INTRODUCTION:-

Management of organic waste in environment friendly manner is becoming

difficult due to rapid increase in population and urbanization. Organic waste materials

are available in huge amounts in the form of farm waste, city waste, poultry litter and

the industrial wastes (food, sugar, cotton, and rice industry). The continuous

accumulation of these materials is becoming a potential source of land, water and air

pollution. Recycling of organic waste is one of the major options, which could be

effective for reducing huge piles of organic wastes.

Composting is an alternative technology for a sustainable solid waste

management. It is a biological decomposition of organic matter by micro-organisms

(Golue ke 1972; Rynk et al., 1992; Beffa et al., 1996; Tiquia et al., 1996). During

composting, the starting material is transformed through a variety of biological and

biochemical processes in which enzymes play a role [Garcia et al., 1992, 1993;

Vuorinen 1999, 2000]. A big significance for the process of composting represents the

cell wall of micro organisms through which mass transfer is possible. Low molecular

weight and water soluble molecules can easily pass through the cell wall where they

take part in the cell metabolism, providing energy and being built up into larger

polymers, with the help of intracellular enzymes. To attack high molecular weight

components, which cannot pass through the cell wall, micro organisms secrete

extracellular enzymes. They break molecules down into the fragments that can be

assimilated, while the rest is converted into a stable product, humus or compost

[Haug, 1980; Biddlestone, J., Gray, 1985].

Xylanases (E.C.2.8.1.8) a group of hemicellulolytic enzymes, are required for

the hydrolysis of β 1,4- xylans present in lignocellulosic materials [Kheng, Omar,

(2005)] . Xylanases are the microbial enzymes that have aroused great interest

recently due to their potential application in many industrial processes viz; nutritional

improvement of lignocellulosic feed stuff (Wallace et al., (2001), production of

hydrolysates from agro-industrial wastes (Kheng and Omar, (2005). Applications of

microbial enzymes extend from food and beverage manufacturing to biomass

conversion, and waste treatment.

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The present study was undertaken in the following aspects

Xylanase production from Bacillus sp, Pseudomonas fluorescens, Rhizopus

nigricans and Trichoderma viride by using different agro residues.

Compost production using EM solution, crude xylanase, and mixing of crude

xylanase and EM solution.

To find out the macro and micro nutrient status of EM treated organic waste,

crude xylanase treated organic waste and mixing of EM and xylanase treated

organic waste .

To compare the seed germination, growth parameters of greengram under

different treated organic composts.

To analyse the carbohydrate and protein content in green gram grown in

different treatments.

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REVIEW OF LITERATURE:-

XYLANASE PRODUCTION:-

Abundance of xylan:-

Xylan together with hemicelluloses forms the second most abundant

renewable polysaccharide in the biosphere. It has been estimated that 500 million tons

of such materials could be annually available from the residues of major crops

[Detroy, 1981].In some higher plants and agricultural wastes, xylan constitutes from

20-40% of the dry weight [Detroy, 1981 and Petterson, 1984]. Xylan is widely

distributed in plant cell walls and forms a main part of the hemicelluloses fraction

[Holt, Sharpe and Williams, 1989]. Xylans are linear homopolymers that contain D-

xylose monomers linked through β -1, 4- glycosyl bonds [Srinivasan and Rele, 1999].

Types of Xylan:-

There are two types of hemicelluloses, the acetylated xylan of hard wood and

arabinoxylan of soft wood (Timell, 1961).

Hard wood xylan is typical O-acetyl-4-0 methyl flucuronic xylan with

approximately 10% xylose units substituted with X-1, 2 linked 4-0-methyl glucoronic

acid side chain and 70% of xylose residues are acetylated at the C2, or C3 position.

Acetylation occurs more frequently at the C3 and double acetylation of a D-xylose

unit has also been reported (Bouveng, 1967). The presence of acetyl groups makes the

xylan signification soluble in water. It constitutes about 15-30% of the cell wall

content.

Soft wood xylan is commonly arabinoxylan in which 10% of xylose units are

substituted with a-2, 3 linked arabinofuranose residues. It constitutes about 7-10% of

the cell wall content (Whistler, 1970 and Biely, 1985).

Structure of xylan:-

The structure of xylan found in cell walls of plants can differ greatly

depending on their origin but they always contain a β -1,4 linked D-xylose back bone.

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[Ebringerova and Heinze, 2000]. Xylan is heterogenous polysaccharide consisting of

a homopolymeric back bone of 1,4-linked β -Xylopyranose residues and short chain

branches including acetyl, X-L- arabinofuranosyl, -D-glucuronyl residues.

Biodegradation of xylan:-

Biodegradation of xylan requires action of several enzymes, among which

xylanases play a key role (Blanco, et al., 1999).

A complete and efficient biodegradation of xylan depends mainly on 2 types

of enzymes: (1) Endo-β -1, 4-xylanase which hydrolyzes the xylanopyranose of the

central chain and (2) β -xylosidese which hydrolyze other xylooligo saccharides

resulting from the action of endoxylanase. Other enzymes used for biodegradation

were acetyl xylanoasterases, glucuronidase, L-arabinofuranosidase (Maria and Samia,

2005). The xylanases commonly act on the xylans like arabinoxylan, arabino-4-0-

methyl, D-glucuronoxylan and glucuronoxylan D-xylanases of this type have been

assigned the number E.C.3.2.1.8 (1-4)-B-D xylano hydrolase, endoxylanase and

3.2.1.3 (1-4) β -D-xylo hydrolase endoxylanase.

Xylan extraction:-

The method of Panbangred et al., (1983) was followed for xylan extraction

from agro wastes. To 50 gm. of finely powdered agro wastes 100 ml. of 3% NaOH

was added and incubated at 121o C for one hour. 50 ml. ethanol was added and mixed

thoroughly with glass rod. Xylan was precipitated and the precipitate was washed

many times with tap water and dried in an oven at 50o C.

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Table 1

Xylan extraction method for various authors.

S.No. SUBSTRATEAUTHOR &

YEAR

ALKALI

METHOD

ACID

METHOD

1.Sugarcane

baggase

Bocchini et al.,

(2005)

0.5ml of

H2So4

2.

Cornhusk,

sugarcane

baggase, Corn

cob, Rice straw,

Rice bran

Tachaapaikoon et

al.,(2006)1N NaoH

3. Corn cob Yang et al., (2008) 1.0g /1 H2So4

4. Barley strawRezaeian et al.,

(2005)3 ml. of NaOH

Xylanase:-

Xylanase (E.C.3.2.1.8- endo- β, 1-4-D-xylanase) is mainly responsible for the

hydrolysis of xylan with β -1-4 xylanolytic linkages. During the last decades

xylanases have been received a great deal of attention mainly due to their various

industrial applications such as pulp, paper, food and feed industries. Xylanase is

responsible for hydrolysis of xylan, a major hemicellulose of plant cell wall (second

most abundant). This enzyme is extensively used in food processing, chemical and

pulp industries (Seyis and Aksoz, 2005). The major uses of this enzyme are in

biopulping, biobleaching, clarifying and liquefying fruit and vegetable juices. In paper

and pulp industries, use of xylanase causes decrease in consumption of chlorine,

Absorbable Organic Halogen (AOH), Chemical Oxygen Demand (COD) and

improves thereby the quality of waste water.

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Occurrence of xylanase:-

Xylanase are widely distributed. They occur in both prokaryotes and

eukaryotes (Dekker and Richards, 1976) and have been demonstrated in higher

eukaryotes including protozoa insects, snails and germinating plant seeds (Taiz and

Horingman, 1976). In general, the level of xylanase in fungal culture is typically

much higher than those from yeasts or bacteria (Singh et al., 2003).

Occurrence of Multiple xylanases in Micro organisms:-

Multiple xylanases have been reported in numerous micro organisms (Dekker,

1985) three different xylanases have been reported in purified form from the culture

filterate of Clostridium stercorarium and in streptomyces sp (Marui et al,. 1985).

Xylanase Production:-

The basic factors for efficient production of xylanolytic enzymes are the

choice of an appropriate inducing substrate and an optimum medium composition.

The important of cellulose free xylanase systems in the paper and pulp industry has

initiated research into the correlation between the production of xylanase and

cellulases by micro organisms.

Xylanases production from micro organisms:-

Xylanases are produced by microorganisms including bacteria, fungi and

actinomycetes (Subramaniyam and Prema, 1999; Nascimento et al., 2002)

Xylaneses have been reported from fungi [ Ishihara et al., 1997; [Icjart et

al.,1999] bacteria [(Gessesse and Mamo., 1998; Inagaki et al., 1998)], and

actinomycetes [ Fernandez et al., 1998; Garg et al., 1998].

Most commercial xylanases are produced by Trichoderma, Bacillus,

Aspergillus, Penicillium, Aureobasidium and Talaromyles species [Li et al., 2000;

Polizeli et al., 2005]

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A large number of bacteria and fungi are known to produce xylanases

(Kulkarni et al., 1999). Among the bacterial sources, Bacillus is an industrially

important source of xylanases [Shah et al., 1999].

Bacterial xylanases:-

Bacterial xylanases hydrolyse xylan to xylotriose and higher oligosaccharides.

Intensive investigations have been performed with xylanolytic enzymes derived from

bacteria, both aerobic and anaerobic. Very few bacterial xylanases have been well

characterized and most have been found to be endoxylanases producing xylobiose as

the main end products.

Table 2:-

List of xylanolytic bacteria studied by various authors.

Bacteria References

Aureobasidium pullulans Tanaka et al., 2005

Bacillus circulansDhillon et al., 2000; Qureshy et al.,

2002; Bocchini et al., 2005.

B. coagulans Heck et al., 2005; Chauhan et al., 2006.

B. licheniformis Damiano et al., 2003; 2006

B. pumilusMoriya et al., 2005; Battan et al.,

2007 ;Kapoor et al., 2008.

B.subtilis Oakley et al., 2003.

B.firmis Chang et al., 2004

Bacillus sp

Aleksandrova et al., 2000; Senthil

kumar et al., 2005; Avcioglu et al.,

2005.

Bacillus Virupakshi et al., 2005.

Cellulomonas flavigenia Hernandez et al., 2007.

C. flavigenaEstrada et al., 2002; Delgado et al.,

2006.

Clostridium sp Marichamy and Mattiasson, 2005.

Clostridium absonum Swaroopa Rani and Krishnanand, 2000.

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Eschericia coli Liu et al., 2003; Yin et al., 2007.

Geobacillus sp Wu et al., 2006.

Paenibacillus Harada et al., 2008.

Pseudomonas sp Xu et al., 2005.

Streptomyces cyaneusNinawe and Kuhad, 2005; Ninawe et al.,

2008.

Streptomycs spTechapun et al., 2001; Petrosyan et al.,

2002; Chungool et al., 2006.

S.violaceroruber Khurana et al., 2007.

S.olivaceoviridis Ding et al., 2004; Ai et al., 2005.

Staphylococcus sp Gupta et al., 2000.

Thermotoga maritimeKittur et al., 2003; Xue and Shao, 2004;

Tan et al., 2008.

Fungal xylanases:-

From an industrial point of view, filamentous fungi are interesting produces of

these enzymes due to xylanases releasing and their easy cultivation [ Wong, Tas and

Saddler., 1988; Dekker., 2003]. Most commercial xylanases produced by

Trichoderma, Bacillus, Aspergillus, Penicillium, Aureobasidium and Talaromyces sp.

[Polizeli, et al., 2005; Azadi, et al., 2000]

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Table 3:

List of xyanolytic fungi studied by various authors.

Fungi Authors.

Aspergillus nidulans Mac cabe et al., (1996).

A.niger Ferreira et al (1994).

A.flavus Arunachalam et al. (2001).

A.fumigatus Bailey and Viikari (1993).

Trichoderma sp. Gomes et al., (1992).

Chaetomium globosum and A. niger Wiacekzyclinka et al., (1994).

Candida utilis Vilas Boas et al., 2002.

A.niger

Deschamps et al., (1984); Smith and

Wood (1991); Youn and Rungyu (1999);

Kansoh et al., (2001); Lenartoviez et al.,

(2002).

Pleurotus sps. Reddy et al., (2003).

PRODUCTION OF XYLANASE BY CHEAPER HEMICELLULOSIC AGRO

WASTES:-

Agricultural by products that contain cellulose, hemicellulose and ligain could

serve as inexpensive sources for xylanase production [ Nascimento et al., 2002;

Techapun et al., 2003] Sugarcane baggase, wheat bran, rice bran, maize bran, gram

bran, wheat straw, rice husk, rice straw, soy hull, sago hampas, grape vine trimmings

dust, saw dust, corn cobs, coconut coirpith, banana waste, cassava waste, palm oil

mill waste, paper pulp, sugar beet pulp, sweet sorghum pulp, apple pomace, peanut

meal, rape seed cake, coconut oil cake, mustard oil cake, cassava flour, wheat flour,

corn flour, steamed rice, stream pretreated willow, starch etc. Cheaper hemicellulosic

substrates namely cotton fibre, corn cob, wheat bran, paddy straw, paddy husk,

sugarcane baggase, corn stalk, tamarind seed, saw dust and wheat straw were used as

substrates for xylanase production. Wheat straw served as a good substrate for

xylanase production in Cryptococcus adeliue (Gomes et al., 2000).

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Wheat bran served as a good carbon source for xylanase production in

Thermomyces lanuginosus (Haq and Deckwer, 1995), and Bacillus licheriformis

(Archana and Satyanarayana, 1998) and wheat straw enhanced maximum xylanase

production in Cryptococcus adeliue (Gomes et al., 2000), Cochliobolus sp. (Bakri et

al., 2008), and maize straw and jowar straw in Trichoderma viride (Goyal et al.,

2008).

Corncobs supported maximum enzyme production in Aspergillus feetidus

(Christov et al., 1999), Aspergillus flaviceps (Ruckmani and Rajendran, 2001) and

Rhizopus oryzae (Bakir et al., 2001). Corn fibre xylan supported more xylanase

production in Fusarium verticillioidies (Saha, 2001).

Elisashvili et al., (1999) reported that the synthesis of cellulases and xylanases

were induced when grown on medium containing crystalline cellulose and plant raw

materials.

A number of studies have been already done on lignocellulosic wastes mainly

wheat bran [(Ninawe (1994); Gawande and Kamat., (1999); Kuhad et al., (2006)].

Sugar cane baggasse (Gutierrew-correa., 1998] and treated wheat straw [ Alfani et al.,

2000] using solid state fermentation or submerged culture fermentation.

Agricultural waste material or by products like corn cobs, sugarcane baggasse,

rice husk, rice straw and oat straw have been used by many scientists for xylanase

synthesis [Siedenberg et al., 1998; Christov et al., 1999; Gawande & Kamat, 1999;

Haq et al., 2002)

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Table 4:

Use of agro residues as substrates for bacterial xylanase production.

S.No. Substrates Organism Authors

1. Wheat bran Bacillus sp

Bataillon et al., 1998; Shah et al., 1999; Senthilkumar et al., 2005.

B. pumilusBattan et al., 2007; Kapoor et al., 2008

Streptomyces spB.subtilisPseudomonas sp

Beg et al., 2001.Yuan et al., 2005Xu et al., 2005

2. Wheat strawB.coagulansPaenibacillua

Chauhan et al., 2006Harada et al., 2008

3. Rice bran

ThermoascusaurantiacusBacillus sp

Santos et al., 2003.

Virupakshi et al., 2005

4. Lemon peelStreptomyces spCellulomonas flavigen

Petroysan et al., 2002Estrada et al., 2002;Delgado et al., 2006; Hermandez et al., 2007.

5.Sugarcane baggasse

B. circulansB.pumilus

Bocchini et al., 2005; Moriya et al., 2005.

6. Corn cob

Streptomyces spS. oliva leoriridisBacillus spThermotoga maritima

Techapun et al., 2003Ninawe and Kuhad, 2005 ; Ai et al., 2005Avciglu et al., 2005; Tan et al., 2008.

7. Grass B. circulars Bocchini et al., 2005.8. Corn husk Streptomyces sp. Churgool et al., 2006.

9. Rice straw B.coagulansDhillon et al., 2000.Chauhun et al., 2006;

10. Corn straw B.licheniformis Damiano et al., 2003.

11.Soyabean residue

B.coagulans Heck et al., 2005.

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Xylanase Assay Method:-

Methods used for the assay of xylanase are reported as many workers. Most of

them report xylanase activities based on the release of reducing sugars from partially

soluble xylan substrates (Tan et al., 1985).

Sugar detection by the DNS methods 9-Dinitro Salicyclic Acid, was chosen by

many workers rather than Somogyi-Nelson (SN) method. This is because SN method

is giving a lower result than DNS (Breull and Saddler, 1985).

Composting

Composting is an aerobic process by which organic materials are degraded

through the activities of successive groups of microorganisms; it is environmentally

sound way to reduce organic wastes and produce organic fertilizer or soil conditioner

(Gajdos, 1992).

Compost is prepared by biological degradation of plant and animal residues

under controlled, aerobic conditions. (Eghball et al., 1997).

The biological or natural degradation of organic waste to compost results in

the production of material which has lost its original identity through reduction in

particle size and the loss of volatile organic materials. The end product is humic,

earthy material that can be utilized in sustainable agricultural production without

damage to the environment.

Basic principles of composting:-

Composting is a naturally occurring, controlled bio-oxidative process that:

1. Requires a solid, heterogeneous organic substrates and both mesophyllic

and thermophyllic organisms.

2. Produces CO2 , Water vapour, nutrients, minerals and reasonably stable

organic matter.

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Basic elements of the composting process:-

(i) Micro organisms:-

Different microbial communities predominate during the various composting

phases (mesopilic and thermophillic), each of which being adapted to a particular

environment.

Composting is a microbiological process, little is known about micro

organisms involved and their activities during specific phases of the composting

process.

Bacteria, fungi, and actinomycetes are the micro organisms that are primarily

responsible for the decomposition of organic material.

Traditional composting process involves an initial stage conducted at

moderate temperature (10 – 40o C), during which the liable organic matter is rapidly

consumed by mesophilic micro organisms, followed by a stager when thermophilic

micro organisms drive the temperature up to 60o C at which lipids, proteins and

complex carbohydrates are consumed and broken down. During the final curing stage

when the materials cools down, mesophilic organisms are able to recolonize and

break down the remaining recalcitrant organic matter (Chefetz et al., 1996).

(ii)Nutrients and organic matter:-

Products rich in carbon, nitrogen, phosphoric and oxygen such as protein,

complex sugars and fats are excellent sources of carbon and nitrogen for micro

organisms. Carbon, nitrogen and phosphorous must be available to build protein.

These nutrients are required for a composting operation and to facilitate microbial

growth.

Proteolysis or the enzymatic break down of protein:-

This is accomplished by micro organisms that release extra cellular enzymes

called proteinase which convert long chains complex protein into smaller amino acid

groups called polypeptides which then further degrade to individual amino acids.

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Fungi and actinomycetes are extremely proteolytic and valuable in initial stages of

solid waste composting operations and in the later stages to further degrade resistant

organic compounds.

(iii)Oxygen:-

Aerobic conditions are essential for efficient composting operations. Aerobes

are micro organisms that predominate in an air – rich environment. Air is to be

supplied in order to provide oxygen facilitating the growth of micro organisms and

also to drive away the hot gases (Haug, R.T, 1993).

To increase the porosity of the mass for effective aeration in all methods of

composting, this is normally done by mixing the wet and dry materials (Sincero and

Sincero, 1996).

Oxygen should reach to the all points of the mass (Sincero and Sincero, 1996 ;

Tcho-banoglous et al., 2003). Aerobic conditions must be maintained by turning the

compost pile or forcing air through it (Tchobanoglous et al., 2003).

(iv) Moisture:-

Moisture is essential to provide a nutrient – rich source for micro organisms.

From this source, micro organisms derive the nutrients they require for protein

synthesis and growth. The synthesis of protein is necessary to promote rapid

microbial population growth which hastens decomposition of organic material. To

assure proper rate of bio degradation, moisture should be in the range of 50% - 70%

throughout the degradation process. The ranges for optimum moisture content greatly

depend on the type of feed stock, its particle size and the rate of composting desired

(Landreth and Rebers, 1996). Generally ranges of 50 – 60% are desirable (Landreth

and Rebers, 1996; Davis and Cornwell, 1998).

(v)Temperature:-

This is a key environmental factor which influences biological activity within

a composting operation. The phases which can be distinguished in the composing

processes according to temperature patterns are: lag, active and maturation (curing)

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phases (Kreith, 1994) or latent, growth, thermophilic and maturation phases

(Polpraset, 1996). The purpose of curing phase is to ensure that compost is stabilized

allowing the remaining nutrients to be metabolized by the available micro organisms

(Landreth and Rebers, 1996).

The temperature change during the composting has a profound effect on the

efficiency of the composting process. Heat is generated by decomposing process of

the organic matter by micro organisms (Hagerty, et al., 1973).

Different types of organic waste degradation accelerators:-

(i) Cellulolytic Microbial Activator (CMA):-

CMA is a group of micro organisms which is highly capable of decomposing

agricultural or organic washes to produce fertilizers in a short period of time. It has

been discovered by the land development department of Thailand since 1986. The

CMA is a combination of eight micro organisms from bacteria, actinomycetes, and

fungi that is able to produce high decomposed cellulose enzyme. The CMA can

increase itself very fast in the high organic matter soil preventing other harmful micro

organisms to grow.

The CMA is more affective when mixed with the rubber factory waste, water

hyacinth and sludge since a good fertilizer is achieved. (Thaniya kaosol, Srinithrar

Wandee, 2009).

(ii) Biodegradation of leather waste by enzymatic treatment:-

Extra cellular alkaline proteases enzyme produced by Bacillus subtilis and it is

treated for leather degradation. (Muhammad Nauman Aftab et al., 2006).

Enzymatic activities during composing:-

Several enzymatic activities have been measured to describe organic matter

decomposition in two microbial –driven processes, composting and vermicomposting

(Garcia et al., 1994, 1995; Benitez et al., 2002, 2005). Enzyme activities have been

used widely as an index of soil fertility or ecosystem status because they are involved

15

in the biological transformations of native and foreign compounds in soils (Tate,

2000).

Usually there is not any correlation between the enzyme activities and

microbial biomass and respiration, and this may depend on the fact that enzymatic

activity is due to enzymatic which may be in a living or dead cell, cell debris, free

enzymes and or enzymes absorbed by clay or immobilized in humic complexes

(Ceccanti and Garcia, 1994; Alef and Nannipieri, 1995 a; Nannipieri et al., 2002).

Earthworm bioreactors have an in house supply of enzymes such as amylase,

cellulose, nitrate reeductase, and acid and alkaline phosphates. These enzymes

biodegrade the complex biomolecules into simple compounds. The digestive enzymes

of earthworms are responsible for the decomposition and humification of organic

matter.

The biological decomposition of organic matter is mediated by a variety of

biochemical processes in which enzymes play a key role (Garcia et al., 1992

Vuorinen, 1999). The major constituents like cellulose, hemicelluloses, lignin, starch

and different protein compounds present in waste are degraded by specific enzymes.

There fore, the quantification of enzyme activity during composting can reflect the

dynamics of composting process in terms of decomposition of organic matter and

nitrogen transformation. In correlation with enzyme activity the changes in microbial

number and types also helpful in providing information about the maturity of the

composted product (Tiqwa, 2002, 2005).

Reports about the evolution of particular enzyme activities during composting

are very rare. Some attention was paid to cellulose (Myrback, K., 1961; Stuzenberger,

F., 1971; Godden, B., 1985).

Specific examples of enzymes important in soil microbiology include,

cellulases, which degrade the polymer cellulose into smaller components; nitrogenase,

which converts dinitrogen gas into biologically available ammonia; sulphatases,

which release protein and certain organic compounds; and phosphatases, which

remove phosphate groups from organic compounds (Burn 1978; Tate 1995;

Nannipieri et al., 1996). Enzymes that catalyse the degradation of polymeric

16

substances, such as cellulose, hemicelulose and lignin, are extra cellular because the

polymer is too large to be transported across the cellular membrane ( Priest, 1984).

However, once the polymer has been reduced to its smaller units, subsequent

catabolism may proceed intra cellular (Skujjins, 1976). Inter cellular and extra cellular

enzymes cannot be distinguished in soil and compost suspensions.

Characterizing and quantifying the enzymatic activity during composting can

reflect the dynamics of the composting process in terms of the decomposition of

organic matter and nitrogen transformations, and may provide information about the

maturity of the composed product.

Biodegradation:-

Microbial biodegradation of the waste is generally considered to be a safe

effective and environmentally process, and certain mushrooms have showed good

potentials for degrading coir pith. For example, Polyporous versicolor (Crawford and

Crawford 1976), Pleurotus sajor-caju (Bisaria et al., 1987) and Pleurotus sp. (Lovie

1988) have been found to be effective in lignin and cellulose degradation, particularly

from the straw of rice and cotton.

Lignocellulose bio degradation:-

The problem of increasing the utility of lignocelluloses wastes has been

known for decades. In addition to the growing demand for traditional applications

(paper manufacture, biomass fuel, composting , animal feed, etc.).

Ligno cellulose is a complex substrate and its biodegradation is not dependent

on environmental conditions alone, but also the degradative capacity of the microbial

population (Wal drop et al., 2000). The composition of the microbial community

charged with ligno cellulose bio degradation determines the rate and extent there of.

Cellulose and hemicelluloses / Enzymes and degradation:-

The efficient hydrolysis of cellulose requires the concerted action of at least 3

enzymes: (i) endo-glucanases to randomly cleave intermonomer bonds; (2)

17

exoglucanases to remove mono and dimmers from the end of the glucose chain; and

(3) β -glucosidase to hydrolyse glucose dimmers (Tomme et al., 1995; Deobold and

Crawford 1997). The concerted actions of these enzymes are required for complete

hydrolysis and utilization of cellulose. The rate limiting step is the ability of

endoglucanases to reach amorphous regions with in the crystalline matrix and create

new chain ends, which exo-cellobiohydrolases can attack.

Although similar types of enzymes are required for hemicelluloses hydrolysis,

more enzymes are required for its complete degradation because of its greater

complexity compared to cellulose. Of these, xylanase is the best studied enzyme

(Kuhad et al., 1997).

A fundamental different exists in the mechanism of cellulose hydrolysis

between aerobic and anaerobic fungi and bacteria (Leschine 1995 ; Tomme et al.,

1995). Aerobic fungi and bacteria characteristically comprise non-complexed

cellulase systems, which entail the secretion of the cellulose hydrolysis enzymes into

the culture medium

Complexed cellulase systems allow greater co ordination between the different

hydrolyzing enzymes. Their close association will restrict loss of degradation

intermediates due to dynamic environmental conditions. In aerobic systems, where

active aeration and agitation is required, loss of the secreted enzymes and their

degradation intermediates due to dynamic environmental conditions.

Bacterial and fungal feruloyl and p-coumaroyl esterases are relatively novel

enzymes capable of releasing feruloyl and p-coumaroyl play an important role in

biodegration of recalcitrant cell walls in grasses (Kuhad et al., 1997). These enzymes

are synergistically with xylanases to distrupt the hemicellullose-lignin association,

without mineralization of the lignin (Fillingham et al., 1999). Therefore,

hemicellulose degradation is required before efficient lignin removal can commence.

Lignocellulose biodegradation by prokaryotes is essentially a slow process

characterized by the lack of powerful ligno cellulose degrading enzymes, especially

lignin peroxidases. Grasses are more susceptible to actinomycete attack than wood

(Antai and Crawford 1981; Mc Carthy 1987). Together with bacteria, actinomycetes

18

play a significant role in the humification process associated with soils and composts

(Trigo and Ball 1994).

Biodegradation by fungi:-

Most fungi are capable cellulose degraders. Anaerobic fungi (Piromyces sp.,

Neo calli-mastix sp. Orpinomycess sp.) from part of the rumen microflora. These

fungi produce active polymer degrading enzymes, including cellulases and xylanases

(Hodrova et al., 1998).

Compost:-

Compost, a nutrient-rich, organic fertilizer and soil conditioner is a product of

humification of organic matter. This process is aided by a combination of living

organisms including bacteria, fungi, and worms which transform and enhance

lignocellulosic waste into humic like substance (Eyheraguibel et al., 2008).

Composting Materials:-

Substrates suitable for making humus rich compost include cereal straw and

bran (Hort et al., 2003); water hyacinth (Chatterjee et al., 2003), Urban wastes (Taiwo

and Oso, 2004); lemon tree prunnings, cotton waste and brewery waste (Garcia-

Gomez et al., 2005); horticultural wastes (Lopez et al., 2006); Olive palm and grape

wastes (Alburquerque et al., 2006: Cayuela et al., 2006; Aravanitoyannis et al., 2007

a).

Benefits of Compost:-

Compost as an effective much surface to increase the amount of soil organic

matter, improves the soil structure and the water storage capacity of the soil, controls

the growth and spread of weeds and provides a long term source for nutrient supply of

the farm (Gonzales, et al., 1990; Sikora and Enkiri, 1999; He , et al.,2000; Chodak et

al., 2001; Levy and Tylor, 2003; Aoumare, et al., 2003).

19

Benefits of compost application in agriculture mainly result from its content of

organic matter, plant nutrients, promoting plant growth and inhibiting root pathogens

or soil-borne diseases (Hoitink, 1980; Alvarez et al., 1995; Perner et al., 2006).

Types of Organic Composts:-

EM Compost:-

EM stands for effective micro organisms. EM is not specific type of micro

organisms. It is a mixed liquid culture solution containing lactic acid bacteria,

photosynthetic and nitrogen fixing bacteria, yeast, ray fungi and molds making about

5 families, 10 genera and 80 different species (Higa and Wididana, 1991 a). All of

these are mutually compatible with one another and can co exist in liquid culture for

extended periods (Higa, 1991). Wididana and Higa (1999) investigated the beneficial

aspects of integrated recycling of urban organic waste with EM technology.

Application of EM is known to increase the microbial diversity of soil and plants,

improve soil quality, and enhance growth and increase yield and quality of crops

(Higa and Parr, 1994; Kishore, 2000).

The microbial solution has the ability to break down organic matter, thereby

producing plant nutrients and enhancing physical and chemical properties (Yadav,

2002).

Effective micro organisms is a technology now widespread around the globe

and known for its versatility and effectiveness under a wide range of environmental

situation (Higa, 2002). Originally developed for enhancing the soil and promoting

growing conditions for food crops, it has also gained a reputation as a very effective

tool in waste management.

EM can be used in agriculture via a number of methods EM is inoculated into

the rhizosphere with the intention to regenerate soil, raise yields, and improve the

nutrient content of foods. EM can be drip fed or sprayed in dilution on to crops and

soil. Alternatively EM can be used as a means of processing organic waste to create a

rich compost to facilitate crop growth.

20

Principles of Effective Micro organisms:- (Anibal, et al., 2007)

The principle of activity of the EM is by increasing the bio diversity of the

micro flora increasing the yield of the crop. Photosynthetic bacteria are the back bone

of the EM, working synergistically with other micro organisms to provide the

nutritional requirement to the plant and also reduce the disease problem.

There are primarily 5 types of bacteria used to prepare EM solution.

Photosynthetic bacteria (Phototrophic bacteria):- are independent self supporting

micro organisms. These bacteria synthesize aminoacids, nucleic acids, bioactive

substances and sugars, substances from secretions of roots, organic matter (carbon) by

using sunlight and the heat of soil as sources of energy. They can use the energy from

infra red band of solar radiation from 700 nm to 1200 nm to produce the organic

matter, while plants cannot. So the efficiency of the plants is increased. These

metabolites are absorbed into plants directly and also cut as substrates for bacteria

increasing the biodiversity of the micro flora. Adding photosynthetic bacteria in the

soil enhances other effective micro organisms. For example, VA (Vesicular –

arbuscular) mycorrhiza in the rhizosphere is increased due to the availability of

nitrogenous compounds (amino acids) for use as substrates secreted by photosynthetic

bacteria. VA mycorrhiza increases the solubility of phosphates in soils thereby

supplying unavailable phosphorous to plants. VA mycorrhiza can coexist with

Azotobactor as nitrogen fixing bacteria and enhance nitrogen fixing ability by

legumes.

Lactic acid bacteria:- Produces lactic acid from sugars. Food and drinks such as

yogurt and pickles have been made by using lactic acid bacteria. However, lactic acid

is a strong sterilizer. It suppresses harmful micro organisms and increases rapid

decomposition of organic matter. More over lactic acid bacteria enhances the break

down of organic matter such as lignin and cellulose, and ferment these materials

which normally take plenty of time. Lactic acid bacteria have the ability to suppress

Fusarium propagation which is harmful micro organism that causes disease problem

in continuous cropping.

21

Yeasts:- Synthesize antimicrobial and useful substances for plant growth from amino

acids and sugars secreted by photosynthetic bacteria, organic matter and plant roots.

Bioactive substances such as hormones and enzymes produced by yeasts promotes

active cell and root division. Their secretions are useful substrates for effective micro

organisms such as lactic acid bacteria and actinomycetes.

Actinomycetes:- are the structure of which is intermediate to that of bacteria and

fungi, produces antimicrobial substances from amino acids secreted by photosynthetic

bacteria and organic matter. These antimicrobial substances suppress harmful fungi

and bacteria. Actinomycetes can coexist with photosynthetic bacteria. Thus, both

species enhance the quality of the soil environment, by increasing the antimicrobial

activity of the soil.

Fermenting fungi:- Such as Aspergillus and Penicillium decompose organic matter

rapidly to produce alcohol, esters and antimicrobial substances. These suppress odors

and prevent infestation of harmful insects and maggots.

Benefits of EM compost:-

The use of EM compost made from a free resource, our organic waste, has

many benefits for soil health and agriculture, whether carried out domestically or as

large scale systems. Effective micro organisms can become established in soil as an

associative group of positive interactions.

The benefits are immense, including:

Decomposition of residual agrochemicals in soils (Higa, 1993).

Greater mineralization of carbon (Daly and Stewart, 1999)

Greater resistance to water stress (Xu 2000, Hui-lian et al., 2000).

More efficient release of nutrients from organic matter (Sangakkara and

Weerasekera, 2001).

Enhanced protein activity.

Enhanced soil fertility, increase crop yield and crop quality, helps to correct

nutritional and physiological crop disorders.

22

Accelerates the decomposition of organic waste, reduces adverse effect of

continuous cropping, enhances soil physical characteristics, increases

beneficial micro organisms in the soil and helps control pathogens by

competitive exclusion.

Vermicompost:-

Vermicomposting, or composting with earth worms, is an excellent technique

for recycling food waste in the apartment as well as composting yard wastes in the

backyard (Bowen, 1969). Earthworm castings contain abundant essential elements

that plants need for healthy growth. Application of both compost and vermicompost

decreased the soil bulk density and increased the water holding capacity of compost

media and this was also significant and proportional to the rate of compost application

(Smith et al., 2000).

Domiquez et al., (1997) reported that solid wastes may be converted into

useful products by composting and or vermicomposting. Chaoui et al., (2003) defines

vermicoposting as the digestion of organic materials by earthworms known as casts.

Vermicomposting involves the bio-oxidation and stabilization of organic

matter through the joint action of earth warms and micro organisms. The

transformations in physiochemical and properties (Dominguez, 2004) and the short

time in which they can occur, make vermicomposting a suitable system for studying

microbe – earthworm interactions (Aira et al.,2006 a).

Decomposition systems, like vermicomposting, are characterized by having a

control donor dynamics, that is, decomposer and detritivores do not control the rate of

regeneration of their resources (Pimm, 1982).

Vermiculture farming involves the use of earth worms as versatile natural

bioreactors for cleaning up the environment with cost-effective waste management

technology for sustainable agriculture (Suryawanshi, Lohar & Killedar, 1997).

Earth worm are physically aerators, crushers and mixers; chemically

degraders; and biologically stimulators in the decomposer system. They effectively

23

harness the beneficial soil microflora, destroy soil pathogens and convert organic

wastes into vitamins, enzymes, antibiotics, growth hormones and protein rich casts.

Organic wastes, broken down and fragmented rapidly by earth worms, result

in a stable non-toxic material with good structure, which has a potentially high

economic value as soil conditioner for plant growth (Prabha, Jeyaraj & Jeyaraj ,

2005).

24

MATERIALS AND METHODS

Xylanase production:-

General Mycological Techniques.

Mycological techniques followed were generally as described in methods in

Microbiology, Vol.4 (Booth, 1971).

Cultures used:-

The bacteria used in these studies were Bacillus sps, Pseudomonas

flouroscense and fungi used in these studies were Rhizopus nigricans and

Trichoderma viride. These cultures were obtained from the laboratory of P.S.G.R.

Krishnammal College for women, Coimbatore, Tamilnadu, India. Bacterial cultures

were maintained on nutrient agar slants and fungal cultures were maintained on potato

dextrose agar slants.

Glass ware:-

Sterilized glassware of “Borosil glassware” was used.

Production of Xylanase

Substrates:-

Wheat bran, Rice bran, Pine apple fibre, Corn stover, Saw dust, Sugarcane

baggase, Groundnut shell, Coir pith, Tea waste, Corn cob, Waste cotton.

Chemicals:-

All chemicals used in the present study were of analar grade from Galaxo

company except the following: Oat spelt xylan was obtained from Sigma chemical

Co., USA.

25

Culture media:

The following culture media were used. Above said agro residues were used

as carbon source for Bacterial and fungal growth.

Horikoshi II Basal medium (liquid medium) for Bacterial growth.

Peptone - 5.0 gm

Yeast extract - 5.0 gm

K2 HP02 - 1.0 gm

Mgso4 7H2O - 0.2 gm

Distilled water - 1000 ml.

pH - 7.5

Nutrient Agar medium:-

Peptone - 5.0 gm

Beef extract - 3.0 gm

Sodium chloride - 5.0 gm

Agar - 15 gm

Distilled water - 1000 ml.

pH - 6.8

Carter and Bulls medium for fungal growth.

Urea - 1.40 gm

MgsO4 - 0.24 gm

Cacl2 - 0.50 gm

Znso4 - 0.20 gm

Cuso4 - 0.005 gm

Feso4 - 1.0 gm

EDTA - 0.6 gm

NaH2 Po4 - 1.56 gm

Peptone - 7.0 gm

Yeast - 2.5 gm

26

Sodium sulphate - 1.0 gm

MnS04 - 0.20 gm

Distilled water - 1000 ml.

pH - 7.0

PDA medium :-

Potato - 200 gm

Agar - 15gm

Dextrose - 15 gm

pH - 7.0

Extraction of xylan from various agro wastes:-

The method of Panbangred et al., (1983) was followed for xylan extraction

from eleven agro wastes (wheat bran, Rice bran, Pine apple fibre, corn stover, Saw

dust, Sugarcane baggase, Groundnut shell, Coir pith, Tea waste, Corn cob, Waste

cotton). 100 ml. of 3% NaOH was added to 50 gm of finely powdered agro wastes

and incubated at 1210 C for one hour. 50 ml of ethanol was added and mixed

thoroughly with glass rod. Xylan was precipitated and the precipitate was washed

many times with tap water and dried in an oven at 500 C.

Sterilization:-

Distilled water, media were sterilized in autoclave at 15 psi for 15 min. Glass

wares were sterilized in a hot air oven.

Source of inoculums:-

Four days old cultures on nutrient agar medium, in petridishes were used as a

source of inoculums. Bacteria from growing front were removed using sterile loop. In

the same way, four day old fungal cultures were used as a source of inoculums.

Liquid state fermentation:-

Conical flasks (150 ml) containing 50 ml of liquid media were used for liquid

state fermentation. Three replicates were maintained for each experiment.

27

Inoculation of liquid culture:-

A single loop of the bacteria and fungi were inoculated into the liquid medium

for separate experiments.

Incubation periods:-

The conical flasks were incubated at room temperature under lab conditions

for 2 days for bacterial growth, 4 days for fungal growth.

Enzyme assay:-

The production of extra cellular xylanase by various isolates was determined

as follows:-

Liquid state fermentation:-

The method of Johri and Pandey, (1982) was followed. Bacterial mat and

fungal mat grown in conical flasks (150 ml) containing 50 ml of liquid media used for

liquid state fermentation and it was filtered through double layered muslin cloth and

the filtrate was centrifuged at 5000 rpm for 30 min. The supernatant was used as

crude enzyme preparation. When necessary the enzyme extracts were stored at 4o C

till use (Shewale and Sadana, 1978).

Storage of enzyme:-

The crude enzyme was stored at 4o C in refrigerator till use.

Enzyme assay:-

The amount of xylanase was estimated by following method of Bailey et al.

(1992). Oat spelt xylan used as the substrate for xylanase and the amount of xylose

released was measured by DNS method of Miller, (1959).

Preparation of oat spelt xylan substrate for xylanase assay (Bailey et al., 1992).

One gram of oat spelt xylan was homogenized in 50 ml. of 0.05 M phosphate

buffer pH 7.0. It was heated to boiling point and cooled with continued stirring. The

28

volume is made up to 100 ml with the same buffer and stored at 4o C, for a maximum

period of one week.

Assay mixture:-

The assay mixture contained

Oat spelt xylan (1%) - 1.8 ml.

Crude enzyme - 0.2 ml.

Phosphate buffer - 0.2 ml.

(0.05 M pH 7.0)

Reagent blank and substrate blanks were maintained:-

The reagent blank contained distilled water instead of enzyme and the

substrate blank contained distilled water instead of substrate.

The test tubes containing assay mixtures and the 2 blanks were incubated at

50o C in a water bath for 10 min. The reaction was stopped by adding 0.5 ml. of 10%

Trichloroacetic acid (TCA). The contents were centrifuged at 3000 rpm for 10 min.

To the supernatant was added 3 ml of DNS reagent and the tubes were cooled to room

temperature and the absorbance was measured at 530 nm in a calorimeter.

Reagent blank was used to set zero in the calorimeter. The difference in OD

between substrate blank and the enzyme mixture was noted and the amount of xylose

released was calculated using a xylose standard graph.

Unit of enzyme activity:-

Reducing sugar released was calculating using a xylose standard and the

activity is expressed in international units.

One international unit of xylanase is the amount of enzyme required to liberate

reducing equivalent to 1 µ mol of D-xylose per min per ml. (Bailey et al., 1992).

29

Calculation:-

The amount of reducing sugars presents at 0 time (of enzyme assay) in the

enzyme extract was measured and this was deducted while calculating enzyme

activity.

Enzyme activity is expressed in International units and expressed as U/ml of

culture filtrate. One international unit of enzyme activity is 1µ mol of reducing sugars

released per minute. For each experiment three replicates were maintained and the

average value of enzyme produced was calculated.

Compost treatments:-

Effective micro organisms:-

EM stock solution (Maple EM.1) was brought from Sri Raam biotech,

Coimbatore, TN.

Preparation of activated EM solution:-

1 litre of EM stock solution and 1 Kg. of organically grown jaggery are to be

mixed with 20 litres of water. The water has to be clean and free form chlorine. The

container should be of good grade plastic. It should be clean, not contaminated with

chemicals and have an airtight lid. As gas pressure will develop, the lids have to be

opened every day for a second release it. During the period of activation, a white layer

of actinomyeetes was formed on the top of the solution companied by pleasant smell.

The pH of the EM should be below 4.0. Activated EM can be diluted up to 1:200 to 1:

1000.

Compost preparation:-

Biodegradable wastes can be composted with the help of EM and crude

enzyme of xylanase. The raw material used for the compost includes vegetable

wastes, coir pith, leaf litter, cow dung and water. For compost preparation 4

treatments were done. For each treatment 2 feet pit was prepared.

30

Treatments:-

T1 - Control

T2 - EM treated organic waste

T3 - Crude xylanase enzyme treated organic waste

T4 - Mixing of EM and crude xylanase treated organic waste.

For each treatments, in a pit biodegradable wastes are spreaded over that cow

dung slurry was sprinkled. In this way alternative layer of agro wastes and cow dung

slurry was laid up to 2 feet. For EM treatment (T2) the activated EM solution should

be sprayed the fresh waste. For 6 kg. of wastes 2 liter of activated EM solution will be

sprayed.

In the same way for T3 experiment 1 liter of xylanase enzyme solution were

sprinkled. For T4 experiment mixing of 500 ml of EM and 500 ml of crude xylanase

were sprinkled. Then the pits was covered with polythene sheet to maintain moisture

content. The wastes were turned twice in a week for aeration. Watering also done for

moisture content.

The composts will be ready after 60 days and it can be used after sieving.

Estimation of Micro, Macro nutrients:-

The composted organic waste obtaned from different treatments were analysed

for the following important parameters.

Total nitrogen:-(Vogel, 1961)

A quantity of 10 g. of the sample was transferred into a clean dry kjeldahl

flask. 30 ml. of conc. Sulphuric acid containing 1 g of salicyclic acid was added to it.

The contents were mixed well and were allowed to stand for 15 min. 5g of sodium

thio sulphate was added and mixed well and allowed to stand for 30 min. Then 10 g of

potassium sulphate – copper sulphate mixture was added and the contents were

digested over a bunser burner until the contents became apple green or colourless.

31

The kjeldahl flask was cooled and 50 ml of distilled water was added. The

contents were mixed well and the supernatant liquid alone was transferred into the

distillation flask. The process was repeated for atleast 6 times to ensure the complete

transfer of the digested material to the distillation flask. 25 ml of 0.1 N sulfuric acid

was taken in a beaker and 2-3 drops of methyl red indicator was added to it. The

beaker was kept at the delivery end, which was well immersed inside the acid. 120 ml

of 40% sodium hydroxide was added till the contents became distinctly alkaline and

then, 10 ml of 10% sodium sulphate was added.

The mouth of the flask was immediately closed and the distillation was

started. The ammonia evolved was collected in 0.1N sulphuric acid. The completion

of the distillation was noted by collecting a drop of the distillate on a moist red litmus

paper, when the red litmus retained the red colour. The distillate with excess acid was

then titrate with 0.1 N potassium hydroxide. The end point was the charge of colours

from pinkish red to straw yellow.

The percentage of nitrogen present in the sample was calculated using the

formula.

0.0014* (a-b) * (100/10)* [100/(100-M)]

a = Volume of 0.1 N sulphuric acid taken

b = Volume of 0.1 N potassium hydroxide

M = Moisture percentage

0.0014 is constant.

Total phosphorus:-

A quantity of 200 ml of hydrochloric acid was pipette out into a 400 ml.

beaker and evaporated into a small bulk. It was transferred into a silica basin using hot

water and evaporated to dryness over a water bath. Then the basin was kept in a hot

air oven at 105o C for 3 hrs. to dehydrate the silica and thus it was made insoluble.

The residue was dissolved again by adding a small quantity of 1:1 hydrochloric acid

and evaporated to dryness over a water bath, followed by dissolving again 1:1 nitric

acid. Then the insoluble silica was allowed to settle overnight. The residue was

filtered with small quantities of 1:4 nitric acid until no yellow colour was left, either

32

in the basin or in the filter paper. The extract was made alkaline with concentrated

ammonium hydroxide and distinctly acidic with conc. nitric acid. 5g solid ammonium

nitrate was added and kept on a thermostat at 65o for 15 min.

A quantity of 10 ml. of the precipitant mixture was added to the beaker in the

thermostate, drop by drop. The beaker was kept in the thermostate for another 30 min,

to allow complete precipitation. Then it was filtered by decantation, pouring only the

supernatant liquid onto the filter paper, retaining the precipitate in the beaker itself.

The precipitate was washed with cold distilled water until the filtrate become acid

free. The filter paper with the precipitate was transferred to the beaker in which

precipitation was already done, and then it was made up into a pulp. 0.1619N

potassium hydroxide was added to it until the yellow precipitate completely dissolved,

to make a colourless solution. A quantity of another 5 ml. of 0.1619N potassium

hydroxide was added to keep the alkali I fair excess quantity. The volume of the alkali

added was noted. A drop of phenolphthalein indicator was added and titrated against

0.1619N nitric acid. The end point was disappearance of pink colour.

The percentage of phosphorus on the sample was calculated using the formula

0.0005 * (a-b)* (500/200)* (100/W)* [100(100-M)]

M= Moisture content of the soil.

W= Weight of the sample.

1ml. of 0.1619M potassium hydroxide = 0.0005 g phosphorus.

Total potassium:-

The standard solution was prepared by dissolving 1.907 g of potassium

chloride in 11 ml. distilled water. It gave 1000 ppm of potassium, 100 ml. of 1000

ppm diluted to 11 gave 100 ppm potassium solution. From that various standards

prepared ranging from 10-100 ppm.

The atomizer was fixed in its place and distilled water was introduced. The

compressor was treated and the pressure was adjusted 19o 1016S, the burner was

lighted with distilled water set zero by using the zero adjustment knob, 100 ppm

potassium solution was introduced the knob was adjusted to read 100 on the scale.

Again, distilled water was introduced and adjusted to read zero. The process was

33

repeated until the meter reading showed 0 with distilled water and 100 with 100 ppm

potassium solution, without any zero adjustments. Then various standard solutions

were introduced and the readings were recorded. They were plotted to draw a standard

curve. The filtrate obtained from sesquioxide estimation was taken in small vial and

introduced through the atomizer. The reading was recorded and the percentage of

potassium was calculated using the standard curve.

The percentage of potassium was calculated using the formula

(A.106)* (500/50)* (100/W)* [100(100-M)]

M= Moisture content of the soil.

W= Weight of the sample.

Estimation of organic carbon content:-

Weight 0.5 gm of the sample. Add 10 ml. of potassium dichromate and 20ml.

of conc. Sulphuric acid. This solution is kept at room temperature for 30 min. Then

200 ml. of distilled water is added. This solution is titrated against ferrous ammonium

sulphate using ferroin indicator. Blank sample also titrated against ferrous ammonium

sulphate using ferroin indicator indicator. The end point is brown colour.

The percentage of organic carbon present in the sample were calculated using

the formula.

{[(B-T)* 1/B]* 0.003* 100} /0.1

B = Blank titrate value

T = Sample titrate value

1ml. of ferrous ammonium sulphate = 0.003 g. of organic carbon.

C:N ratio:

The C:N ratio was calculated by dividing organic carbon by total nitrogen of

the organic waste sample.

34

Calclium:-

To 5 ml. of digested sample, 4 ml. hydroxylamine hydrochloric (5%), 3 ml. of

KOH (25%) and a pinch of patrons readers indicators was added and made up to 30

ml. with distilled water and titrated against 0.01N EDTA solution. The titrant value

was noted. End point was the appearance of Blue colour. The calcium content was

calculated and expressed in percentage.

Magnesium:-

5 ml. of digested sample was taken and 10 ml. calcium Magnesium buffer, 15

ml. distilled water and Erichrome black indicator was added and titrated with 0.01N

EDTA. The titrant value was noted. End point was the appearance of blue colour. The

magnesium content was calculated and expressed in percentage.

Sodium:-

5 ml. of digested sample was added to 25 ppm sodium chloride solution and

50 ppm distilled water. The reading was calculated in flame photometer.

Calculation

Sodium content =

Where, Factor was measured from the reading of sodium chloride solution

(25/50 ppm) from the flamephotometer.

pH: (Jackson, 1973)

A quantity of 10g of the sample was taken in a conical flask and added with

100 ml of distilled water, kept in a rotary shaker for 30 minutes. After 30 min it was

filtered through muslin cloth. The filterate was used for pH determination.

EC: (Jackson, M.L. 1973)

35

The EC of the sample was estimated by conductivity bridge using soil water

ratio 1:10 based on the method suggested by Jackson(1973)

Moisture content:

A known quantity of sample was taken in a pre-weighed moisture bottle and

kept in an oven at 110°C for 12h. After drying, the moisture bottle along with the

sample was weighed and the difference in weight due to moisture loss was calculated.

Field Experiment:-

Testing plant:-

The plant chosen for this study was Green gram (Vigna radiata, (L) wilczek. It

is one of the most whole some among pulses in India. It is free from the heaviness and

tendency to flatulence, which is associated with other pulses. It is an erect or sub erect

herb.

Field preparation:-

The experimental field was ploughed well and narrowed. Then the field was

divided into four equal plots, for 4 different treatments. Ridges and furrows were

constructed in the field. Totally 100 seeds were taken at the rate of 25 seeds for each

treatments. The seeds were sown on the ridges of the plots.

1st plot was kept as T1 (control)

2nd plot was kept as T2 (EM)

3rd plot was kept as T3 (crude xylanase)

4th plot was kept as T4 (EM and crude xylanase)

Replicates was constructed for all the treatments.

Seed Viabilty

The viability of the seed accession is a measure of how seeds are alive and

could develop into plants which were reproduce themselves, given the appropriate

condition. Viability of the seed is tested by seed germination method.

36

Between paper method

A random sample of seeds is selected from the accession about 100 seeds were

selected and that is divided into 4 replicates then an absorbent paper is taken and it is

cut into equal on the outside of the paper, the name of the seed replicate of the test

and date of test started is noted. Then the paper is moistened seeds are arranged at

regular intervals on the paper. At the edge 2cm space is leaved, to fold the paper

without damage. Another absorbent paper is taken, and it is moistened. Wet absorbent

paper is kept over another absorbent paper, which contain seeds. Paper is rolled

loosely toward the end and it is kept on a ventilated plastic box. Oxygen is essential

for respiration during germination, so the container used should be adequately

ventilated and the moisture content of the paper is also maintained. Days later unroll

the paper carefully. Avoid tearing of the paper or damaging the roots and record the

number of seeds germinated in each replicate. If it is not germinated again roll the

paper and kept in a ventilated container and it was cheeked next day.

Number of seeds taken: 100

Replicate 1:25

Replicate 2:25

Replicate 3:25

Replicate 4:25

Number of seeds germinated

Replicate 1:23

Replicate 2:24

Replicate 2:24

Replicate 2:24

The mean percentage viability of the accession is considered next day it is

above 90%, so the seeds are viable .

37

Growth Attributes:-

Germination percentage of the plants grown in different treatments:-

Germination percentage of all the seeds sown were observed using the

formula.

Shoot Length of Green Gram Under Different Treatments:-

The shoot length of green gram plant was measured from ground level to tip of

the topmost leaf on 7th, 14th, 21st, 28th, 35th, 42nd, 49th, 56th, days after germination

respectively. 5 plants were taken at random at each stage and the mean value was

expressed in cm.

Root Length of Green Gram Grown Under Different Treatments:-

The root length of green gram grown under different treatments were

measured on 56th day. 5 plants were taken at random and the mean value was

expressed in cm.

Number of Leaves in Different Treatments:-

The No. of leaves of green gram under different treatments were measured on

56th day. 5 plants were taken at random and the mean value was expressed in cm.

Length of Leaves:-

The length of green gram under different treatments were measured on 56 th

day. 5 plants were taken at random and the mean value was expressed in cm.

38

Biochemical Analysis:-

Estimation of carbohydrate content (Hedge and Hofreiter, 1962)

Preparation of anthrone reagent:-

A quantity of 1 gm. of anthrone was dissolved in 100 ml. of Conc. sulphuric

acid.

Procedure:-

A quantity of 1 gm. of seed material was taken and it was ground in pestle and

mortar using 80% ethanol. The extract was centrifuged and the supernatant was taken.

To 1 ml. of the extract 6 ml. of anthrone and 3 ml. of distilled water added and shaken

well.

The O.D values were calculated at 620 nm in spectrometer for each sample

(T1, T2, T3, T4). Hence the carbohydrate content of the seeds was calculated.

Estimation of protein. (Lowry et al., 1951).

Preparation of phosphate buffer:-

A quantity of 1.2 gm. of sodium hydrogen phosphate and 0.9 gm. of potassium

dihydrogen phosphate were dissolved in 100 ml. of distilled water and the pH was

corrected to 6.5.

Preparation of Reagent A:-

A quantity of 0.4 gm of NaOH was dissolved in 100 ml of distilled water. To

that 2 gm sodium carbonate was added .

Preparation of Reagent B:-

A quantity of 1 gm. of sodium potassium tartarate was dissolved in 100 ml of

water. To that 0.5 gm of copper sulphate was added.

39

Preparation of alkaline copper sulphate reagent.

A quantity of 50 ml Reagent A mixed with 1 ml. of Reagent B.

Preparation of Folin phenol reagent:-

Commercially available folin phenol reagent was diluted with water in the

ratio of 1:1.

Procedure:-

A quantity of 1 gm. of plant material was homogenized in 20 ml. of buffer and

centrifuged. The volume of the supernatant was made up to 20 ml using phosphate

buffer. 1ml. of supernatant was pipette out and 1 ml. of 10% tricarboxylic acid was

added. The precipitate obtained was dissolved in 1 ml. of 1N NaoH. After 5 min, 5

ml. of alkaline copper sulphate reagent was added. After 10 min, 0.5 ml. of folin

phenol reagent was added. The tubes were placed in water both for 10 min. and then

cooled. The O.D values were calculated at 620 nm in spectrophotometer for each

sample and then from the standard graph, the value of proteins present in the sample

taken was estimated for each treatment. Hence, the protein content of the taproot was

calculated.

STATISTICAL ANALYSIS:

The results were expressed as means of standard deviation and the data were

analysed using ANOVA. The groups were compare by DUNCAN multiple range test

by the method suggested by Bennet and Franklin,(1967).

40

EXPERIMENTAL RESULTS

XYLANASE PRODUCTION

EXPERIMENT I

Production of xylanase from various agro wastes by Bacillus sp and

Pseudomonas fluorescens in liquid state fermentation.

Various agricultural residues have been screened for xylanase production.

Rice bran enhanced maximum xylanase production in Thermoascus aurantiacus

(Santos et al., 2003) Bacillus pumilus (Battan et al., 2007), orange bagasse in

Streptomyces cyaneus (Ninawe et al., 2008) and corn cob in Thermotogo maritime

(Tan et al., 2008).

Hence an experiment was carried out to find out the effect of various agro

wasters namely, Rice bran, Wheat bran, Corn cob, Sugarcane bagasse, Corn stover,

Tea waste, Saw dust, Groundnut shell, Pine apple fibre, Waste cotton and Coir pith on

xylanase production by Bacillus sp and Pseudomnonas fluorescens in liquid state

fermentation.

Xylans from all these agro residues were extracted (as mentioned in materials

and methods) and used as substrates as the only carbon source for liquid state

fermentation.

A single loop of bacterial inoculum was grown in conical flasks (150 ml)

containing 50 ml of liquid media (Horikoshi II basal medium) used for liquid state

fermentation.

Enzyme was extracted and assayed as mentioned in materials and methods.

The results are presented in Table 1 and Fig. 1, Plate 1.

From Table 1 and Figure 1, Plate I, it can be seen that in Bacillus sp and

Pseudomnonas fluorescens of the various agro residues tried, rice bran was found to

support maximum xylanase production in liquid state fermentation in these two

species.

41

Table: 1

Production of xylanase from various agro wastes by Bacillus sp and

Pseudomonas fluorescens in liquid state fermentation.

S.No Substrate Bacillus spPseudomonas

fluorescens

1 Wheat Bran 5.27 h 7.09 f

2 Sugarcane baggase 0.67 f 0.08 b

3 Corn cob 0.06 b 0.05 ab

4 Corn stover 0.06 b 0.04 a

5 Coir pith 0.03 a 0.04 a

6 Groundnut shell 0.26 e 0.17 d

7 Pineapple fibre 2.58 o 5.18 e

8 Tea waste 0.13 c 0.04 a

9 Rice bran 8.25 i 9.18 o

10 Cotton waste 0.16 d 0.13 c

11 Saw dust 0.02 a 0.02 a

Observation values are mean of three replicates.

Means followed by a common letter are not significantly different at the

5% level by DMRT

42

EXPERIMENT II

Production of xylanase from various agro wastes by Trichoderma viride and

Rhizopus nigricans in liquid state fermentation.

Various agricultural residues have been screened for xylanase production.

Wheat bran enhanced maximum xylanase production in Aspergillus japonicus

(Simoes and Tank – Tornisielo, 2006), sugarcane bagasse in Trichoderma harzianum

(Rezende et al., 2002), wheat straw in Cochliobolus sativus (Yasser Bakri, et al.,

2008).

Hence an experiment was carried out to find out the effect of various agro

wastes namely, Rice bran, Wheat bran, Sugarcane bagasse, Corn cob, Corn stover,

Coirpirth, Tea waste, Saw dust, Groundnut shell, Pineapple and Waste cotton.

Xylans from all these agro residues were extracted (as mentioned in materials

and methods) and used as the only carbon source for liquid state fermentation.

Enzyme was extracted and assayed as mentioned in materials and methods.

The results are presented in Table 2 and Fig 2, Plate 2.

From Table 2 and Fig 2, plate II it can be seen that in Trichoderma viride and

Rhizopous nigricans of the various agro residue tried, wheat bran was found to

support maximum xylanase production in liquid state fermentation in these two

species.

43

Table 2:

Production of xylanase from various agro wastes by Trichoderma viride and

Rhizopous nigricans in liquid state fermentation.

S.No SubstrateTrichoderma

viride

Rhizopus

nigricans

1 Wheat Bran 8.51 f 9.23 f

2 Sugarcane baggase 0.17 cd 0.82 d

3 Corn cob 0.14 ab 0.21 b

4 Corn stover 0.61 f 0.52 c

5 Coir pith 0.15 bc 0.12 a

6 Groundnut shell 0.12 a 0.12 a

7 Pineapple fibre 0.14 ab 0.18 ab

8 Tea waste 0.18 de 0.17 ab

9 Rice bran 3.28 h 7.11 e

10 Cotton waste 1.05 o 0.11 a

11 Saw dust 0.20 e 0.13 ab

Observation values are mean of three replicates.

Means followed by a common letter are not significantly different at the

5% level by DMRT

44

EXPERIMENT III

Macronutrient status in different treated organic wastes

Pati and Chandra (1981) reported that nitrogen-fixing bacteria on leaf surface

could markedly increase crop yield. When EM was applied to soil or plant leaf, the

population of nitrogen fixing and photosynthetic bacteria increase dramatically (Reid.,

1979).

In general, there is immobilization of nitrogen due to decomposition of cereal

straw. However, with farmyard manure (FYM) or compost application no such effect

is observed. The addition of FYM and cereal residues result in improvement of total

soil nitrogen(Bhardwaj and Gaur,1985).

An experiment was carried out to find out the macro nutrients content of

control (untreated), EM treated organic wastes, xylanase treated organic wastes and

mixing of EM and crude xylanase treated organic wastes was analysed and percentage

of NPK was found out. After and before composting treatments are represented Plate

3.

Nitrogen was estimated by kjeldahl method, where the samples were subjected

to sulphuric acid, salicyclic acid mixture and potassium sulphate, copper sulphate

mixture, followed by titration. Phosphorous was estimated in nitric acid medium,

followed by titration. Potassium was using atomizer by sequioxide estimation method.

The results are tabulated in Table 3 and presented in Fig 3.

From Table 3 Fig 3 it can be seen that in maximum nitrogen content was

noticed in EM treated organic wastes. Maximum phosphorous content was seen in

mixing of EM and crude xylanase treated organic waste and maximum potassium in

xylanase treated organic wastes.

45

Table: 3

Macronutrient status in different treated organic wastes. (in Percentage)

S. No ParametersComposts

T1 T2 T3 T4

1 Nitrogen 5.85 7.02 6.82 6.20

2Phosphorou

s0.21 0.39 0.44 0.49

3 Potassium 0.392 0.450 0.470 0.452

46

EXPERIMENT IV

C:N ratio of different treated organic wastes

Although the values of macronutrients are important for assessing the quality

of compost, C:N ratio plays an essential part. According to Padmaja and Adlene

Sangeeth. 2008, C:N ratio of EM compost was narrowed down drastically to 16:1

from 43:7.

Hence, C:N ratio was analysed in the treated organic wastes. The carbon was

estimated with Erlenmayer flask when the samples were subjected to the reaction with

potassium dichromate and sulphuric acid, followed by titration. Estimation of 'N'

content was already estimated.

C:N ratio was presented in Table 4 and Fig 4 and from this results crude

xylanase treated organic wastes showed the minimum C:N ratio, Which was a good

indicator of the compost.

47

Table: 4

C/N Ratio of status in different treated organic wastes

S. No Parameters

Composts

T1 T2 T3 T4

1. C/N Ratio 2.87 1.54 1.46 1.63

48

EXPERIMENT V

Micronutrients of different treated organic wastes:

According to Stoeppler-Zimmer and Petersen(1997), the organic compost

contains high levels of phosphorous, magnesium and calcium.

Hence micronutrients content of control, EM treated organic waste, crude

xylanase treated organic waste and mixing of EM and crude xylanase treated organic

waste was analysed and percentage of sodium, calcium, organic carbon, moisture, pH

and EC was found out.

Calcium was estimated by adding of hydroxylamine hydrochloric acid to

sample and KOH, patrons readers indicators and titrated against EDTA solution.

Sodium was estimated by adding sodium chloride solution and reading was calculated

in flamephotometer. pH, mositure and EC also carried out in treated organic waste.

The results are tabulated in Table 5 and represented in Fig 5. From this results

EM treated organic waste (T2) showed maximum sodium content and calcium

content. Organic carbon was found to be maximum in T1 control , and minimum in

xylanase treated organic waste (T3). Alkaline pH showed in T1 control and (T2) EM

treated organic waste , where as acidic pH showed in crude xylanase treated organic

waste and mixing of EM and crude xylanase treated organic waste. Moisture content

was maximum in xylanase treated organic waste. EC was maximum in control

followed by xylanase treated organic waste.

49

Table: 5

Micronutrient status in different treated organic wastes

S. No Parameters Composts

T1 T2 T3 T4

Sodium 0.32 0.70 0.50 0.40

Organic Carbon 16.82 10.86 10.0 10.14

Calcium 1.82 2.40 2.20 2.0

Moisture 13.56 12.26 14.51 12.50

pH 8.6 8.2 6.5 6.8

EC 116.19 104.12 114.0 107.11

50

Growth attributes of the green gram

EXPERIMENT VI

Germination of ratio of green gram:

Determination of germination ratio of green gram in control, EM

compost, xylanase compost, mixing of EM and xylanase compost treated plants.

Sangakkara and Higa, (1994) states that EM can stimulate seed germination

and early growth of food crops.

The germination rate of green gram seeds was calculated. The results are

tabulated in Table 6 and Fig 6, and maximum germination ratio was seen in mixing of

EM and xylanase compost treated plants (T4).

51

Table: 6:

Germination rate of green gram under different compost treatments

S. No Treatment Germination rate(%)

1. T1 92 a

2. T2 94 b

3. T3 94 b

4. T4 96 c

Observation values are mean of five replicates.

Means followed by a common letter are not significantly different at the

5% level by DMRT.

52

EXPERIMENT VII

Effect of EM treated organic waste, xylanase treated organic waste and mixing

of EM and xylanase treated organic waste on shoot length of green gram.

The shoot length of the plants determine the healthy nature of the plants. This

attributes to the increased nutrient uptake of the plants. Calderia (2000) reported that

seedling growth increased with increasing properties of the vermicompost.

Hence, the shoot length of the crops was taken at random for all the

treatments, at an interval of 7 days from the date of germination. The results were

taken and the mean value were found out .

The final values are tabulated in the Table 7 and represented in Fig 7, plate 4.

From the Table 7, it can be seen that the maximum value of the height of the

plant was observed in T4 (71.5cm) and least height was recorded in T1 (41.5cm)

53

Table 7:

Shoot length of green gram under different treatment.

S. No. Days Shoot length(cm)

T1 T2 T3 T4

1. 7th 6.1 h 8.1 h 8.3 h 8.8 h

2. 14th 8.1 g 15.2 g 15.3 g 16.1 g

3. 21st 11.1 f 23.2 f 24.6 f 25.9 f

4. 28th 15.3 e 32.7 e 34.7 e 36.7 e

5. 35th 25.3 d 41.3 d 41.8 d 42.2 d

6. 42nd 33.1 c 50.4 c 50.9 c 53.0 c

7. 49th 40.4 b 60.4 b 61.3 b 61.8 b

8. 56th 41.2 a 69.3 a 70.2 a 71.5 a

Observation values are mean of five replicates.

Means followed by a common letter are not significantly different at the

5% level by DMRT.

54

EXPERIMENT VIII

Effect of EM treated organic waste , xylanase treated organic waste, EM+

xylanase treated organic waste on root length of green gram.

Bhowmik, et al., 2004 reported that the root volume of plants increased by

adding biofertilizers.

The root length of the crops was taken at random for all treatments at the end

of the 56th day. The results were noted and the mean value was found out.

The final values were tabulated in Table 8 and represented in Fig 8, Plate 5

From the Table 8 it can be noticed that the maximum root length was observed in T3

(15.7cm) the least value in T1 (7.9cm).

55

Table 8:

Root length of green gram under different treatments (cm)

S. No. Treatment Root Length (cm)

1. T1 7.57 a

2. T2 15.20 c

3. T3 15.68 d

4. T4 12.28 b

Observation values are mean of five replicates.

Means followed by a common letter are not significantly different at the

5% level by DMRT.

56

EXPERIMENT IX

Number of leaves of green gram / plant.

Abdel-Mouty et al., (2001) reported the number of leaves/branches was

increased by using organic fertilizer application. The increasing of leaf yield was

studied by Kipkosgei, et al., (2003) in Solanum villosum by using organic fertilizer.

Number of leaves of the crops was observed for all treatments. EM+xylanase

compost treated plants (T4) showed the maximum number of leaves / plant.

The values were tabulated in Table 9 and represented in Fig 9.

57

Table 9:

Number of leaves of green gram / plant

S. No. Treatment No.of leaves/Plant

1 T1 36 a

2 T2 50 b

3 T3 60 c

4 T4 75 d

Observation values are mean of five replicates.

Means followed by a common letter are not significantly different at the

5% level by DMRT.

58

EXPERIMENT X

Length of green gram leaves under different treatments:

Hameeda et al.,(2007) found that rice straw compost applied at 2.5 t ha-1

showed significant improvement in shoot length, leaf area, plant biomass, root volume

and mycorrhizal colonization of sorghum plant.

The length of the green gram leaves was taken at random for all treatments.

The results were noted and the mean value was found out.

The final values were tabulated in Table 10 and represented in Fig 10. From

the Table 10 the maximum value for the length of leaves was observed in T4 (15.8cm)

and the least value in T1 (9.9cm)

59

Table 10:

Length of green gram leaves under different treatments

S. No Treatments Length of leaves (cm)

1. T1 9.8 a

2. T2 15.2 b

3. T3 15.3 b

4. T4 15.7 c

Observation values are mean of five replicates.

Means followed by a common letter are not significantly different at the

5% level by DMRT.

60

Biochemical analysis:-

The biochemical analysis was done to determine the quality, following the

attributes of the crop.

EXPERIMENT XI

Estimation of protein:-

According to Wange et al., (1997), the protein content of pea plant increased

with organic and bio fertilizers application.

The sample was extracted in phosphate buffer and to it alkaline copper

sulphate and folin phenol reagent were added the protein content of the sample were

determined using spectrophotometer.

The observation was presented in Table 11 and represented in Fig 11. From

the table 11, it was observed that protein content of the sample was found to be

maximum in T4 for green gram.

61

Table 11

Estimation of Protein content of green gram under different treatments. (mg/g).

S. No Treatments Protein Content (mg/g)

1. T1 18.16

2. T2 20.75

3. T3 20.78

4. T4 20.82

62

EXPERIMENT XII

Estimation of Carbohydrates:-

Uher et al., (2006) reported that, the improvement of nutritive values in pea

plants by adding organic manures.

Carbohydrate contents of the seed were estimated by anthrone method. The

samples were extracted in 80% ethanol and anthrone reagent was added to the extract

followed by water. The value of carbohydrate content of the samples was determined

with spectrophotometer.

The observation results are tabulated in Table 12 and represented in Fig 12.

From the Table 12, it was observed that carbohydrate content of the sample was found

to be maximum in T4 for green gram.

63

Table 12

Estimation of Carbohydrate content of green gram under different treatments.

S. No Treatments Carbohydrate Content (mg / g)

1. T1 18.70

2. T2 20.25

3. T3 20.23

4. T4 20.80

64

Discussion

This study aims to investigate the effect of different treated organic waste

composts on the growth and biochemical parameters of seedlings of Vigna radiate,

(L) wilczek. The results of this study are discussed here.

Xylanase production

xylanases are produced by diverse genera and species of bacteria and fungi

(Subramanian and Prema, 2000). Recently the interest in thermostable enzyme has

increased and thermo stability has become a desirable property of enzyme used in

many application.

Bacterial xylanase production

Bacteria showing high xylanase activity were isolated from 300 soil samples

(Panbangred et al., 1983). The bacteria used in there studies ware Bacillus sp and

Pseudomonas flourescens.

Various agricultural wastes like sugarcane bagasse, wheat bran, rice bran,

maize bran, gram bran, wheat straw, rice straw, rice husk, soy hull, corn cobs, banana

wastes, tea wastes, cassava waste, apple pomace used for xylanase production

(Balasubramanium et al., 2001)

There is an urgent need to manage the bulk wastes effectively and

economically. At the same time, it is also necessary to generate value added products

from these wastes. The use of purified xylan as substrate for uneconomical for large

scale production of xylanases. Therefore, several natural agro residues were tested as

substrates (wheat bran, rice bran, pine apple fiber, corn stover, sawdust, sugarcane

bagasse, groundnut shell, coir pith, tea waste, corn cob and waste cotton) for

xylanases production using Horikoshi basal medium. Among the 11 substrates tested,

rice bran was found to be the most suitable substrate xylanase production. It enhanced

the enzyme production up to 8.25 (U/ml) in Bacillus sps and 9.18 (U/ml)in

Pseudomonas fluorescens.

65

Rice bran neutral xylan contains 46% xylose, 44.9% arabinose, 6.1%

galactose, 1.9% glucose and 1.1% hydrouronic acid (Shibuya et al., 1985)

The highest production of xylanase on rice bran may be possible due to very

low lignin and silica content as compared to other substrates rice bran is a complete

nutritious feed for micro organisms having all in gradients and also remains loose,

even under moist conditions providing a large surface area (Babu and Satyanarayana,

1996). Lequart et al. (1999) reported that the enhanced production of xylanase may be

due to the presence of considerable amount of soluble sugars like glucose (42.5%

dry), xylose (15.4% dry), arabinose (3.1% dry) and galactose (2.7% dry). Rice bran

supported maximum xylanase production in a number of organisms namely in

Bacillus pumilus (Poorna and Prema, 2007) Paenibacillus sp (Harada et al., 2008) and

in Arthobacter sp (Khandepatker and Bhosle., 2008)

Fungal xylanase production:

The fungi used in these studies were Trichoderma viridae and Rhizopus

nigricans. Among the 11 substrate tested, wheat bran was found to be the most

suitable substrate for xylanase production. It enhanced the enzyme production up to

8.15 (U/ml) in Trichoderma viridae and 9.23 (U/ml) in Rhizopus nigricans.

Wheat bran consists of 30% cellulose, 27% hemicelluloses, 21% lignin and

8% ash (Gawande and Kamat, 1999)

Wheat bran supported maximum xylanase production in a number of

organisms in Schizophyllm radiatum (Cavazzoni et al., 1989), Aureobasidium

pullulans (Karani et al.,1998), and in Aspergillus tamari (Ferreira et al., 1999;

Panagiotu et al., 2003,).

Alam et al. (1994) reported that Thermoascus lanuginosus and T.aurantiacus

produced xylanase activity when grown on various lignocellulosic substrates,

maximum xylanase production was obtained with wheat bran.

66

Composting:

The modern concept of environment is based on the recycling of waste in this

context, composting appear to be a safe form of treatment of some waste and the

reclamation of the nutrients contained in them (Iranzo et al., 2004). During the last

few years, composting has gained wide acceptance as a key component of integrated

solid waste management. It has been promoted as an eco-friendly and sustainable

solution to urban waste management. It encourages the production of beneficial micro

organisms which in turn breaks down organic matter to create humus.

In this study, the organic wastes were decomposed by different treatments like

EM, xylanase and mixing of EM and xylanase. The organic materials are broken

down by microbes and enzymes, and finally composts or manure produced. In

vermicomposting, the complex organic molecules are digested through a mutualist

earthworm micro flora-digestion system. Amylase, cellulose, xylanase,

endoglucanase, cellobiase, acid phosphatase, alkaline phosphatase and nitrate

reductase produced jointly by earthworms and gut microflora are supposed to play a

central role in the process of digestion and gentrification of soil organic matter.

Enzyme activity in earthwoms is regionally specialized and influenced by

physiological state, age and micro organisms (Lakshmi prabha et al., 2007) It shows

the enzymes are one of the factor to breakdown the organic materials.

Bacteria may also attack more complex materials, or may exploit substances

released from the less-degradable substances due to extra cellular enzyme activities of

other organisms (Epstein, 1997)

Role of enzymes in composting:

Enzymatic parameters also reflect the activity of the microbial community and

indicate the ability of composting to degrade a wide range of common organic

substrates (Mondini et al., 2004).

Enzymatic activities play an important role during the composting process, as

they are implicated in the biological and biochemical processes through which the

initial organic substrates are transformed into the end product (Tiquia, 2002). As a

67

consequence, specific enzymatic activities could provide a way of characterizing the

composting process with relation to the rate of transformation of organic residues and

the stability of the end product. Mondini et al., (2004) reported that change in the

location of enzymes throughout the composting process, i.e. from extra cellular to

complexed with humic-like substances, might be useful at the moment of evaluating

compost stability. Despite of the fact that measurement of enzymatic activities is easy,

quick and in expensive, it is difficult to establish general threshold values to apply

enzymatic activities as indicators of compost stability due to the widely different

organic substrates involved in the composting processes. Thus, for compost

characterization, it is necessary to follow the dynamics of enzymatic activities over

time.

EM (Effective Microorganisms) can be applied to turn over organic and food

waste and even our effluent into beneficial microbe and nutrient rich compost that has

the capacity to assert a powerful regenerative effect on soil (Higa, 1994), helping to

re-establish a balanced soil ecology, raise yields in organic agriculture, and to combat

oxidative stress in plants and humans.

Micro organisms decompose and ferment organic matter into humus,

containing nutrients and hormones that facilitate plant growth.

Composted organic material can used as a source of important nutrients for

sustainable crop productivity.The composted organic wastes cannot only act as

supplement to chemical fertilizers but may also improve the organic matter status and

physico-chemical properties of soil (Harmsen et al., 1994).

The micro, macro nutrient of the compost was analysed. The N, P, K contents

showed a wide difference in the 3 composts. The N, P, K contents are found to

increased in organic waste treatments than in untreated control.

The increase of total nitrogen in compost was caused by the decrease of

substrate carbon resulting from the loss of CO2 (because of the decomposition of the

organic matter which is chemically bound with nitrogen) (Soumare, et al., 2002,

Zorpas, et al., 2003).

68

Phosphorus is not volatilized during the composting process (Warman and

Termeer, 1996). Levels of phosphorus along with quality of nitrogen and potassium

are important to determine. Insam et al., 2002 reported that, Actinomycetes compete

with other organisms for nutrients and can inhibit microbial growth due to the

production of antibiotics, lytic enzymes or even by parasitism. They play an important

role in the degrading natural polymers process and colonize organic materials after

bacteria and fungi have consumed easily degrade fractions: their enzymes enable

them degrade tough debris such as: woody stems, bark or newspaper. The interaction

between various functional groups of micro organisms depends on nutrient resources

and biochemical mechanisms of organic and in organic matter transformation

changes.

Micro nutrients like sodium and calcium increased in EM treated organic

waste organic carbon was decreased in xylanase treated organic waste.The total

organic carbon decreased substantially in all treated composts the decrease might be

attributed to the mineralization of organic matter to CO2 and H2O by

microorganisms.(Sunita Gaind et al., 2006).

The C:N ratio being an important parameter to judge the maturity of compost.

In this experiments, the xylanase treated organic waste shows low C:N ratio.

According to Michel et al., (1996) C/N ratio is among one of the important factor

affecting compost quality. The initial content of carbon however gradually decreased

as the decomposition progressed. The decrease in total N content was due to

conversion of organic nitrogen to ammonia and the subsequent loss of ammonia. This

is depends up on the type of material being composted than in high C:N ratio.

The decrease of C:N ratio corresponded to stable from of the organic matter.

So this C/N ratio is regarded as a criterion of maturity of compost. (Hardy et al.,

1993). Chefetz, et al., 1998 reported that the decrease of the C/N ratio is explained by

the transformation of carbon into CO2 followed by lower decrease in the concentration

of organic acids.

pH level of the 3 treatment composts was studied. The increase of pH values

can be indicator of maturity. The compost alkalinity can against some pathogenic

69

fungi since a large number of fungi grow only under acid conditions (Saidi, et al.,

2006).

In our experiment, alkaline pH was noticed in untreated (control) and EM

treated organic waste, xylanase treated organic waste showed acidic pH. An increase

in the pH values was recorded during the active phase, suggesting the alkalinization of

the manure as a consequence of the release of ammonia from the degradation and

mineralization of organic compounds. The pH of compost plays an important role in

the availability of nutrients. High alkaline pH can reduce the availability of P,fe, mn

(Sunita gaind et al., 2006).

The EC also affects the quality of composts in a large way because it reflects

their salinity and suitability for crop growth. Among 4 treatments, the xylanase treated

organic waste showed the maximum EC.The addition of liquid animal wastes to the

maturation piles and the turning of the piles could have a been responsible for the

increase in EC. This increase the EC of compost obtained after 270 days of

maturation did not exceed the limit value of 3ms cm-1 indicating a material that could

be safely applied to soil. (Soumarie et al., 2002).

Growth parameters:

The coincident application of organic manures and biofertilizers is frequently

recommended for improving soil properties and obtaining clean agricultures products

(Gomaa, 1995).

Recently a great attention was paid towards the application of bio-organic

farming to avoid the heavy use of agrochemical that resulted in numerous

environmental troubles (Lampkin, 1999). This study demonstrated the effectiveness

of different bio-composts for promoting growth and nutrition in green gram under

field conditions.

In this study, among all treatments mixing of EM and xylanase (T4) compost

showed maximum seed germination and shoot length. This may be due to the

nutrition content of the compost. In our study xylanase treatment (T3) and mixing of

70

EM and xylanase treatment (T4) compost showed the maximum phosphorous and

potassium content.

Phosphorous was maximum in mixing of EM and xylanase treatment compost

(T4) and potassium was maximum in xylanase treatment compost (T3). According to

Espinosa et al., 1999 phosphorous (P) enhances seed germination and hastens

maturity.

Somida., (2002) states that, potassium is a essential plant nutrient. It plays an

important role in growth, yield and quality of crops. Potassium acts as catalyst for

many enzymatic processes, regulates water use in the plant.

There was a noticeable increase in plant height from 8.1cm to 69.3cm in EM

compost (T2), 8.3 cm to 70.2 cm in xylanase compost (T3) and 8.8cm to 71.5cm in

mixing of xylanase and EM compost (T4) over the absolute control from 6.1 cm to

41.2 cm. Savithri et al., 1999 attained an increased yield of radish with coir pith

compost plants grown in composted coir pith recorded significant increase in plant

growth.

EM fertilizer increased yields in maize, lettuce, onion, rice, papaya, herbage

grasses, vegetables and apples with application of EM fertilizer (Daly and Stewart

1999, Sangakkara and Higa 2000, Fujita 2000, Chagas et al., 2001, Bruggenwart

2001, Hader 2001, Prinsloo 2002).

A maximum increase in the root volume was recorded in (T3) xylanase

compost treated plants (15.68 cm), (T2) EM compost treated plants (15.2 cm) and the

minimum root volume was observed in the control T1 (7.57cm).The results were on

par with the results of Thenmozhi (2003) who registered a sharp increase of 0.05 cu

mm to 0.49 cu mm from 30 to 90 days after seedlings by treating chilies with EM-soil

application treatment.

The effect of compost or vermicompost on plant growth depends on the source

of material used for compost or vermicompost preparation role of micro organisms

and nutrient content (Jack and Thies, 2006).

71

Number of leaves / plant increased significantly in T4 which shows 75 number

compared to absolute control, T1 (36). Length of green gram leaves increased in T4

(15.7cm) over than control T1 (9.8cm).

There was maximum increase in plant height, root volume, number of leaves,

leaf length by application of combined EM and xylanase treated compost. This

increase might be due to the activities of micro organisms which decompose or

ferment the compost and enriched the nutrient portion of the compost, which helped

to build up growth parameters of the cotton crop (Higa & Wididana, 1991 a). The

effectiveness of those microorganisms was also studied to decompose wastes by

anaerobic decomposition which helps to maintain the C/N ratio in the waste to supply

the nutrients to the crop. In this present study the combination of EM and xylanase

was a good catalyst for decomposing the wastes in to compost.

Biochemical analysis:

Protein content:

A significant increase in protein content was observed in T4 (20.82 mg /g)

over than T1 (18.16mg/g). The result is in accordance with the result of Saxena et al.,

(2001) who observed that the application of N and P increases the protein content in

soyabean.

Carbohydrate content:

A maximum increase in total carbohydrate content was noticed in T4 which

showed 20.80 mg/g and the control (T1) showed 18.70 mg/g. Manonmani and Anand

(2002) observed an increase in biochemical constituents such as carbohydrate in leaf

tissue of lady's finger by the application of vermicompost.

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Summary:

The effect of compost derived organic wastes which was treated by EM, crude

xylanase, and combination of EM and crude xylanase on the growth of greengram has

been studied under field conditions.

The crude xylanase enzyme was produced by bacteria and fungi. The bacteria

used for this study was Bacillus sp and Pseudomonas fluroscencs. The fungi used for

this study was Trichoderma viridae and Rhizopus nigricans. Among 11 substrates rice

bran was showed maximum xylanase production in bacterial species, where as in

fungal species wheat bran showed maximum xylanase production.

The EM solution was brought from Sri Ram biotech, Coimbatore. It was

activated using jaggary under anaerobic conditions for 1 week.

The EM solution, crude xylanase solution was applied to the organic wastes

under different treatments like EM treatment, xylanase treatment, combination of EM

and xylanase treatment. The control was also maintained, (untreated). The treated

organic wastes are turn into compost after 60 days.

The treated organic wastes are evaluated to micro, macronutrients, pH, EC and

moisture. Nitrogen, calcium, and sodium content was found to be maximum in EM

treated organic wastes. Potassium, moisture content was maximum found in xylanase

treatment. Minimum C/N ratio content was found in xylanse treated organic waste

(T3) where as phosphorous content maximum in combination of EM and xylanase

treated organic wastes(T4).

The growth parameters like seed germination, shoot length, number of leaves,

and length of leaves were observed maximum in combination of EM and xylanase

compost treated plants (T4). Root length was maximum in xylanase treated compost

(T3).

The biochemical contents of protein and carbohydrate was maximum in

combination of EM and xylanase treated plants (T4).

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CONCLUSION

Composting process proved to be a useful device for the reduction of organic

domestic waste. The inoculation of EM and crude xylanase could be very useful to

improve the composting process. So composting is a best waste management tool to

reduce environmental pollution.

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