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BIODEGRADATION OF CELLULOSIC WASTE

SUBMITTED BY: - SHIV BHATIA

Abbreviations

AFEX BMCC BSA β C °C C6H8O7.H2O CBM CD CMC Cu DAP DNS DP EC

Ammonia Fiber Explosion Bacterial Microcrystalline Cellulose Bovine serum albumin Beta Carbon Degree Celsius Citric acid Carbohydrate-binding modules Catalytic domain Carboxymethyl cellulose Copper Diammonium phosphate 3,5-dinitroslicylic acid Degree of polymerization Enzyme Commission

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et al FAS g GHs h H HEC HPLC IMTECH K2Cr2O7 kb kg KMnO4 M MAW min mL MTCC Na – K tartarate NaF NaOH

et alii Ferrous ammonium sulphate Gram Glycoside hydrolases Hour Hydrogen Hydroxyethyl cellulose High performance liquid chromatography Institute of Microbial Technology Potassium dichromate Kilobase Kilogram Potassium per magnate Molarity Mixed Agriculural Waste Minute(s) Mililiter Microbial Type Culture Collection Sodium potassium tartarate Sodium flouride Sodium hydroxide

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nm nmole N O O .D. PAHBAH PDB ppm pH ppt rpm RS s SF sp. TLC TSS μg μL %

Nanometer Nanomole Normality Oxygen Optical density Para hydroxyl bezoylhydrazine Potato Dextrose Broth Part per million Potential of hydrogen Precipitation Rotation per minute Reducing sugar second Synergy factor Species Thin layer chromatography Total soluble sugars Microgram Microliter Percent

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List of Tables
TABLE TABLE TYPE PAGE NO.

Table 1 Table 2

Sources and types of Solid Wastes Genera of Microorganisms Capable of Utilizing Different Components of Organic Matter

16 21

Table 3 Table 4. Table 5 Table 6 51

Sources and quantities of organic wastes Experimental set up for degradation of mixed agricultural waste % Organic Carbon of Treated mixed agricultural waste Reducing Sugar in PPM from 0 to 90 days

40 42 49

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INDEX

Sr. No.
1. Introduction 2. Review of literature 3. Materials and Methods 4. Results and Discussion

Page nos. 8 12 41 47 53

5. References

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INTRODUCTION
Waste Waste, rubbish, trash, garbage or junk is unwanted or undesired materials. “Waste” is the general term; though the other terms are used loosely as synonyms, they have more specific meanings: rubbish or trash are mixed household waste and including paper and packaging; food waste or garbage is kitchen and table waste and junk or scrap is metallic or industrial material. There are other categories of waste as well: sewage, ash, manure and plant materials from garden operations, including grass cuttings, fallen leaves and pruned branches Some components of waste can be recycled once recovered from the waste stream, e.g. plastic bottles, metals, glass or paper. The biodegradable component of the wastes (paper & food waste) can be composted or anaerobically digested to produce soil improvers and renewable fuels. If it is not dealt within a suitable manner, biodegradable waste can thus contribute to greenhouse gas emissions and by implication climate change. There are some definitions of wastes:Merriam-Webster defines waste as "refuse from places of human or animal habitation." The World Book Dictionary defines waste as "useless or worthless material; stuff to be thrown away." Unfortunately, both definitions reflect a widespread attitude that does not recognize waste as a resource. Zero Waste America defines waste as "a resource that is not safely recycled back into the environment or the marketplace." This definition takes into account the value of waste as a resource, as well as the threat unsafe recycling can present to the environment and public health. United Nations Statistics Division (UNSD) defines waste as " Wastes are the materials that are not prime products for which the generators has no further use in terms of his/her own purposes
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of production, transformation or consumption and of which he/she wants to dispose. Wastes may be generated during the extraction of raw materials, the processing of raw materials into intermediate and final products, the consumption of the final products and other human activities. Residues recycled or reused at the place of generation are excluding” United Kingdom defines waste:-The UK’s Environmental Protection Act, 1990, indicated waste includes any substance which contributes a scrap material, an effluent or other unwanted surplus arising from the application of any process or any substance or article which requires to be disposed of which has been broken, worn out, contaminated or otherwise spoiled; this is supplemented with anything which is discarded otherwise dealt with as if it were waste shall be presumed to be waste unless the contrary is proved. This definition was amended by the Waste Management Licensing Regulations, 1994 defining waste as: “ Any substance or object which the producer or the person in possession of it, discards or intends or is required to discard but with exception of anything excluded from the scope of the Waste Directive”(www.google.com). SOME OTHER DEFINITIONS OF WASTE:"Any substance or object that in the categories set out in part 2 of Schedule 4 of the EPA 1990 which the holder discards or intends or is required to discard.. an item is discarded when it is no longer part of the normal commercial cycle or part of the chain of utility." "A product which is no longer used in its primary role. which the holder then intends to, or is required to, discard." (SEPA. Waste Management Licensing Regulations, 1994.) Construction waste specifically is "materials resulting from the construction, remodelling, repair or demolition of buildings, bridges, pavements and other structures." The word 'waste' and the act of 'wasting' are human inventions. Waste doesn't exist in nature. In nature, everything has a purpose. Waste was created by humans for short-term convenience and short-term profit. Wasting results in long-term harmful consequences for both humans, nature, and the economy.

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Growth of population, increasing urbanization, rising standards of living due to technological innovations have contributed to an increase both in the quantity and variety of solid wastes generated by industrial, mining, domestic and agricultural activities. From going further what is waste.

List of waste types
 Animal by-products  Biodegradable waste  Biomedical waste  Chemical waste  Clinical waste  Commercial waste  Construction and demolition waste (C&D waste)  Controlled waste
 Composite, demolition, domestic, E- waste

 Farm waste  Hazardous waste
 Household & industrial waste

 Inert waste

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 Municipal solid waste  Radioactive waste (nuclear waste) o Low level waste o High level waste o Spent nuclear fuel
o

Mixed waste (radioactive/hazardous) (www.google.com)

CELLULOSE Cellulose is an organic compound with the formula (C6H10O5), a polysaccharide consisting of a linear chain of several hundred to over ten thousand β(1→4) linked D-glucose units. Cellulose is the structural component of the primary cell wall of green plants, many forms of algae and the oomycetes. Some species of bacteria secrete it to form biofilms. Cellulose is the most common organic compound on Earth. About 33 percent of all plant matter is cellulose (the cellulose content of cotton is 90 percent and that of wood is 50 percent). For industrial use, cellulose is mainly obtained from wood pulp and cotton. It is mainly used to produce cardboard and paper; to a smaller extent it is converted into a wide variety of derivative products such as cellophane and rayon. Converting cellulose from energy crops into biofuels such as cellulosic ethanol is under investigation as an alternative fuel source.

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Some animals, particularly ruminants and termites, can digest cellulose with the help of symbiotic micro-organisms that live in their guts. Cellulose is not digestible by humans and is often referred to as 'dietary fiber' or 'roughage', acting as a hydrophilic bulking agent for feces
(www.google.com).

Review of literature
Organic wastes Organic waste is produced wherever there is human habitation. The main forms of organic waste are household food waste, agricultural waste, human and animal waste. In industrialized countries the amount of organic waste produced is increasing dramatically each year. Although many gardening enthusiasts compost some of their kitchen and garden waste, much of the household waste goes into landfill sites and is often the most hazardous waste. The organic waste component of landfill is broken down by micro-organisms to form a liquid 'leachate' which contains bacteria, rotting matter and maybe chemical contaminants from the landfill. This leachate can present a serious hazard if it reaches a watercourse or enters the water table. Digesting organic matter in landfills also generates methane, which is a harmful greenhouse gas,
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in large quantity. Human organic waste is usually pumped to a treatment plant where it is treated, and then the effluent enters a watercourse, or it is deposited directly into the sea. Little effort is made to reclaim the valuable nutrient or energy content of this waste ( www.wikipedia.org). In developing countries, there is a different approach to dealing with organic waste. In fact, the word 'waste' is often an inappropriate term for organic matter, which is often put to good use. The economies of most developing countries dictates that materials and resources must be used to their full potential, and this has propagated a culture of reuse, repair and recycling. In many developing countries there exists a whole sector of recyclers, scavengers and collectors, whose business is to salvage 'waste' material and reclaim it for further use. Extension education is significant range of fields like Agriculture, Natural Resources, Environmental and Bio Diversity Conservation, Rural Development, Home Management Skill Development, Disaster Management, Waste Management, Value Adding Management. Among them, waste management extension is highly significant because of the millions of tons of annual waste in vegetal, animal, environmental and natural resources products as well as millions of hectors of land degradation. Waste management extension deals with raising the efficiency and productivity of the agricultural industry, intellectually and/ or economically. Both producers and consumers should be fully aware of the mechanism by which waste in agricultural commodities diminishes to a considerable level. In agriculture, knowledge and decision-making capacity determine how production factor (i.e. oil, water, capital, chemicals, etc) are utilized. Agricultural extension is a focal issue in formulating and disseminating knowledge and helping farmers to be competent decision makers. Literature review, content analysis and modeling through utilizing contingency tables were employed to conduct the study. Different experiences in this regard have been collected and results show that the greater the use of AWMEE(Agricultural Waste Management Extension Education), the less agricultural waste, the higher the agricultural productivity and lower the land degradation. SOLID WASTE, THEIR TYPE AND NATURE:Agro waste(organic nature):- Its important sources includes Cotton stalk, Rice & Wheat straw and husk, Baggage, sisal and vegetable residues , Ground nut shell, Banana stalk and jute, Saw mill waste.
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Industrial waste (inorganic nature):- It may include Coal consumption residues, steel slag, bauxite red mud, Coal washeries waste, construction debris. Hazardous waste: - It includes metallurgical residues, galvanizing waste, tannery waste etc. Non-hazardous other process waste:- Waste gypsum, lime sludge, lime stone waste, marble processing residues, broken glass and ceramics, kiln dust etc. Hospital waste: - It is generated during the diagnosis, treatment, or immunization of human beings or animals or in research activities in these fields or in the production or testing of biological. It may include wastes like sharps, soiled waste, disposables, anatomical waste, cultures, discarded medicines, chemical wastes, etc. These are in the form of disposable syringes, swabs, bandages, body fluids, human excreta, etc. The above mentioned waste are recycled or utilized by different methods according to the product such as Particle boards, insulation boards, wall panels, printing paper and corrugating medium, roofing sheets, fuel, binder, fibrous building panels, bricks, acid proof cement, cement, blocks, tiles, paint, aggregate, concrete, wood substitute products, ceramic products bricks, lightweight aggregates, fuel, gypsum plaster, fibrous gypsum boards, bricks, blocks, cement clinker, super sulphate, hydraulic binder , and board etc. Disposal Methods of wastes There are a few ways to dispose of waste materials. The two main methods of disposing of waste materials is landfills and incineration. Each method has their advantages and disadvantages. Landfills involves burying the waste to get rid of it. This method if done properly can be very inexpensive and hygienic. Many people probably think this method would be very unhygienic but that really depends if it is done properly or not. There are some countries that do not do this method properly and it can cause such issues as wind-blown litter, attraction of vermin, and generation of liquid leachate (the liquid that drains from a landfill). Another issue that might arise from landfills is gas (usually methane and carbon dioxide) when the waste breaks down over time. Usually landfills are established in disused quarries (type of open-pit mine), mining voids or borrow pits (an area where soil, gravel or sand has been dug for use in another location).

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Although there are a lot of negative effects of landfills if poorly designed most new ones are designed in a way to prevent negative effects. Incineration is the second method of disposing waste. This method involves the combustion of waste materials. With this method the waste material is heated to very high temperatures and is converted into materials such as: heat, gas, steam and ash. Incineration can be done on a small scale by individual people such as in a fire and also done on a much large scale by an industry. This method of waste management is considered beneficial for such materials as medical waste. This method however is also a very controversial method of waste disposal because of the emission of gaseous pollutants (green house effect). Recycling Methods Recyling refers to the reuse or recover of materials that would normally be considered waste. There are a few different methods of recycling such as: physical reprocessing, biological reprocessing, and energy recovery. People are always looking for new ways as well to recycle materials because of the constant issues we are having with waste in our environment. One of the most popular method of recyling is physical reprocessing. This is common in most countries. This is the method of taking waste materials such as empty beverage containers and using the material to create new materials. Normally waste materials that can be physically reprocessed are usually collected by the local government and are then reprocessed into new products. Some common materials that are physically reprocessed include: aluminum beverage cans, steel food cans, glass bottles, newpapers, magazines, and cardboard. Biological reprocessing is another common method of recycling that many people do. Materials such as plants, food scraps, and paper products can be decomposed into the organic matter. The organic matter that is produced from this type of recycling can then be used for such things as landscaping purpose or agricultural uses. Usually this method of recycling is done by putting the materials in a dedicated container and let to stay there until it decomposes. The final method of recycling is Energy recovery. This method harnesses directly and indirectly the combustion fuel and other types of fuel produced from waste. These types of fuel can be produced by thermal treatment of the waste and used for such things as cooking or heating. The thermal treatment is usually done under very high pressure in a sealed vessel.

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Avoidance and Reduction Methods The avoidance and reduction of waste is a very important part of waste management. By reducing waste it helps the environment and everyone in it. Some methods of avoidance include the reuse of second-hand products, repairing broken items and using them again, and designing products that are resusable. As well consumers are encouraged to not use disposable products and use products are designed to help the environment. Transportation of Waste There are a few way waste is handled. In most countries waste dispose is handled by the local government authorities. In other countries there are no such systems in place and disposal of waste is more difficult. In countries such as Canada and the United States curbside collection is common and occurs weekly. In some countries in Europe they have a collection system known as Envac which conveys refuse via underground conduits using a vacuum system.

Table 1. SOURCES AND TYPES OF SOLID WASTES Source Residential Typical waste generators Single and multifamily dwellings Types of solid wastes Food wastes, paper, cardboard, plastics, textiles, leather, yard wastes, wood, glass, metals, ashes, special wastes (e.g., bulky items, consumer electronics, white goods, batteries, oil, tires), and household hazardous wastes.). Housekeeping wastes, packaging, food wastes, construction and demolition materials, hazardous wastes, ashes, special wastes.

Industrial

Light and heavy manufacturing, fabrication, construction sites, power and chemical plants.

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Commercial

Stores, hotels, restaurants, markets, office buildings, etc.

Paper, cardboard, plastics, wood, food wastes, glass, metals, special wastes, hazardous wastes. Same as commercial. Wood, steel, concrete, dirt, etc.

Institutional Construction and demolition Municipal services

Schools, hospitals, prisons, government centers. New construction sites, road repair, renovation sites, demolition of buildings Street cleaning, landscaping, parks, beaches, other recreational areas, water and wastewater treatment plants. Heavy and light manufacturing, refineries, chemical plants, power plants, mineral extraction and processing.

Process (manufacturing, etc.)

Street sweepings; landscape and tree trimmings; general wastes from parks, beaches, and other recreational areas; sludge. Industrial process wastes, scrap materials, off-specification products, slay, tailings.

Agriculture

Crops, orchards, vineyards, dairies, feedlots, farms.

Spoiled food wastes, agricultural wastes, hazardous wastes (e.g., pesticides).

Agricultural Waste
Agricultural wastes include both natural (organic) and non-natural wastes. Main non-natural waste arisings include packaging, non-packaging plastics (e.g. silage and horticultural films); agrochemicals; animal health products (e.g. used syringes); waste from machinery (e.g. oil, tyres and batteries) and building waste (e.g. asbestos sheeting). Total quantity of non-natural waste is estimated as 500,000 tonnes per year of which approximately 225,000 tonnes pesticide washings and spent sheep dips (Environmental Agency). Therefore, there is a need to put in place a waste management policy as regards to agricultural waste. Currently, farmers use a variety of recovery and disposal methods, depending on circumstances. They include reuse on farm, take back by suppliers, inclusion with household waste, stockpiling, and the most common are burial or burning (especially packaging and plastic films). According
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to the research carried out by Environmental Agency, some two thirds of all wastes are buried or burnt on farm. This wide ranging practices of disposal/recovery methods reflects the long term exclusion of agricultural wastes from the controlled waste regulations. Although the quantity of wastes produced by the agricultural sector is significantly low, the pollution potential of these wastes are high on a long term basis. For instance, the land spreading of manures and slurries can cause nutrient and organic pollution of soils and waters, if relevant guidelines are not followed or applications are inappropriately timed or excessive. Animal excreta are also a potential source of Cryptosporidium and other pathogens including Salmonella and Campylobacter (Environmental Agency). According to the EC Framework directive, the some of the current practices are no longer viable. The government is expected to bring some agricultural wastes under the Waste Management Regulations (DEFRA). A public consultation document will be published early 2004 seeking stakeholders’ views on proposed regulations.

Cellulosic agro-waste Cellulosic biomass constitutes a huge and renewable resource that can be converted to compost and fuel feedstocks. More efficient means for conversion of agricultural and forest waste are sought so that useful biomass-derived products can not only compete with or eventually replace petroleum based products but also supplement and complement the use of petroleum based fuels as additives to promote more efficient burning and lower emissions. Using these cellulosic resources efficiently can thus reduce the disposal problems and pollution resulting from accumulation of these wastes. Cellulose is a polymer of glucose and is the most abundant organic material in nature. It is however, resistant to decomposition. The annual production of cellulose is estimated to be about 100 X 109 tons. A large number of bacteria, fungi and actinomycetes are known to degrade cellulose .When cellulose is associated with pentosans such as xylans and mannans it undergoes rapid decomposition.
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On the other hand, when associated with lignin, the decomposition rate is very slow. The degradation of cellulose is by enzymes and the enzyme system that converts cellulose to glucose consists of at least three enzymes, an exoglucanase, an endoglucanase and a β-glucosidase (cellulase complex). Biocycling Due to the development of civilization, vast amount of organic waste is being produced. Its return to the environment is highly desirable, as it is characterized by significant amount of macro- and micronutrients. However, some of the organic waste cannot be used directly, and needs improvement in its physical and chemical properties. One way of organic waste utilization is composting with usage of the appropriate biopreparations. Biological methods of decomposition of solid waste and liquid waste are known all over the world. They are regarded, to high extent, as effective, relatively cheap and most environmentally friendly way for utilizing most of the organic waste, in which organic fraction is usually represented by proteins, fats and carbohydrates. However, one of the biggest problems in this kind of process is a proper choice of micro-organisms, in terms of both quantity and quality, which determines the utilization effects of the bio preparation as well as the speed of the process. The complete biodegradation of organic waste cannot be assumed, as it may contain compounds that cannot be decomposed, i.e. hemicelluloses, but biodegradation of most proteins, fats and carbohydrates in organic waste, originating from for example households seems to be possible (Bujak and Targoński 1998). Organic manure obtained in this process is characterized by better properties in comparison with the initial waste and can be used in agriculture, if its chemical composition (content of heavy metals) and health and sanitary properties are unquestioned.

ROLE OF VARIOUS MICROBES IN BIO-CYCLING
Fungi Most fungi are capable cellulose degraders. However, their ability to facilitate rapid lignocellulose degradation attracted attention from scientists and entrepreneurs alike. White-rot fungi comprise powerful lignin degrading enzymes that enable them in nature to bridge the lignin
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barrier and, hence, overcome the rate-limiting step in the carbon cycle (Elder & Kelly 1994). Phanerochaete chrysosporium is the best studied. New information regarding the identities of the cellulose, hemicelluloses or lignin degrading enzymes, their unique catalytic capabilities, the physiological conditions required for optimum secretion or activity etc. is constantly being added to an already impressive volume of work and varies between fungi and bacterial genera, species and even strains. Anaerobic fungi (Piromyces spp., Neocalli-mastix spp. and Orpinomyces spp.) form part of the rumen microflora. These fungi produce active polymer degrading enzymes, including cellulases and xylanases. Their cellulases are among the most active reported to date and able to solubilise both amorphous and crystalline cellulose (Wubah et al., 1993). These fungi can be used in situations where process principles and design necessitate anaerobic conditions. Several brown rot fungi, including Gloeophyllum sepiarium (Mansfield et al., 1998), Gloeophyllum trabeum (Herr et al., 1978a; Mansfield et al., 1998), Polyporus schweinitzii , Serpula incrassata (Kleman-Leyer & Kirk, 1994) and Tyromyces palustris (Ishihara & Shimizu, 1984), Trichoderma koningii and Phanerochaete chrysosporium, fungi of the genera Neocallimastix, Piromonas and Sphaeromonas (Leschine 1995; Tomme et al., 1995) white rot fungi P. chrysosporium, Pleurotus ostreatus and Trametes versicolor (Kuhad et al., 1997; Leonowicz et al., 1984) are also capable to degrade the cellulosic biomass.

Bacteria Lignocellulose biodegradation by prokaryotes is essentially a slow process characterized by the lack of powerful lignocellulose degrading enzymes, especially lignin peroxidases. Grasses are more susceptible to actinomycete attack than wood (Crawford et al., 1981; McCarthy 1987). Together with bacteria, actinomycetes play a significant role in the humification processes associated with soils and composts (Trigo & Ball, 1994). The enzymatic ability to cleave alkylaryl ether bonds enable bacteria to degrade oligomeric and monomeric aromatic compounds released during fungal lignin degradation (Vicuna et al., 1993; White et al., 1996). Bacillus subtilis, Bacillus macerans, Pseudomonas fluorescens, Pseudomonas fragi, Serratia liquefaciens, Acinetobacter junii, Acinetobacter lvoffii, Cytophaga sp. (Bujak and Targoński, 1998) have the ability to degrade the lignocellulosic waste. Microbial degradation of organic
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matter was investigated using culture of heterotrophyc bacteria enriched from solid samples collected with waste heap. A mixture of bacteria containing Pseudomonas fluorescen, Areomonas hydrophilia, Oligella sp. Bacillus megaterium, Streptomonas maltophilia, Stenotrophomonas maltophilia, Bacillus amyloliquefaciens, Bacillus circulans, Bacillus pumilus and Burkhordelia capacia was used for the biodegradation experiments (Vicuna, 2000).

Table2. Microorganisms capable of utilizing different components of organic matter F. Alternaria, Aspergillus, Chaetomium, Coprinus, Fomes, Fusarium, Cellulose Myrothecium, Penicillium, Polyporus, Rhizoctonia, Rhizopus, Trametes, Triclwderma, Trichothecium, Verticillium, Zygorynchus B. Achromobacter, Angiococcus, Bacillus, Cellfalcicula, Cellulamonas, Cellvibrio, Clostridium, Cytophaga, Polyangium, Pseudomonas, Sorangium, Sporocytophaga, Vibrio A. Micromonopora, Nocardia, Streptomyces, Streptosporangium F. Alternaria, Fusarium, Trichothecium, Aspergillus, Rhizopus, Zygorynchus, Hemicellulose Chaetomium, Helminthosporium, Penicillium, Coriolus, Fames, Polyporus B. Bacillus, Achromobacter, Pseudomonas, Cytophaga, Sporocytophaga, Lactobacillus, Vibrio A. Streptomyces

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F. Clavaria, Clitocybe, Collybia, Flammula, Hypholoma, Lepiota, Mycena, Lignin Pholiota, Arthrobotrys, Cephalosporium, Humicola B. Pseudomonas, Flavobacterium F. Aspergillus, Fomes, Fusarium, Polyporus, Rhizopus B. Achromobacter, Bacillus, Chromobacterium, Clostridium, Cytophaga A. Micromonospora, Nocardia, Streptomyces

Starch

P ectin

F. Fusarium, Verticillium B. Bacillus, Clostridium, Pseudomonas F. Penicillium, Aspergillus, Fusarium B. Pseudomonas, Flavobacterium, Beneckea, Micrococcus, Cytophaga, Clostridium F. Fusarium, Mucor, Mortierella, Trichoderma, Aspergillus, Gliocladium, Penicillium, Thamnidium, Absidia B. Cytophaga, Achromobacter, Bacillus, Beneckea, Chromobacterium, Flavobacterium, Micrococcus, Pseudomonas A. Streptomyces, Nocardia, Micromonospora

Inulin

Chitin

AGRICULTURAL WASTE AS AN ENERGY SOURCE IN DEVELOPING COUNTRIES The main problems facing rural villages in developing countries are agricultural waste, sewage and municipal solid waste. However, few studies have been conducted on the utilization of agricultural waste for composting and/or animal fodder. Most of the proposed solutions have not been implemented because they did not meet the basic elements of sustainability: social progression, technical and technological improvements, environmental protection and economic development. This poster presents a sustainable solution by combining all wastes generated in rural areas in one complex: EBRWC (Environmentally Balanced Rural Waste Complex) that has been developed to produce valuable products with zero pollution. Such a complex is generated from agricultural waste, disposal of sewage in water canals and disposal of municipal solid waste. Several compatible techniques are located together within this agricultural complex:
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utilizing briquettes as a renewable energy source; using anaerobic digestion (biogas) to produce energy and fertilizer; composting for soil conditioner; animal fodder and other recycling techniques for solid waste. The main outputs of EBRWC are fertilizer, energy, animal fodder and other recycled materials depending on the availability of wastes, and according to demand and need. (S.M. El-Haggar, 2000)
 ENVIRONMENTALLY BALANCED RURAL WASTE COMPLEX

The EBRWC can be defined as a selective collection of compatible activities located together in one area (complex) to minimize (or prevent) the impact on the environment and treatment costs for sewage, municipal solid waste and agricultural waste. A typical example of such a rural waste complex consists of several compatible techniques such as briquetting, anaerobic digestion (biogas), composting, animal fodder and other recycling techniques for solid wastes located together. Thus, EBRWC is a self-sustaining unit that draws all its inputs from rural wastes, achieving zero waste and pollution. Some emissions may be released into the atmosphere, but the emission level would be significantly less than that of the raw waste fed into the EBRWC. A typical rural waste complex would operate to utilize all agricultural waste, sewage and municipal solid waste as sources of energy, fertilizer, animal fodder and other products depending on the constituents of the municipal solid waste. In other words, all the wastes will be used as raw material for a valuable product according to demand and need within the rural waste complex. Thus a rural waste complex will consist of a number of compatible activities, the waste of one being used as raw material for others, with no external waste generated from the complex. This technique will produce different products as well as keep the rural environment free of pollution from sewage, agricultural and solid waste. The main advantage of the complex is that it helps the sustainable development of the national economy in rural areas.  AGRICULTURAL WASTE AS AN ENERGY SOURCE Agriculture biomass resources in Egypt are estimated to be around 25 million tonnes (dry matter) per year. Fifty percent of the biomass is used as fuel in rural areas by direct combustion in low efficiency traditional furnaces (figure1. Mixing agricultural residues for drying and using as
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energy source). The traditional furnaces are primitive mud stoves and ovens that produce large quantities of air pollution and are extremely energy inefficient. The agriculture biomass waste (resources) consist mainly of cotton stalks, rice straw, etc.

Fig. 1 Mixing of agricultural residues by a tractor

BRIQUETTING SYSTEM The briquetting process is the conversion of agricultural waste into uniformly shaped briquettes that are easy to use, transport and store. The idea of briquetting is to use materials that are otherwise not usable due to a lack of density, compressing them into a solid fuel of a convenient shape that can be burned like wood or charcoal. The briquettes have better physical and combustion characteristics than the initial waste. Briquettes will improve the combustion efficiency of existing traditional furnaces. In addition to killing all insects and diseases they reduce the risk of fire in the countryside. The idea of briquetting is to use materials that are not otherwise usable due to a lack of density, compressing them into a solid fuel of a convenient shape that can be burned like wood or charcoal (figure 2). Briquettes were discovered to be an
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important source of energy during the First and Second World Wars for heat and electricity production using simple technologies. One of the recommended technologies is lever operating press (mechanical or hydraulic press). Briquetting allows ease of transportation and safe storage of wastes as they have a uniform shape and are free of insects and disease carriers.

Advantages of briquetting :
 Gets rid of insects  Decreases the volume of waste  Efficient solid fuel of high thermal value  Low energy consumption for production  Protects the environment  Provides job opportunities  Less hazardous.

Fig. 2. Briquettes generated from agricultural waste

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PRETREATMENT OF AGRICULTURAL WASTES (MAINLY CELLULOSIC AND LIGNIN COMPONENTS) The effect of pretreatment of lignocellulosic materials has been recognized for a long time (McMillan, 1994). The purpose of the pretreatment is to remove lignin and hemicellulose, reduce cellulose crystallinity and increase the porosity of the materials

There are various requirements which are to be fulfilled:  Cost effective treatment.  Avoid the formation of byproducts inhibitory to the subsequent hydrolysis and fermentation process and degradation or loss of carbohydrate.  Improve the formation of sugars or the ability to subsequently form sugars by enzymatic hydrolysis.

Physical pretreatment  Mechanical comminution Waste materials can be comminuted by a combination of chipping, grinding and milling to reduce cellulose crystallinity. The size of the material is usually 10-30 mm after chipping and 0.2-2 mm after milling and grinding. Vibratory ball milling has been found to be more effective in breaking down the cellulose crystallinity of spruce and aspen chips and improving the digestibility of biomass of the biomass than ordinary ball milling (Millet et al., 1976). The power requirement of the mechanical comminution of agricultural wastes depends on the final particle size and the waste biomass characteristics (Cadoche and López, 1989).
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 Pyrolysis Pyrolysis has also been used for pretreatment of lignocellulosic materials. When the materials are treated at temperatures greater than 300 ºC, cellulose rapidly decomposes to produce gaseous products and residual char (Kilzer and Broido, 1965; Shafizadeh and Bradbury, 1979). The decomposition is much slower and less volatile products are formed at lower temperatures. Mild acid hydrolysis (1 N H2SO4, 97 ºC, 2.5 hr) of the residues from pyrolysis pretreatment has resulted in 80-85% conversion of cellulose to reducing sugars with more than 50% glucose (Fan et al., 1987). The process can be enhanced with the presence of oxygen (Shafizadeh and Bradbury, 1979). When zinc chloride or sodium carbonate is added as catalyst, the decomposition of pure cellulose can occur at a lower temperature. Physico-chemical pretreatment
 Steam explosion (Auto hydrolysis)

Steam explosion is the most commonly used method for the pretreatment of lignocellulosic materials (McMillan, 1994). In this method, chopped biomass is treated with high-pressure saturated steam and then pressure is swiftly reduced, which makes the materials undergo an explosive decompression. Steam explosion is typically initiated at a temperature of 160-260 ºC (corresponding pressure 0.69-4.83 MPa) for several seconds to a few minutes before the material is exposed to atmospheric pressure. The process causes hemicellulose degradation and lignin transformation due to high temperature, thus increasing the potential of cellulose hydrolysis. The factors that affect steam explosion pretreatment are residence time, temperature, chip size and moisture (Duff and Murray, 1996). Optimal hemicellulose solubilization and hydrolysis can be achieved by either high temperature and short residence time (270 ºC, 1 min) or lower temperature and longer residence time (190 ºC, 10 min)( Duff and Murray, 1996). Recent studies indicate that lower temperature and longer residence time are more favorable. Addition of H2SO4 or CO2 in steam explosion can effectively improve enzymatic hydrolysis decrease the production of inhibitory compounds and lead to more complete removal of hemicellulose. The advantages of steam explosion pretreatment include the low energy requirement compared to mechanical comminution and no recycling or environmental costs. The

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environmental mechanical methods require 70 % more energy than steam explosion to achieve the same size reduction. Limitations of steam explosion It causes destruction of a portion xylan fraction, incomplete disruption of the lignincarbohydrate matrix and generation of compounds that may be inhibitory to microorganisms used in downstream processes (Mackie, 1985). Because of the formation of degradation products that are inhibitory to microbial growth, enzyme hydrolysis and fermentation, pretreated biomass needs to be washed by waster to remove the inhibitory materials along with water soluble hemicelluloses. Biological pretreatment In biological pretreatment processes, microorganisms such as brown, white and soft –rot fungi are used to degrade lignin and hemicellulose in waste materials (Schurz, 1978). Brown rots mainly attack cellulose, while white and soft rots attack both cellulose and lignin. White-rot fungi are the most effective basidiomycetes for biological pretreatment of lignocellulosic materials (Fan et al. 1987). In order to prevent the loss of cellulose, a cellulase less mutant of Sporotrichum pulverulentum was developed for the degradation of lignin in wood chips (Ander and Eriksson, 1977). The white rot fungus P. chrysoporium produces lignin degrading enzymes, lignin peroxidase and manganese dependent peroxidases, during secondary metabolism in response to carbon to nitrogen limitation. Both enzymes have been found in the extracellular filtrates of many whiterot fungi for the degradation of wood cell wall (Kirk and Farrell, 1987; Waldner et al., 1988). Other enzymes including polyphenol oxidases, laccases, H2O2 producing enzymes and quinonereducing enzymes can also degrade lignin (Blanchette, 1991). The advantages of biological pretreatment include low energy requirement and mild environmental conditions. However, the rate of hydrolysis in most biological pretreatment processes is very slow.

Enzymatic hydrolysis

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Enzymatic hydrolysis of cellulose is carried out by celullase enzymes which are highly specific (Béguin and Aubert, 1994). The products of hydrolysis are usually reducing sugars including glucose. Utility cost of enzymatic hydrolysis is low as compared to acid or alkaline hydrolysis because enzyme hydrolysis is usually conducted at mild conditions (pH 4.8 and temperature 45-50 ºC) and does not have a corrosion problem (Duff and Murray, 1996). Both bacteria and fungi can produce cellulases for the hydrolysis of the lignocellulosic materials. These microorganisms can be aerobic or anaerobic, mesophilic or thermophilic. Bacteria belonging to Clostridium, Cellulomonas, Bacillus, Thermomonospora, and Streptomyces etc. can produce cellulases (Bisaria, 1991). Because anaerobes have a very low growth rate and require anaerobic growth conditions, most research for commercial cellulase production has focused on fungi (Duff and Murray, 1996). Fungi has been reported to produce cellulases include Sclerotium rolfsii, P. chrysoporium and species of Tricoderma, Aspergillus and Penicilium (Sternberg, 1976; Fan et al., 1987; Duff and Murray, 1996). Of all these fungal genera, Tricoderma has been most extensively studied for cellulase production (Sternberg, 1976). Cellulases are usually a mixture o several enzymes. At least three major groups of cellulases are involved in the hydrolysis process: (1) endoglucanase which attacks regions of low crystallinity in the cellulose fiber, creating free chain-ends; (2) exoglucanase or cellobiohydrolase which degrades the molecule further by removing cellobiose units from free chain ends; (3) βglucosidase which hydrolyzes cellobiose to produce glucose (Coughlan and Ljungdahl, 1988). During the enzymatic hydrolysis, cellulose is degraded by the cellulases to reducing sugars that can be fermented by yeast or bacteria to ethanol. Value added products of agricultural wastes Many agricultural by-products from agricultural activities and agro-based processing litter the environments and constitute waste problems. This research is aimed at converting some of these unwanted agricultural waste or by-products to commercially useful products such as pulp and cellulose acetate. This work has the potential of providing new market and applications for low value and utilized agricultural wastes by converting into cellulose acetate. The principle behind the research is that most agricultural by-products are composed of cellulose in the plant
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cell walls. Cellulose (C6H10O5)n is a large chain polymeric polysaccharide carbohydrate of betaglucose. The principal functional groups in pure cellulose are hydroxy (-OH) making cellulose a polyol with primary and secondary alcohol functional groups (-CH2OH, -CHOH). The multiple hydroxy groups of cellulose in cellulosic materials (agro-waste) can be partially or wholly modified by reacting with various chemicals to produce a wide range of end products referred to as ‘cellulose derivatives’. These derivatives such as cellulose nitrates, acetates, xanthates, ethers, rayon and cellophanes have far reaching industrial applications. Cellulose nitrate has the largest industrial use as lacquer coating for decorative and protective purposes. It is also useful in the photographic industry, in explosives and propellants1-4. Among all cellulose derivatives, cellulose acetate as by far been recognized as the most important organic ester of cellulose owing to its extensive industrial and commercial importance. It has many uses such as in the production of plastics films, lacquers, photographic films, thermoplastic moulding, transparent sheeting, camera accessories, magnetic tapes, combs, telephone, and electrical parts 5-7. The production of cellulose acetate from agro-waste such as jute stick, cotton linters, woodchips, rice straw, wheat hull and corn fiber have been reported in literature8,1,7. In Nigeria, enormous volumes of agricultural wastes that contain cellulosic fibers are generated annually. Many of these agro-wastes are allowed to rot away utilized. For example, tonnes of maize stem, cobs from Zea mays, plantain stem, raffia from Raphia hookeri are allowed to rot away yearly and eventually polluting the environment. These agricultural wastes can be gainfully utilized in the production of pulp for papermaking and conversion to cellulose derivatives for manufacture of plastics, photographic films etc. This will not only encourage local farmers as new market is developed for materials but will also boost the Nigerian economy. Also the problem of environmental pollution arising from the decay of these materials will be minimized. Recently, our research group has reported on the conversion of some agricultural wastes to cellulose derivatives7. The present work is another trial aimed at practically exploiting these biodegradable agricultural wastes materials as a potential alternative for the production of pulps and subsequent conversion to cellulose derivatives.

1. Production of Cellulosic Polymers from Agricultural Wastes

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Cellulosic polymers namely cellulose, di-and tri-acetate were produced from fourteen agricultural wastes; Branch and fiber after oil extraction from oil palm (Elais guineensis), raffia, piassava, bamboo pulp, bamboo bark from raphia palm (Raphia hookeri), stem and cob of maize plant (Zea mays), fruit fiber from coconut fruit (Cocos nucifera), sawdusts from cotton tree (Cossypium hirsutum), pear wood (Manilkara obovata), stem of Southern gamba green (Andropogon tectorus), sugarcane baggase (Saccharium officinarum) and plantain stem (Musa paradisiaca). They were subjected to soda pulping and hypochlorite bleaching system. Results obtained show that pulp yield from these materials were: 70.00, 39.59, 55.40, 86.00, 84.60, 80.00, 40.84, 81.67, 35.70, 69.11, 4.54, 47.19, 31.70 and 52.44% respectively. The pulps were acetylated with acetic anhydride in ethanoic acid catalyzed by conc. H2SO4 to obtain cellulose derivatives (Cellulose diacetate and triacetate). The cellulose diacetate yields were 41.20, 17.85, 23.13, 20.80, 20.23, 20.00, 39.00, 44.00, 18.80, 20.75, 20.03, 41.20, 44.00, and 39.00% respectively while the results obtained as average of four determinations for cellulose triacetate yields were: 52.00, 51.00, 43.10, 46.60, 49.00, 35.00, 40.60, 54.00, 57.50, 62.52, 35.70. 52.00, 53.00 and 38.70% respectively for all the agricultural wastes utilized. The presence of these cellulose derivatives was confirmed by a solubility test in acetone and chloroform.

2. Composting

Composting is biological burning of biomass without losing the nitrogen and phosphorus to air (figure3).Composting requires moisture, low temperatures and proper microbial power for converting organic matter into compost. Composting is one of the best known recycling processes for organic waste to close the natural loop. The major factors affecting the decomposition of organic matter by micro-organisms are oxygen and moisture. Temperature, which is a result of microbial activity, is also an important factor. The other variables affecting the process of composting are nutrients (carbon and nitrogen), pH, time and the physical characteristics of the raw material (porosity, structure, texture and particle size). The quality and decomposition rate depends on the selection and mixing of raw materials. Composting is the nature’s way of recycling. Composting biodegrades the organic waste i.e. food waste, manure, leaves, grass trimmings, paper, wood, feathers, crop residue etc., and turns it into a valuable organic fertilizer. Because compost materials usually contain some biological resistant
[30]

compounds, a complete stabilization (maturation) during composting may not be achieved. The time required for maturation depends on environmental factors within and around the composting pile. Some traditional indicators can be used to measure the degree of stabilization such as decline in temperature, absence of odour, and lake of attraction of insects in the final products. Composting is a natural biological process, carried out under controlled aerobic conditions. In this process, various microorganisms, including bacteria and fungi break down organic matter into simpler substances. The effectiveness of the composting process is dependent upon the environmental conditions present within the composting system i.e. oxygen, temperature, moisture, material disturbance, organic matter and the size and activity of microbial populations. Composting is relatively simple to manage and can be carried out on a wide range of scales in almost any indoor and outdoor environment and in almost any geographical location. It has a potential to manage most of the organic matter in the waste stream including restaurant waste, leaves and yard wastes, farm wastes, animal manure, animal carcasses, paper products, sewage sludge, wood etc. and can be easily incorporated into any waste management plan (www.soil.ncsu.edu). In 1876 Justus von Liebig, a German chemist calculated that North African lands that were supplying two thirds of the grains consumed in Rome were becoming less fertile and loosing their quality and productivity. He found, on conducting research, the reason behind this phenomenon: when crops are exported from North Africa to Europe, their wastes do not go back to North Africa but are flushed into the Mediterranean. Agricultural waste is rich in organic matter. This matter is derived from the soil and the soil needs it back in order to continue producing healthy crops. However, this was not the case and, in von Liebig’s opinion, was a breaking of the natural loop that gives the land back its nutrients. He called this phenomenon the “direct flow”. The German scientist proposed artificial fertilizers, which were meant to compensate the soil for loss of organic matter, but they were not the same as natural fertilizers. Since approximately 55-55% of the waste stream is organic matter, composting can play a significant role in diverting waste from landfills thereby conserving landfill space and reducing
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the production of leachate and methane gas. In addition, an effective composing program can produce a high quality soil amendment with a variety of end uses. Composting is applied microbiology at its most complexes, involving the interaction of thousands upon thousands of different species of microorganisms in a highly complex ecosystem. The composting process kills weed seeds and suppresses human and plant pathogens that doesn’t happen when leaves and other detritus rot down on their own. Once applied, compost “balances” the soil flora that is for each of the scores or more of disease organisms that can affect each species of plant at least12 to 15 different species of bio-control microorganisms need to be present, with the food and conditions they require, if the plant is to be healthy. Composting accomplishes that among other things (www.soil.ncsu.edu). The essential elements required by composting microorganisms are carbon, nitrogen, oxygen, moisture. If any of these elements are lacking or if they are not provided in the proper proportion, the microorganisms will not flourish and will not provide adequate matter into stable compost that is odor and pathogen free and poor breeding substrate for flies and other insects. In addition, it will significantly reduce the volume and weight of organic waste as the composting process converts much of the biodegradable component to gaseous carbon dioxide.

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Fig. 3. Composting of agriculture waste.

India emphasize composting for biodegradable waste in its municipal solid waste management and handling rule in 2000. Special funding is arranged to construct large-scale composting plants (100-700 ton/day), which is contracted to be operated by private company. However the success is seen in community and household composting which initiated by NGO and local government.

Earthworm Biotechnology in composting
Earthworms comprise a group of invertebrates which live in the ground, eat decomposing organic material and produce a rich earthy substance referred to as castings. More technically, the worms are terrestrial annelid worms which are any of a family of numerous widely distributed hermaphroditic worms that move through the soil by means of setae. Of these several varieties, the red worm has been found to be a hearty, rapidly multiplying organism capable of eating significant amounts of decomposing organic material.

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Worms have long been used for bait and for the conversion of some materials into soil like material. Consequently, they have been raised purposely toward these ends. The rate of reproduction of such animals is spectacular, increasing by as much as 1,200 times in a year. A need has been established for the conversion of organic agricultural waste to a neutral or useful product. The conventional method of burning such waste material as rice straw, almond shells and the like is coming into disfavor and legislative prohibition. An environmentally neutral or advantageous process is needed.

Bio-combustion:Composting

Composting is biological burning of biomass without loosing the nitrogen and phosphorus to air

composting requires moisture, low temperatures and proper microbial power for converting organic matter into compost

Vermicomposting
 Commonly called as vermiculture  In this process earthworms are introduced in the decomposition web.  Earthworms act as bioreactors.  They provide aerobicity, semi digested food for the microbes to act upon.  Excreta of earthworm- Vermi cast and other organic matter in digested form (in a

Vermibed) together is called vermicompost (fig.4).

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Earthworms in Vermicomposting  Surface dwellers  Humus formers  Tolerating high organic matter content  Continuous breeders  Varieties:Eisenia spp, Eudrilus spp,Perionyx spp are common varieties used in vermicomposting

Wormicomposting for waste treatment: Design considerations  Quantify waste  Type of waste  Waste collection  Infrastructure  Processing bin /container – Process Area  Watering arrangement  Drainage
 Equipments and machinery viz; shredder

 Plantation  Manpower
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Transverse section of Vermibed

Fig. 4. Vermibed

Options for Composting
 Pit composting: garden and floral refuse (figure 5)  Windrow composting: dung's manures and agro refuse (figure 6)  Bin composting: food waste, faunal refuse (figure 7)  Composter planter: for garden and food waste refuse together - City farming (figure 8)  Root zone composting for cellulosic garden refuse – Urban forest (figure 9)

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Fig. 5. Pit Composting

Fig. 6. Windrow Composting
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Fig. 7. Bin composting

Fig. 8. Composter Planter
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Source: www.inoraindia.com

Fig. 9. Root Zone Composting

Compost microbes are tremendously diverse and their ecologies are extremely complex. Methods used to research the microbial community succession include traditional plate-count method (Hassen et al. 2001; Boulter et al. 2002), community level physiological profiles (CLPPs) (Laine et al. 1997; Mondini and Insam 2003), phospholipid fatty acid (PLFA) analysis (Herrmann and Shann 1997; Klamer and Ba°a°th 1998; Steger et al. 2005), molecular technologies and quinone profile method (Tang et al. 2004). It is well known that less than 10% of bacteria existing in ecosystems could be culturable when using plating culture techniques. Clearly, the conventional approaches are not suitable for the analysis of microbial community structure in many ecosystems (Hu et al. 1999). Different advantages and limitations were found by using the other methods. Quinone profile method entails direct analysis of respiratory quinones in cell membranes to quantitatively reveal community profiles according to quinine molecular types. It is superior to molecular technologies because it correlates quantitatively to the microbial biomass. It also gives more information on taxonomy compared with the PLFA
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method, because most of the microorganisms contain a major quinone species (Tang et al. 2004). Quinone profile method is widely used in different ecosystems including soil (Song and Katayama 2005), activated sludge and bioreactor (Nozawa et al. 1998; Hu et al. 1999; Okunuki et al. 2004). The method was also used in composting. (Tang et al. 2003) detected the microbial community structure in various compost products. Quinone profiles and physico-chemical properties were measured to characterize the microbial community structure during a 14-day thermophilic composting of cattle manure mixed with rice straw as a bulking agent by (Tang et al. 2004) www.inoraindia.com

Table3: Sources and quantities of organic wastes Organic waste source Agricultural waste Municipal solid waste Sewage treatment plants Quantity 25 million tonnes of dry material/year 6.6 million tonnes of dry organic waste/year 4.3 million tonnes of dry sludge/year

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MATERIAL AND METHODS

 Microorganisms 1. Bacillus sp. AS3 (EU754025)
2. Trichoderma reesei (MTCC164) 3. Bacterial inoculum

Bacillus sp. AS3 was used for the biodegradation of Mixed Agricultural Waste (MAW). 1% inoculum was added to 100 mL of Nutrient Broth media and incubated at 37ºC for 24 h at 160 rpm. After 24 h of incubation, the inoculum was mixed with the 1 kg of wet biomass of waste.
4. Fungal inoculums

The fungi used for the degradation of Mixed Agricultural Waste (MAW) was Trichoderma reseei (MTCC 164), procured from Institute of Microbial Technology (IMTECH), Chandigarh. For the suspension of fungal spore 0.1% Tween 80 solution was used and the spore count of the fungal suspensions was set to approximately 5 x 106 spores/mL using haemocytometer. 1% spore suspension is added to 100 mL of Potato Dextrose Broth (PDB) for mass cultivation and incubated at 28 ± 2 ºC for 48 h at 120 rpm. After 48 h of incubation, the inoculum was mixed with the waste. 5. Collection of agricultural wastes Agricultural wastes from different plant/tree species i.e. Tylophora indica, Hevea brasilienosis, Eucalyptus, Polyalthia longofolia, Tectona grandis, Mangifera indica, Bamboosa vulgaris and grass cuttings, weeds and mixed waste were collected from the different places of Thapar University, Patiala, and were placed in the pits present at Building centre, Thapar University, Patiala. After complete air drying the waste material was chopped/ crushed using grass cutting machine (Toka machine).

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6. Biodegradation of mixed agricultural waste (MAW) The experiment was set by using eight different round plastic tubs of diameter 14.6 inches and depth of 6.5 inches, in duplicates. One kilogram of untreated chopped Mixed Agricultural Waste (MAW) was put into each tub under shade condition. Chopping was done 2-3 times with grinding machine (Toka machine). Moisture was maintained to 40-50% in each tub and regular mixing of waste was done after a regular interval of 3-4 days. Prior to the experimental set up, a suitable environment was created for the growth and survival of microorganisms by maintaining the appropriate moisture content only for 10 days. Whenever the suitable environment was created, experimental setup was done as explained in table 5. The experimental setup was for 90 days and every 15 days of interval the samples were withdrawn from the tubs and analyzed for the different parameters in the laboratory. DAP is used as a fertilizer and it positively helps in growth of fungi. Fast decomposition of rice straw by fungal inoculant (Trichoderma sp.) along with DAP, and then incorporation of its product (rice straw manure) into the soil may help to restore soil structure and fertility for paddy field (Man, 2000) Table 4. Experimental set up for degradation of Mixed Agricultural Waste Tub No. MAW Trichoderma reesei Bacillus sp. (MTCC 164) 1 2 3 4 1 kg as control 1 kg 1 kg 1 kg ------------------------------------------100 mL 100 mL AS3 -----------100 mL 100 mL -------------------------1g DAP

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 Estimation of pH (Potentiometry)

Procedure  Weighed 2g of air dried waste sample into a 100 ml beaker.  Added 20 ml of distilled water to it.  Thoroughly stirred it for 10 seconds using a glass rod.  Further stirred the suspension four five times during the next 30 minutes.  Allowed suspension to settle for 30 minutes.  In the meanwhile, rinsed the electrodes with distilled water and carefully wiped with filter paper.  Measured pH of the sample by immersing the combination electrode in supernatant solution.  Recorded pH value when the reading had stabilized.

Estimation of percentage organic carbon (Walkley & Black, 1934)

Reagents:

1 N K2Cr2O7 was prepared by dissolving 49.04 gm of Potassium dichromate in 1000 ml of distilled water.

 0.5 N Ferrous ammonium sulphate was prepared by adding 198 g salt in 1000 ml distilled water and also added 20 ml of concentrated sulphuric acid.

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 Diphenylamine indicator was prepared by adding 0.5 g of diphenylamine in a mixture of 20 ml distilled water and 100 ml concentrated sulphuric acid.  Concentrated sulphuric acid.  Orthophosphoric acid (85%) was prepared by mixing 15 ml distilled water to orthophosphoric acid.  Sodium fluoride.

Procedure
 Took 0.1 g of dried waste sample in a 500 ml conical flask and added 10 ml of 1N

K2Cr2O7 to it. Swirled the flask for mixing the waste and reagent.
 Added 20 ml of H2SO4 and allowed the flask to stand undisturbed for 30 minutes after

which added 200 ml of distilled water.  To the mixture, added 10 ml of 85% Orthophosphoric acid, 0.5 g of NaF and I ml of diphenylamine indicator.  Ultimately titrated with 0.5 N ferrous ammonium sulphate till the end point was observed from violet blue to green. Also a blank was taken without waste sample.

Calculation Organic Carbon (%) = 10* (B-T) * 0.003 * 100/(B * weight of sample taken) Where B = volume of ferrous ammonium sulfate consumed for blank titration T = volume of ferrous ammonium sulfate consumed for sample titration

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 Reducing sugar estimation by Dinitrosalicylic Acid (DNS) method (Ghose,

1987) Reagents
1. Citrate Buffer was prepared by dissolving 210 g of Citric acid monohydrate

(C6H8O7.H2O) in 750 ml of distilled water. It was diluted to 0.05 M buffer and the desired pH was set using NaOH
2. DNS Reagent was prepared by mixing 10.6 g of 3, 5 - Dinitrosalicylic acid and 19.8 g

NaOH in 1416 ml of distilled water. To this solution 306 g of Rochelle salt (Na – K tartarate), 8.3 g of Na metabisulfite and 7.6 ml of Phenol (melt at 50°C) were added. 3. Substrate: CarboxyMethyl Cellulose (1 %) 4. Blank consisted of 1 ml citrate buffer, 3 ml DNS Boiled for 5 min. 5. Enzyme blank consisted of 0.5 ml citrate buffer, 0.5 ml enzyme solution, 3 ml DNS boiled for 5 min. Procedure 1. Standard curve a. Different dilutions of glucose solution were prepared by mixing stock glucose solution (10 mg/ml) and Distilled water in the test tube. b. The range of glucose concentration was 2.0 to 6.7 mg/ml. c. To this 3 ml of DNS was added & mixed. d. This solution was boiled for exactly 5 min in a vigorously boiling water bath. e. Sample was diluted & OD was measured at 540 nm against the spectro zero. 2. Sample Analysis a. 0.5 ml enzyme was taken. b. To this 0.5 ml of substrate CMC was added. c. This solution was mixed well & incubated at 50ºC for 30 min. Same procedure was followed as for the standard curve.
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Sample digestion 0.5 g of dried sample were added to 10 ml of acetic nitric reagent and mouth of tubes were covered with marbles to avoid evaporation. Tubes were placed in boiling water bath for refluxing for 4-5 hours. Further tubes were centrifuged at 8000-10000 rpm for 10 minutes and supernatant was discarded. To the residue 10 ml of distilled water was added, again centrifuged as above and again supernatant was discarded. Same procedure was repeated for 2-3 times. 10 ml of 67% H2SO4 was added in two installments of 5 ml each by mixing well on vortex mixer. The reaction mixture was incubated at room temperature for 1 hour and diluted to 100 times for color measurement.

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RESULTS AND DISCUSSION

1. Biodegradation of Mixed Agricultural Waste (MAW) Biodegradation of Mixed Agricultural Waste (MAW) comprising of leaves of different plant/tree species i.e., Tylophora indica, Hevea brasiliensis, Eucalyptus, Polyalthia longofolia, Tectona grandis, Mangifera indica, Bamboosa vulgaris and grass cuttings, by microbes involving Bacillus sp. AS3 in combination with Trichoderma reesei (MTCC 164) and activators such as diammonium phosphate (DAP) was studied. The experiment was set by using tubs of diameter 14.6 inches and depth of 6.5 inches, in duplicates. One kilogram of untreated chopped Mixed Agricultural Waste (MAW) was put into each tub under shade condition. Chopping was done 2-3 times with grinding machine (Toka machine). Moisture was maintained to 40-50% in each tub and regular mixing of waste was done at an interval of 3-4 days. Four different treatments were given to Mixed Agricultural Waste (MAW) by using Bacillus sp. AS3, Trichoderma reesei (MTCC 164), and DAP along with control for biodegradation. Biomass was treated for a period of 90 days and samples were withdrawn at 15 days of interval from the tubs and analyzed for different parameters such pH, oxidisable carbon, and reducing sugar(figure 12.a.b.c.d). 2. pH Analysis of Mixed Agricultural Waste (MAW) indicated that pH was 6.86±0.4 at 0th day. There was a gradual increase in pH in all the treatments over a period of 90 days and it varied from 7.68±0.5 to 8.12±0.5. pH fluctuations permit cellulose hydrolysis to occur at pH values below those supporting growth of the cellulolytic population. Studies of leaf litter decomposition in aquatic
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environments have shown a clear inhibition of leaf litter mass removal under progressively acidic conditions (Chauvet and Merce´, 1988; Friberg et al., 1980; Mackay and Kersey, 1985). Inhibition of cellulose hydrolysis at low pH has also been observed in soil (Holub et al., 1993). Substantial cellulose hydrolysis occurs by ruminal bacteria at pH below 6.0, once the bacteria have adhered to cellulose, synthesizes a glycocalyx, and initiates bacterial growth at higher pH .

3.

Organic Carbon In all treatments gradual decrease in oxidisable carbon was observed over a period of 90

days (table 5, figure 10) .Mixed Agricultural Waste (MAW) as control had oxidisable carbon of 26.92% at 0 day of treatment, 6.68% decrease in oxidisable carbon was also observed in control. The maximum i.e. 11.94% decrease in oxidisable carbon was observed with treatment comprising of Bacillus sp. AS3 in combination with Trichoderma reesei (MTCC 164) followed by 10.67% decrease in treatment comprising of Trichoderma reesei (MTCC 164) along with activator diammonium phosphate Among various treatments biodegradation by co-inoculation of Bacillus sp.AS3 and Trichoderma reesei (MTCC 164), as judge by decrease in organic carbon percentage in 90 days was highest followed by treatment by Trichoderma reesei (MTCC 164) along with DAP (1 g/kg of wet biomass). DAP is used as a fertilizer and it positively helps in growth of fungi. Fast decomposition of rice straw by fungal inoculant (Trichoderma sp.) along with DAP, and then incorporation of its product (rice straw manure) into the soil may help to restore soil structure and fertility for paddy field . The organic carbon decreased with the progression of decomposition. As the decomposition progressed due to the losses of carbon mainly as carbon dioxide, the carbon content of the agricultural waste decreased with time (Goyal et al., 2005). Cellulose and hemicelluloses degradation occur during primary metabolism, whereas lignin degradation is a secondary metabolic event triggered by limitation of carbon, nitrogen, or sulfur.
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Table 5: % Organic Carbon of Treated MAW (Mixed Agricultural Waste) Material 0 day 15 days 30 days 45 days 60 days 75 days 90 days

Control

26.62 ± .91 26.36 ± .07

25.45 ±.7 24.07±.06 23.74 ± .07 20.99± .02 19.97 ± .11

MAW+ B.AS3

26.62 ± .91 25.95 ± .05 24 ± .03

22.8 ± .03 21.88 ±.14 19.39 ±.35 18.25 ± .35

MAW+B.AS3+ 26.62 ± .91 20.79 ± .27 18.92±.1 T.reesei

17.9 ± .03 17.02 ±.01 14.93 ±.27 14.17 ± .11

MAW+B.AS3+ 26.62 ± .91 24.61 ± .29 23.41±.57 20.45±.53 17.77 ± .21 16.72 ±.14 15.88 ± .10 T.reesei+DAP

Fig. 10. % Organic Carbon of MAW (Mixed Agricultural Waste) in different treatments at different time intervals

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C: Control; MAW: Mixed Agricultural Waste; B.AS3: Bacillus sp. AS3; T.reesei: Trichoderma reseei(MTCC 164); DAP: Diammonium phosphate.
4.

Reducing sugars Estimation of reducing sugars in all treatments showed gradual increase over a period of

90 days (table 6, figure 11). However after 15 days of treatment there was a slight decrease in sugar content and then onwards gradual increase in reducing sugar content was observed due to hydrolysis of cellulose. Mixed Agricultural Waste (MAW) as control had reducing sugar content 0f 240.3 ppm at 0 day of treatment, 14 ppm increases in reducing sugar content was also observed in control. The maximum i.e. 89.9 ppm increases in reducing sugar content was observed with treatment comprising of Bacillus sp. AS3 (EU 754025) in combination with Trichoderma reesei (MTCC 164) followed by 74.4 ppm increase in treatment comprising of Trichoderma reesei (MTCC 164) along with activator diammonium phosphate (Plate 1 . A/B/C/D) Enzymes are the main mediators of various degradative processes which help to degrade the nonstarch polysaccharides in substances to reducing sugars. Thus with the decrease in the amount of cellulose, a corresponding increase in reducing sugars was obtained. A. niger and T. reesei were able to bring the highest percentage in cellulose degradation due to its vigorous growth and therefore ability to produce more cellulolytic enzymes within the short period of time.

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Table 6: Reducing Sugar in PPM from 0 to 90 days
Material 0 day 15 days 30 days 45 days 60 days 75 days 90 days

Control

241.54±1.5 220.99±0.02 225.1±.03

231.55±.61

240.88±.14

252±1.16

254.75±.61

MAW+B.AS3

241.54±1.5 191.21±.53

227.79±.94 245.52±.57

262.585±2.27 273.13±0

276.97±.22

MAW + B.AS3+T.reese i MAW+B.AS3+ T.reesei+ DAP

241.54±1.5 150.05±.10 250.65±.67

283.04±.20

305.85±1.18

324.47±.49 330.9±1.1

241.54±1.5 162.25±.06 242.99±.02

272.10±.03

294.95±.09

311.97±.21 315.18±.75

350 Reducing sugar (ppm) 300 250 200 150 100 50 0 15 30 45 60 75 90 Time (days) MAW+B.sp. AS3+T.reesei MAW+B.sp. AS3+T.reesei+DA P C MAW+B.sp. AS3

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Fig. 11. Reducing sugar content (ppm) of mixed agricultural wastein different treatments in different time intervals

C: Control; MAW: Mixed Agricultural Waste; B.AS3: Bacillus sp. AS3; T.reesei: Trichoderma reseei(MTCC 164); DAP: Diammonium phosphate

Fig. 12. A. Raw Mixed Agricultural Mixed Agricultural Waste (0 day) 30 days)

Fig. 12. B. Raw Waste (after

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Fig. 12. C. Raw Mixed Agricultural Mixed Agricultural Waste (after 60 days) (after 90 days)

Fig. 12. D. Raw

Waste

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