by repelling or by

suppressing them (Anonymous, 2001; Rodriguez et al., 2000; Edwards and Arancon, 2004).

Many studies have shown that the presence of earthworm and its vermicompost resulted in

advantages as explained below.

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High levels of bio-available nutrients for plants

Earthworms mineralize the nitrogen (N), phosphorus (P), and all essential organic and

inorganic elements in the compost to make it bio-available to plants as nutrients (Buchanan et

al., 1988). They recycle N in soil in very short time, ranging from 20 - 200 kg N/ha/year and

increase nitrogen contents by over 85% (Patil, 1993). After 28 weeks the soil with living worms

contained 75 ppm of nitrate nitrogen (N), compared with the controlled soil which had only 45

ppm (Barley and Jennings, 1959). Worms increase nitrogen levels in soil by adding their

metabolic and excretory products (vermicast), mucus, body fluid, enzymes and decaying tissues

of dead worms (Dash and Patra, 1979; Whalen et al., 1999). Lee (1985) suggested that the

passage of organic matter through the gut of worm results in phosphorus (P) converted to more

bio-available forms. This is done by both worm‟s gut enzyme „phosphatases‟ and by the

phosphate solubilizing microorganisms in the worm cast (Satchell and Martin, 1984).

High level of beneficial and biologically active soil microorganisms

Among beneficial soil microbes stimulated by earthworms are nitrogen-fixing and

phosphate solubilizing bacteria, the actinomycetes and mycorrhizal fungi. Suhane et al. (2007)

found that the total bacterial count was more than 1010/gr of vermicompost. It included

Actinomycetes, Azotobacter, Rhizobium, Nitrobacter and Phosphate Solubilizing Bacteria,

ranging from 102 - 106 per g of vermicompost.

Humus

Vermicompost contains „humus‟ excreted by worms which makes it markedly different

from other organic fertilizers. It takes several years for soil or any organic matter to decompose

21

to form humus while earthworms secrete humus in its excreta. Without humus plants cannot

grow and survive. The humic and fulvic acids in humus are essential to plants in four basic ways:

1) Enables plant to extract nutrients from soil; 2) Help dissolve unresolved minerals to make

organic matter ready for plants to use; 3) Stimulates root growth; and 4) Helps plants overcome

stress. Presence of humus in soil even helps chemical fertilizers to work better (Kangmin, 1998;

Kangmin and Peizhen, 2010). This was also indicated by Tomati et al. (1987) and Canellas et al.

(2002) found that humic acids isolated from vermicompost enhanced root elongation and

formation of lateral roots in maize roots. Humus in vermicast extracts „toxins‟, „harmful fungi

and bacteria‟ from soil and protects plants.

Plant growth hormones

Edwards and Burrows (1988) and Atiyeh et al. (2000) speculated that the growth

responses of plants from vermicompost appeared more like „hormone-induced activity‟

associated with the high levels of nutrients, humic acids and humates in vermicompost.

Researches show that vermicompost use further stimulates plant growth even when plants are

already receiving „optimal nutrition‟. It consistently improved seed germination, enhanced

seedling growth and development, and increased plant productivity significantly much more than

would be possible from the mere conversion of mineral nutrients into plant-available forms.

Neilson (1965), Tomati et al. (1987, 1995) and Suhane et al. (2007) have also reported that

vermicompost contained growth promoting hormone „auxins‟, „cytokinins‟ and flowering

hormone „gibberellins‟ secreted by earthworms.

22

Soil enzymes

Vermicompost contain enzymes like amylase, lipase, cellulase and chitinase, which

continue to break down organic matter in the soil (to release the nutrients and make it available

to the plant roots) even after they have been excreted (Tiwary et al., 1989; Chaoui et al., 2003).

They also increase the levels of some important soil enzymes like dehydrogenase, acid and

alkaline phosphatases and urease. Urease play a key role in N2-cycle as it hydrolyses urea and

phosphates bioconvert soil phosphorus into bio-available form for plants.

Controlling pest and disease without pesticides

Earthworms are both „plant growth promoter and protector‟. There has been considerable

evidence in recent years regarding the ability of earthworms and its vermicompost to protect

plants against various pests and diseases either by suppressing or repelling them or by inducing

biological resistance in plants to fight them or by killing them through pesticidal action.

Furthermore, the actinomycetes fungus excreted by the earthworms in their vermicast produce

chemicals that kill parasitic fungi, such as Pythium and Fusarium (Edward and Arancon, 2004).

Yardim et al. (2006) reported that application of vermicompost reduced the damage by striped

Cucumber beetle (Acalymma vittatum), spotted Cucumber beetle (Diabotrica undecimpunctata)

and larval hornworms (Manduca quinquemaculata) on tomatoes in both greenhouse and field

experiments. There are several plant protection abilities of earthworms. Recently, Newington et

al. (2004) have implicated earthworms as influencing the abundance of above-ground herbivores

and their natural enemies (crop pests) which they devour.

23

Ability to induce biological resistance in plants

Vermicompost contains some antibiotics and actinomycetes which help in increasing the

„power of biological resistance‟ among the crop plants against pest and diseases. Pesticide spray

was significantly reduced where earthworms and vermicompost were used in agriculture (Singh,

1993; Suhane et al., 2007).

Ability to repel crop pests

There seems to be strong evidence that worms varmicastings sometimes repel hard-

bodied pests (Anonymous, 2001). Edwards and Arancon, (2004) reports statistically significant

decrease in arthropods (aphids, buds, mealy bug, spider mite) populations, and subsequent

reduction in plant damage, in tomato, pepper, and cabbage trials with 20 and 40% vermicompost

additions. George Hahn, doing commercial vermicomposting in California, U.S., claims that his

product repels many different insects‟ pests. His explanation is that this is due to production of

enzymes „chitinase‟ by worms which breaks down the chitin in the insect‟s exoskelton (Munroe,

2007).

Ability to suppress plant disease

Arancon et al. (2002) reported that vermicompost application suppressed 20 - 40%

infection of insect pests that is, aphids (Myzus persicae), mearly bugs (Pseudococcus spp.) and

cabbage white caterpillars (Peiris brassicae) on pepper (Capiscum annuum), cabbage (Brassica

oleracea) and tomato (Lycopersicum esculentum). Furthermore, Edwards and Arancon (2004)

have found that the use of vermicompost in crops inhibited the soil-born fungal diseases. They

also found significant suppression of plant-parasitic nematodes in field trials with pepper,

24

tomatoes, strawberries and grapes. The explanation behind this concept is that high levels of

agronomically beneficial microbial population in vermicompost protects plants by out-competing

plant pathogens for available food resources that is, by starving them and also by blocking their

excess to plant roots by occupying all the available sites.

In addition, Edwards and Arancon (2004) also reported the disease suppressing effects of

applications of vermicompost, on attacks by fungus Pythium on cucumber, Rhizoctonia on

radishes in the greenhouse, by Verticillium on strawberries and by Phomposis and Sphaerotheca

fulginae on grapes in the field. In all these experiments vermicompost applications suppressed

the incidence of the disease significantly. They also found that, the ability of pathogen

suppression disappeared when the vermicompost was sterilized, convincingly indicating that the

biological mechanism of disease suppression involved was „microbial antagonism‟. Meanwhile,

Edwards et al. (2007) reported considerable suppression of root knot nematode (Meloidogyne

incognita) and drastic suppression of spotted spider mites (Tetranychus spp.) and aphid

(M. persicae) in tomato plants after application of vermicompost teas (vermiwash liquid). They

are serious pests of several crops.

Vermiwash - A growth promoting and plant protecting

The brownish-red liquid which collects in all vermicomposting practices is also

„productive‟ and „protective‟ for farm crops. This liquid partially comes from the body of

earthworms (as worm‟s body contain plenty of water) and is rich in amino acids, vitamins,

nutrients like nitrogen, potassium, magnesium, zinc, calcium, iron and copper and some growth

hormones like „auxins‟, „cytokinins‟. It also contains plenty of nitrogen-fixing and phosphate

25

solubilising bacteria (nitrosomonas, nitrobacter and actinomycetes). Vermiwash has great

„growth promoting‟ as well as „pest killing‟ properties. Buckerfield and Webster (1998) reported

that weekly application of vermiwash increased radish yield by 7.3%. Thangavel et al. (2003)

also observed that both growth and yield of paddy increased with the application of vermiwash

and vermicast extracts.

Farmers from Bihar in North India reported growth promoting and pesticidal properties

of this liquid. They used it on brinjal and tomato with excellent results. The plants were healthy

and bore bigger fruits with unique shine over it. Spray of vermiwash effectively controlled all

incidences of pests and diseases significantly reduced the use of chemical pesticides and

insecticides on vegetable crops and the products were significantly different from others with

high market value (Suhane et al., 2007; Sinha et al., 2009). George et al. (2007) studied the use

of vermiwash for the management of „Thrips‟ (Scirtothrips dorsalis) and „Mites‟

(Polyphagotarsonemus latus) on chilli amended with vermicompost to evaluate its efficacy

against thrips and mites. Vermiwash was used in three different dilutions e.g. 1:1, 1:2 and 1:4 by

mixing with water both as „seedling dip‟ treatment and „foliar spray‟. Six rounds of vermiwash

sprays were taken up at 15 days interval commencing at two weeks after transplanting.

Among the various treatments, application of vermicompost at the rate of 0.5 ton/ha with

6 sprays of vermiwash at 1:1 dilution showed significantly lower incidence of thrips and mites

attack. The treatment resulted in very low mean population of thrips and mites. In addition, the

application of vermicompost gave a highest yield (2.98 quintal/ha). Giraddi (2003) also reported

significantly lower pest population in chilli applied with vermiwash (soil drench 30 days after

26

transplanting, and foliar spray at 60 and 75 days after transplanting) as compared to untreated

crops. Suthar (2010 a) has reported hormone like substances in vermiwash. He studied its impact

on seed germination, roots and shoots length in Cyamopsis tertagonoloba and compared with

urea solution (0.05%). Maximum germination was 90% on 50% vermiwash as compared to

61.7% in urea solution. Maximum root and shoot length was 8.65 and 12.42 cm on 100%

vermiwash as compared to 5.87 and 7.73 cm on urea. The seedlings with 100% vermiwash foliar

spray showed the maximum level of total protein and soluble sugars in their tissues.

Studies on the role of Vermiculture Biotechnology (VBT)

There have been several reports that earthworms and their excretory products (vermicast)

can induce excellent plant growth and enhance crop production. Baker et al. (1997) found that

the earthworms (Aporrectodea trapezoids) increased growth of wheat crops (Triticum aestivum)

by 39%, grain yield by 35%, lifted protein value of the grain by 12% and also resisted crop

diseases as compared to the control. Baker et al. (2006) also reported that in Parana, Brazil

invasion of earthworms significantly altered soil structure and water holding capacity. The grain

yields of wheat and soybean increased by 47 and 51%, respectively, Palanisamy (1996) reported

that earthworms and its vermicast improve the growth and yield of wheat by more than 40%.

Bhatia et al. (2000), Sharma (2001) and Suthar (2010a, 2010b) also reported better yield and

growth in wheat crops applied with vermicompost in soil. Kale et al. (1992) who studied on the

agronomic impacts of vermicompost on rice crops (Oryza sativa) reported that greater population

of nitrogen fixers, actinomycetes and mycorrhizal fungi inducing better nutrient uptake by crops

and better growth. Jeyabal and Kuppuswamy (2001) studied the impact of vermicompost on rice-

legume cropping system in India.

27

They showed that the integrated application of vermicompost, chemical fertilizer and

biofertilizers (Azospirillum and Phosphorbacteria) increased rice yield by 15.9% over chemical

fertilizer used alone. Guerrero and Guerrero (2008) also reported good response of upland rice

crops grown on vermicompost. Buckerfield and Webster (1998) found that worm worked waste

(vermicompost) boosted grape yield by two-fold as compared to chemical fertilizers. Treated

vines with vermicompost produced 23% more grapes due to 18% increase in bunch numbers.

Furthermore, a study on grapes carried out on „eroded wastelands‟ in Sangli district of

Maharashtra, India, treated with vermicasting at the rate of 5 tons/ha showed that the grape

harvest was normal with improvement in quality, taste and shelf life. The soil analysis showed

that within one year pH came down from 8.3 - 6.9 and the value of potash increased from 62.5 -

800 kg/ha. There was also marked improvement in the nutritional quality of the grape fruits

(Sinha et al., 2009).

Arancon et al. (2004) studied the agronomic impacts of vermicompost and inorganic

(chemical) fertilizers on strawberries (Fragaria ananasa) when applied separately and also in

combination. Significantly, the „yield‟ of marketable strawberries and the „weight‟ of the „largest

fruit‟ was 35% greater on plants grown on vermicompost as compared to inorganic fertilizers in

220 days after transplanting. Also, there were 36% more „runners‟ and 40% more „flowers‟ on

plants grown on vermicompost. Also, farm soils applied with vermicompost had significantly

greater „microbial biomass‟ than the one applied with inorganic fertilizers. Singh et al. (2008)

also reported that vermicompost increased the yield of strawberries by Sinha et al. 32.7% and

also drastically reduced the incidence of physiological disorders like albinism (16.1 - 4.5%), fruit

malformations (11.5 - 4%), grey mould (10.4 - 2.1%) and diseases like Botrytis rot. By

28

suppressing the nutrient related disorders, vermicompost application increased the yield and

quality of marketable strawberry fruits up to 58.6%.

Webster (2005) studied the agronomic impact of vermicompost on cherries and found

that, it increased yield of „cherries‟ for three years after „single application‟ inferring that the use

of vermicompost in soil builds up fertility and restore its vitality for long time and its further use

can be reduced to a minimum after some years of application in farms. Studies on the production

of important vegetable crops like tomato (Lycopersicum esculentus), eggplant (Solanum

melangena) and okra (Abelmoschus esculentus) have yielded very good results (Guerrero and

Guerrero, 2006; Gupta et al., 2008; Sinha et al., 2009). Agarwal et al. (2010) studied growth

impacts of earthworms (with feed materials), vermicompost, cow dung compost and chemical

fertilizers on okra (A. esculentus). Worms and vermicompost promoted excellent growth in the

vegetable crop with more flowers and fruits development. But the most significant observation

was drastically less incidence of „Yellow Vein Mosaic‟, „Color Rot‟ and „Powdery Mildew‟

diseases in worm and vermicompost applied plants. Meena et al. (2007) studied the growth

impacts of organic manure (containing earthworm casts) on garden pea (Pisum sativum) and

compared with chemical fertilizers. It produced higher green pod plants, higher green grain

weight per plant, higher percentage of protein content and carbohydrates and higher green pod

yield (24.8 - 91%) as compared to chemical fertilizer.

Baker et al. (2006) reported a study of earthworms on soil properties and herbage

production in a mined field micro-plot experiment in Ireland. The presence of earthworms had

little effect on herbage production in the first year. But total herbage yield was 25% greater in the

29

second year and 49% greater in the third year in plots receiving annual topdressing of cattle

slurry with earthworms compared to similarly-treated plots with cattle slurry but without

earthworms. The conclusion drawn from such study is that earthworms in soil are paramount in

plant productivity. In the first year, it took the worm to restore and condition the mined soil. By

second year, enough nutritive „vermicast‟ got accumulated in soil and improved soil fertility

which promoted higher herbage yield (25 %). In the third year, the worm population in soil

increased significantly leading to higher excretion of vermicast, higher soil fertility and higher

plant production (49%). In a bucket experiment they found that the cumulative herbage yields

over a period of 20 months was 89% higher in buckets with earthworms added with cattle

manure as compared to those without earthworms but only with cattle manure, and only 19%

higher in buckets receiving exclusive chemical fertilizers.

Ansari (2008) studied the production of potato (Solanum tuberosum) by application of

vermicompost in a reclaimed sodic (alkaline) soil in India. With good potato growth, the sodicity

(ESP) of the soil was also reduced from initial 96.74 - 73.68 in just about 12 weeks. The average

available nitrogen (N) content of the soil increased from initial 336.00 - 829.33 kg/ha. Sinha et

al. (2009) reported that farmers at Phaltan in Satara district of Maharashtra, India, applied live

earthworms to their sugarcane crop grown on saline soils irrigated by saline ground water. The

yield was 125ton/ha of sugarcane and there was marked improvement in soil chemistry. Within a

year, there was 37% more nitrogen, 66% more phosphates and 10% more potash. The chloride

content was less by 46%. Earthworms and its vermicompost works like „miracle growth

promoter‟ and is nutritionally superior to the conventional compost and chemical fertilizers.

Reduced incidence of „pest and disease attack‟, and „better taste of organic food products

30

especially „fruits and vegetables‟ grown with vermiculture are matter of great socioeconomic and

environmental significance (Hand, 1988; Lee, 2003). Presence of earthworms in soil particularly

makes a big difference in growth of flowering and fruit crops and significantly aid in fruit

development. The 18% increase in yield of wheat crops over chemical fertilizers in their farm

studies made in India has great economic and agronomic significance. Use of vermicompost over

the years build up the soil‟s physical, chemical and biological properties restoring its natural

fertility.

Subsequently, reduced amount of vermicompost is required to maintain productivity.

VBT will truly bring in „economic prosperity‟ for the farmers, „ecological security‟ for the farms

and „food security‟ for the people. With the growing global popularity of „organic foods‟ which

became a US $ 6.5 billion business every year by 2000, there will be great demand for

earthworms and vermicompost in future (Sinha et al., 2010b). The „natural control of crop pests‟

influenced by earthworms seems particularly fruitful research area to be pursued. More study is

required to develop the potential of „vermiwash‟ as a sustainable, non-toxic and environmentally

friendly alternative to the „chemical pesticides‟. Earthworms are justifying the beliefs and

fulfilling the dreams of Charles Darwin who called earthworms as „friends of farmers‟ and that

of Anatoly Igonin of Russia who said „Earthworms create soil and improve soil‟s fertility and

provides critical biosphere‟s functions: disinfecting, neutralizing, protective and productive‟.

Anti-cancer activity

Earthworm has been recorded with a long history. Five hundred years ago, Shizhen Li

compiled the famous medical book Compendium of Material, in which the earthworm (Earth

31

dragon) was recorded as a drug prescribed for antipyretic and diuretic purposes in the form of

dried powder in clinic. Now the remedy is still used in the folk. In the end of 19th century,

Fredericq [1878] discovered one enzyme secreted from the alimentary tract of earthworm. Then

several proteases were separated from the earthworm in 1920 (Keilin, 1920). They could

dissolve casein, gelatin, and albumin. This was the preliminary research about the earthworm

proteases. Large-scale research about earthworm protease began in 1980. Mihara et al. [1983]

isolated a group of proteases with fibrinolytic activity from the earthworm Lumbricus rubellus.

Subsequently different purification methods were applied to isolate the enzymes, including gel

filtration, affinity chromatography, ion exchanging chromatography, and high-pressure liquid

chromatography (HPLC). More proteases have been obtained from different species, such as

lumbrokinase (Mihara et. al., 1983), earthworm-tissue plasminogen activator (Wu and Fan,

1986), earthworm plasminogen activator (Yang and Ru, 1997; Yang et al., 1998a; 1998b; 1998c;

1998d; 1998e) component A of EFE (EFEa) (Tang et al., 2000; Tang et al., 2002), and

biologically active glycolipoprotein complex (G-90) [Popovic et al., 2001; Popovic et al., 1998;

Hrzenjak et al., 1998a; 1998b; Hrzenjak et al., 1992; Grdisa et al., 2001).

Clinical application and medical research- the earthworm protease as a fibrinolytic agent

The formation of thrombus in the blood causes many devastating diseases such as stroke

and myocardial infarction. Several enzymes have been used as the thrombolytic agents including

urokinase (UK), streptokinase, recombinant tissue-type plasminogen activator, staphylokinase,

and recombinant prourokinase (Verstraete M. 2000; Zhao J. and Li D. 2002). These agents are

administered via intravenous injection generally. Some of them are effective, but they also have

some limitations such as fast clearance, lack of resistance to reocclusion, bleeding complications,

32

and other adverse effects (Verstraete M. 2000). The earthworm protease functions in the

fibrinolysis and plasminogen activation, distinct from those enzymes (UK, tissue-type

plasminogen activator, etc.) (Kasai et al., 1985; Madison et al., 1995; Kim et al., 1993).

Therefore they have been used to treat the thrombosis. The proteases during oral experiments

both in animals and clinics show significant fibrinolytic efficacy. A distinct amelioration is

observed in the treatment of blood high-viscosity syndrome and thrombocytosis (Cong et al.,

2000). In addition, the proteases are stable during a long-term storage at room temperature

(Nakajima et al., 2000), in the form of oral capsule. Earthworm is easily raised, which renders

the isozymes into a relatively inexpensive thrombolytic agent. So far, the earthworm proteases

have been used as an orally administered fibrinolytic agent to prevent and treat clotting diseases,

such as myocardial infarction and cerebral thrombus (Jin et al., 2000).

Antitumor

Cancer has a reputation of being an incurable disease. Although some methods such as

surgery, chemotherapy, radiation therapy, and immunotherapy are available, they are far from

reaching the goal of complete removal of the cancer cells without damage to the rest of the body.

It is demonstrated that the earthworm crude extract has the ability to kill the cancer cells directly

in vitro (Zhang and Wang 1987; Zeng et al., 1995) and inhibit the occurrence and development

of tumor in vivo (Wang et al., 1986). Furthermore, it has been proved that the earthworm

proteases enhance the curative effects by both radiation therapy and chemotherapy (Zhang et al.,

1991; Zhang et al., 1992). The most malignant tumors secrete urokinase-type plasminogen

activator (u-PA). In order to inhibit the hyperactivity of the u-PA, inhibitors of plasminogen

activators are synthesized by the surrounding cells for tissue protection, resulting in a high

33

concentration of fibrin locally. The glycolipoprotein mixture (G-90) was isolated from the

homogenate of E. fetida (Popovic et al. 1998; Hrzenjak et al., 1998a; 1998b; Grdisa et al., 2001),

which is assayed in a euglobulinic test applied to fibrin clot from blood plasma of patients who

suffered from malignant tumors. The effect of G-90 on the fibrinolysis rate is related to not only

its concentration, but also to histological type where the malignant tumors invade. The blood

with the fibrin clots derived from the dogs with cardiopathies and the dogs with malignant

tumors was examined for the time of coagulation and fibrinolysis by adding different substances

including G-90. The clotting time in the presence of G-90 shows dogs with malignant tumors

healthy dogs with cardiopathies (Popovic et al., 2001).

Recently, a glycosylated component is separated from the earthworm E. fetida by Xie and

coworkers (Xie et al., 2003), which has relations with apoptosis of tumor cells. It is highly

homologous to LrP-I-1 and LrP-I-2. It is identified to be a plasmin and also a plasminogen

activator. From the results of the phase-contrast microscopy observation of apoptotic cells and

the localization of fluorescent antibodies in cell nucleus, the antitumor activity is observed. The

earthworm protease possesses obvious anti-tumor activity in the hepatoma cells. The

proliferation of the hepatoma cell treated with the proteases is inhibited in proportion to the

concentration of the proteases. The growth of tumor xenograft in nude mice is significantly

suppressed after being fed with the earthworm protease for four weeks. At the same time, it has

been found that the earthworm protease can induce apoptosis of hepatoma cells and down-

regulated the expression of matrix metal protease-2. As described above (Chen et al., 2007), the

earthworm protease is a potential candidate for treating some kind of tumors (Rong et al., 2010).

34

Earthworm mediated bioremediation

Bioaugmentation of soil with xenobiotic-degrading micro-organisms is generally

hindered by the poor transport and dispersal of soil inoculants (Elsas and Heijnen, 1990), and has

been criticized as an ineffective strategy for treating contaminated soils (Goldstein et al., 1985).

In situ bioremediation is also limited by the supply of suitable electron acceptors (Harding, 1997;

Margesin et al., 2000). While anaerobic conditions can lead to removal of halogenated

substituted xenobiotics, such as the PCB‟s, textile dyes etc (Wiegel and Wu, 2000), their

mineralization is exclusively aerobic (Furukawa, 1982; Robinson and Lenn, 1994).

Consequently, in oxygen limited sites, such as the soil subsurface, mineralization of toxic and

other synthetic dyes (xenobiotics) can be limited without manual mixing of the contaminated soil

(McDermott et al., 1989) or the introduction of oxygen from forced air, pure oxygen or hydrogen

peroxide (Alexander, 1999; Harkness et al., 1993).

In nature, the movement of soil animals and earthworms can enhance the transport and

distribution of bacteria. Earthworms have been shown to improve the dispersal of soil inoculants

through bioturbation (Daane et al., 1997; Doube et al., 1994; Hampson and Coombes, 1989;

Hutchinson and Kamel, 1956; Singer et al., 1999; Stephens et al., 1994; Thorpe et al., 1996), and

transport of the microbial inoculant into the burrows via bypass Flow (Bouma et al., 1982;

Edwards et al., 1992; Ehlers, 1975; Farenhorst et al., 2000; Lee, 1985; Madsen and Alexander,

1982; Pivetz and Steenhuis, 1995). Earthworm activity and burrowing also has been shown to

increase soil aeration (Kretzschmar, 1978; Kretzschmar, 1987; Lee, 1985; Schack-Kirchner and

Hildebrand, 1998). Through their mucilaginous secretions, earthworms `prime' the soil, thereby

increasing microbial activity and mineral nutrient availability (Wolters, 2000).

35

Despite the abundance of evidence suggesting earthworms could contribute significantly

to improving in situ xenobiotic remediation through mixing, aeration, and improved soil fertility,

there has been surprisingly little research on their use in a bioremediation strategy and especially

the textile bioremediation.

36

Vermi‟ stands for earthworm, which are regarded as „Farmer‟s friend‟ from the time

immemorial. Earthworms are known for their beneficial role in the soil system and form a major

component of the soil ecosystem. They have been efficiently ploughing the land for millions of

years and are also known as biological indicators of soil fertility (Batt and Steiner, 1992). They

support adequate growth of bacteria, fungi, and actinomycetes and protozoan, which are essential

for sustaining a healthy soil (Desetir, 1991). They act on organic debris and accelerate the

decomposition process in natural manure known as “vermicomposting”.

Vermiculture is a mixed culture containing soil bacteria mixed and an effective strain of

earthworms (NIIR Board, 2008). Earthworm has efficiency to consume all types of organic rich

waste material including vegetable waste, industrial and other organic waste. Vermicroposting

refers to the production of plant nutrient rich excreta of worms.

Earthworms play a vital role in plant growth. It is a quite possible to effect quick change

over for sustainable agriculture by harnessing brand new vermicompost technology to the soil.

An earthworm‟s body consists of a series of cylindrical segments, and earthworms move by

changing the dimensions of each segment (Gray and Lissmann 1938). A spacious fluid-filled

cavity, the coelom, runs the length of the earthworm, providing the animal with support. This

fluid is called as the „Coelomic fluid‟. The coelom acts as a hydrostatic skeleton, transferring the

forces generated by the muscles to the animal‟s environment (Alexander 1983). Coelomic fluid

does not flow from one segment to the next because an earthworm‟s coelom is divided internally

by walls called septa. A segment can be regarded as a cylinder of constant volume, because the

coelomic fluid cannot be compressed, and experiments by Newell (1950) demonstrated that

37

coelomic fluid does not flow from one segment to the next. A decrease in segment length must

be accompanied by an increase in segment circumference, and vice versa. When circular muscles

contract, the segment lengthens and axial pressure is produced. When longitudinal muscles

contract, the segment shortens and radial pressure is produced. In normal movement the activity

of the earthworm‟s muscles is co-ordinated to give waves of segment elongation and contraction

which pass from the anterior to the posterior. Earthworms use axial pressure to thrust a segment

forwards. Radial pressure is used to widen the burrow, and it also plays a role in enabling the

earthworm to grip the burrow walls. The thickness of the circular muscles led Chapman (1950)

to propose that radial pressure was the more important pressure for a burrowing earthworm.

Chapman suggested that earthworms burrowed primarily by using radial pressure to widen

existing crevices in the soil, and he called this concept “crevice burrowing”. Radial pressures

have been found to be higher than axial ones in burrowing earthworms (McKenzie and Dexter

1988a, 1988b; Keudel and Schrader 1999), as Chapman (1950) predicted. Earthworms can ingest

soil to create crevices, and then widen the crevices using radial pressure (Kemper et al. 1988). In

addition to widening a burrow, earthworms may use radial pressure to reduce stresses in the soil

around the anterior of the body.

In recent times, the commercial vermin culturists have started promoting a product called

vermiwash. This vermiwash would have enzymes, secretions of earthworms which would

stimulate the growth and yield of crops and even develop resistance in crops receiving this spray.

Such a preparation would certainly have the soluble plant nutrients apart from some organic

acids and mucus of earthworms and microbes (Shivsubramanian and Ganeshkumar, 2004). But

so far there are no experimental evidences or standardized method for collecting the coelomic

38

fluid – the so called „Vermiwash‟. Neither is there any report on the physicochemical properties

of this fluid nor any characterization recorded so far in its original concentrated form.

The present study was carried out to evaluate the composition of coelomic fluid by

considering different collection methods. The fluid was then subjected to physical and

biochemical characterization. For this, following objectives were set.

Standardization of the procedure for collecting coelomic fluid from E. eugeniae

Determination of the biochemical constituents of coelomic fluid.

Characterization of proteins present in the coelomic fluid by SDS PAGE.

Characterization of protease activity of the coelomic fluid by gelatin diffusion assay.

Characterization of endogenous metabolite profile of Coelomic fluid.