1. General Introduction

1. GENERAL INTRODUCTION

Conservation, reuse, recycling and composting are the solid waste management philosophies of the 21st century. Earthworms are Annelidan Oligochaetes, forming the major terresterial macrofauna which constitute more than 80 per cent of the soil invertebrates biomass. Earthworms are nature’s own tiller, aerator, crusher, composter, moisture builder of the top soil and above all soil ultimate friend and benefactor (Watanabe, 1975; Lal, 1988). Earthworms have 600 million years of experience as a biomanager of soil (Bhawalkar, 1994). Aristotle and Darwin have referred earthworms as “the intestine of the earth” and the “nature’s plough to man”. At present earthworms are extensively used for production of vermicompost from organic wastes, vermiprotein, soil reclamation, soil detoxification and abatement of environmental pollution (Garg et al., 2005). Earthworms have increasingly been used for organic wastes such as animal waste, plant waste, industrial waste, municipal garbage and industrial sludges management and production of nutrient rich organic manure-vermicompost. The earthworms and/or vermicompost are used to: degrade organic waste, indicate environmental pollution, detoxicate polluted soil, turnover the soil, prepare protein rich animal feed, produce medicine, improve soil physico-chemical properties, restore soil fertility, induce plant growth, increase plant productivity and reduce pathogenic microbes. In a nutshell, earthworms may referred as waste stabilizer, compost manufactures, protein producers, pollution preventers, ecosystem engineers and in short Biogold. 2

1.1. Earthworm Reynolds (1994) reported worldwide occurrence of 3,627 terrestrial earthworms. So far, 402 species and subspecies of earthworms belonging to 66 genera and 10 families are known from India. Indian earthworm’s fauna is represented by 357 native and 45 exotic peregrine species (Julka, 2001). On the basis of morphological nature and ecological strategies, earthworms have been classified into three groups by Bouche (1977). He has classified earthworms into epigeic or surface dwelling forms (eg. Perionyx excavatus, Eisenia fetida, and Eudrilus eugeniae), anecic (or) top soil dwelling forms (eg. Lampito mauritii and Pheretima diplotetratheca) and endogeic or bottom soil dwelling (deep burrowing) forms (eg. Lumbricus terrestris and Allolo bophora longa).

Among the three functional groups, epigeic species have greater potentiality for degrading organic wastes, and endogeic species have better capacity of protein conservation and soil turn over whereas anecic species remain inbetween these two species (Dash and Senapati, 1980). Edwards (1988) characterized seven species of earthworms which are utilized for vermicomposting in different parts of the world. They are E. eugeniae, P. excavatus, E. fetida, E. andrei, L. rubellus, Dendrobaena veneta, and Polypheretima elongata. Indian earthworms other than Eudrilus eugeniae and Eisenia fetida that could possibly be utilized for vermicomposting of organic wastes are Lampito mauritii, Dendrobaena repaensis, Metaphise houlleti (Julka and Sanapati, 1993; Kaushik et al., 1999).

1.2. EARTHWORM IN ORGANIC WASTE MANAGEMENT In recent years, the problem of efficient disposal and management of organic solid wastes has become more vigorous due to rapidly increasing populations, intensive agriculture and industralization. Production of large quantities of organic wastes all over the world poses major environmental (offensive odor, contamination of ground water, and soil) and disposal problems (Edwards 1985). According to Vimal and Talashilkar (1983) 22683 lakh tons of organic waste are annually available in India, with gross NPK content of 13967 thousand tons. Recently, Bhattacharjee (2002) reported that India produces about 3000 million tons of organic waste annually which could be utilized for recovering important resources like fertilizer, fuel, food and fodder. This huge amount of organic waste also has the potentiality to produce 400 million tons of plant nutrients besides biogas and alcohol.

The generation of solid waste in India is increasing in an alarming way leading to the great environmental degradation due to the putricible matter present in these wastes. The complex structural composition of organic wastes resist their breakdown, so the natural decomposition becomes a slow process, resulting in the accumulation of these wastes in large quantities which in turn, lead to environmental pollution and cause hygenic problem. Under the present conditions of acute energy crisis and environmental degradation with the growth of industries, cities, and ever increasing human population, it has become essential to develop

4 appropriate technology for recovery of energy from non-conventional sources like organic wastes which are once thought to be of no use.

Actually wastes are nothing but misplaced and mismanaged wealth of organic resources and wastes become a source of renewable energy if properly utilized. Proper utilization of organic wastes can improve soil physical condition and environmental quality as well as provide nutrients for plants (Bhardwaj, 1995). Now the major problem is concerned with the method of utilization or recycling of organic wastes. The present method of utilization of organic wastes by land spreading, open land dumping and traditional composting are inefficient and yet there remains a major problem of utilizing these wastes profitably in order to realize their full potential and avoid pollution. Further, traditional method of composting resulting in losses of about 55% of organic matter and from 30 to 50% of nitrogen (Ketkar, 1993).

The employment of earthworms for waste management as an efficient alternative method is suggested by many researchers (Kale, 1994; Ramalingam, 1997; Parthasarathi and Ranganathan, 1999; Atiyeh et al., 2000; Arancon et al., 2003; Manivannan et al., 2004; Suthar, 2006). Because of their food and feeding habits the earthworms should be considered nature’s most useful converters of organic wastes into organic manure.

1.3. THE GROWTH AND REPRODUCTION OF EARTHWORM The growth of earthworms is most commonly measured in terms of increase in weight (biomass). The increase in biomass of the whole body (growth rate) is expressed as in mg or gram per worm per day or in terms of percentage (Maynard et al., 1983). Growth in earthworms according to one view is by elongation of predestined segments (Kale, 1994) and the other considers it due to addition of new segments during post-emergence growth. Reinecke and Viljoen (1988) by their study on Eudrilus eugeniae found the latter view to be applicable.

The feasibility of using earthworm for waste management is dependent on a fundamental knowledge about the basic parameters like the survival, growth and reproduction of earthworm species. Environmental conditions and population density are known to affect growth and reproduction of earthworms. The potential of earthworms as the waste processor has been well estabilized (Neuhauser et al., 1979; Kale, 1994; Elvira et al., 1998; Parthasarathi et al., 1999; Karmegam and Daniel, 2000; Ramalingam, 2004; Norman et al., 2007; Herlihy, 2007). But the wastes decomposing potentiality depends upon biomass production and reproduction capacity of those earthworm species selected for vermiculture.

Earthworms’ growth, maturation, cocoon production and reproduction potential are not only influenced by the environmental conditions alone but also strongly affected by the quality and availability

6 of food (Reinecke and Viljoen, 1990; Garg et al., 2005). Hence, to understand the full potentiality of compost worms as waste decomposer, it is essential to study the growth and reproduction of earthworms that could be cultured on variety of organic wastes. Earthworms have been shown to require food rich in nitrogen, cellulose, microorganism for their growth and reproduction (Hartenstein and Bisesi, 1989; Ranganathan and Parthasarathi, 1999). Earthworms continue to grow throughout their lives, but the rate of growth declines following sexual maturity (Reinecke et al., 1992; Edwards and Bohlen, 1996; Singh, 1997; Kale, 1998). However, reports on the chemical changes during breakdown of the organic waste by the activity of the earthworm are scarce (Albanell et al. 1998; Ghosh et al., 1999). The aim of the present investigation is to document the stepwise chemical changes during the composting of kitchen waste by an indigenous species of earthworm. Such study is necessary to determine the time of vermistabilization for harvesting of quality compost from a particular type of waste.

1.4. VERMICULTURE AND VERMICOMPOSTING TECHNOLOGY “Vermiculture” is the rearing of earthworms in a suitable substrate (food medium) in order to raise their population and biomass. Vermiculture is practiced for the mass production of earthworms with the multiple objectives of waste management, soil fertility, detoxification and vermicompost production for sustainable agriculture (Edward, 1988). Vermicomposting is defined as “the biodegradation of organic matter

7 occurring when earthworm feeds on them under control conditions” (Hervas et al., 1989). 1000 kg of moisture organic wastes can be converted into 300 kg of vermicompost by earthworms in 45 to 60 days. One kg of earthworm could produce 10 kg of vermicompost in 45-60 days (Gunathilagaraj, 1994). Vermiculture through vermicomposting involves hundred times higher resource generation when compared to the conventional composting. For example vermicomposting of 100 kg of organic wastes will give 2 kg of NPK, but through conventional composting, only one kg of NPK could be obtained. Earthworms also have a greater impact on nitrogen transformations in manure, by enhancing nitrogen mineralization, so that mineral nitrogen was retained in the nitrate form (Atiyeh et al., 2000).

Pre–requisite for vermicomposting

Optimum pH (6), moisture (60-70%), temperature (20-30C), C:N ratio (less than 35:1), proper aeration, best quality and availability of the food and its partial decomposition by microbes, protection from direct sunlight, rain and predators, enough living space and avoidance of toxic conditions are the important pre-requisites for vermicomposting.

Phases of vermicomposting There are many methods of composting such as indoor method, pit method, heap method, Berkly method, Nadep method and windrow method (Jambhekar, 1994). According to Senapati et al. (1984) pit method of vermicomposting involves three phases as follows:

Phase 1: The first phase include the process of collection of organic wastes, mechanical separation of non-degradable materials, shredding (crushing) and storage of organic wastes. Phase 2: The second phase known as vermicomposting phase, involves preparation of worm beds or wastes mixtures, inoculation of appropriate worm species (generally epigeic worms) and their maintenance. Phase 3: The third phase is known as vermicomposting collection and worm harvesting phase, involves the separation of earthworms from the compost by mechanical (hand sorting sieving) or photo or thermal methods besides the collection of vermicompost.

Mechanism and advantage of vermicomposting During vermicomposting, the organic wastes undergoes physical, chemical and biological changes. Generally, the worms feed daily 1 to 20 times of their live weight (Senapati and Dash, 1984). Earthworms, the secondary decomposer, while feeding on partially degraded (by microbes) organic matter, make them further get disintegrated into finer particles by the grinding action of the gizzard in its alimentary canal. By this mechanical action of gizzard particle size is decreased thereby increasing the surface area available for growth of microbes. This promotes further breakdown of organic matter on the one hand and provide greater supply of food for worms on the other hand (Neuhauser et al., 1980; Edwards et al., 1985).

Vermicompost are the rich source of macro and micro nutrients, vitamins, enzymes, antibiotics, growth hormones and microflora with low C:N ratio (Bhawalkar, 1991). Further, vermicomposts are finely divided peat like materials with a high porosity, aeration, drainage and water holding capacity and usually contain more mineral elements (Norman et al., 2007). Many of these elements (nutrients) are changed into forms that can be taken up more readily by the plants, such as nitrates exchangeable phosphorus and soluble potassium, calcium and magnesium (Edwards and Burrows, 1988; Norman, 2007).

Vermicomposting of organic wastes has numerous benefits (Sabine, 1978; Bano and Kale, 1992). It produces useful and marketable organic fertilizer and vermiprotein for live stock food or even human food by increasing the biomass and population of worms during vermicomposting of organic wastes. It also reduce obnoxious qualities of organic wastes such as bad smell, harmful microbes, decreases bulk density of waste and thus helps to manage environmental pollution and safe disposal of wastes in a beneficial way. In addition, it also creates job opportunities and generates income for rural masses.

1.5. MICROBIAL POPULATION OF VERMICOMPOST The microorganisms (mainly bacteria, fungi, actinomycetes) are the primary decomposer of organic wastes. The microorganisms not only mineralize complex substances (organic waste) into plant available form but also can synthesis whole series of biologically active substances

(Pramanik et al, 2007). Vermicomposting is a principle originates from the fact that earthworms by the process of feeding, fragmenting the substrate thereby, increasing the surface area for further microbial colonization and faster bio-degradation (Chaioui et al., 2003). Microbes are responsible for the biochemical degradation of the organic matter. Earthworms are the important drivers of the process, conducting the substrate (organic wastes), producing congential conditions for the activities of microbes and altering biological activity (Aira et al., 2002). Earthworms primes the symbiotic gut microflora with secreted mucus and water to increase their degradation of ingested organic matter and the release of assimilable metabolites (Pramanik et al., 2007). Thus the micro-organisms and earthworms act symbiotically to accelerate and enhance the decomposition of organic matter and as a consequence, mineralization and humification takes place resulting in the availability of nutrients for plants (Lee, 1985; Edwards and Bohlen, 1996; Chaioui et al., 2003). Earthworm’s gut harbours specific symbiotic microflora (Lavelle et al., 1983; Wallwork, 1983; Parthasarathi and Ranganathan, 2000).

Earthworms derive nutrients from the decomposing organic matter and also from the proliferating microorganisms. Though earthworms are saprophytes or detritivores, they actually require a combination of microorganisms, cellulose and grit (soil) for the maximal growth (Flack and Hartenstein, 1984). Fungi and bacteria are the sources of protein rich food for earthworms. Many earthworm species have been found predominantly to utilize soil fungi and bacteria as the food (Edwards and

Bohlen, 1996; Ranganathan and Parthasarathi, 1999). Bacterial and fungal feeding by earthworms have been reported by many investigators (Ponomareva, 1962; Parle, 1963; Dash et al., 1979; Cook and Luxton, 1980; Parthasarathi et al., 1997; Parthasarathi and Ranganathan, 1999; Ramalingam, 2004).

Vermicomposting and microorganisms Microorganisms occurred in an increased level in the vermicompost. Among various types of microorganisms, fungi and bacteria are predominant in vermicompost and soil. Various researchers by comparative analysis of earthworms casts and soil reported that passage of soil or organic matter through the worm’s gut usually resulted the following in the cast: an increased level of microbial population (Ponomareva, 1962; Dash et al., 1979; Satchell, 1983; Mulongoy, 1986; Tiwari et al., 1989; Peterson and Hendriksen, 1993); microbial activity (Mulongoy, 1986; Parthasarathi and Ranganathan, 1999); microbial respiration (Parle, 1963); nitrification (Syers et al., 1979; Scheu, 1987); denitrification (Elliott et al., 1990); enzyme activity and NPK enrichment (Lee, 1985; Mulongoy, 1986; Tiwari et al., 1989); VAM spores (Reddell and Spain, 1991) and production of polysaccharide gum by bacteria (Tomati and Galli, 1995). Further, muco-polysaccharides with vermicast act as an important substrate for aerobic free-living beneficial microbes. So cellulolytic, lignolytic, nitrifying and nitrogen fixing microorganisms are found to establish on worm cast (Satchell, 1983; Hartenstein, 1983; Kale et al., 1988).

1.6. IMPORTANCE OF SOIL Soil performs wide range of functions which sustain either directly or indirectly the human population. It consists of inorganic mineral particles, organic matter and living organisms. Soil plays a vital role in food production and provides nutrients for plant growth and yield. It act as a reservoir of water and store house for various minerals. In India, the area of productive agricultural land is about 143 million hectares of which 83 million hectare suffer from various degrees of soil degradation. Hence conservation of soil fertility, plant productivity and wide use of soil are very essential today.

Chemical fertilizer The chemical fertilizers contains only 5 or 6 element in high concentration (N P K are the prime elements). The continuous use of large quantities of chemical fertilizers to increase the yield and the use of improved crop varieties and pesticides cause several hazards such as: heavy loss of macronutrients (Prasad and Singh, 1981); deficiency of micronutrients (Kanwar and Radhawa, 1978); nutrient imbalance, reduction in organic matter content (Padmaja et al., 1996) and as a result soil gets deteriorated, which in turn affects the plant productivity. As a consequence, organic farming has evolved and this could maintain simultaneously both soil fertility and plant productivity. In organic farming, organic manures and biofertilizers are mainly used. In the era of sustainable organic farming, vermicomposting for the production of

13 organic manure and the use of vermicompost in agricultural lands is a break through in the field of agriculture.

Organic manure (i) Farm yard manure Organic manures serve as cheap and effective substitute for chemical fertilizers. The multitude of literatures indicated that FYM application to soil has improved physical, chemical properties of soil, organic matter content, availability of minerals and nutrients, uptake of nutrients by plants, microbial population, plant growth and yield etc. (Yoshida, 1980). At present the availability of common organic manures like FYM and green manure are limited due to various reasons. So utilization of available organic wastes for production of organic manure using earthworms and recycling them in agriculture in the form of vermicompost is gaining importance in recent years (Edwards et al., 1985; Kale, 1994; Aira et al., 2007).

(ii) Vermicompost Vermicompost has been shown to have higher level of organic matter, organic carbon, total and available NPK, micronutrients, microbial and enzyme activities and growth regulators (Lee, 1985; Edwards and Bohlen, 1996; Bhawalkar, 1999; Parthasarathi and Ranganathan, 1999, 2000; Ramalingam and Thilagar, 2000; Ramalingam and Ranganathan, 2001; Christy et al., 2005). Vermicompost have less soluble salts, neutral pH, greater cation exchange capacity and humic acid content (Albanell et

14 al., 1998; Ramamoorthy., 2004). They contain nutrients in forms that are taken by the plant readily such as nitrates, exchangeable phosphorus and soluble potassium, calcium, magnesium (Edwards and Burrows, 1988; Orazco et al., 1996). Apart from this, it contains plant growth promoting substances such as cytokinins, gibberllins, IAA etc. Vermicompost are usually more stable than their parent materials with increased availability of nutrients and improve physico-chemical and biological properties of soil (Edwards and Burrows, 1988; Orazco et al., 1996).

1.7. EFFECT OF VERMICOMPOST ON SOIL FERTILITY AND PLANT PRODUCTIVITY Vermicompost promotes soil physical properties: such as porosity, aeration, drainage, granulation, water holding capacity and root penetration (Edwards and Burrows, 1988; Anburani, 2000). Vermicompost can increase soil organic matter, soil water retention and transmission, and other physical properties of soil, decrease bulk density, penetration resistance and increase soil aggregation (Biswas et al., 1970; Turner et al., 1994; Zebarth et al., 1999). Vasanthi and Kumarasamy (1999) found that application of vermicompost has not only enhanced soil fertility but also increased plant productivity. Long term organic manure application is recommended to maintain soil fertility (Galli et al., 1992).

1.8. COWDUNG (CD) Waste from animals is one of the major under utilized resources in many countries including India. Generally animal wastes referred mainly to excreta (dung etc.) and urine are used along with bedding and mixed soil. The other by-products / animal wastes include hairs, feathers, hides, skins, bones, horns, hoofs, tallow, blood and non-edible meat wastes (Jain and Sushil kumar, 1995). The present population of bovines in India consists of 201-206 million cattle and 76-80 million buffaloes. It is projected to be 282 million heads by the end of this century. Cattle and buffaloes make up 60% of the total live stock population, but are the source of 91% of the total dung produced. Dung production per animal, per year (dry weight basis) is calculated as 1.1 tons for cattle (cow/ox) and 1.35 tons for buffaloes (Jain and Sushil kumar, 1995). The live stocking excreta consists chiefly of undigested food. It also contains residue from digestive fluids, waste mineral matter, worn-out cells from the intestinal lining, mucus, bacteria, undigested protein, calcium, magnesium, iron, and phosphorus. Animal excreta and their composition differ due to various factors such as animal age, species, protein and fibre content of the food, environment and productivity (Jain and Sushilkumar, 1995). Cowdung at present, is used as manure, soil conditioner, feed stock for biogas plant, fuel etc. Earthworms are known to feed on cowdung and it is apparently a highly nutritious food (Lee, 1983). Cowdung is known to be one of the best natural feed for earthworms and also usually used for vermicomposting practices.

1.9. KITCHEN WASTE Household kitchen waste is one of the major sources of municipal solid waste. In India, domestic waste is mostly of organic nature and contributes 70% to 80% of urban solid wastes (Kale, 1994). Each household of four family members generates 0.5-0.75 kg kitchen wastes per day (Kale, 1994). Under the present condition of environmental degradation vermicomposting technology is a process of production of vermicompost through stabilization of organic waste by earthworm activity. The process of vermistabilization is due to microbial decomposition of organic matter within the gut of earthworms and thus the undigested released excreta of earthworm does not undergo rapid decomposition (Mitchell et al., 1980). Research on vermicompositing of a variety of wastes is gaining momentum throughout the world (Reinecke et al., 1992; Elvira et al., 1997; Singh, 1997). However, reports on the chemical changes during breakdown of the organic waste by the activity of the earthworm are scarce (Albanell et al., 1998; Ghosh et al., 1999). The aim of the present investigation is to document the stepwise chemical changes during the composting of kitchen waste by an indigenous species of earthworm. Such study is necessary to determine the time of vermistabilization for harvesting of quality compost from a particular type of waste.

1.10. Tea Plant (Camellia sinensis) (L.) O. Kuntze Tea is the cheapest beverage in the world next to water. It is obtained from the tea plant (Camellia sinensis) (L.) O. Kuntze var., which belongs

17 to family Theaceae. Tea is a crop of wide adaptability to different climates and soils in various parts of the world. It is known to have originated in South-East China, and grown in countries with climates ranging from the Mediterranean type to hot, humid tropics (Carr and Stephens, 1992).

Among the tea producing countries in the world, India holds the second largest acreage under this crop next to China. Tea in India had been introduced as a crop as early as in the seventeenth century and has since then been in existence. Tea holds 30 per cent of agriculture in various parts such as Assam, West Bengal, Karnataka, Kerala and Tamil Nadu. The climatic conditions in these places are the main factor, which encourages the development of tea cultivation. High altitude, widespread rainfall and rich mineral content soil contribute to the growth of this crop.

India is one of the largest tea producer and is the biggest consumer in the world. India produces about 800 million kilogram (mkg) of tea. In India, tea is grown on 4,30,888 hectares of land. The world production of tea increased by 2.45 per cent in 2010 to reach 4011.9 mkg compared to 3,939.9 mkg in 2009. India maintained its leading producer status in tea with the share of 28.3 per cent. India’s contribution in world tea production was 1053.7 mkg in 2010 compared to 1046.5 mkg in 2009 (Annual Report, 2010).

In South India, tea is grown in Karnataka, Kerala and Tamil Nadu. It is grown over an area of 85,100 hectares of land out of which Tamil

Nadu constitutes 48,429 hectares of tea cultivation in Anamallais, Kanyakumari, Madurai, Nilgiris, Nilgiris Wynaad and Tirunelveli districts. Among South Indian States, Tamil Nadu has the highest productivity. Tamil Nadu State has maintained its contribution of 55 per cent in South Indian tea production. Out of the 200 mkg of tea produced in South India, the contribution from the small grower sector in Nilgiris is nearly 60 mkg which is 29 per cent of the total production.

Tea planting on commercial scale started in 1853 at Nilgiris. Tea occupies the first position in agriculture in Nilgiris mainly contributed by small growers. Due to widespread rainfall from the North East Monsoon and South West Monsoon in Nilgiris and the temperature in the elevation which does not rise above 25oC tea is grown well. In Nilgiris, tea is cultivated in about 50,000 hectares of land out of which nearly 30,000 hectares are owned by small growers. The average land holding is below 1.00 hectare. India produces a wide range of commercial varieties of tea. In Tamil Nadu, the UPASI varieties such as UPASI-1 – UPASI-27 were cultivated in Nilgiris district. Camellia sinensis (L.), O. Kuntze var. UPASI-9 is one of the important cash crop grown in Nilgiris district of South India (Status Report, 2010).

1.11. INDUSTRIAL TEA WASTE Tea is one of the major drinks in the world. Consumption of ready- made tea, which packed into cans and bottles, has been increasing remarkably in recent years. Beverage companies manufacturing various

19 tea drinks produce tons of tea-leaf waste annually, most of which is burned, dumped into landfills or used as compost. Tea waste may be considered a valuable protein source consisting of 22-35% of crude protein (CP) (Kondo et al., 2004). While tea waste contains high CP, it is known to contain a high proportion of tannins (Kondo et al., 2004). Thus, due attention must be given to tannin of tea wastes when it is used instead of commercial feed in livestock production. India produces about 4,000 million metric tons of organic wastes annually which are disposed by ocean dumping, incineration and land application. Tea and vegetable are important food crops of the world which is cultivated over an area of more than seven million hectares and its annual production is more than eighty million tones. India is one of the second leading producers of tea, which are mostly grown in Niligiris, Tamil Nadu State. The tea waste, cow dung and kitchen waste mixture is very easy to degrade in agricultural field. It is intented to test whether these wastes could be used for vermiculture and vermicomposting. Hence, a series of investigations were carried out to convert tea waste, cow dung and kitchen waste into vermicompost using the earthworms, Eudrilus eugeniae (Kinberg) and Eisenia fetida (Savigny) and to study the efficacy of the application of the vermicompost on crop productivity.

OBJECTIVES OF PRESENT STUDY 1. To study the vermicomposting of industrial tea waste, cowdung and kitchen waste in different proportions by the two different earthworm species Eudrilus eugeniae and Eisenia fetida for a period of 90 days. 2. To determine the growth and multiplication of Eudrilus eugeniae and Eisenia fetida during vermicomposting of tea waste in combination with CD+KW for a period of 90 days. 3. To estimate NPK (macro nutrients) and organic carbon in control compost and vermicompost originated from different proportions of TW+CD+KW substrates by the action of Eudrilus eugeniae and Eisenia fetida for a period of 90 days. 4. To determine the microbial population quantitatively in control compost and vermicompost originated from different proportions of TW+CD+KW by the action of Eudrilus eugeniae and Eisenia fetida. 5. To determine and compare the effect of application of NPK, FYM, 100% vermicompost and 50% vermicompost supplemented with 50% NPK on the growth and yield of tea plant (C. sinensis) in red soil. 6. To findout the changes in the physico-chemical parameters of red soil of Tea plant

2. REVIEW OF LITERATURE

2.1. EARTHWORM Earthworms are classified in the phylum Annelida, class Oligochaeta, order Opisthopora. Earthworm is the common name for the largest members of the Oligochaeta (which is either a class or subclass depending on the author) in the phylum Annelida. In classical systems they were placed in the order Opisthopora, on the basis of the male pores opening to the outside of the body posterior to the female pores, even though the male segments are anterior to the female. Cladistic studies have supported placing them instead in the suborder Lumbricina of the order Haplotaxida. Folk names for earthworm include "dew-worm", "rainworm", "night crawler" and "angleworm". To the casual observer,it may appear that there is only one species of earthworm. However, there are world-wide some 3000 species (Sims and Gerard, 1985).

Earthworms have a dense nutritional content because of their soil- based origin. Previous studies on earthworm have shown its antipyretic, antispasmodic, detoxic, diuretic, antihypertensive, antiallergic, antiasthmatic, spermatocidal, antioxidative, antimicrobial, anticancer, antiulceral and anti-inflammatory activities (Ismail, 1997). Earthworm Lampito mauritii, found throughout India, especially in south India, has been earlier reported to have antimicrobial, anti-inflammatory, antioxidative and antiulceral properties (Balamurugan et al., 2004). E. eugeniae converted the food waste into odour free and nutrient rich 22 material. Similar kind of result was reported by Suthar (2009) using Eisenia fetida in vegetable wastes.

Earthworms are hermaphrodites and sexually matured worms have a distinctive epidermal ring-shaped clitellum, which has gland cells that secrete materials to form the cocoon (Edwards and Lofty, 1977; Edwards and Bohlen, 1996; Gajalakshmi and Abbasi, 2004). The application of different types of species is not reliable unless it is compatible with the specific substrate being studied. The species good for one region may not necessarily be good in another. This is especially true for the deep burrowers as they require more time to acclimatize to a new environment (Singh 1997). Earthworms accelerate the mineralization rate and result in castings with a higher nutritional value and degree of humification, suggesting that this kind of industrial waste can be used in vermicomposting (Albanell et al., 1998).

Eudrilus eugeniae is an exotic and epigeic earthworm species used in India and other countries for management of organic wastes. Kale et al. (1982) have reported that Eudrilus eugeniae is to be the Indian equivalent to Eisenia fetida. Earthworm cast amendment has been shown to increase plant dry weight (Edwards, 1995) and plant N uptake (Tomati et al., 1994).

According to Edwards et al. (2004) earthworms can act as bio-concentrators for heavy metals and toxic materials. The resinous

23 substances excreted by earthworms together with humus produced help to increase the water-retaining capacity of soils. Besides, bulk density of soil was reduced by 30%, which provides vast internal spaces to accommodate air and moisture and an enormous surface upon which hydrolytic and oxidative catalyses can be affected by soil micro-organisms, enzymes and humid substances (Kolher, 1995).

2.2. GROWTH AND REPRODUCTION Growth and reproduction of Eudrilus eugeniae using various organic wastes (as feed materials) had been studied by various researchers. The reproductive potential of earthworm was influenced by the quality and availability of food (Neuhauser et al., 1979; Bhattacharjee, 2002; Julka et al., 2009). Various authors have contributed to our knowledge of the relationship between earthworm’s reproduction with factors such as moisture (Reinecke and Venter, 1987; Julka et al., 2009), temperature (Reinecke and Kriel, 1981; Julka et al., 2009) and food (Neuhauser et al., 1979; Dash and Senapati, 1980; Elvira et al., 1998).

A survey of literature showed reports on growth and cocoon production (Reinecke and Hallatt, 1989); growth and life cycle (Hallatt et al., 1990); growth and reproduction (Loehr et al., 1985; Reinecke et al., 1992; Ismail, 1997).

Elvira et al. (1998) studied growth and reproduction of Eisenia andrei using sludges from paper and textile mill. The growth and

24 reproduction of Eudrilus eugeniae studied by Thilagar (1999) using sugarcane trash; Gajalakshmi et al. (2001) using cowdung spikled paper waste; Chaudhuri et al. (2002) using kitchen wastes; Priyashankar (2005) using rubber leaf; Christy et al. (2005) using sago industrial waste - pressmud mixture, Suthar (2007) using agriculture wastes, FYM and urban solid waste, Earthworms have been shown to require food rich in nitrogen, cellulose and microorganisms for growth and reproduction (Hartenstein and Bisesi, 1989; Ranganthan and Parthasarathi, 1999; Lores et al., 2006; Lazcano et al., 2008).

Manivannan et al. (2004) has studied the growth, reproduction and life cycle of E. eugeniae cultured in sugar industry waste. Many authors have studied the life cycle of the composting earthworm species E. fetida (Edwards et al., 1998; Tripathi and Bhardwarj, 2004; Garg and Kaushik, 2005; Gundai et al., 2002; Suthar, 2009). E. eugeniae with wide choice of habitat and food preference has the highest frequency of distribution (Gajalakshmi et al., 2001; Suthar, 2008; Kale and Bano, 2009; Khawairakpam and Bhargava, 2009), able to withstand wide range of temperature, soil moisture and various other physical factors (Kale and Krishnamoorthy, 1978; Kale et al., 1988; Edwards and Bholen, 1996; Manivannan et al., 2004).

Kale (1994) initial observations of bacterial cells in the mucus of mating earthworms suggested that transmission might occur by direct incorporation into the egg capsules. During earthworm mating, a

25 gelatinous mucus sheath is formed externally around both worms, sperm is exchanged, and a precapsule forms around the clitella of the individual worms. The eggs, sperm, and albumin are expelled through pores into this precapsule, which then slides off the anterior end to form the mature egg capsule with a sealed chitinous shell.

Dominguez et al. (2001) studied the population dynamics of E. eugeniae and the effect of soil moisture and temperature on the hatching process and emergence pattern of juveniles in the cattle waste solids, paper waste (Gajalakshmi et al., 2001; Gupta and Garg, 2009), Kitchen wastes (Chaudhuri et al., 2000; Adi and Noor, 2009), sewage sludge (Gupta and Garg, 2008; Suthar, 2009; Khawairakpam and Bhargava, 2009).

2.3. VERMICOMPOSTING Vermicomposting is an effective biological process for conversion of organic wastes into a stable end product, where in microbial activity plays an essential role. Increasing civilization and urbanization has led to an increase in the generation of wastes, there by polluting environment from various sources. Disposal and environmental friendly management of these wastes has become a serious global problem. Much attention has been paid in recent years to develop efficient low input technologies to convert nutrient rich organic wastes into value-added products for sustainable land practices (Kale et al., 1982; Padma et al., 2002; Garg and Kaushik, 2005).

The potentiality of Eudrilus eugeniae for utilization of organic wastes was studied by Kale et al. (1982); Edwards et al. (1988); Suthar et al. (2007). Further, the growth and population dynamics of P. excavatus, E. eugeniae and E. fetida in different kinds of organic wastes were investigated by Kale (1994); Kaushik et al. (2004); Chaudhuri et al. (2002).

Epigeic earthworms such as Eudrilus eugeniae, Eisenia fetida, Perionyx excavatus, and other composting earthworms are used all over the world for composting of various kinds of organic wastes under seminatural conditions. Researchers from various part of the world have contributed to the knowledge of vermicomposting technology and benefits of vermicomposting organic wastes originated from animals, plants, agriculture, agroindustries, plant based industries, urban sewage etc. The vermicomposting of different organic wastes by diverse variety of earthworms was carried out by Ranganathan (2006).

Reinecke et al. (1992) reported that E. fetida had a wider tolerance for temperature than Eudrilus eugeniae and P. excavatus. It tolerates as high as 42˚C as well as low soil temperature below 5˚C. The quality and amount of food material influences not only the size of earthworm population but also the species present and their rate of growth and fecundity (Dominguez et al., 2000, Chaudhuri and Battacharjee, 2002). Hendriksen (1990) suggested that C:N ratio and particularly polyphenol

27 concentration are the most important factor determining litter palatability in detrivorus earthworms.

Vermitechnology utilizes earthworms as versatile natural bio-reactors to convert organic waste into value-added products, the vermicasts (EPA 1980), at a faster rate and can be applied to industries producing organic wastes through the synergistic effect of microorganisms and earthworms (Roig, 1993; Bhawalkar, 1995; Bhattacharya, 2007; Surekha and Mahadev Kumar, 2007). Zajone and Sidor (1990) reported that greatest weight increase in E. fetida was obtained when 50 g of soil was mixed with 150 g cellulose waste. Nayak and Rath (1996) claimed that organic residues comprising city, industrial, agricultural farm, household and kitchen waste with dead or decaying materials can be used as bedding materials for vermicomposting. Joshi (1997) suggested that animal manure, dairy and poultry waste, food industry waste; slaughterhouse waste or biogas sludge could be used for recycling through vermicompositng. The best results of vermicomposting were obtained from paper and food manufacturing industries when treated with E. fetida, E. andrei and P. excavatus (Piccone et al., 1986).

Vermicomposting system have been developed to utilize high moisture content organic wastes from agricultural, industrial, municipal sources, paper mill sludge, biosolids, aquaculture effluent and food wastes etc. (Neuhauser et al., 1979; Elvira et al., 1995, 1998; Hand et al., 1988; Ndegwa and Thompson, 2000; Suthar, 2006, 2007). Four endemic species

28 of earthworms viz, P. excavatus, Lampito mauritii, Dichogaster bolaui and Drawida willsi are recommended by Senapati (1993) for vermicomposting practices in India along with E. eugeniae and E. fetida.

The review of literature indicated that many researchers have analysed the chemical composition of vermicompost or vermicast and reported: reduction in pH, (near neutral pH) (Haimi and Huhta, 1987); narrow down of C:N ratio (Bhawalkar and Bhawalkar, 1993; Suthar, 2006); decrease in C:P ratio (Mba, 1983; Pore et al., 1992); increase in crude protein, amino acids and vitamins (Vimal and Talashilkar, 1983); reduction in organic carbon content (Mba 1983; Jambhekar, 1992; Ramalingam, 2001) and increase in the levels of total nitrogen (Lee, 1985; Orozco et al., 1995; Gunadi et al., 2002; Christy and Ramalingam, 2005); total phosphorus (Satchell and Martin, 1984); total potassium (Pramanik et al., 2007); available N, P, K, Ca, Mg, Na and Zn (Edwards and Bohlen, 1996) and available Fe, Cu, Mn and Zn; (Hervas et al., 1989) in vermicast.

Bird and Hale (1982) undertook work on sludge contaminated with heavy metals from industrial sources and found an increase in heavy metal concentration in vermicompost applied soil above the prescribed limits, suggesting that vermicomposting could be used for treating waste contaminated with metals, etc. The best result of cotton waste with cattle manure was reported by Zazone and Sidor (1990) tried vermicomposting using urban and industrial sources. Madhukeshwar et al. (1996) claimed

29 that any kind of organic waste generated in an agro-based industry or biotechnology unit when treated with earthworms would be a resourceful vermicompost. Waste from the fruit pulp, biscuit and sugar industries were bio-degradable in field designs using E. eugeniae, E. fetida and P. excavatus for waste management. The wastes were bio-converted to compost in 40-90 days. The quality of the compost obtained had increased micro and macronutrients. Waste from the olive oil industry, either alone or mixed with cattle manure, was a suitable substrate for vermicompost (Elvira et al., 1998; Moreno et al., 2000).

Vermicomposting technology using earthworms can very well be adopted for converting waste into wealth. The viability of using earthworms as a treatment or management technique for numerous organic waste streams has been investigated by a number of workers (Hand et al., 1988; Madan et al., 1988; Logsdon, 1994; Singh et al., 2002). Similarly a number of industrial wastes have been vermicomposted and turned into nutrient rich manure (Sundaravadivel et al., 1995).

Hand et al. (1988) defined vermicomposting as a low cost technology system for the processing or treatment of organic wastes. The activity of earthworms along with micro organisms have brought out a rapid mineralization process and generation of the nutrients for plant growth. Karmegam et al. (2000) also observed more NPK in the vermicompost than in the control.

Vermicomposting of pre-treated pig manure using E. fetida produced a humus rich odour free vermicast (Chan, 1988). Pheretima asiatica could stabilize most of the solids arising from the treatment waste including raw pig manure (Wong, 1991). Various workers have suggested that E. fetida could be used in sludge management (Mitchell et al., 1980; Neuhauser, 1980).

The research publication by Elvira et al. (1997) on paper mill sludge composting by Eisenia andrei revealed a noticeable reduction in organic carbon level, C/N ratio, C/P ratio and an increase in N and P at the end of the experiment. Large amount of humic substance were produced during the vermicomposting and these have been reported to have positive effects on plant growth independent of nutrition (Chen and Ariad, 1990; Atiyeh et al., 2002). The vermicomposts have more available nutrients than the organic waste from which they are produced.

A comparative study, on the quality of organic matter and heavy metal in different mixtures of paper mill sludge and sewage sludge before and after vermicomposting (E. endrei) reported that a 1:6 mixture of paper mill sludge to sewage sludge was the most effective mixture for increasing the weight of E.andrei during the vermicomposting period. vermicomposting of pulp mill sludge mixed with garbage sludge,cowdung, pig slurry and poultry slurry at different ratios showed highest growth and highest mortality of E.andrei in all the mixtures considered (Elvira et al., 1998).

Ghosh et al. (1999) reported higher level of transformation of phosphorus from organic to inorganic state, and thereby into available forms during vermicomposting compared to ordinary composting. Rise in the level of P content during vermicomposting is probably due to mineralization and mobilization of P due to bacterial and faecal phosphatase activity of earthworms (Krishnamoorthy 1990). The studies on the effects of vermicomposting on some components of organic waste showed that vermicompost enhances degree of polymerization of humic substances along with a decrease of ammonium N and an increase of nitric N (Cegarra, 1992).

Vermicompost is homogenous, with desirable aesthetics and possess plant growth hormones and high levels of soil enzymes, while enhancing microbial populations and tending to hold more nutrients over longer periods without adverse impacts on the environment (Ndegwa and Thompson, 2001). Butt (1993) utilized solid paper mill sludge and spent yeast as a feed for soil dwelling earthworms (lumbricus terrestris). Zharikov et al. (1993) reported work on utilizing the wastes of the microbiology industry (Sewage sludge, husks, and low quality bacterial preparations) by red earthworms.

The importance of vermicompost in agriculture, horticulture, waste management and soil conservation has been reviewed by many workers (Riggle, 1994, Edwards, 1995; Kaviraj et al., 2003). Edwards (1995) reported that in a Rothamsted study with 25 types of vegetables, fruits or

32 ornamentals, earthworm casts (EW) performed better than compost or commercial potting mixture amendments. Galli et al. (1992) reported an increase of 30% in protein synthesis in Lactuca sativa seedling following the application of vermicompost.

Vermicomposting involves bio-oxidation and stabilization of organic material through the interactions between earthworms and microorganisms. Although microorganisms are mainly responsible for the biochemical degradation of organic matter, earthworms play an important role in the process by fragmenting and conditioning the substrate, increasing the surface area for growth of microorganisms, and altering its biological activity (Dominguez, 2004; Dominguez and Edwards, 2001).

During the vermicomposting process, earthworms can modify the diversity and abundance of the micro flora directly, by selective feeding, or by stimulation of particular group of microorganisms (Pedersen and Hendriksen, 1993; Devliegher and Verstraete, 1995; Wolter and Scheu, 1999; Tiunov and Scheu, 2000). Earthworms exert other indirect effects on microbial communities, such as microbial dispersion and the release of additional food resources in their cast. For all these reasons, better knowledge of the changes in the chemical and biochemical properties of organic wastes during the vermicomposting process is required to understand the effect of the earthworms activities on the process of biodegradation.

Monsoon et al. (2007) reported an increase in nutrients of kitchen waste vermicomposted by E. eugeniae: in N, from 1.31 to 2.12%; in P, from 0.121 to 0.7%; in K, from 0.45 to 0.48% and the C:N ratio decreased from 32.45 to 13.66% and a significantly higher number of microbes were observed. A higher microbial load was also observed in paddy fields to which vermicompost was applied (Kale et al., 1992). Meena and Renu (2009) reported a increase in nutrients when press mud was blended with saw dust and treated using three different earthworm species E. fetida, E. eugeniae and P. excavatus individually (monocultures) and in combination (polycultures). E. fetida vermicasts from sheep manure alone and mixed with cotton wastes were analyzed for their properties and chemical composition every 2 weeks for 3 months and compared with the same manure without earthworms (Kale et al., 1992).

Vermicompost process will progress properly by starting the process with a C:N ratio around 25-30 and it will decrease during the process. Carbon reduces because heterotrophic bacteria use organic material as source of electron and carbon is oxidized to CO and releases to atmosphere (Tchobanoglous et al., 1993). Increasing in number of worms can be effective in maintenance of pH around neutral range. It is important for obtaining vermicompost to be at the standard range of A class’s range, 6.5-8.4, (Brinton, 2000)

2.4. MICROBIAL STUDIES Microorganisms are essential part of biodiversity and play significant role in structuring and functioning of the ecosystem on the environment. The microorganisms (mainly bacteria, fungi, actinomycetes) are the primary decomposer of organic wastes. The microorganisms not only mineralize complex substances (organic waste) into plant available form but also can synthesis whole series of biologically active substances (Pramanik et al., 2007). Microbes are responsible for the biochemical degradation of the organic matter. Earthworms are the important drivers of the process, conducting the substrate (organic wastes), producing congential conditions for the activities of microbes and altering biological activity (Aira et al., 2002).

Earthworms primes the symbiotic gut microflora with secreted mucus and water to increase their degradation of ingested organic matter and the release of assimilable metabolites (Pramanik et al., 2007). Thus the micro-organisms and earthworms act symbiotically to accelerate and enhance the decomposition of organic matter and as a consequence, mineralization and humification takes place resulting in the availability of nutrients for plants (Lee, 1985; Edwards and Bohlen, 1996; Chaioui et al., 2003). Earthworm’s gut harbours specific symbiotic microflora (Lavelle et al., 1983; Wallwork, 1983; Parthasarathi and Ranganathan, 2000).

Lavelle et al. (1983), have proposed a symbiotic relationship between earthworms and their gut microflora. Earthworms are ubiquitous

35 soil invertebrates that ingest large amounts of mineral soil and organic material containing variety of microorganism (Pedersen and Hendriksen, 1993). Many earthworm species have been found to predominantly utilize soil bacteria (Daniel and Andrson, 1992; Pedersen and Hendriksen, 1993) and soil fungi (Cooke and Luxton, 1980; Edwards and Bhohlen, 1996).

Many authors have studied the microbial community in the gut of earthworms (Fisher, 1995; Karsten, 1997). It is well known that Gram negative bacteria are common inhabitants of the intestinal canal of earthworms (Reyes, 1976). The total microbial load in the different regions of the gut of worms has also shown more intense colonization of microbes in the anterior part of the intestine than the other region. The presence of fungal propagules in the earthworm gut and in cast material has been known for some time (Parle, 1963) and earthworm have been implicated in both the reduction and dispersal of soil borne animal and plant fungal disease and the spread of beneficial group such as mycorrhizal fungi (Gange, 1993). Parle (1963) reported that population of yeast and fungi did not proliferate during passage through the gut, although actinomycetes and bacteria did.

Tiwari and Mishra (1993) have observed that the worm castings are also rich in nutrients and that encourages in the growth of microorganisms in their experiments. Many of these effects are associated with the symbiotic relationships between earthworms and microorganisms, which mainly occur in the earthworm gut, casts burrows, and middens. It is

36 generally accepted that microbial biomass and respiration are greater in earthworm casts than in the parent soil (Tiunov and Scheu, 2000; Aira et al., 2002). However, earthworms can feed on these selectively (Moody et al., 1995; Edwards et al., 2004), resulting in an increase in culturable aerobic micro organisms in the gut contents of earthworms, are seen with studies on Lumbricus terrestris and Lumbricus rubellus (Kristufek et al., 1992; Fisher et al., 1995; Schonholzer et al., 1999).

Microorganisms occurred in an increased level in the vermicompost. Among various types of microorganisms, fungi and bacteria are predominant in vermicompost and soil. Various researchers by comparative analysis of earthworms casts and soil reported that passage of soil or organic matter through the worm’s gut usually resulted the following in the cast: an increased level of microbial population (Ponomareva, 1962; Dash et al., 1979; Satchell, 1983; Mulongoy, 1986; Tiwari et al., 1989; Paderson and Hendriksen, 1993); microbial activity (Mulongoy, 1986; Parthasarathi and Ranganathan, 2001); microbial respiration (Parle, 1963); nitrification (Syers et al., 1979; Scheu, 1987); denitrification (Elliott et al., 1990); enzyme activity and NPK enrichment (Lee, 1985; Mulongoy, 1986; Tiwari et al., 1989); VAM spores (Reddell and Spain, 1991) and production of polysaccharide gum by bacteria (Tomati and Galli, 1995).

Earthworms derived nutrients from the decomposing organic matter and also from the proliferating microorganisms. Though earthworms are

37 saprophytes or detritivores, they actually require a combination of microorganisms, cellulose and grit (soil) for the maximal growth (Flack and Hartenstein, 1984). Fungi and bacteria are the sources of protein rich food for earthworms. Many earthworm species have been found predominantly to utilize soil fungi and bacteria as the food (Edwards and Bohlen, 1996; Ranganathan and Parthasarathi, 1999). Bacterial and fungal feeding by earthworms have been reported by many investigators (Ponomareva, 1962; Parle, 1963; Dash et al., 1979; Cook and Luxton, 1980; Parthasarathi et al., 1997; Parthasarathi and Ranganathan, 1999; Ramalingam, 2004).

2.5. PLANT AND SOIL Tea is the cheapest beverage of the world next to water. It is obtained from the tea plant (Camellia sinensis) (L.) O. Kuntze var. which belongs to family Theaceae. Tea is crop of wide adaptability to different climates and soils in various parts of the world. It is known to have originated in South-East China, and grown in countries with climates ranging from the Mediterranean type to hot, humid tropics (Carr and Stephens, 1992).

Tea waste may be considered a valuable protein source consisting of 22-35 % of crude protein (CP) (Kondo et al., 2004). While tea waste contains high CP, it is known to contain a high proportion of tannins (Kondo et al., 2004). Animals feed tannin rich diets showed decreased feed intake (Silanikove et al., 1994), increased faecal N excretion (Nune-

Hernandez et al., 1991), reduced digestibility and less ruminal degradability (Woodward and Reed, 1997). All living plants require a range of essential nutrients to allow them to function and grow. Nitrogen is used in the production of protoplasm, protein and chlorophyll and is the primary building block for all plants parts. Nitrogen is the main nutrient requirement of tea. Yeilds increase with increasing use of nitrogen up to high levels with good economic returns. Rates of 200-500kg/ha/year are widely quoted in the literature for high yielding tea (Bomheur and Willson, 1992).

Positive effect of adding vermicompost to soil for tomato had shown by Federico et al. (2006). The ability and play on active role of Eisenia fetida to convert waste to vermicompost has been proven in many studies (Bansal and Kapoor, 2000). Other species of red worms or red wigglers such as Lumbricus rubellus, Perionyx sansibaricus, Perionyx excavates, Eisenia andreii and some other species successfully are used in vermicompost production. In vermicomposting the worms are sensitive to pH and they don’t tolerate a wide range of pH and they prefer neutral pH. Although, some studies showed that the worms can be alive in some higher or lower pH, but the recommended pH for vermicomposting is around 6-7 (Dickerson, 2001).

Tea is the most widely consumed beverage in the world. It has been reported that worm casts enhance nutrients uptake by plants (Tomati et al., 1988); stimulate plant root initiation, development and increased

39 root biomass (Grappelli et al., 1985); enhance plant growth, increase crop yield and plant productivity (Edwards, 1983; Grappelli et al., 1985) and increase protein synthetic activity of plants (Tomati et al., 1995). The increased growth and yield of paddy, wheat, maize, tomato, rose, citrus, guava, cumy leaf, turmeric, ornamental plants, cereals, radish, pulses, oil seeds, spices, vegetable fruits and sorghum have been reported by many investigators (Kulkami et al., 1996; Ramalingam, 1997; Sevugaperumal et al., 1998; Atiyeh et al., 1999; Buckerfield et al., 1999; Garg and Bharadwaj, 2000). In recent years, worldwide awareness to increase production and use of vermicompost has been felt in order to reduce environmental pollution, to improve public health and to reduce use of inorganic fertilizer in agriculture.

Tea waste is known to contain a higher percentage of nitrogen than most of other organic manures (Krishnapillai, 1981). The nutrient content of compost depends on the consistency of the ingredients that had been used in its preparation and the level of its degradation. Therefore, the nutrient content of compost is highly variable from sample to sample. Hence, differences could be seen in terms of the treatments (Sharma, 2002).

Cantazaro (1998) demonstrated the importance of the synchronization between nutrient release and plant uptake and showed that slower release of fertilizers can increase plant yield and reduce nutrient leaching.

A large number of investigation have demonstrated the beneficial effects of vermicomposted organic wastes on the growth of a variety of plants (Kale et al., 1992; Atiyeh et al., 2000; Chaoui et al., 2003; Singh et al., 2008). The integration of vermicompost with fertilization tended to increase the yield of crop viz, potato, rape seed, mulberry and marigold (Ghosh et al., 1999; Arancon et al., 2006).

Vasanthi and Kumaraswamy(1999) reported that paddy grain yields were significantly higher in plots treated with vermicompost plus NPK than in the treatment that received NPK alone. Earlier investigations have shown that the worm caste had enhanced nutrient uptake by plants (Tomati et al., 1998; Atiyeh et al., 2002), stimulated plant root intiation, increased root biomass (Grappelli et al., 1985; Atiyeh et al., 2000) and increased crop yield and plant productivity (Kale et al., 1991). Albanell et al. (1998) and Tomati et al. (1990) also reported that vermicompost contains higher amount of humic acid content and biological substances such as plant growth regulators. Edwards (1995) reported that in a Rothamsted study with 25 types of vegetable, fruits and ornamental plants, tea plants, earthworm casts performed better than compost or commercial potting mixture amendments.

Plant nutrients such as nitrogen, potassium, phosphorus and calcium present in the feed material are converted through microbial action into forms that are much more soluble and available to the plants than those in the parent substrate (Ndegwa, 2001). Soil microorganisms play an

41 important role in improving soil fertility and crop productivity due to their capability of fixing atmospheric N, solubilizing insoluble P and decomposing farm wastes resulting in the release of plant nutrients (Joshi and Kelkar, 1952; Tewatia et al., 2007).

3. MATERIALS AND METHODS

3.1. Study area and its topography Nilgiris is a mountain district situated at the junction of Eastern and Western Ghats stretching upto 2,54,055 hectares of land of Tamil Nadu State, India. The elevation of the district ranges from 1000 m to 2640 m. The district of Nilgiris is divided into six taluks viz., Coonoor, Gudalur, Kotagiri, Kundah, Ooty and Pandalur. The main crop in Nilgiris district is tea which occupies considerable acreage of cultivated area. This district is bounded by Karnataka on the North, Coimbatore district on the South, Kerala State on the West and Erode district on the East. It is geographically located between 76o14’ and 77o02’ East longitude and 11o10’ and 11o42’ North latitude. In Ooty taluk, tea occupies 50 per cent of the total cultivated area, in Kundah taluk tea occupies 50 per cent of the total cultivated area, in Coonoor taluk tea occupies 80 per cent of the net cultivated area, in Kotagiri taluk tea occupies 74 per cent of the total cultivated area, in Gudalur taluk tea is cultivated in 31 per cent of the total area and in Pandalur taluk tea is cultivated in 26 per cent of the total area. The twelve study sites (Fig.2) were located at Nilgiris district of Tamil Nadu State at an elevation of 2,640 m. The Doddabetta, at the elevation of 2,640 m above mean sea level, represents the highest peak of the district.

3.2. Biology of Eudrilus eugeniae (Kinberg) 3.2.1. Distribution Eudrilus eugeniae is an epigeic, exotic, detrivorous, tropical, compost earthworm. E. eugeniae has originated from West Africa (Graff, 43

1981) and are popularly called as “African nightcrawler”. They are also found in Sri Lanka and the Western ghats of India, particularly in Travancore and Poona. The biology, growth, reproduction and life cycle of Eudrilus eugeniae have been studied by various researchers (Viljoen and Reinecke, 1994; Ramalingam, 1997; Vinotha, 1999; Manivannan et al., 2004).

3.2.2. Classification Phylum : Annelida Class : Chaetopoda Order : Oligochaeta Suborder : Terricolae Family : Megascolecidae Genus : Eudrilus Species : eugeniae

3.2.3. Habit and Habitats E. eugeniae lives on the surface layer (epigeic) of most soil and are also found wherever organic matter is accumulated (Bouche, 1977). It is nocturnal and lies in the surface layer during the day.

3.2.4. Morphology and growth The worm is reddish brown with convex dorsal surface and pale white, flattened ventral side. The clitellum is paler than the rest of the body. The adult worms are about 25-30 cm in length, 5-7 mm in diameter,

44 consist about 250-300 segments and weigh 5600 mg of maximum individual biomass (Viljoen and Reinecke, 1994) (Plate I). The rate of growth in oligochaetes is relatively proportional to nutritional level (Avel, 1959). Age of the worm, organic matter content, moisture (65 – 75%) and temperature (28 – 34°C) are other factors influencing the growth rate of worms (Lavelle et al., 1983).

3.2.5. Maturation and cocoon production According to Viljoen and Reinecke (1994) the first indications of clitellum development appear between 35-45 days. The formation of cocoon starts within 24hours after copulation and continued upto ± 3days in E. eugenia. The cocoons of E. eugenia have an irregular oval shape and are sharply pointed with fibrous tips at the two ends. The cocoons are soft and greyish-white in color immediately after formation, but harden rapidly with the color changing to orange brown. Finally, the cocoons become dark brown in color immediately before hatchling. The mean length of the cocoon is 6.02 mm (range 4.3 – 7.8 mm), diameter between 2.1 – 4.0 mm and mean mass of 16.99 mg (Reinecke and Viljoen, 1988). A mean production of 1.3 cocoons/worm/ day was observed by Viljoen and Reinecke (1994).

3.2.6. Incubation and hatchlings Incubation period of E. eugenia cocoon is 16.6 days at 25°C in cattle manure with a hatchling success of 84% and 2.5 mean number of hatchlings per viable cocoon (Reinecke and Viljoen, 1988). Upon

45 emergence, the hatchlings have a pink yellowish to red colour with the hinder most segments still not fully differentitated.

3.3. Biology of Eisenia fetida (Savingny) The Chinese epigeic earthworm Eisenia fetida is a surface feeding earthworm (Tsukamoto and Watannbe, 1977; Graff, 1981). The life cycle has been thoroughly investigated and reported by Venter and Reinecke (1988). The compost worm E. fetida lives mainly on dead plant material (Neuhauser et al., 1980).

3.3.1. Classification Phylum : Annelida Class : Chaetopoda Order : Oligochaeta Suborder : Terricolae Family : Lumbricidae Genus : Eisenia Species : fetida

3.3.2. Morphology and growth E. fetida is dark brown in colour with yellow colour in the tip of the tail. The adult worms are about 5-7 cm in length, 3-5 mm in diameter and 500-600 mg of biomass (Venter and Reinecke, 1988) (Plate I). It has a temperature tolerance of 29°C and high level of moisture. It initially grow

46 very slowly during the first 30 days, then the growth rate of the worm is increasing steadily (Venter and Reinecke, 1988).

3.3.3. Maturation and cocoon production In E. fetida the first indication of clitellum development appear 50 days after hatchling. It starts producing cocoon at the mean age of 55days. The rate of cocoon production was 0.35/worm/day. The newly formed cocoon is usually light white in colour and immediately the colour changed into light brown (Venter and Reinecke, 1988). The length of the cocoon is 3.2-4.00 mm, diameter 2.0-2.7 mm and mean biomass of 12.65 mg. The cocoon is oval shape and is sharply pointed with fibrous tips at the ends. A mean production of 1.6 cocoon / worm / day was observed by Viljoen and Reinecke (1994).

3.3.4. Incubation and hatchlings The mean incubation period of E. fetida is 23 days. The average hatching success is 73%. The number of hatchlings/cocoon vary from 1-9 with a mean of 2.7/cocoon (Venter and Reinecke, 1988). The hatchlings are red in colour with the hinder most segments still not fully differentiated.

3.4. Selection of Eudrilus eugeniae and Eisenia fetida for the present study The exotic earthworm Eudrilus eugeniae and Eisenia fetida has been selected for the present study due to the following reasons:

i) The prevailing climatic conditions in South India is ideal for the activity of Eudrilus eugeniae and Eisenia fetida (Bano and Kale, 1988). Since the worms are tropical in origin they perform well at tropical and sub tropical conditions. Further, Edwards (1996) has recommended it as an ideal species for vermicomposting in tropical conditions. ii) It works through its substrate 24 hr. a day and has high through put capacity for organic matter and soil (Hartenstein and Bisesi 1989). It also has shorter transit time for food than the endogeic worm (Kale et al., 1992). It shows higher feeding rate and rapid growth rate than other epigeic worms such as Eudrilus eugeniae and Eisenia fetida. iii) It is a voracious feeder on organic matter and prefers food richer in nitrogen, cellulose and microorganisms (Hartenstein and Bisesi, 1989) and all kinds of organic wastes having these qualities from the diet to this worm. iv) The biomass turnover is the most important factor in the species selection for vermicomposting. Though India is rich in earthworm fauna the majority of them are smaller in size, deep dwellers, humus feeders, characterized by long life cycle and lesser biomass. On the other hand, Eudrilus eugeniae and Eisenia fetida attains higher biomass within shorter period of time with higher fecundity than any other species of earth worms (Hartenstein and Bisesi, 1989). Hence, it is highly effective in working on organic waste (Kate et al., 1992)

Further, it is a rich source of animal protein (Vermiprotein for fish, poultry and pigs) (Sabine, 1978). v) It has a shorter incubation period, higher fecundity and shorter life cycle with longevity more than one year. vi) The castes of this species are easily collectable from the surface. vii) These are easily maintainable in the laboratory on cow dung substance.

3.5. Procurement and rearing of earthworms Eudrilus eugeniae and Eisenia fetida was obtained from the breeding stock maintained in the Department of Tea research foundation (UPASI) of coonoor, Nilgiris, India. The worms were stocked in cement tanks containing urine free, sun dried and powdered cowdung. The cement tanks (with Eudrilus eugeniae and Eisenia fetida stock) were covered by wooden framed iron mesh and maintained at room temperature (27  2C) with 60-70% moisture. Once in 15 days the surface layer of used up cowdung was removed and replaced with fresh cowdung.

3.6. Selection and collection of industrial Tea wastes, cowdung and kitchen waste 3.6.1. Tea Waste (TW) Accumulation of Tea Waste near the industrial premises in landfill sites or incineration of derived Tea Waste leads to environmental pollution, loss of nutrients and has environmental and economical

49 disadvantages. Therefore, biological treatment methods have received much attention and are considered as low cost efficient treatments. One such method is vermitreatment. Vermicomposting could be an adequate technology for the transformation of waste into valuable products (Kondo et al, 2004). Hence waste from the tea industry is collected for vermiculture and utilized for production of organic manure vermicompost in an eco-friendly way. The tea waste with moisture (Plate-II) was collected from waste pond of the Indico Tea Factory at Ooty Tamilnadu premises and brought to the laboratory. In the laboratory tea waste was exposed to the sunlight for two days to kill undesirable organisms and to reduce the foul smell. After two days the tea waste was used for the present study.

3.6.2. Cowdung (CD) Cowdung is deemed as highly suitable natural feed for earthworms (Graff, 1981; Hatanaka et al., 1983; Lee, 1985). Hence, cow dung is selected for the present study to provide nitrogen and stimulate biodegradation of tea waste by the action of Eudrilus eugeniae and Eisenia fetida. Urine and straw free cow dung was collected from dairy yard at the Agricultural Farm, Ooty. It was sundried powdered and stored in jute bags and it is used for the present study.

3.6.3. Kitchen Waste Household kitchen waste is one of the major sources of municipal solid waste. In India, domestic waste is mostly of organic nature and

50 contributes 70% to 80% of urban solid wastes (Kale, 1998). Each household of four family members generates 0.5-0.75 kg kitchen wastes per day (Kale, 1998). Under the present condition of environmental degradation vermicomposting technology is a process of production of vermicompost through stabilization of organic waste by earthworm activity. The kitchen wastes were collected from the houses at Ooty, stored in jute bags and it is used for the present study.

3.7. Growth and reproduction of Eudrilus eugeniae and Eisenia fetida in organic wastes 3.7.1. Preparation of experimental media In the present study, Industrial Tea waste, Cow dung and Kitchen waste mixture were prepared in the following manner. T1 - 1000 (g) soil T2 - 400(g) TW + 200(g) CD + 400(g) KW T3 - 500(g) TW + 100(g) CD + 400(g) KW T4 - 600(g) TW + 100(g) CD + 300(g) KW The vermicomposting experiments were performed for 90 days. Tea Waste (TW), kitchen waste (KW) and Cow dung (CD) were weighed (dry weight) in specific concentration as given in Table 3 and mixed using well water, so as to have 60-70% moisture. The feed mixtures were transferred to separate plastic troughs (35 diameter x 12 cm depths). The Plate II represents the treatments T1-T4. Since initial decomposition was found to improve food acceptability by worms (Edwards and Bohlen,

1996), the feed substrates in the troughs were allowed 15 days for initial decomposition.

3.7.2. Inoculation of Earthworms After 15 days of initial decomposition, 10 newly emerged hatchlings of Eudrilus eugeniae (Kinberg) and Eisenia fetida (Savingny) were collected from the stock culture and introduced after recording their initial weight into each plastic trough T1-T4 each containing one kg of feed substrate). The plastic troughs were covered by nylon net to avoid water loss, to prevent predators and also to allow free aeration. The troughs

(T1-T4) were kept under laboratory conditions of 28  2C in the vermiculture lab and maintained with 60-70% moisture. For each treatment three replicates were maintained.

3.7.3. Growth and reproduction of compost worms Earthworm growth is usually measured in terms of weight (biomass) gain. Once in 15 days up to 90 days weights of earthworms were recorded. Every time hatchlings and adults were collected and weighed separately and the adults were reintroduced into the respective plastic troughs, but the hatchlings were hand sorted and counted. Hand gloves were used to avoid physical damage to earthworms by nails. Handling of worms every 15 days did not cause any damage to worm. Once in a month the newly prepared (Plate III) feed mixtures were added and surface layer of vermicast was removed from the experimental troughs and discarded after checking cocoons and hatchlings.

The growth rate of worms (for specific periods) was calculated using the following formula specified by Mazantseva (1982).

W - W Worms growth rate (mg/ worm / day) = 2 1 T2 - T1

W1 and W2 = Body weight of the earthworm at the beginning

(W1) and at the end (W2)

T1 and T2 = Age of worms at the beginning (T1) and at the end

(T2) of specific periods.

3.8. Statistical analysis Earthworm mean biomass, standard error (SE), biomass increase or decrease percentage over control values were calculated. Further, significance of the data was also tested applying on one way analysis of variance (ANOVA) and Duncan’s multiple-ranged test was used as a posthoc analysis to compare the means.

Chemical analysis For the purpose of chemical analysis the sample were collected from all (T1 – T4) initial mixture (0 days), control (natural compost – after 90 days) and vermicompost at 90th day. Then the samples were air dried in the shade at room temperature, ground in a stainless steel blender and stored in plastic vials for further chemical analysis.

Determination of pH and Electrical conductivity (EC) pH and EC were determined by the method described by ISI Bulletin (1982). 5 gram of dry sample was taken in a 100 ml beaker and 50 ml of distilled water was added. The content was mixed well using a glass rod. After 30 min the pH and EC were determined by using digital pH and electrical conductivity meter (moded-Global DPH500).

Estimation of total nitrogen (N) The total nitrogen of compost sample was estimated by Kjeldahl method, as detailed by Tandon (1993). This method was carried out by two steps (i) digestion of the sample to convert the N compound in the

+ + compost sample NH4 form and (ii) distillation and determination of NH4 in the digest.

(i) Digestion of the sample To a 100 ml Kjeldahl flask, 0.5 g dried sample was transferred. Twenty ml of the sulphuric salicylic acid mixture was added and swirled gently so as to bring the dry sample in contact with the reagents. It was permitted to stand over night. Next day 5 g of sodium thiosulphate was added and heated gently for about 5 minutes. Care was taken to avoid frothing. The contents were cooled to which 10 g of sulphate mixture was added and digested on Kjeldahl apparatus at full heat for 1 hr. Bumping during the digestion can be avoided by adding glass beads. When the digestion was completed, the digest was cooled, diluted and distilled as detailed below.

(ii) Distillation by Kjeldahl method To vaccum jacket of micro-Kjeldhal distillation apparatus, 10 ml of digest was transferred. In a conical flask, 10 ml of 4% boric acid solution was taken, containing bromocresol green and methyl red indicators, to which the condenser outlet of the flask was dipped. After completion of distillation the colour of the solution changed into green. Then this

solution was titrated against 0.1 N standardized H2SO4; appearance of pink colour was the end point.

(iii) Calculation Weight of the sample = 0.5 g Volume of digestion = 100 ml; Aliquot taken = 5 ml Titre value (TV) = Sample TV = Blank TV

TV x 0.00007 x 100x100 N (%) (1 ml of N/10 H2SO4 = 0.00014 gN) 0.5 x 5.0 (Or) N (%) = TV x 0.28]

Estimation of total phosphorus (P) Total phosphorus content of the sample was estimated as per Tandon (1993) by colorimetric method.

(i) Diacid digestion

This method was carried out using a 9:4 mixture of HNO3: HClO4. The procedure was as follows: 1gm of ground sample was taken in 1000 ml of volumetric flask. To this, 10 ml of acid mixture was added and the

55 contents of the flask were mixed by swirling. The flask was placed on low heat hot plate in a digestion chamber. Then, the flask was heated at higher temperature until the production of red NO2 – fumes cease. The contents were further evaporated until the volume was reduced to about 3-5 ml but not to dryness. The end of digestion was confirmed when the liquid become colourless. Then cooling the flask to which the condenser outlet of the flask was dipped. After adding the aliquot digest, the funnel of the apparatus was washed with 2.3 ml of deionised water and 10 ml of 40% NaOH solution was added. 5 ml aliquot was distilled to the flask containing 10 ml of boric acid. After completion of distillation, the boric acid was titrated against N/200 H2SO4. Blank was also carried out to the same end point as that of the sample.

(ii) Determination of phosphorus

(i) Reagents Ammonium molybdate – ammonium vandate in HNO3 In 400 ml of distilled water 22.5 g ammonium molybdate was dissolved and also ammonium vandate was dissolved in 300 ml boiling distilled water and added to molybdate solution and cooled to room temperature. After that 250 ml of conc. HNO3 was added and diluted to 1 litre.

(ii) Phosphate standard solution

In distilled water 0.2195 g of analytical grade KH2 – PO4 – potassium dihydrogen orthophosphate was dissolved and diluted to 1 litre. This solution contains 50 ppm of phosphate.

(iii) Preparation of standard curve To obtain 0, 1, 2, 3, 4 and 5 ppm standard phosphorus solution was pipetted out into 25 ml volumetric flask. Then 10 ml of vanadomolybdate reagent was added to each flask. The volume was made up with deionized water and mixed thoroughly. In about 30 minutes, yellow colour developed. The absorbance of the solution was read at 420nm in the UV – VIS spectrophotometer (Model SL – 159 Elico). Then the standard curve was prepared by plotting absorbance (in Y axis) against concentration P (in x axis).

(iv) Phosphorus content of sample The aliquot from sample digestion (5 ml) was pipetted out to 25 ml volumetric flask and the procedure detailed for standard curve preparation was followed. Twenty ml of deionized (or) glass distilled water was added and the solution was made up to the mark with deionized water. After that it was filtered through whatman No.1 filter paper. (v) Calculation

1 P2O5 in % = sample concentration (ppm) x weight of sample

100 Final volume (ml) x aliquot (ml) 1000

Estimation of total potassium (K) Total potassium content of the substrate was determined by Flame Photometric method as described by Tandon (1993).

(i) Standard stock solution of K To prepare a standard stock solution of K, in a 1000 ml standard flask, 1.91 g of analytical grade KCl salt was dissolved in distilled water and made up to 1 litre. This contains 1000 ppm of K. From this stock, 10, 20, 30, 40 and 50 ppm of K solutions were prepared by appropriate dilutions.

(ii) Preparation of standard curve The flame photometer (Model C1 – 22D) was started and distilled water was first atomized, the galvanometer reading was adjusted to 0.0 (zero). Again fed 50 ppm solution and galvanometer reading was adjusted to 100. Then 10, 20, 30, and 40 standard K solutions were subsequently fed and corresponding galvanometer readings were recorded. A standard curve was drawn by plotting flame photometer reading on Y axis and K concentrations on X – axis.

(iii) K content of sample The unknown sample was atomized into the flame photometer and the reading was recorded. The potassium concentration was determined using the prepared standard curve, and multiplied with dilution factor.

Calculations

x 100 100 K (%) = x x x 100 106 10 1 Here, volume of extract = 100 ml Aliquot taken = 10 ml

100 = dilution factor 1

Estimation of sodium (Na) and calcium (Ca) Sodium and calcium contents of the samples were determined by following the procedure of Tandon (1993) and the method is similar to that of K estimation.

(i) Standard stock solution of Na To prepare a standard stock solution of Na, in a 1000 ml standard flask 2.54 g of NaCl (AR) salt was dissolved in distilled water and made upto 1 litre. This solution contains 1000 ppm of Na. From this stock 5, 10, 15 and 20 ppm of Na solution were prepared by appropriate dilutions.

(ii) Standard stock solution of Ca To prepare a standard stock solution of Ca, 2.25 g of CaCO3 was dissolved in 5 ml of deionized water in a 1000 ml standard flask. Approximately 10 ml of HCl was added drop wise, to dissolve CaCO3 completely. Then diluted to 1 litre with deionized water. This solution contain 1000 ppm of Ca. Standard curve preparation and estimation of Na and Ca concentrations in unknown sample solution were done by the following procedure as described for potassium estimation.

Estimation of magnesium (Mg), zinc (Zn), iron (Fe), copper (Cu) and manganese (Mn) Mg, Zn, Fe, Cu and Mn content were estimated using atomic absorption spectrophotometer (AAS) according to the procedure outlined by Tandon (1993).

Calculation of Mg (%) = a/10 x 100/1 x 100/1 x100 Calculation of micronutrients = a x 100/1 (Zn, Fe, Cu and Mn) (ppm) = a x 100

Estimation of organic carbon (OC) The determination of organic carbon was carried out as per the procedure of ISI Bulletin (1982).

1g of oven dried sample (at 105C) was placed in constant mass

silica crucible and heated in an electrical furnace (muffle furnace) at

550C for 2hr. The crucible was allowed to cool down and again weighed.

Initial mass - Final mass Total carbon (%) = x 100 Initial mass taken

Total carbon (%) Organic carbon (%) = A A = a constant 1.724 (Walkely and Black – 1934)

Calculation of C: N ratio The ratio of the percentage of carbon to that of nitrogen i.e., C/N ratio was calculated by dividing the percentage of carbon estimated for the manure sample with the percentage of nitrogen estimated for the same manure sample.

Statistical analysis

By using computer, mean values ( x ) with standard error (SE) were obtained from the data. The statistical significance between treatments

60 was analyzed using one way analysis of variance (ANOVA). Duncan’s multiple-ranged test (DMRT) was performed to identify the homogenous type of treatments for the various parameters. A significant difference between any pairs of means that was subjected to ANOVA was indicated by CD value.

Determination of microbial population in vermicompost and control compost For the determination of total microbial population (bacteria, fungi and actinomycetes), the fresh samples were collected from control (WU) (i.e. 90 days maintained worm unworked compost) and vermicompost (WW) (i.e. 90 days wormworked compost) of E. eugeniae and E. fetida produced from different concentrations of TW + CD + KW feed mixtures.

Culture and determination of total microbial population The total number of bacteria, fungi and actinomycetes were estimated by “serial dilution plate method” (Allen, 1953). It is assumed that each developing colony in the plate is coming up from a single cell or spore or hypae (Plate V). 10 grams of manure sample was transferred to a 250 ml conical flask with 100 ml of deionized water and thoroughly shaken in a rotary shaker for 10 minutes. 10 ml of the mixture was pipetted out and transferred to water blanks with 90 ml of sterile water. The serial dilutions of each mixture were made by using sterile deionised water and dilution of the manure sample viz. 10-4, 10-5, and 10-6 were prepared (Plate IV).

Appropriate dilution viz., 10-4 for fungi, 10-5 for actinomycetes and 10-6 for bacteria were chosen for respective organisms. An aliquot of 1 ml of the respective dilution were spread in sterile petriplate aseptically and dispersed with respective media viz., Rose Bengal Agar medium for fungi, Nutrient Agar medium for bacteria, and Kenknight’s Agar medium for actinomycetes. Plates were rotated gently three times in clockwise and anticlockwise direction to ensure uniform distribution of the manure mixture. The petriplates were incubated at room temperature (28C). The colonies were developed. The colonies were counted on 3rd, 5th, and 11th day for bacteria, fungi, actinomycetes respectively using colony counter and expressed the population per gram of oven dried sample.

(i) Cleaning and sterilization of glass wares All the glass wares were soaked in cleaning solution for 12 hrs, then in soap water for few minutes and washed in tap water. Finally they are cleaned in distilled water, dried and used. All the media were sterilized

in an autoclave. The glass wares were sterilized at 180C for 3 hrs in an

oven.

(ii) Incubation Thermostat controlled “Hereaus” incubators were used with constant humidity. To maintain a constant humidity the inner walls of the incubator were lined with whatman No. 1 filter paper. In the bottom most rack, petridishes containing distilled water were kept with filter paper wick to continuously irrigate the paper linings.

(iii) Chemicals All the chemicals used in preparing media and reagents were of analytical reagent quality.

(iv) Adjustment of pH The pH of the media was tested by universal pH indicator paper either N’ NaOH or NaOH or N’ HCl was used to adjust the pH.

Composition of microbial culture / growth medium (i) Bacteria Nutrient Agar (Anonymous, 1977) Content Gms / litre Peptic digest of animal tissue 10.00 Beef extract 10.00 Sodium chloride 5.00 Agar 12.00 Distilled water 1000 ml pH 7.3

(ii) Fungi Rose Bengal Agar (Emmon et al., 1970) Content Gms / litre Papaic digest of soyabean meal 5.00 Dextrose 10.00 Monopotassium 0.50

Phosphate 0.50 Rose bengal 0.50 Agar 15.00 Distilled water 1000 ml pH 7.2

(iii) Actinomycetes (Emmon et al., 1970) Content Gms / litre Beef heart infusion, solids 10.00 Tryptose 10.00 Casein enzyme hydrolysate 4.00 Yeast extract 5.00 Dextrose 5.00 L-cysteine hydrochloride 1.00 Starch soluble 1.00 Sodium chloride 5.00 Monopotassium phosphate 15.00 Ammonium sulphate 1.00 Magnesium sulphate 0.20 Calcium chloride 0.20 Agar 20.00 Distilled water 1000 ml pH 6.9

Determination of microbial activity (dehydrogenase activity) To determine the microbial activity (in terms of dehydrogenase activity), samples were collected from initial substrate, worm unworked natural compost (control) and vermicompost prepared with E. eugeniae and E. fetida of all the treatments (T1-T4). Dehydrogenase activity was determined according to the method described by Stevenson (1959).

Reagents preparation 5 ml substrate sample

50 mg CaCO3 1% Triphenyl tetra sodium chloride (TTC) 5 ml methanol

Procedure 5 grams of substrate sample was mixed in a 50 ml beaker with 50 mg of dry CaCO3 and brought to 90% water holding capacity with water containing 0.5 ml of 1% solution of Triphenyl Tetra Sodium chloride (TTC) and the substrate was then thoroughly mixed. The sample was then incubated at 30°C in a humified incubator for 24 hrs. Following incubation, 5 ml of methanol was added to the beaker and stirred for 5 minutes. The resulting slurry was washed into Buchner funnel and washed with successive aliquot of methanol. Volumes of extractants used for individual sample were then recorded. The density of colored extract was determined in UV-VIS spectrophotometer (SL 159) at 485nm, using methanol as the reference blank. Concentrations were determined by

65 comparison with a standard curve of TPF (Triphenyl formazan) in methanol. Results were recorded in volumes of hydrogen transferred during the reduction of TTC to TPF in 5 g of substrate according to the equation: 2, 3, and 5 – TTC + 2H→TPF + HCL. The formation of 1 mg of TPF requires 150.35 µlH. The calculated dehydrogenase activity was expressed in terms of µl/5 g of substrates.

Quality analysis of microbes For the qualitative analysis of microbes the samples were collected from initial, worm unworked natural compost (control), and the vermicompost of (E. eugeniae and E. fetida) T4 treatment. The T4 treatment was chosen in this experiment because this treatment was found to be more suitable for growth and reproduction of the earthworms, higher nutrient contents and more population of microbes than other treatments

(T1, T2, T3 and T4).

Species of fungus, bacteria and actinomycetes isolation from test samples were done according to the method described by Mackie and McCartney (1989).

Isolation of fungi Preparation of Lactophenol cotton blue stain The following components were used to prepare the Lactophenol cotton blue stain.

Lactic acid - 20.0 g Phenol crystals - 20.0 g Glycerol - 40.0 ml Cotton blue - 2.0 ml (1% aqueous) Distilled water - 20.0 ml

Lactic acid and glycerol were added to the distilled water. To this, phenol crystals were added and mixed well. It is heated gently in hot water with frequent agitation until the crystals dissolved completely. Then the cotton blue dye was added and mixed thoroughly and stored in a brown bottle.

Staining process A drop of lactophenol cotton blue was placed on a clean slide. A small tuft of the fungus, usually with spore and spore bearing structures was transferred into the drop, using a flamed, cooled needle. Gently teased the material using the two mounted needles. The stain was gently mixed with the mold structures. A cover glass was placed over the preparation taking care to avoid trapping air bubbles in the stain. The slide was allowed to dry.

After staining, the structure of the fungus was photographed under the Nikon Microscope.

Isolation of bacteria Inoculating needles and loops were used to transfer the microorganisms aseptically from the culture to the slide and stained.

Preparation of s bacterial smear A fresh clean slide was taken and marked the smear area on the underside of the slide with a marking pencil. The slide was flamed and placed on the table. A few amount of sample was taken from the culture medium containing bacteria culture with the help of sterilized needle and transferred to the clean glass slide. The bacteria suspension was spreaded thinly over the slide with the help of another clean slide. The smear was allowed to dry.

Staining of bacteria The gram stain is the most useful and widely employed differential stain in bacteriology. It divides bacteria into two groups namely gram negative and positive.

Preparation of gram stain Gram’s Iodine Iodine - 1.0 g Potassium iodide - 2.0 g Distilled water - 300 ml Iodine and potassium iodide was dissolved in distilled water and stored in a sterilized bottle.

Ethyl alcohol (95%) Ethyl alcohol (100%) - 95.0 ml Distilled water - 5.0 ml 95 ml of ethyl alcohol was mixed with 5 ml of distilled water and stored in a clear bottle.

Safranin Safranin (2.5% solution in 95% ethyl alcohol) - 10.0 ml Distilled water - 100.0 ml 10 ml of Safranin was mixed with 100 ml of distilled water and stored in a clean bottle.

Staining process Prepared bacterial smear was taken and covered with crystal violet for 30 seconds. The slide was washed with distilled water for few seconds using wash bottle. Then the smear was covered with Gram’s iodine solution with 95% ethyl alcohol. Ethyl alcohol was added drop by drop until no more color flows the smear (The gram-positive bacteria are not affected while all gram negative bacteria are completely decolorized).

Then the slide was washed with distilled water and drained. Finally, safranin was applied to smears for 30 seconds. Again the smear was washed with distilled water and blot dried with absorbent paper and allowed to dry. The safranin stained the colorless gram negative bacteria

69 into pink but does not altered the dark purple color of the gram positive bacteria.

After staining process, the bacterial cell on the slide was photographed under the Nikon Microscope.

Statistical analysis

The estimated microbial populations were expressed as the mean 

S.E. Percent changes over control values were also calculated. The difference in the mean values of control (WU) and experimental (WW) microbial population were tested for their statistical significance using one way analysis of variance (ANOVA) and Duncan’s multiple-ranged test at P < 0.05.

Application of vermicompost on the tea plant (C. sinensis (L.) O. Kuntze var. To understand the comparative effect of inorganic fertilizer NPK, organic manure (FYM) and vermicompost on the growth and yield components of tea plant, NPK (inorganic fertilizer) and FYM (Farm yard manure (traditional organic manure) were also selected for the present investigation. NPK is used in the form of urea (N), superphosphate (P), murate of potash (K). Urea, superphosphate and murate potash were purchased from local shop. FYM was collected from dairy yard at the Agricultural Farm, Ooty the Nilgiris.

Treatment Design T1 : Control (without application of inorganic, NPK or vermicompost) T2 : 100% recommended dose NPK T3 : 100% recommended dose vermicompost T4 : 100% recommended dose FYM T5 : 50% NPK and 50% vermicompost

Experimental details of the field The field experiment was conducted at Nunthala village, Niligiris Dt. Tamil Nadu, India. The experiment was conducted from June 2009 to Nov 2010 (18 months). This experiment was laid out in randomized block design with three replications. Minimum and maximum temperature

ranged between 15.6 to 31.1C and relative humidity ranged between 80%

to 95%. These data were periodically collected from the Agricultural Metrological Observatory, Nilgiris.

Cultivation details (i) Field preparation The field was prepared through repeated ploughing upto a satisfactory tilth condition. The clods were broken and field was levelled. The fields were formed as per the layout.

(ii) Plantation Plantation of 3500 plant at a rate of 5 ha-1 was followed. The inter row spacing of 45 cm with an intra row spacing of 45 cm was maintained. Holes were first opened with a marker stick to a depth of 10 to 15 cm and the plants were planted in the holes and covered with soil. Irrigation was given immediately after plantation and care was taken to avoid excess soaking of water.

Biometric observations Three sample plants for each treatment field were selected at random and labelled for biometric observations. The observations on growth and yield parameters were recorded at 6 months, 10 months, 14 months and 18 months (harvest).

a. Growth components i) Shoot length (height) Shoot length of the plant was measured from the base of the main stem to the tip of the terminal leaf in cm. The mean value was calculated and recorded.

ii) Root Length Root length of the plant was measured from the cotyledonary node to the tip of the primary root and expressed in cm.

72 iii) Dry weight of shoot and root

The root and stem were dried at 80C for 48 hrs in an hot air oven and then weighed in electronic balance and expressed in mg. iv) Fresh weight of shoot and root The root and stem were collected fresh and weighed in electronic balances and expressed in gram.

Number of leaves The number of leaves in experimental plants were counted and recorded.

Statistical analysis For growth and yield components of tea plant Camellia sinensis (L). The mean value Standard error and F values were determined. One way analysis of variance (ANOVA) and Duncan’s multiple-ranged test.

Analysis of physico-chemical parameters of the soil Before commencement of the experiment, (ie. before plantation of tea plant) soil samples were collected at random from the experimental field up to 15 cm depth, shade dried, powdered and sieved. The samples were used to analyse physico-chemical parameters viz., Pore space, Bulk density, Particle density, Water holding capacity and available Organic carbon, N P K as per the standard analytical procedures. Likewise the post harvest soil samples were also collected in the respective treatment field,

73 shade dried, powdered, sieved and used to analyse physico-chemical parameters.

Determination of Physical parameters 1. Pore space (PS) The porosity of the soil sample was determined by the method followed by Kanwar and Chopra (1980). 20 g of the sample was weighed and transferred in small quantities to a 100 ml measuring cylinder with the glass stopper by gently tapping the cylinder. The volume of sample was noted. A known volume of 50 ml of water was added to the sample for complete soaking of the sample. The cylinder was kept undisturbed for some time for proper filling of the pore space, with water. The volume of sample and water added were recorded at the end of the experiment and the percentage of pore-space was calculated. Volume of the sample taken : Wg Volume of the sample : p ml Volume of the water added : q ml Volume of the sample and water : p + q ml Volume of the sample and water at the end of the experiment : r ml Pore space (p + q) – r : t ml Percentage of pore space : t/p x 100.

2. Bulk density (BD) BD of the sample was calculated from the following formula,

W BD = Mgm-3 V where W – weight of sample taken V – volume of the sample

3. Particle density (PD) Particle density was measured by using the formula,

W PD = Mgm-3 V - P where W _weight of sample taken V – volume of the sample P – pore space of the sample.

4. Water holding capacity (WHC) WHC of the sample was measured by the method of Baruah and Barthakur (1999). 30 gm of the sample was taken and transferred in a funnel (dia. 35cm). The lower end of the funnel was closed by wet cotton (or) nylon mesh to prevent the soil sample escape from the funnel. The lower part of the funnel was connected by a small tube for collection of water. 100 ml of water was poured into the funnel containing the soil, the excess of water from the soil was collected by a measuring cylinder. The

75 volume of the water was noted on the measuring cylinder at the end of the experiment. WHC of the soil, was calculated from the following method.

Calculation Weight of the sample taken (W) = 30 g

Volume of water added (V1) = 100 ml Volume of the excess water at the

end of the experiment (V2) = x ml

WHC of 30 g sample (V3) = V2 – V1 ml

WHC of 1g sample (V4) = 30  V3 ml

 WHC of 100 g sample = V4 + 100

Chemical parameters 1. Estimation of available nitrogen (N) Available nitrogen present in the soil sample was estimated by alkaline potassium permanganate method as described by Subbiah and Asija (1956).

(i) Reagents preparation

A) 0.32% KMNO4 solution

About 3.2 g of reagent grade KMNO4 was dissolved in distilled water, made up to one litre in volumetric flask and mixed well.

B) 2.5% NaOH 25 g of sodium hydroxide was dissolved in distilled water made upto one litre in volumetric flask and mixed well.

C) Standardised sulphuric acid, 0.02N (N/50) Three ml of conc. sulphuric acid was mixed in one litre distilled water to make N/50 sulphuric acid.

D) Mixed indicator 10 gm of bromocresol green and 0.07 gm of methyl red were dissolved in 100 ml of ethanol.

E) Boric acid-indicator solution 20 gm of boric acid was dissolved in about 900 ml of hot water, cooled and added 20 ml of mixed indicator solution. 0.1 sodium hydroxide solution was added drop wise until colour was reddish purple and diluted to one litre with distilled water.

(ii) Procedure 20 gm of soil was transferred to a distillation flask. 20 ml of distilled water was added and one ml of liquid paraffin wax was added to

control frothing and then 100 ml of 0.32% KMNO4 and 100 ml of 2.5% NaOH solutions were added. Distilled the contents in a Kjeldahl assembly at a steady rate collecting the liberated ammonia in 250 ml beaker containing 20 ml of boric acid with mixed indicator. Distillation was

77 continued for about 30 minutes, and then the contents of the beaker were titrated against the standardized sulphuric acid till pink colour appeared. A blank was run without soil samples.

Calculations Weight of soil taken = 20 g

Volume of standardized H2SO4 consumed in the sample titration = X ml

Volume of H2SO4 consumed in the blank titration= Y ml

Therefore actual volume of H2SO4 used = (X-Y) ml

X – Y mol of N/50 H2SO4 =0.00028  X-Y 0.00028  X- Y kg of N This is present in 20 g of soil = 0.00028  X- Yg of N

Therefore, 2,000,000 kg of soil will contain = 0.0028  X-Y

2 x 106  kg of N 20 Since 1 hectare furrow – slice = 2  106 kg soil Therefore 28 X titrate value = kg. ha-1

2. Estimation of available phosphorus (P) Available phosphorus content in the soil was estimated by the method as described by Olsen et al. (1954).

(i) Reagents preparation

1. NaHCO3 About 84 g of sodium bicarbonate was dissolved in distilled water and made up to 2 litres. The solution was thoroughly mixed and adjusted to pH 8.5 with 1N sodium hydroxide.

2. Reagent – A 12 g of ammonium molybdate was dissolved in 250 ml of distilled water. 0.2908 g of antimony potassium tartarate was dissolved in 100 ml of distilled water. Then these two solutions were added to 1000 ml of 5N sulphuric acid and it was mixed thoroughly and made to 2 litres with distilled water.

3. Reagent – B 1.056 ascorbic acid was dissolved in 200 ml of reagent–A and mixed well. The reagent was prepared fresh before starting the experiment.

(ii) Procedure 5 g of soil was transferred to a 100 ml polythene shaking bottle. A pinch of charcoal was added and 50 ml of 0.5N sodium bicarbonate was added. Then these solutions were shaken in a reciprocatory mechanical shaker for thirty minutes. Then the solutions were filtered through whatman No. 4 filter paper and filtrate was collected in a clean beaker. 5 ml of the filtrate was pipette out into a 25 ml volumetric flask; the solution

79 was diluted to about 16 ml with distilled water. Then 4 ml of reagent – B was added and made up to the mark (25 ml). After ten minutes the blue colour was developed and the intensity of the blue colour was observed at 660nm in UV-VIS spectrophotometer (SL 159 model).

Calculations Weight of the soil taken = 5 g

Volume of 0.5 N NaHCO3 used = 50 ml Volume of the extracted solution taken for P estimation = 5 ml Final volume made upto = 25 ml Therefore amount of available

X  25 50 2 106 P in the soil =  =kgha-1 106 55

3. Estimation of available potassium (K) Available potassium in the soil was estimated by neutral normal ammonium acetate method as described by Standford and English (1949).

i) Reagent

Neutral in NH4OAC

About 77 g of ammonium acetate (NH4OAC) was dissolved in one litre of distilled water.

(ii) Procedure 5 g of soil was transferred to a 100 ml polythene shaking bottle. 25 ml of neutral normal ammonium acetate was added and shaken in a mechanical shaker for five minutes. The solution was filtered through dry whatman No. 4 dry filter paper collecting the filtrate in test tube. The amount of K in the filtrate was determined using flame photometer.

Calculation Weight of the soil taken = 5 g

Volume of neutral 1N NH4 OAC used = 25 m Concentration of K = ppm.

a  25 2 106 Therefore available K in the soil =  =kg. ha-1 106 5

Statistical analysis The significant difference between treatments were analysed statistically, using one way analysis of variance (ANOVA) and Duncan’s multiple-ranged test. A significant difference between any pairs of means that was subjected to ANOVA was indicated by CD value.

4. RESULTS

The growth (in terms of biomass) and reproduction (hatchling production) of compost earthworm Eudrilus eugeniae and Eisenia fetida cultured in different concentrations of tea waste + cowdung + kitchen waste (T1 – T4) for a period of 90 days are presented in table 1 and 2. These tables include data of Eudrilus eugeniae and Eisenia fetida. Growth (biomass), mean growth rate (g/w/day), biomass increase or decrease over control, total number of hatchlings as well as statistical significance on the basis of one way analysis of variance (ANOVA) is also given in table 1 and 2. It showed the existence of significant differences (p>0.05) between T1 control (soil alone), T2, T3 and T4 experimental groups with reference to weight gain for most of the periods of study.

Changes in biomass (wet weight) of Eudrilus eugeniae and Eisenia fetida cultured in different concentrations of Tea Waste + Cow dung + Kitchen waste (T1–T4) are presented graphically in fig.1. Further, the number of hatchlings produced by E. eugeniae and E. fetida cultured in treatments T1, T2, T3 and T4 are graphically presented in Fig.1; Plate V.

Growth performace of E. eugeniae and E. fetida in different concentrations of TW + CD + KW mixture The mean biomass of inoculated E. eugeniae and E. fetida was about 0.080 gm. The different concentrations of TW + CD + KW feed mixtures differentially influenced the growth of E. eugeniae and E. fetida. Generally the body weight of E. eugeniae increased continuously upto 82

90days, in all the four treatments. E. eugeniae registered 1.293 ± 0.027 g; 1.483 ± 0.017 g; 1.542 ± 0.023 g and 1.575 ± 0.01 g in T1, T2, T3 and T4 treatment respectively. Whereas, on the same day E. fetida recorded 1.117 ± 0.011 g; 1.261 ± 0.011 g; 1.321 ± 0.018 g and 1.425 ± 0.010 g in T1, T2, T3, T4 treatments respectively (Table 1 and 2). The efficiency of four treatments to support the growth of E. eugeniae and E. fetida could be ranked in following order: T4 – (TW 60% + CD 10% + KW 30%) > T3-(TW 50% + CD 10% + KW 40%) > T2-(TW 40% + CD 20% + KW 40%) > T1- control soil.

Among the four treatments the net individual weight gain by E. eugeniae were in the following order: TW+CD+KW 6:1:3 ratio (T4) > TW+CD+KW 5:1:4 ratio(T3) > TW+CD+KW 4:2:4 ratio (T2) > control soil (T1), and for E. fetida the net individual weight gain ranked in following order: TW+CD+KW 6:1:3 ratio (T4) > TW+CD+KW 5:1:4 ratio (T3) > TW+CD+KW 4:2:4 ratio (T2) > control soil (T1).

The calculated mean growth rate of E. eugeniae and E. fetida cultured in different concentrations of TW+CD+KW feed mixture indicate the following points. Both species of worm showed maximum growth rate in T4 followed by T3, T2, T1 treatment (Table 1 and 2; Fig.1)

Hatchling rate of E. eugeniae and E. fetida The total number of hatchling production after 90days in different treatment has been presented in table 1 and 2. In T4 treatment maximum

83 number of hatchlings 656 in E. eugeniae and 605 in E. fetida and minimum number of hatchlings at T1 treatment 575 in E. eugeniae and 388 in E. fetida were observed.

In all the treatment E. eugeniae produced more numbers of hatchlings than E. fetida during experimentation. The maximum number of hatchling production by E. eugeniae was recorded in T4 (656 nos) followed by, T3 (638 nos), T2 (526 nos) and T1 (402 nos) treatment and E. fetida in T4 (605 nos) followed by T3 (543 nos), T2 (480 nos) and T1 (308 nos) treatment (Fig.1).

The average vermicompost recovery was higher in all the treatment than compost recovery of natural compost. Among the different treatment T3 and T4 recovered higher rate of vermicompost recovery than the treatment T1 (100% soil) and T2 (40% TW + 20% CD + KW 40%) (Table 1, 2).

Nutrient changes in E. eugeniae and E. fetida during vermicompost of industrial tea waste with cow dung and kitchen waste The industrial tea wastes with cow dung and kitchen waste mixture are used in this study for the period of 90 days. The industrial tea waste, cow dung and kitchen waste physico-chemical properties are given in the (Table 3 and 4).

The pH, EC, TOC, C:N ratio, macro and micro nutrients in different concentrations of industrial tea waste, cow dung and kitchen waste mixtures, initial, worm unworked natural compost (control) and vermicompost of E. eugeniae and E. fetida were analysed. The change in nutrient level are graphically presented (Fig.2-5). The observations of chemical analysis of the different mixtures of industrial tea waste, cow dung and kitchen waste before vermicomposting revealed N, P, K, Ca, Mg, Na, Zn, Fe, Cu and Mn to be more in T4 and T3 treatment than the other treatments (T1 –T2). Statistical significance on the basis of one way analysis of varience (ANOVA) in also given in (table 5-12).

pH The pH level of vermicompost were decreased in all the treatments. Especially T4 treatments of E. eugeniae (4.9 ± 0.01) and E. fetida (4.8 ± 0.04) showed significantly (P<0.05) decreased pH level than T3, T2 and T1 treatments (Table 5 and 6).

TOC, C:N ratio TOC content was reduced in vermicompost of all treatment. Especially T4 treatments in E. eugeniae (11.0 ± 0.04) and in E. fetida (11.2 ± 0.03) showed significantly (P<0.05) reduced TOC contents by the end of vermicomposting than T3, T2 and T1 treatments (Table 5 and 6). The C:N ratio significantly (P<0.05) varied between the treatment T1-T4. The C:N ratio of vermicompost was reduced in T4 treatment of E.

85 eugeniae (18.1 ± 0.12) and in E. fetida (18.0 ± 0.11) The T4 treatment was significantly lower than T3, T2 and T1 (Table 7 and 8)

When compared to control worm unworked natural compost (WU) the maximum pH, TOC and CN ratio reduction was observed in vermicompost obtained from T4 treatments minimum in T1 followed by T2 and T3 of both species.

EC The EC of vermicompost obtained from different treatments T1-T4 of E. eugineae and E. fetida range from 0.6 ± 0.04 to 1.9 ± 0.04 and from 0.7 ± 0.05 to 1.7 ± 0.05 respectively. EC of T4 and T3 treatments significantly increased than T1-T2 treatment of both species (Table 5 and 6).

Macro Nutrient The macro nutrients, N,P,K were found to be increased significantly (P<0.05) in vermicompost obtained from all the treatments T1-T4 of E. eugeniae and E. fetida. Among the different treatment T4, T3 treatment showed higher macro nutrient than T2, T1 treatments (Table 7 and 8).

Micro Nutrient The micro nutrients (Ca, mg, Na, Zn, Fe, Cu and Mn) of vermicompost produced by E. eugeniae and E. fetida showed significantly

86 different in all treatments T1-T4. However, the T4 and T3 treatment showed significantly (P<0.05) higher level of micro nutrients than T2, T1 treatments (Table 9-12).

In this present analysis the pH, TOC and CN ratio significantly (P<0.05) decreased in the all the treatments (T1-T4). On the other hand EC, N, P, K and micro nutrients (Ca, mg, Na, Zn, Fe, Cu and Mn) were found to be increased significantly (P<0.05) in all treatments.

In general the physico-chemical analysis of vermicompost indicate that among the parameters tested the level of pH, TOC and CN ratio were significantly (p<0.05) reduced than that of the values of control (natural compost) and on the other hand the level of EC, N, P, K, Ca, mg, Na, Zn, Fe and Mn were significantly (P<0.05) increased than the control.Of the two worms the vermicompost of E. eugeniae exihibits more nutrients than E. fetida.

Estimation of microbial population in vermicompost The population of bacteria, fungi and actinomycetes in the worm unworked control compost (WUW) and worm worked vermicompost (WW) samples collected after 90 days from different concentrations of tea waste, cow dung and kitchen waste mixtures.

Total microbial population The total microbial population of vermicompost of E. eugeniae and E. fetida ranged from 3.38 ± 0.21 to 5.10 ± 0.23 and from 3.31 ± 0.21 to

3.84 ± 0.28 respectively in all the treatments (T1-T4). Among the different treatments T4 and T3 were found to have significantly (P<0.05) higher microbial population than T2 and T1 treatments (Table 13 and Fig.6).

Microbial activity In the present analysis the microbial activity of vermicompost obtained from all the treatments T1-T4 were increased significantly (P<0.05) and especially in T4 of E. eugeniae (7.32 ± 0.31) and E. fetida (6.92 ± 0.59) and T3 of E. eugeniae (6.60 ±0.05) and E. fetida (5.95 ± 0.63) treatments were found to be significantly (P<0.05) higher than T2, T1 treatments (Table 14 and Fig.7)

Quantitative analysis of microbes Fungal isolation The list of fungal flora isolated from the vermicompost of E. eugeniae and E. fetida in industrial tea waste, cow dung and kitchen waste mixtures are recorded in (Table 15) which shows a total of 10 fungal species belonging to 7 genera. This includes two phytocomycetes (Rhizopus nigricans, Mucrozygospore sps.) only one Ascomycetes (Chaetomium globossum) Seven Deutromycetes (Aspergillus flavus, A. niger, A. nidulans, Cladosporium herbarium, Penicillium janthinellum, Aspergillus fumigatus, Penicillum citrinum (Plate VI-VII). Of these six fungul species such as Aspergillus flavus, A. nidulans, Cladosporium herbarium, Penicillium janthinellum, Chaetomium globossom, A. nidulans

88 were identified in worm unworked natural compost (Control) and 8 species in vermicompost of E. eugeniae and 5 species in vermicompost of E. fetida were identified.

Bacterial isolation Table 16 shows the isolation of bacteria from worm unworked natural compost found to contain six species such as Pseudomonas aeruginos, Enterobacter aerogens, Proteus vulgaris, Escherichia coli, Klebsiella pneumoniae and Streptococcus pyogens. In vermicompost of E. eugeniae nine species such as Klebsiella pnemoniae, Pseudomonas aeruginosa, Enterobacter aerogens, Morganella morgarii, Proteus vulgaris, Escherichia coli, Streptococcus pyogens, Bacillus subtilis and Serratia marcescens in vermicompost of E. fetida five species such as Klebsiella pnemoniae, Websiella phemoniae, Pseudomonas aeruginosa, Staphylococcus annus and Bacillus subtilis. Totally eleven bacterial species were identified of these eight gram negative bacteria and three gram positive bacteria (Plate VIII and IX).

Isolation of Actinomycetes Actinomycetes such as Streptomyces albus, S. griseus are identified in worm unworked natural compost. Streptomyces albus and Nocordia caviae present in vermicompost of E. eugeniae and only one species Streptomyces albus in vermicompost of E. fetida were identified (Table 16 and Plate X).

Influence of vermicompost on Tea plant Camellia (sinensis (L.) O. Kuntze var. productivity and soil fertility The effects of the application of inorganic fertilizers NPK, organic manure FYM, vermicompost and 50% vermicompost supplemented with 50% NPK on the growth components of tea plant at 6 months, 10 months, 14 months and 18 months (harvest) are determined and presented in Table 17-18 and Figs.8-10. Further yield components are also determined and presented in Table 17-18 and Fig.8-10. The growth components include shoot length (height), root length, dry weight of shoot and root. The yield components include number of leaves, fresh weight of shoot and fresh weight of root. To understand more clearly about the observations, recorded on growth components at 6 months, 10 months, 14 months and 18 months (harvest). The results of individual growth parameter viz shoot length, root length, number of leaves, shoot dry weight and root dry weight fresh weight of shoot and fresh weight of root are tabulated and presented in Table 17 and 18.

The statistical significance of the data on the basis of one way analysis of variance (ANOVA) are also given in Tables 17 and 18. ANOVA shows the existence of statistically significant (P<0.05) differences between the control and experimental data for all the growth and yield components are studied. Further, for each parameter, minimum significant difference or critical difference (CD) at 5% level is also calculated and incorporated in the tables.

Plate XI shows the tea plant cultivation in the experimental field applied with NPK, FYM, 100% vermicompost, 50% NPK supplemented with 50% vermicompost. Plate XII shows the photograph of the root development of the tea plants in the experimental fields (T5) of 6th month, 10th month, 14th month and 18th month. This photograph provides visual evidence for the differential development of root in tea plants due to the application of NPK, VC, FYM and NPK + VC. The photographs clearly show the lengthy primary root with more number of secondary (lateral) roots in VC supplemented with NPK (T5) and VC alone (T3) treated plants compared to plants grown in other field (T1, T2, and T4).

Plate XIII shows the shoot development of tea plants collected on 6th month, 10th month, 14th month and 18th month from NPK + VC treated experimental field (T5). This photograph provide visual evidence for the maximum height, more number of branches in vermicompost treated (T3) as well as vermicompost supplemented with NPK (T5) treated plants and minimum growth, less number of yield in control (T1) and NPK (T2) treated plants.

Generally in all the treatments, for all the growth components studied, the tea plants show an increasing trend from 6 months to 18 months (harvest). Among the treatments in T5 (50% NPK supplemented with 50% vermicompost) and T3 (VC) applied field the tea plant show conspicuous effects and maximum response both in growth and yield components. On the other hand in field applied with NPK (T2) tea plant

91 shows moderate response and in field treated with FYM (T4) and control field (T1) tea plant shows least response with reference to growth and yield components for all the periods of investigation.

Growth components such as shoot length, root length, number of leaves, shoot fresh weight and root fresh weight, shoot dry weight, and root dry weight are all significantly enhanced in T5 (50% NPK + 50% VC) as well as in T3 (VC) for all the periods of study (Table 17 and 18). The comparison of per cent change values between the treatments T1-T5 clearly prove that plants grown in recommended doses of vermicompost alone (T3) and 50% vermicompost supplemented with 50% NPK (T5) applied field, show roughly 30-100% (some parameters more than 100%) enhancement in each growth parameters investigated. For example shoot length of tea plants cultivated in T5 increased 17.5 cm on 6th month, 26.2 cm on 10th month, 48.2 cm on 14th month, 64.2 cm on 18th month, over control T1. Root length of tea plants biomass grown in T5 increased 12.0 cm on 6th month, 23.4 cm on 10th month, 30.8 cm on 14th month and 35.2 cm on 18th month over the control. Dry weight of shoot of tea plants biomass grown in T5 increased 2.06, 2.60, 3.99, 6.98 gm on 6th month, 10th month, 14th month, and 18th month over control. Dry weight of root of tea plants grown in T5 increased 0.83, 0.92, 1.99, 3.53 gm on 6th month, 10th month, 14th month and 18th month respectively and fresh shoot weight of tea plant grown in T5 incresed 4.14, 4.58, 8.60 and 19.45 gm on 6th month,10th month,14th month and 18th month respectively and fresh root length of tea plant grown in T5 incresed in 1.94, 2.14, 3.46 and 8.17

92 on 6th month, 10th month, 14th month and 18th month respectively. The total number of leaves of tea plant grown in T5 treatment are 7, 11, 16, and 24 numbers on 6th month, 10th month, 14th month and 18th month respectively.

The comparison of percent change over control values between T1 to T5 treatments clearly prove that tea plant cultivated in recommended dose of T3 (vermicompost alone) and T5 (50% NPK supplemented with 50% vermicompost) field show more number of leaves per plant, maximum number of seeds per capsule and higher capsule weight. For example on 18 months in T5 field (50% NPK + 50% VC applied field) the number of leaves / tea plant increased to 46%; leaves weight increased to 27.4%; leaves weight / plant increased to 65.3% over control.

The efficiency of NPK, VC, FYM, and NPK +VC treatments (T2- T5) to support or enhance the growth and yield components of tea plant could be ranked in the following order: T5 (50% NPK + 50% VC) > T3 (VC at the rate of 5t ha-1) > T2 (NPK at the rate of 35:23:23 kg/ ha-1) > T4 (FYM at the rate of 12.5t/ha-1).

Soil fertility Effect of vermicompost on soil fertility The effects of the application of inorganic fertilizer (T2), organic manure FYM (T4), vermicompost (T3) and vermicompost supplemented with NPK (T5) on the physico-chemical properties of red soil before

93 sowing (initial soil) and post harvest soil of tea plant are determined and presented in the Tables 19-21; Figs.11-12. The statistical significance of the data on the basis of one way analysis of varience (ANOVA) are given in Tables 19-21. ANOVA indicates the existence of statistically significant (P < 0.05) differences between control and experimental data for all the physical and chemical parameters investigated. Further, for each physico-chemical parameters critical difference (CD) is also calculated at 5% level. The physical parameters include pore space (PS), partical density (PD) bulkdensity (BD) and water holding capacity (WHC). The chemical parameters include organic carbon (OC), available nitrogen (N), available phosphorus (P) and available potassium (K).

Physical properties The results indicated that the physical properties such as, pore space, water holding capacity (WHC) show an increasing trend over control. On the contrary, particle density and bulk density showed decreasing trend over control. The comparison of the results between (T1) control and (T2) NPK, (T3) VC, (T4) FYM, (T5) NPK + VC indicated that among the physical parameters porosity and water holding capacity recorded the highest values of the soil collected from the experimental field applied with 50% vermicompost supplemented with 50% NPK (T5) and vermicompost (T3) applied field. Field applied with FYM (T4) show moderate increase in the above said parameters when compared to control (T1). On the other hand, the soil treated with NPK (T2) was found to be on par with control (T1) (Tables 20-21; Fig.12-13). The efficiency of

NPK (T2), VC (T3), FYM (T4) and VC NPK (T5) treatments to increase the porosity, water holding capacity, and EC of the post harvest soil could be ranked in the following order: T5 (NPK+VC) > T3 (VC) > T4 (FTM) >

T2 (NPK).

The comparison of results between control (T1) and manures treatments (T2-T5) indicated that particle density (PD) and bulk density (BD) decreased to a maximum extent (3.21-1.53; 1.93-1.01%) in the soil applied with vermicompost (T3) and NPK + VC (T5). Soil applied with FYM (T4) showed moderate decrease in particle density (PD) and bulk density (BD) over control (T1). On the other hand soil treated with NPK (T2) is found to be on par with control (T1) (Table 20) (Fig.12). The influence of NPK (T2), VC (T3), NPK+VC (T5) treatments decreased the particle density (PD) and bulk density (BD) of post harvest soil and were in the order of VC (T3) > NPK + VC (T5) > FYM (T4) > NPK (T2).

Chemical properties In general, the results indicated that all the chemical parameters such as OC, available N, P, and K, showed an increasing trend over control. The comparison of results between control (T1) and manure treatments (T2-T5) indicate that all the chemical parameters tested registered significant highest value in the post harvest soil applied with 50% VC supplemented with 50% NPK (T5) followed by 100% vermicompost treatment (T3) and it ranked next to T5. Plots applied with FYM (T4), showed moderate increase in all the chemical parameters

95 tested. On the other hand soils treated with NPK (T2) showed minimum increase in all the chemical parameters studied (Table 20; Fig.12-13). The influence or efficiency of NPK (T2), VC (T3), FYM (T4), NPK + VC (T5) to increase the OC, available NPK could be ranked in the following order: 50% NPK + 50% VC (T5) > VC (T3) > FYM (T4) > NPK (T2).

5. DISCUSSION

Efficacy of industrial tea waste (TW), cowdung (CD) and kitchen waste (KW) mixture on the growth and reproduction of Eudrilus eugeniae and Eisenia fetida.

The growth rate of biomass and reproduction of earthworm in Eudrilus eugeniae and Eisenia fetida were maximum in T4 and T3. The growth rate of Eudrilus eugeniae cultured in different organic wastes were reported by many investigators. Eudrilus eugeniae cultured on cowdung (for a period of one year) increased at the rate of 3.5 mg/worm/day (Reinecke et al., 1992); Eisenia fetida (for total period of 90 days) cultured on sludges from paper and pulp industries increased 8.4 mg/ worm/ day (Elvira et al., 1998) and Eudrilus eugeniae (for a total period of 105 days) cultured on kitchen wastes increased 2.5 mg/worm/days (Chaudhuri et al., 2002).

Murchie (1960) proved experimentally the existence of a significant relationship between weight increase and substrate type, which may reasonably be attributed to nutritional quality of the substrate. Such correlation between increased growth, cocoon production, reproduction rates etc. and quality of various organic wastes used as feed was reported in a variety of earthworms: E. eugeniae, E. fetida, and P. excavatus on cattle dung (Kale et al., 1982; Reinecke et al., 1992); L. mauritii on cattle dung (Kale and Bano, 1992); E. eugeniae on sugar factory refuse (Kale, 1994); E. andrei on sludge from paper and pulp industries (Elvira et al., 97

1998); E. eugeniae, L. mauritii on sugar mill waste pressmud (Ramalingam, 2004); E. eugeniae and E. fetida on sugar factory refuse (Ramamoorthy, 2004); P. excavatus on various waste such as sheep dung, biogas sludge, poultry manure (Kale et al., 1982), pig solid waste, turkey wastes and horse solid waste (Edwards et al., 1988), vegetable waste (Atiyeh et al., 2002) gurghum industry waste (Suthar, 2006) were reported.

Different types of organic wastes originated from different sources have been used for vermiculture and vermicomposting (Edwards et al., 1985; Haimi and Huhta, 1986; Kale, 1994; Ramalingam, 2004; Bhattacharjee, 2002; Loh et al., 2005; Christy and Ramalingam, 2005; Suthar, 2006). But all the wastes are neither readily acceptable to worms nor equally support worms growth and reproduction. The quality and quantity of feed, various physico- chemical parameters influenced earthworm’s growth and fecundity (Reinecke and Hallatt, 1989; Reinecke and Viljoen, 1990; Kale and Bano, 1992; Kale et al., 1992; Aira et al., 2006; Suthar, 2006). Falling in line with these observations, in the present study (restricted to 90 days) among the different proportions of TW + CD + KW tested, in (T1-T4) Eudrilus eugeniae recorded maximum growth rate of 6.4 g /w / day in T4 (TW60% +CD10%+KW30%) and 5.9 g/w/day in T3 (TW50% + CD10% + KW40%). At the same time Eseniae fetida recorded maximum growth rate of 6.4 g/w/day in T4 (TW60% +CD10%+KW30%) and 5.9 g/w/day inT3 (TW50% + CD10%+KW40%).

The perusal of literature indicated higher growth rate of Eudrilus eugeniae during pre-reproductive period (up to 45 days) and a decline in growth rate (even though biomass continued to increase) during reproductive period (45 to 90 days). The present findings of accelerated growth rate during the pre-reproductive phase of Eisenia fetida is in accordance with the findings of Ramalingam (1997). The decline in worm growth rate during the reproductive phase is due to the onset of cocoon production since copulation and cocoon production need large amount of energy (Graff, 1981; Viljoen and Reinecke, 1994). Hence, the observed higher growth rate during pre-reproductive period reflects the active growth of the worms, due to availability of more energy. On the contrary, the decline in growth rate recorded during reproductive period is an indication of onset of reproduction and utilization of energy for cocoon production besides growth.

A close observation of the results revealed that among different proportions of TW + CD + KW mixture (T1-T4) treatment Eudrilus eugeniae gained maximum hatchling production of 604 in T4 followed by T3,T2 and T1.At the same time Eisenia fetida gained maximum hatchling production of 504 in T4 and minimum in T1 treatment.

The maximum weight gain by earthworm was reported by Gangadar et al. (1995) in 25% sulphur waste residues and cowdung mixture and Kavian et al. (1991) in 25% soyabean oil sludge-cowdung substrate. Tiwari et al. (1993) reported that addition of organic matter had

99 resulted in an increased population density and biomass of worm which coincides with the present study. Kavian et al. (1996) vermicomposted paper mill sludge in combination with cowdung using the earthworms Lumbricus rubellus and reported that 25% concentration of sludge enriched with required nutrients was an ideal substrate for the growth and reproduction of the earthworm.

In nutshell, presently under-utilized organic matter rich tea waste in balanced combination with cowdung and kitchen waste (TW+CD+KW) could be used as a culture medium to raise E. eugeniae and other species of earthworms and production of organic manure ‘vermicompost’ besides abating environment pollution. Further, the present investigation proved that tea waste and cowdung is equivalent to kitchen waste in supporting growth and reproduction of E. eugeniae and E fetida, hence cowdung and kitchen waste can also be utilized in combination with tea waste and converted into nutrient rich vermicompost.

Among the two species, E. eugeniae exhibits better growth and reproduction than E. fetida in different treatment (T1-T4). E. eugeniae grew significantly (p<0.05) and produce more hatchlings in all the treatments. From the observation it is recommended that the industrial tea waste could be better vermicomposted by E. eugenia and could be used for vermiculture practices.

100

Nutrients changes during vermicomposting of industrial tea waste with cow dung and kitchen waste pH The earthworm activity significantly contributed towards neutral soil pH conditions through the production of casts. The availability of several plant nutrients and level of other elements depend upon the pH levels of the organic manure. Barois et al. (1999), recording of higher pH level in soils in the organic farm site compared to the integrated farm site is likely due to the addition of organic matter and the non use of chemical fertilizers in the farm site. Earthworm are very sensitive to pH and in general are neutrophilic in nature (Edwards and Bholen, 1996).The soil quality includes soil reaction (pH), mineral nutrient elements, water content, composition of soil atmosphere and biotic factors. Mature compost when added to soil directly affected almost all of these factors

(Marinari et al., 2000). The production of NH4, CO2 and organic acids during microbial metabolism in vermicompost may be contributed to the decrease in soil pH (Albanell et al., 1998). Falling in the line with this observation it is found that vermicomposting process has reduced the pH from 6.0 to neutral pH in the vermicomposted mixture of industrial teawaste, cow dong and kitchen waste. Most of the other reports on vermicomposting (Gunadi and Edwards, 2003; Garg and Kaushik, 2005) have also reported similar results.

101

Electrical Conductivity (EC) Soil electrical conductivity (EC) is a measurement that correlates with soil properties that affect crop productivity, including soil texture, cation exchange capacity (CEC), drainage conditions.The soil analysed with vermicompost had significantly (p<0.05) higher EC than the untreated soils. The increased EC during the period of the composting and vermicomposting processes is in consistence with that of earlier workers (Kaviraj and Sharma, 2003), which was probably due to the degradation of organic matter releasing minerals such as exchangeable Ca, Mg, K, and P in the available forms, that is, in the form of cations in the vermicompost and compost (Tognetti et al., 2005). The soils EC increased with increasing application rate of vermicompost in soil as reported by Atiyeh et al. (2001). Lazcano et al. (2008) reported that the EC values ranged between 0.72 ± 0.02 ds/m in the vermicompost from different organic waste. The electrical conductivity of vermicompost depends on the raw materials used for vermicompost and their ion concentration (Atiyeh et al., 2002). In the present study the EC has increased in all the worm worked vermicompost (T1-T4) than control. In the present study, EC was increased in the range of 0.6 ± 0.06 to 1.9 ± 0.05 for different treatments after vermicomposting of both species.

Total organic corbon (TOC) The organic carbon is the main source of energy for plants and animals, and the value of organic matter are very important for soil health. The deficiency in organic carbon reduces storage capacity of soil.

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Nitrogen, phosphorus and sulphur lead to reduction in soil fertility (Kale et al., 1993). The chemical analysis of vermicompost indicated that during the process of vermicomposting, the level of organic carbon was reduced in the vermicompost obtained from various treatments (T1-T4) when compared to control. The results revealed that during the process of vermicomposting, the level of OC was reduced to a lesser extent. The final product, vermicompost obtained from various treatments, retained the quantity of OC ranging between 26.8 - 28.5%. Many earlier investigators had reported and confirmed the reduction of OC content in organic wastes after vermicomposted into vermicompost (Satchell, and Martin 1984; Ramalingam and Thilagar, 2000; Karmegam and Daniel, 2000; Ramalingam and Ranganathan, 2001 and Loh et al., 2005; Suthar, 2006). The observed reduction in the level of OC in the present study falls in line with the earlier reported results. Drop in the level of OC due to combined action of earthworm and microbes during vermicomposting revealed that earthworm accelerate the decomposition of organic matter.

At the same time, the maintenance of high level of OC in the vermicompost represents an important source of organic carbon for carbon depleted soil. Organic matter encourages the formation of topsoil and soil aggregates in the surface soil horizon (Brady, 1988). So it can be concluded that OC in the vermicompost form the main source of energy both for soil organisms and plants and is helpful in the formation of topsoil and soil aggregates.

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C:N ratio The significant reduction (-22% to -50%) (T1-T4) and narrow range of C:N ratio below 20:1 in the present study reflected the efficient worm activity, leading to accelerated the rate of organic matter decomposition and mineralization there by resulting in nutrient rich good quality vermicompost, particularly from the treatments T3 and T4. The observed significant reduction in the levels of C:N ratios in the vermicompost obtained from treatments, T3-T4 were in accordance with the work of Mba (1983) who found that in E. eugeniae worked cassava feel compost C:N ratio decreased whereas total nitrogen increased. Many researchers had reported narrowing down of C:N ratio in the vermicompost produced from different types of organic wastes (Syers et al., 1979; Kale, 1994; Kale et al., 1994; Ramalingam et al., 2000; Karmegam and Daniel, 2000; Bhattacharjee, 2002; Christy and Ramalingam, 2005; Pramanik et al., 2007).

The reduction in carbon and lowering of C:N ratio in the worm worked compost could be achieved on one hand by the combustion of carbon during worm respiration and worm gut microbial utilization (Edwards and Bohlen, 1996; Chaudhuri et al., 2000) and on the other hand an increase in the level of nitrogen due to loss of dry matter (OC) as

CO2, as well as water loss by evaporation during microbial mineralization of organic matter (Syers et al., 1979) coupled with the addition of worm’s nitrogenous wastes through excretion and mucus secretion (Curry et al., 1995). The decrease in C: N ratio over time might also be attributed to

104 increase in the earthworm population which led to rapid decrease in organic carbon due to enhanced oxidation of the organic matter (Nedgewa and Thompson, 2000). So, from the present investigation it can be concluded that the reduction in C:N ratios of vermicompost indicated enhanced biodegradation process of the organic matter in the TW + CD + KW substrates. Further, reduction in C:N ratios of vermicompost are the indices for the effective biodegradation of organic wastes such as TW, CD and KW and production of good quality compost during the vermicomposting process.

Nitrogen, phosphorus, potassium (N,P,K.) The significantly increased levels of NPK in the vermicompost obtained from the treatments T1-T4, specifically in T3 and T4 over control indicated effective decomposition of organic wastes (TW + CD+KW) by the combined action of earthworm and microbes. The increased levels of NPK in the vermicompost were in conformity with the results of earlier workers. Edwards et al. (1985) stated that by the combined action of worms and microorganisms on the waste materials most of the NPK, Mg were converted into available form. Kale (1994) reported a significant increase in available NPK in worm worked cowdung and sheep dung. Tiwari et al. (1989) have recorded higher value of organic carbon, NPK in the cast than in the top soil samples. Suthar, (2006) reported that the C:N ratio was reduced and also NPK contents were increased during vermicomposting process. Haimi and Huhta (1990) and Haimi and Boucelman (1991) have demonstrated that the feeding

105 activity of worm significantly enhanced mineralization of macro and micro nutrients of birch litter. Jambhekar (1992) noticed considerable increase in available N, P, and K in the worm worked wastes than that of the original wastes. Ramalingam (1997) reported a significant increase in the level of N, P, K, Ca and Mg in E. eugeniae and E. fetida worked vermicompost.

Balamurgan and Vijayalakshmi (2004) indicated increased level of N, P, K in composted pressmud and coir waste using microbes and earthworm E. eugeniae. Karmegam and Daniel (2000) reported a significant hike in the level of N, P, K in E. fetida worked on leaf litters. Ramalingam and Thilagar (2000) reported increased levels of N, P, K, Ca and Mg and other micronutrients in the sugarcane trash compost produced by P. excavatus. Ramalingam and Ranganathan (2001) reported significant increase in the levels of N, P, K, Ca, Mg and other micronutrients in the vermicomposted pressmud by the action of E. eugeniae.

The presence of large number of microflora in the vermicomposting of the earthworms might play an important role in increasing P and K content by acid production for solubilisation of insoluble P and K during organic matter decomposition as reported by Sharma (2003). Significant rise in nitrogen (N) content during vermicomposting process compared to control is probably contributed by

+ earthworms through excretion of NH4 (ammonia), secretion of mucus

106 and addition of nitrogen by microbes. Rise in the levels of phosphorus and potassium content during vermicomposting is probably due to the mineralization, solubilisation and mobilization of phosphorus and potassium, because of bacterial and earthworm activity (Krishnamoorthy, 1983).

Suthar (2006) investigation support the hypothesis that earthworm can enhance the mineralization of N P K during their inoculation in waste system. In the present study, the significantly increased levels of NPK in the vermicompost generated from TW+CD+KW substrates (T1-T4) and also in other treatments could be due to the combined action of microorganisms and Eudrils eugeniae and E. fetida which increased the mineralization of organic matter when it passes through the gut. Microbial and enzyme activity also contributes to increase the mineral nutrients in the vermicasts through nitrification, phosphate solubilization and mineralization (Edwards and Bohlen, 1996; Ranganathan and Vinotha, 1998). Parthasarathi and Ranganathan (2000) found the enzyme activities involved in mineralization enhanced during the passage of organic matter through the gut of earthworms.

Thus, vermicompost obtained from TW60% + CD10% + KW40% (T4) as well as TW50% + CD10% + KW40% (T3) mixtures evidenced with increased level of NPK and drastically reduced C:N ratios and hence can be considered as good quality vermicompost.

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From the present investigation it could be suggested that the underutilized tea waste, cow dung, kitchen waste can also be profitably utilized for the production of nutrient and microbial rich vermicompost besides acting as a booster for the biodegradation of tea plant. Further, from the nutrient analysis of the vermicompost it could be deduced that not all the concentrations of TW+CD+KW are equally accepted and processed by E. eugeniae and E. fetida as a consequence resulting in differential mineralization rate between the treatment concentrations. Hence, it can be concluded that the quality of vermicompost partly depends upon quality of organic wastes used for vermicomposting and partly upon the rate of degradation of organic wastes by the combined effects of earthworm and microbial activities. The present study proved beyond doubt that TW, CD and KW are served as best raw material for the production of nutrients and microbial rich organic manure. Hence, from the present investigation T4 (TW60% + CD10% + KW30%) feed mixtures can be recommended for vermiculture and vermicomposting and production of organic manure in an ecofriendly way.

Microbial population Microorganisms are the primary decomposers of organic matter. The role of microbial activity in the vermicomposting of earthworms, in the casts and in the soil is very essential for the degradation of organic waste and release of nutrients in available form to plants (Syers et al., 1979). Earthworm gut harbours specific symbiotic microflora (Lavelle, 1983; Wallwork, 1984). During vermicomposting process, the organic

108 matter passes through the worm gut undergoes physical, chemical, and biochemical changes by the combined effect of earthworms and microbial activities. Earthworms not only help the proliferation of microbes by speeding up physical degradation process of organic matter when it passes through the gut but also stimulate other free living aerobic microbial activities in the casts favouring further decomposition (Kale et al., 1991). Organic matter that passes through the gut of earthworms released as vermicast results in an increased level of microbial population, microbial activity, microbial respiration, enzyme activity and NPK enrichment, production of polysaccharide gum by bacteria, establishment of lignocellulolytic, nitrifying and nitrogen fixing microorganisms etc.

Hendriksen (1990) recorded high bacterial population in the earthworm cast. In accordance with the above reports in the present study the bacterial population was found to be significantly greater in the fresh vermicast obtained from the treatments T1-T4 (especially T4 (TW+60%+CD10%+KW30%) T3 & T2). The high population of bacteria may be due to bacterial growth during transit through the gut of earthworms.

The increased occurrence of fungal population in the cast is attributed to high rate of proliferation of fungi even though earthworm selectively feed fungi (Scheu, 1987; Tiunov and Scheu, 2004). Dash et al., (1986) reported that fungal population had increased in fresh casts of L.mauritii and D.calebi. Parthasarathi et al. (1997) found that fresh casts

109 of L. mauritii and E. eugeniae reared on pressmud contain high number of fungi population. Vinotha (1999) also found that fresh casts of P. excavatus and E. eugeniae reared on pressmud contain greater numbers of fungi. In confirmity with the above reported results in the present study also the fungal population is found to be significantly higher in the fresh vermicast obtained from treatments T1-T4. The enhancement of fungal population may be due to the multiplication of fungi during their transit through the worms gut. Further, from the results it could be also suggested that the tea waste and other organic wastes highly support the growth and multiplication of fungi than actinomycetes.

Many earlier researchers have reported high actinomycetes population in the vermicast of earthworms (Jambhekar, 1992; Indra et al., 1996; Pramanik, 2007). The actinomycetes population was found to be significantly greater in the fresh casts obtained from the treatments T4. The enhancement of actinomycetes population in the present study may be due to multiplication of actinomycetes during their transit through the worm’s gut.

Influence of NPK, FYM and vermicompost on the growth and yield components of Tea plant (C. sinensis) (L.) O. Kuntze var. The fertilizing quality of vermicompost is determined by its effect on plant growth and yield. The present study has clearly proved that the application of vermicompost (T3) and vermicompost supplemented with NPK (T5) has a significant stimulative effect on the growth and yield of

110 tea plant (C. sinensis). This may be due to the high nutrients composition of vermicompost over the other organic manure (FYM) and inorganic fertilizer (NPK) as well as due to the influence of vermicompost on the physical properties of soil such as porosity, particle density, bulk density, WHC, CEC etc. The potential of vermicompost to improve growth and yield may be due to changes in the physico-chemical properties of soil, overall increase in microbial activity and also due to the effect of plant growth regulators produced by the microorganism.

Vermicompost also has a positive effect on vegetative growth, stimulating shoot and root development (Edwards et al., 2004). The effects include alterations in seedling morphology such as increased leaf area and root branching (Lazcano et al., 2011). Vermicompost has also been shown to stimulate plant flowering, increasing the number and biomass of the flowers produced (Atiyeh et al., 2002; Arancon et al., 2008), as well as increasing fruit yield (Atiyeh et al., 2000; Arancon et al., 2004; Singh et al., 2008). In addition to increasing plant growth and productivity, vermicompost may also increase the nutritional quality of some vegetable crops such as tomatoes (Gutiérrez-Miceli et al., 2007), spinach (Peyvast et al., 2008), strawberries (Singh et al., 2008), Chinese cabbage (Wang et al., 2010), and sweet corn (Lazcano et al., 2011).

Many researchers have proved that vermicompost is one of the best organic fertilizers and recommended it as a suitable substitute for chemical fertilizers. The present observations ie, the vermicompost

111 increased the plant height and other growth attributes as well as yield attributes falls in line with many reports already made by many researchers. Various studies have shown that the application of vermicompost or vermicast has a significant influence on the growth, root development, root nodules formation and yield of plants (Grappelli et al., 1985; Kale and Bano, 1988; Kale et al., 1992; Tomati and Galli, 1995; Vadiraj et al., 1996; Edwards and Bohlen, 1996; Ramalingam, 1997; Atiyeh et al., 1999; Karmegam and Daniel, 2000; Parthasarathi and Ranganathan, 2002).

Further there have been numerous studies on which plants have been grown in the experimental fields with earthworms or their casts or vermicompost, which resulted in an increase in plant growth and yield. Kale and Bano (1986) found that the vegetative growth of plants was influenced by Eudrilus eugeniae casts in a better way than chemical fertilizers. Kale (1994) had recorded excellent effect of vermicompost on the growth and yield of cereals, pulses, oil seeds, spices, vegetables, fruits, ornamental plants, cash crops and plantation crops. Arulmurugan (1996) had studied the effect of vermicompost on growth, yield, and protein of tea plant. He had recorded increased plant height, root length, root volume, number of seeds / plant, protein and oil content of seeds together with increased uptake of NPK by plants.

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Plant productivity stimulating factors in vermicompost Atiyeh et al. (2002) also suggested that vermicompost contain plant growth regulators, plant growth hormones which may be responsible for the increased germination, growth and yield of plants. It is generally believed that the stimulative effect of vermicompost or vermicast on plant growth and yield could be due to synergistic action of several factors in the vermicompost, but the major claims goes to microbial metabolites, the growth regulators present in it as suggested by Tomati et al. (1995).

Effect of growth regulators Growth regulators influence growth and development of plant, by enhancing plant metabolism. Root initiation, elongation, increased root biomass; enhanced plant growth and development are recorded in cast treated plants (Tomati et al., 1988). The presence of such growth regulators in cast is proved directly by chemical and biochemical analysis (Edwards, 1983; Satchell, 1983; Tomati et al., 1994; Tomati and Galli, 1995) and indirectly by the hormone like effect of worm cast when applied to plant (Tomati et al., 1988).

Vermicast, due to the presence of growth regulators enhances nitrogen utilization and stimulates nitrate reductase activity, the first step in protein synthesis (Dell’ Agnola et al., 1981; Tomati et al., 1995). The accelerated growth observed in the present study in vermicompost treated tea plant may be quite possibly due to the above said effects of

113 vermicompost such as influencing protein synthesis, plant metabolism, growth and development due to the presence of growth hormones.

Effects of nutrient enrichment The fertilizing value of vermicompost and its beneficial effects on plant growth and yield have been related to the presence of active nutrients. Vermicompost has been shown to have higher level of organic matter, OC, total and available NPK, micronutrients etc. (Lee, 1985; Bhawalkar, 1991; Edwards and Bohlen, 1996; Ramalingam and Thilagar, 2000; Parthasarathi and Ranganathan, 2001; Ramalingam and Ranganathan, 2001). NPK substituted vermicompost has higher N content and high microbial activity which favour the plant growth. In the present study also increased levels of NPK were recorded.

Effect of photosynthesis It is now proved that the vermicompost not only stimulate protein synthesis but also enhances the photosynthesis (on which directly or indirectly all plants and animals depend for provision of energy for metabolic reactions) in plants by providing higher level of nitrogen. About 50% of the nitrogen taken by plants is used to form Ribulose 1-6 biphosphate carboxylase the enzyme necessary for photosynthesis, so increased level of nitrogen in cast may improve photosynthesis in plant (Tomati et al., 1995) and this could have influenced plant growth. Leaf area index reflects photosynthetic ability of plants and nitrogen content of

114 leaves and its enhancement is responsible for the higher growth and yield of plants (Lazcano et al., 2008).

Comparison of the growth and yield components of tea plant (C. sinensis) cultivated in NPK, FYM, vermicompost, VC+NPK treated field The comparison of the growth and yield components of tea plant (C. sinensis) cultivated in NPK, FYM, vermicompost, NPK + VC treated fields indicate the nutritional superiority of vermicompost (T3) and vermicompost supplemented with 50% NPK (T5) over 100% NPK and FYM treatments as suggested by UPASI Annual Report (2009). This fact is evident from the observed variation in the growth and yield components of tea plant (C. sinensis) (L.) between T1 (control) T2 (NPK), T3 (VC), T4 (FYM) and T5 (NPK + VC) in all periods of study. The influence of NPK, FYM and vermicompost on the growth and yield parameters of tea plant (C. sinensis) (L.) could be ranked in the following order: T5 (50% NPK + 50% VC) > T3 (VC) > T2 (NPK) > T4 (FYM) > T1 (Control).

The present results show vast difference in the rate of growth for the entire period of study as well as in the quantity of yield between the tea plant (C. sinensis) (L.) cultivated in NPK (T2) FYM (T4) and vermicompost (T3), vermicompost supplemented with NPK (T5) treated field. The plant root system constitutes an integral part of the plant and is important in contributing to the structural and functional integrity and alterations of the shoot (Aung, 1982). Root growth and development are

115 demonstrated to be directly influenced by different types of organic manures. Vermicompost significantly stimulates the growth of a wide range of plant species including several horticultural crops such as tomato (Atiyeh et al., 1999; Atiyeh et al., 2000; Atiyeh et al., 2001; Hashemimajd et al., 2004; Gutiérrez-Miceli et al., 2007), pepper (Arancon et al., 2004, Arancon et al., 2005), garlic (Argüello et al., 2006), sweet corn (Lazcano et al., 2011) and green gram (Karmegam et al., 1999). A number of previous reports confirmed that the growth regulators play an important role in rooting, such as root initiation, root elongation and root development (Grappelli et al., 1985; Tomati et al., 1988, 1990). Further improved root growth has been related to a greater availability of mineral nutrients (Lee, 1983; Satchell et al., 1984).

Ranganathan and Christopher (1994) opined that the vermicompost application promotes plant growth in general and root growth in particular. All these evidences strongly support the current conclusion that the vermicompost and NPK supplemented with vermicompost treated tea plant might have developed roots quickly which observed nutrients promptly and enhanced the growth and yield components profusely than the plants treated with NPK and FYM. Vermicompost application along with recommended dose of fertilizers might have reduced the loss of nitrogen and increased the nutrient uptake. Growth and yield of Tea plant (C. sinensis) (L.) especially in the treatments T5 (50% NPK + 50% VC) and T3 (100% vermicompost) is significantly enhanced. Similar findings were reported by Grapelli et al., (1985), who stated that application of

116 vermicompost significantly increased the dry matter production which might be attributed to increased leaf area index and greater accumulation of nitrogen. Vermicasts form the suitable base for free living beneficial microbes whose activity is essential for the release of nutrients in the soil and also it possesses the growth regulators particularly cytokinins, auxin and gibberellins, which play an important role in the plant growth and development (Tomati et al., 1983).

The increased yield components in the treatment T5 (50% NPK + 50% VC) might be attributed to the application of inorganic fertilizers as well as the beneficial effect of the humus contributed by vermicompost. The humus might have improved the physical conditions of the soil making a favourable environment for increased uptake of nutrient elements by the plants and also had solubilizing effects of fixed form of nutrients. Similar findings were also reported by Sinha et al., (1981) and Shroff and Devasathali (1994). An appropriate combination of vermicompost and inorganic nutrient source was found to enhance the efficiency of nutrients and ultimately increased the yield components. The results were in accordance with the findings of the Kale et al. (1992).

The increased yield was mainly due to improvement in yield attributing characters like number of leaves/plant and number of branches. The prolonged availability of nutrients which increased nutrient absorption by crop during the crop growth period due to application of inorganic fertilizers along with vermicompost inturn might have enhanced

117 the growth and yield of tea plant (C. sinensis) (L.). Significant increase in growth components and seed yield of tea plant (C. sinensis) (L.) crop through integration of organic manure and inorganic nutrients were also reported by UPASI Annual Report (2009).

Application of vermicompost might be attributed mainly to higher content of available nutrients and presence of beneficial microflora such as nitrogen fixers. Bhawalkar (1996) stated that the significant increase in yield due to phosphate solubilizing fungi VAM, higher enzyme activity and biologically active metabolities in vermicompost. These might have increased the availability of both native and applied nutrients in the soil and their uptake by crop. Further the higher values recorded for growth and yield components of tea plant cultivated in 50% vermicompost + 50% NPK (T5) treated plots, might have been due to the more availability of NPK for the plants.

From the ongoing discussion it is evident that vermicompost could easily replace the utility of chemical fertilizer. Vermicompost could be applied for all varieties of cash crops, plantation crop, cereals pulses, oil seeds, spices, vegetables, fruits, ornamental plants, strawberries as suggested by Edwards (1985); Kulkami et al. (1996); Atiyeh et al. (1999); Bucker field (1999); Parthasarathi and Ranganathan (2002); Arancon (2006). At the same time, it is not advisable to stop the use of chemical fertilizers completely. Many hybrid varieties require more NPK and so many workers have proved that combined use of vermicompost along

118 with chemical fertilizers will have more effect on plant growth and yield (Ramamoorthy, 2004). In accordance with the above reports, the present study also proved that combined use of vermicompost along with NPK (T5) has maximum effect on tea plant growth and yield. So it can be concluded that depending upon the crop need, the chemical fertilizer can be used along with vermicompost.

Effects on physical properties of soil Soil aggregation influences structural characteristics of soil such as water infiltration, water holding capacity, porosity and aeration. The porosity depends upon the texture and aggregation of soil. Earthworm contributes to soil aggregation mainly through the production of casts. Earthworm casts contain more water stable aggregation than the surrounding soil (Edwards and Bohlen, 1996). The increased porosity in T5 (VC+NPK) and T3 (VC) field in the present study is probably due to aggregation of the soil particles by the action of microorganisms in the vermicompost which produce polysacchrides providing a cementing action between the soil particles and possibly also by fungal mycelia (Edwards and Bohlen, 1996).

Tomati and Galli (1995) stated that vermicast appeared to be enriched with polysaccharides which act in the soil as cementing substances causing aggregate stability, contributing to create and maintain the soil structure and ensuring better aeration, water retention, drinage and aerobic conditions, very useful for root development and nutrients

119 availability of plants. In the present investigation the results revealed that there was a significant decrease in the particle and bulk density of the soil, in T5 and T3 field, which could be due to the increased porosity of the soil. Similar observations were made by Vasanthi and Kumarasamy (1999) who found a significant reduction in the bulk density of the soils treated with vermicompost plus NPK. The least reduction in bulk density among the five treatments was observed in field treated only with NPK. TW+CD+KW application promotes the WHC and also contributes to an increase in the cation exchange capacity (Jhon, 1990). Tea waste residues compost provides greater nutrient values, soil organic matter, total and active fraction of carbon to the soil, WHC and reduce nitrate leaching (Ramamoorthy, 2004). The increased WHC in T5 and T3 field, in the present investigation are due to increased porosity, and decreased bulk density of the soil as a consequence of vermicompost application and these inturn provide greater aeration and drainage.

The increased CEC in T5 and T3 in the present investigation was mainly due to higher amount of organic matter in the vermicompost. This observation falls in line with Vasanthi and Kumarasamy, (1999) who found significantly increased CEC of the soil treated with vermicompost plus NPK. TW+CD+KW mixture compost amendments should also improve soil properties like bulk density, WHC, aggregate stability and cation exchange capacity, CEC increased by the addition of TW+CD+KW compost. Organic fertilizer provides significant cation exchange capacity to hold cations such as K+. The change in cation exchange capacity of

120 organics by acidification might have enhanced K availability to plants (Vasanthi and Kumarasamy, 1999).

Ramamoorthy (2004) studied the effect of application of NPK (Pressmud, sugarcane, trash, bagasse) vermicompost supplemented with NPK on the growth and yield of black gram and also on the physical and chemical properties of soil and concluded that vermicompost and vermicompost supplemented with NPK applied plots showed increased porosity, WHC, CEC and decreased particle density and bulk density. The present observations such as increased porosity, WHC, CEC and decreased particle and bulk density in the vermicompost (T3) and NPK supplemented with vermicompost (T5) applied plots were falling in line with the above reported results.

Effects on chemical properties of soil The results indicated that the application of vermicompost not only influenced physical properties of soil but also influenced the chemical properties of the soil. The increased organic matter and organic carbon (OC) content (Table 21) in T3 (VC) and T5 (NPK + VC) applied field in the present study were mainly due to higher amount of OC content in vermicompost. The present results corroborated with the observations made by Vasanthi and Kumarasamy (1999) and who reported increased OC content in the soil due to incorporation of various enriched compost, FYM and vermicompost.

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The perusal of results in the present study revealed that there was significant increase and retention of the available NPK of the soil treated with vermicompost (T3) and NPK supplemented with vermicompost (T5). The NPK content remained high even after harvest of tea plant. The increase in the nutrients may be due to the higher content of these nutrients in the vermicompost. Similar increase in available NPK in the soil was observed when soil was treated with enriched compost from different organic wastes, FYM, vermicompost and vermicompost plus NPK after the harvest of rice, ragi and cowpea (Vasanthi and Kumarasamy, 1999; Chaioui et al. 2003). Wide spread adoption and long term application of vermicompost to agricultural lands, which were starved of organic matter, organic carbon, macro and micro nutrients, would facilitate and maintain the structure and fertility of soil for sustained agricultural use for long time.

Thus, the present investigation proved that vermicompost as well as NPK supplemented with vermicompost are superior to inorganic fertilizer NPK and traditional organic manure FYM. The vermicompost is most needed for sustainable agriculture since it enhances both soil fertility and plant productivity.

On the whole the present investigation confirmed the possibility of utilization of tea waste in balanced combination either with CD or KW for vermiculture, production of nutrient rich vermicompost and their stimulative effect on the growth and yield of tea plant (C. sinensis) (L.).

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So, tea waste and cowdung could be used as feed substitute in balanced combination for Eudrilus eugeniae that convert into organic manure- vermicompost (also other composting earthworms) used for agricultural crops, particularly tea plant (C. sinensis) (L.) O. Kuntze var.

6. SUMMARY

The present study mainly focused on vermicomposting of industrial tea waste, cowdung and kitchen waste using two earthworm species namely Eudrilus eugeniae and Eisenia fetida and its effect on crop productivity.

The growth and reproduction rate of two earthworm increased continuously from 0 to 90 days in a industrial tea waste, cowdung and kitchen waste mixture. Particularly, E. eugeniae gain more weight and produced more number of hatchlings than E. fetida.

The physico-chemical analysis of vermicompost of industrial tea waste, cowdung and kitchen waste produced by two earthworms indicated that the level of pH, TOC and C:N ratio were reduced than control and the EC, macro and micro nutrients were increased than the control. Of these two worms the vermicompost of E. eugeniae exhibited more nutrients than E. fetida. The increased mineralization and conservation of nutrients is due to the biocatalytic role of earthworms in the decomposition and conservation mechanism.

The microbial population, microbial activity were high in vermicompost produced by E. eugeniae than E. fetida. The increased level of microbial population, microbial activity in the vermicompost of E. eugeniae could be due to the higher nutrient concentration and quality 124 in the substrate multiplication of microbes while passing through the gut of worms.

Among the different treatment (T1 – T4) of industrial tea waste,

cowdung and kitchen waste mixture, the earthworm species grown on T4 treatment (60% tea waste + 10% cowdung + 30% kitchen waste) exhibited better growth, reproduction rate, microbial population, microbial activity. Of these two worms, E. eugeniae showed better results than E. fetida.

The comparative field evaluation of vermicompost of industrial tea waste, inorganic fertilizer, farmyard manure and inorganic fertilizer supplemented with vermicompost on tea plant showed that the field treated with 50% inorganic fertilizer supplemented with 50% vermicompost of E. eugeniae exhibited the highest yield of tea plant (Camilla sinensis). So, the use of vermicompost in combination with inorganic fertilizer is recommended for improving the crop productivity.

7. CONCLUSION

The progressive increase in the size of the world population resulted large volume of organic wastes all over the world. The growth of industries and ever increasing population has led to an increased accumulation of waste materials. Recycling of waste through vermitechnology reduces the problem of non-utilization of waste.

Vermicompost has higher economic value compared to compost derived from traditional methods. Alternative to chemical fertilizers, locally available organic wastes of anthropogenic and natural products were used as biofertilizers after employing earthworm as decomposers, for degradation and recycling to enhance the production of crops which are free from pollution and health hazard.

Tea is one of the major drinks in the world. India is one of the largest tea producers and it is the biggest consumer in the world. Consumption of ready-made tea, which packed into cans and bottles, has been increasing remarkably in recent years. Vermi-technology is recently popular in conversion of bio-wastes into nutrient rich organic manures because of its simple methodology and low investment without any sophisticated infrastructure.

Therefore, in the present investigation an attempt was made to convert industrial tea wastes into organic manures through 126 vermitechnology using two species of earthworm E. eugeniae and E. fetida.

The tea waste, cow dung and kitchen waste mixture is easily degraded into vermicompost by the E. eugeniae and E. fetida. Among the two species E. eugeniae showed better results.

The application of 50% NPK supplemented with 50% vermicompost on tea plant showed higher rate of growth and yield. Hence, the present study concludes that vermicompost could be prepared from industrial tea wastes with cow dung and kitchen waste and it can be recommended to improve the long-term soil fertility and crop productivity by means of reducing the usage of inorganic fertilizers.