BHAGWAN MAHAVIR COLLEGE OF M.Sc BIOTECHNOLOGY
AFFILIATED TO VEER NARMAD SOUTH GUJARAT UNIVERSITY UNIVERSITY)

ACKNOWLEDGEMENT
I thank the almighty whose blessings have enabled me to accomplish my dissertation work successfully.
I am very much thankful to all my professors and my co-guidance Mr. Naresh butani in our institute who made us work hard, taught us how to manage everything skillfully and made us into confident individuals.
It is my pride and privilege to express my sincere thanks and deep sense of gratitude to my
Project guidance Miss.Priya Bande , Department of Biotechnology and Environmental
Sciences, MITCON, pune for her valuable advice, splendid supervision and constant patience through which this work was able to take the shape in which it has been presented. It was her valuable discussions and endless endeavors through which I have gained a lot. Her constant encouragement and confidence-imbibing attitude has always been a moral support for me.
My sincere thanks to Miss.Neha Vora and Dr.Chandrashekharkulkarni
, Head Department of Biotechnology and Environmental Sciences, MITCON, pune for his immense concern throughout the project work.
I also wish to thank all my friends, for providing the mandatory scholastic inputs during my course venture.
Finally, I wish to extend a warm thanks to everybody involved directly or indirectly with my work.
The whole credit of my achievements goes to my parents and my brothers who were always there for me in my difficulties. It was their unshakable faith in me that has always helped me to proceed further.

DECLARATION
I hereby declared that the work presented in the Project entitled “PRODUCTION OF
DIFFERENT VALUE ADDED PRODUCTS FROM THE AGRICULTURAL WASTE BY
SOLID STATE FERMENTATION METHOD” has been carried out by SUTARIYA
KULDEEP VIRJI under the guidance of PriyaBande, Project Guide, at MITCON, Pune. The entitled Work is original and no partof this work is either published or submitted in any university for the award of any degree or diploma.
Date:
Place: Pune

CONTENTS
Sr.NO. PAGE.NO
1. Abstract
………………………………………………………….
2. Introduction …………………………………………………………
3. Materials & methods ……………………………………………….
4. Results & Discussion ……………………………………………….
5. Conclusions ………………………………………………………….
6. Reference …………………………………………………………..
7. Appendix-I Culture Medium ………………………………………
8. Appendix- II Stains And Reagents ……………………………….

“PRODUCTION OF DIFFERENT VALUE ADDED PRODUCTS FROM THE
AGRICULTURAL WASTE BY SOLID STATE FERMENTATION”
For the fruits processing industry the disposal of fresh fruits waste has become a major concern for many factories. Apple pomace, orange peels& pineapple waste are the major solid by product. These waste contain a high content of pectin, cellulose and hemi cellulose, which make it suitable as fermentation substrate when hydrolyzed.
Fruits processing residues contain both soluble and insoluble carbohydrates. The latter arepresent in the cell walls of the peels, particularly in the form of pectin, cellulose and hemicellulose. These polymers can be hydrolyzed enzymatic ally by cellulase, β-glucosidase andpectinase to their corresponding soluble carbohydrates.
Production of different value added products from fermentable sugars in fruit wastehydrolyzate is an alternative to utilize industrial citrus processing waste and avoids thedisposal-associated problems.
My project deals with the extraction of ethanol& animal feed from the thawed and ground apple pomacewaste as a substrate and SaccharomyceCerevisiae as the organism, production of cellulose enzyme using orange peel as substrates and Trichoderma spp.
as the organism by solid state fermentation.

India has diverse agro-climatic conditions that has enabled her to produce a wide variety of horticultural crops. Among this crops, apple occupies a prominent position in india. Apple processing industries are one of the major industries of Himachal Pradesh, jammu and
Kashmir, and Uttaranchal in India, manufacturing various products like juice, concentrates, wine, cidar, canned slics, etc. Apple pomace is left over residue after juice extraction containing peel, seeds and remaining solid pats and represent about 25-35% of the weight of the fresh apple processed. It is a waste and disposal is major environmental problem, but being a precious resource, its utilization is a challenge and opportunity to the scientists and technologist.
The rapid depleting non-renewable resources has already reached pinnacle. Now there has been an urgent need for a renewable, sustainable energy sources. In recent years ethanol had been a promising renewable source. Ethanol is earning increasing attention as a potentially cleaner, renewable, and domestically produced alternative to fossil fuels for transportation.
Hence ethanol production from different biological sources such as sugar cane, bagasse, miscanthus, sugar beet, sorghum, grain sorghum, switch grass, barley, hemp, kenaf, potatoes, sweet potatoes, cassava, sunflower, fruit, molasses, corn, Stover, grain, wheat, straw, cotton.
Production of ethanol starts with photosynthesis causing a feedstock, such as sugar cane or corn, to grow. These feed stocks are processed into ethanol.
The first generation of ethanol production used corn as a substrate, later corn was considered as a feedstock and so lead to the second generation of production of ethanol which used micro organisms and different wastes as substrates.

2.1 SOLID-STATE FERMENTATION
2.1.1 Definition
Solid-state fermentation, or solid-substrate fermentation (SSF), while difficult to define precisely, is generally referred to as the process in which microbial growth and product formation occur on the surface of solid materials in the absence or near-absence of free water, and the substrate contains certain moisture that exists in absorbed form within the solid matrix (E. Cannel and M. Moo-Young et al 1980, R. E. Mudgett et al 1986).
Solid-state fermentation deals with the utilization of water-insoluble materials for microbial growth and metabolic activities. It is different from surface culture, which uses either a solid or liquid substrate, and refers primarily to the mode of growth (M. Moo-Young, A. R.
Moreira, and R. P. Tengerdy et al 1983). It is also distinguished from submerged liquid fermentation/culture by the fact that microbial growth and product formation occur at or near the surfaces of solid materials with low moisture contents. Microbial activities cease at a certain low level of moisture content, and this establishes the lower limit at which solidstate fermentation can take place (M. Moo-Young, A. R. Moreira, and R. P. Tengerdy et al 1983).
The upper limit for solid-state fermentation is a function of absorbency of the medium which varies with the substrate material type (M. Moo-Young, A. R. Moreira, and R. P. Tengerdy et al. 1983).
Solid-state fermentations are not as well as characterized on a fundamental scientific or engineering basis as are the submerged liquid cultures that have been used almost exclusively in the West for the industrial production of microbial metabolites (R. E. Mudgett et al. 1986).
They are, however, widely used in the orient for thousands of years, and traditional methods used in food processing have been modernized and extended to nontraditional products (E.
Cannel and M. Moo-Young et al. 1980, R. E. Mudgett et al. 1986). Today solid-state fermentation is increasingly gaining interest in the world for development of value-added

products from a variety of cheap materials, and for bioremediation of agricultural and industrial wastes.
2.1.2 History
Solid-state fermentation has been used long before the underlying microbiological or biochemical processes involved were understood. The use of naturally occurring microorganisms in the preparation of foods such as bread and cheese, or directly as food such as mushrooms, dates back many centuries, and these are some examples of traditional solidstate fermentation systems (K. E. Aidoo, R. Hendry, and B. J. B. Wood et al. 1982). As early as 2600 B.C., Egyptians were making bread by methods essentially similar to those of today
(K. E. Aidoo, R. Hendry, and B. J. B. Wood et al. 1982). In Asia, cheese had been prepared as food for several hundred years before the birth of Christ. The preparation of koji for soy sauce and miso production in Japan and Southeast Asia goes back as far as 1000 years ago and probably 3000 years ago in China. Preservation offish, meat, and other animal products by solid-state fermentation goes back about 2500 years (K. E. Aidoo, R. Hendry, and B. J. B.
Wood et al. 1982). Vinegar was produced by solid-state fermentation from fruit pomace in the eighteenth century (A. Pandey et al. 1992). The production of gallic acid is another early example of solidstate fermentation, and its discovery was made in the eighteenth century (K.
E. Aidoo, R. Hendry, and B. J. B. Wood et al. 1982). Solid-state composting was used for sewage treatment in the late nineteenth century. The production of fungal and other microbial enzymes by solidstate fermentation started during the early twentieth century. The new fermentation introduced from 1920 to 1940 was the production of gluconic acid, citric acid, and enzymes, as well as the development of rotary drum fermenter (K. E. Aidoo, R. Hendry, and B. J. B. Wood et al. 1982,5). Between 1940 and 1950, the fermentation industry developed very rapidly, and the first clinically useful antibiotic, penicillin, was produced by

both solid-state fermentation and submerged culture method. During the decade from 1950 to
1960, steroid transformation by fungal spores, which was produced by solid-state fermentation, was developed. From 1960 to 1980, many important new solid-state fermentation processes were developed, which include the production of mycotoxins and the treatment and reuse of animal, plant, and domestic wastes (K. E. Aidoo, R. Hendry, and B. J.
B. Wood et al. 1982, A. Pandey et al. 1992). Since 1980 the developments of solid-state fermentation have been made in every aspect: expansion of microbial types, utilization of wastes as cheaper substrates, improvement of fermenter design, and discovery of new products. The history and recent developments in solid-state fermentation have been reviewed in detail by several authors.
2.1.3 Microbial Types
Although a wide range of microorganisms are able to grow on solid substrates, a relatively few genera and species are employed in the main commercial systems. Filamentous microorganisms are most widely used in solid-state fermentations. The ability of such microorganisms to colonize substrates by apical growth and penetration gives them a considerable ecological advantage over nonmotile bacteria and yeasts, which are less able to multiply and colonize on low-moisture substrates (E. Smith and K. E. Aidoo et al 1988).
Among the filamentous fungi, three classes have gained the most practical importance in
SSF: the phycomycetes such as the genera Mucor, andRhizopus, the ascomycetes with the genera Aspergillus and Penicillium, and the basidiomycetes, especially the white rot fungi such as edible mushrooms (M. Moo-Young, A. R. Moreira, and R. P. Tengerdy et al 1983).
Bacteria and yeasts usually grow on solid substrates at the 40 to 70% moisture levels and can play important roles in some solid-state fermentations (M. Moo-Young, A. R. Moreira, and
R. P. Tengerdy et al 1983, E. Smith and K. E. Aidoo et al 1988). In composting, thermophilic bacteria grow predominantly when the temperature exceeds 6O
0
C, while ensiling processes

are predominated by lactic acid bacteria (D. A. Mitchell 1992). In food fermentations, the best-known yeast genera such as Saccharomyces, Candida, and bacterial genera such as
Lactobacillus and Bacillus, have established their commercial roles (E. Smith and K. E.
Aidoo et al 1988).
Very few actinomycetes were used in solid-state fermentation (Jermini and Demain et al
1988) first developed a solidstate fermentation system for the production of cephalosporin antibiotics by Streptomyces clavuligerus grown on barley, and approximately 300 ug of cephalosporins per gram of substrate was produced under the optimal conditions for seven days (Sircar et al. 1998) recently optimized a solid-state fermentation medium for the production of clavulanic acid antibiotics by Streptomyces clavuligerus grown on a medium consisted of wheat rawa, soya flour, dipotassium hydrogen phosphate, and sunflower oil cake.
2.1.4 Factors Affecting SSF
Solid-state fermentations are in general carried out in fermenters of simple construction and operation and without the range of control units found in liquid fermentation systems.
Laboratory studies are usually carried out in conical flasks, beakers, Rowx bottles, jars, or glass incubators. For a pilot-plant or large-scale SSF, however, the design of bioreactors in batch or continuous mode has been empirical in nature (A. Pandey et al. 1991). For a solidstate fermentation with a selected microorganism, the success of the process will depend on culture condition with the controlled parameters including mainly nutrient supplements, moisture content, temperature, pH, aeration, and agitation.
Nutritional factors usually limit the growth of fungi. In solid-state fermentation, this limitation is much more severe due to the limited diffusion rate of the substrate and the limited access of the fungus to the substrate. An important indicator of nutritional regulation of growth in solid-state fermentation is the C/N ratio. Optimal C/N ratio varies in a wide

range from 10 to 100 or higher in various SSF processes, but the availability of C and N can be more important than the ratio. In most SSF systems, the C source comes from the natural starchy or cellulosic materials while the N source is added. The most commonly used synthetic N sources are NH4Cl, (NH4)SO4, NH4NO3 and urea, while some organic N sources such as soybean cake and yeast extract are often used in solid-state fermentation.
The moisture level of the substrate will have a determining influence on the success of the overall process. A better way of expressing moisture content is water activity (aw), which gives the availability of water for growth of microorganisms. In general, the types of the microorganisms that can grow in SSF systems are determined by the water activity. Most bacteria grow at higher aw values while filamentous fungi and some yeasts can grow at lower aw level. The microorganisms, which can grow and are capable of carrying out their metabolic activities at lower aw level, are suitable for SSF processes. Water activity level in
SSF is governed by the nature of substrate, the type of end product, and the requirement of microorganisms. A high aw level results in decreased porosity or intracellular spaces, lower oxygen diffusion, decreased gas exchange, enhanced formation of aerial mycelium, decreased substrate degradation and an increased risk of bacterial contamination. In contrast, low aw will lead to decreased substrate swelling and decreased microbial growth (B. K. Lonsane et al. 1985).
Temperature is another critical parameter that can affect SSF processes, since every microorganism has its own optimal temperatures for growth and for metabolism, which are often different. During solid-state fermentation, a large quantity of metabolic heat is generated and this is directly related to the metabolic activities of the microorganism (B. K.
Lonsane et al. 1985, B. K. Lonsane et al 1992). It is not difficult to control the temperature for solid-state fermentation in the laboratory; in large scale SSF, however, due to high substrate concentration, microbial heat generation is much greater per unit volume than in

liquid fermentation, and therefore, constant heat removal for temperature control is a major problem (B. K. Lonsane et al 1992).
Although pH is one of the critical factors, the monitoring and control of pH during solid-state fermentation is not usually attempted. It is difficult to monitor and control pH in the solid state fermentation, since pH electrodes able to measure the pH of the moist solids, in the absence of free water, are not available. On the other hand, good buffering capacity of some of the substrates used in SSF helps in eliminating the need for pH control during fermentation. This advantage is, therefore, exploited in the initial adjustment of the pH of the solids in the range of 4.4 to 5.0 during moistening by using water at the desired pH level.
Another approach in counteracting the acidification of the fermenting mass is the use of urea as the nitrogen source rather than ammonium salts (B. K. Lonsane et al. 1985).
For most solid-state fermentations, oxygen is essential and is usually achieved by free air exchange or forcing sterile air under pressure through the fermenting mass. The mechanism of O2 transfer from the circulating air into the aerobic microorganisms is unclear, but most probably it occurs via the O2 dissolved in the water film around the particle surface. Aeration not only provides oxygen but also simultaneously removes carbon dioxide, other volatile metabolites and heat from the fermenter (B. K. Lonsane et al 1992).
Agitation is not employed in many solid-state fermentation processes; however, it is usually an essential part of periodically or continuously agitated aerobic solid-state fermentations.
Agitation of the fermenting mass can ensure homogeneity with respect to temperature and gaseous environment and promote heat and gas transfers. It also helps for the uniform distribution of nutrients during the course of fermentation (B. K. Lonsane et al 1992).

2.1.5 Comparison to Liquid Fermentation
Solid-state fermentation has numerous advantages over the conventional stirred or aerated liquid fermentation both on a laboratory and large scale. First of all, the medium is relatively simple (K. E. Aidoo, R. Hendry, and B. J. B. Wood et al. 1982). For instance, cereal grain, legumes, or other plant and animal products, various agricultural and food processing wastes, are all potential substrates of solid-state fermentation. Usually the only other medium component required in SSF is water, although occasionally other nutrients such as nitrogen sources or minerals may be added (K. E. Aidoo, R. Hendry, and B. J. B. Wood et al. 1982).
Other advantages of solid-state fermentation over liquid fermentation include higher substrate concentration, less probability of contamination, superior productivity, improved product recovery, reduced energy and water requirements and wastewater output, and lower capital investment (K. E. Aidoo, R. Hendry, and B. J. B. Wood, P. E. Cook et al. 1994).
Despite solid-state fermentation being both economically and environmentally attractive, their biotechnological exploitation has been rather limited (A. Pandey et al. 1992). Compared with liquid fermentation, the major problems associated with solid-state fermentation include the limitations on microorganisms, medium heterogeneity, heat and mass transfer control, growth measurement and monitoring, and scale-up problems.
On the whole, SSF may be the exclusive tool, especially when demand for the product is limited, as is the case for some industrial enzymes. Furthermore, SSF has demonstrated a great potential in bioconversion of agricultural and industrial wastes into useful value-added products. In addition to obtaining a variety of value-added products, SSF system can also be used in environmental remediation of agricultural by-products.

2.1.6 Substrates
Both natural and synthetic substances can be used in solidstate fermentation. The main substrates for solid-state fermentation are insoluble in water, and water is absorbed into substrate particles that can be used by microorganisms for growth and metabolic activity.
Bacteria and yeasts grow on the surface of the substrate while fungal mycelia penetrate into the particles of the substrate (A. Pandey et al. 1992). The substrates used are, with the exception of synthetic media, usually cheap agricultural raw materials and its byproducts (A.
Pandey et al. 1991).
The most widely used substrates for solid-state fermentation in practice are mainly materials of plant origin, which include starchy materials such as grains, rice, corn, roots, tubers and legumes, and cellulosic, lignin, proteins, and lipid materials (J. E. Smith and K. E. Aidoo et al. 1988). These organic materials in nature are mixtures of polymeric compounds in structure, which act as a source of carbon, nitrogen, and other nutrients as well as providing anchorage for the microorganisms (A. Pandey et al. 1992). Recent interest in solid-state fermentation has put more weight on cheaper substrates, including various agricultural or agroindustrial by-products. Agricultural and food processing wastes such as wheat bran, cassava, sugar beet pulp, bagasse, citrus peel, corn cob, banana waste, sawdust, wheat and rice straw, and fruit pomace are the most commonly used substrates for solid-state fermentation. Such by-products are usually lignocellulosic and pectinrich wastes in nature and have been used in SSF to produce many value-added products.
2.2 FRUIT PROCESSING WASTES
2.2.1 Apple Pomace
Apple pomace is the residual left after juice extraction and represents about 25% of the original fruit. It is rich in carbohydrate but low in protein and fat contents. The freshly pressed apple pomace has a low pH ranging from 3.1 to 3.8, with a bulk density of 935 kg/m3

(Y. D. Hang et al. 1985, W. J. Jewell and R. J. Cummings et al 1984). Since it is produced wet with high moisture content, it is susceptible to rapid growth of microorganisms. The major composition of apple pomace is shown in Table 1.
It is estimated that nearly 36 million tons of apples are produced annually in the United
States, and approximately 45% of which is used for processing purposes, with a primary by product of apple pomace, which results from processing for juice, cider, applesauce, or slices
(Y. D. Hang et al. 1985). More than 500 apple-processing plants in the United States generate a total of about 1.3 million metric tons of apple pomace each year, and the annual disposal fees for apple pomace alone exceed $10 million in the United States (W. J. Jewell and R. J.
Cummings et al 1984, K. J. Carson, J. L. Collins 1994). The high level of organic contents in apple pomace will result in environmental pollution if they are disposed directly to the environment.
Table 1: Proximate composition of apple pomace (Joshi VK et al. 1998 , Parmar Mukesh et al.2003
, Joshi VK, Pandey A and Sandhu Dk 1999 , Joshi VK and Sandhu DK et al.
1996).
Constituents
Composition
Wet weight basis Dry weight basis
Moisture (%)
Acidity (% malic acid)
Total Soluble Solids(TSS o
B)
Total Carbohydrate (%)
Glucose
Fructose
Sucrose
Xylose
66.4-78.2
NA
NA
9.50-22.0
6.10
13.60
NA
NA
3.97-5.40
2.54-3.28
57.85
48.00-62.00
22.70
23.60
1.80
0.06

pH
Vitamin C (mg/100g)
Soluble Proteins (%)
Protein (%)
Cruid fiber (%)
Fat [ether extract (%)]
Pectin (%)
Ash(%) NA
Polyphenol
Amino acids
Minerals:
Potassium (%)
Calcium (%)
Sodium (%)
Magnesium (%)
Copper (mg/l)
Zinc (mg/l)
Manganese (mgl)
Iron (mg/l)
Calorific value (Kcal/100g)
NA= Not Applicable
3.05-3.80
-
NA
1.03-1.82
4.30-10.50
0.82-1.43
1.50-2.50
1.60
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
0.95
0.06
0.20
0.02
1.10
15.00
8.50-9.00
230.00
295.00
3.90
8.53-18.50
3.29
4.45-5.67
4.70-48.72
3.49-3.90
3.50-14.32
0.99
1.52

2.2.2
Other Fruit and Vegetable Processing Wastes
The food processing industry in the United States generates large amount of various byproducts as wastes each year, a large portion of which comes from fruit and vegetable processing industries. It is estimated that approximately 9 million tons of solid residuals are generated annually from the fruit and vegetable processing industry in the United States (Y.
D. Hang et al. 1979). In addition to apple pomace, other major fruit and vegetable processing wastes come from citrus, grape, cherries, berries, banana, olive, peach, pear, pineapple, kiwifruit, apricot, sweet corn, tomato, potato, asparagus, beans, peas, carrots, beet, pumpkin, squash, spinach, and sauerkraut processing industries (Y. D. Hang et al. 1979-23). The primary sources of solid residues resulting from fruit and vegetable processing operations include sorting, cutting, slicing, peeling, pulping, and pressing (Y. D. Hang et al. 1979).
Traditionally about 79% of the fruit and vegetable processing wastes is used for animal feed, and the remaining 21% is handled as waste (Y. D. Hang et al. 1979). The solid wastes contain mainly soluble sugar and other hydrolyzable materials with small amounts of crude fiber.
Disposal of such wastes may present an added cost to processors, and direct disposal to soil or in a landfill may not continue to be acceptable. Thus, exploration of potential value-added uses for pomace is highly attractive.
2.3 Products
Several products are generated from solid-state fermentation. Some products are directly used as foods. For example, many kinds of higher fungi, edible mushrooms, have been cultivated and used as human food for centuries in China and Japan. They usually grow on agricultural residues, such as wheat straw, wood sawdust, grasses, horse manure, cotton seed crust, and fruit pomaces (E. Moyson and H. Verachtert et al. 1995, S. Ohga and Y. Kitamoto et al.
1997). In ancient time, the bread was made through natural solidstate fermentation by indigenous microorganisms. Today people use the pure culture of baker's yeast to produce

bread (G. Reed et al. 1982). Roquefort and Camembert represent the two important types of mold-fermented cheese. Penicillium roqueforti is the blue mold of Roquefort cheese while
Penicillium camemberti is the white mold of Camembert (C. S. Pederson et al. 1979).
Penicillium species and Mucor species are often used in fermented sausage production (M. D.
Selgas et al 1995, F. K. Lucke et al.1994).
Among many oriental fermented products, soy sauce is probably the most well known in the diet of Western countries (H. L. Wang and C. W. Hesseltine et al. 1982). Soy sauce is made from soybeans and wheat flour fermented by molds. The main microorganisms include
Aspergillus oryzae (koji) and yeasts and lactic acid bacteria. Tempeh, a vegetarian meat analogue and source of vitamin B-12 (generally lacking in vegetarian diets), is yet another product made in the orient by solidstate fermentation (S. Keuth and B. Bisping et al. 1994).
Solid-state fermentation can be used not only as a tool for nutritional enrichment but also as a means of reducing toxins in raw substrates. It was reported that the tempeh mold Rhizopus oligosporus could decrease the aflatoxin content of peanut presscake by 70% during fermentation (A. G. Van-Veen, D. C. W. Graham, and K. H. Steinkraus et al. 1968). A significant reduction in antinutritional and toxic components in breads and other plantderived foods by solid-state fermentation was observed (N. R. Reddy et al. 1994). Biomass production is another important aspect of bioconversion from wastes to value-added feed or food through solid-state processes (A. Noomhorm, S. Ilangantileke, and M. B. Bautista et al.
1992). A recent study shows that carotenoid could be produced from cornmeal by solid-state fermentation using Penicillium species, with a maximum yield of 5.26 mg of carotenoid per
100 g of substrate (M. Gutierrez-Rojas et al. 1996).
Solid-state fermentation has been used for producing several industrially important products today. Production of many kinds of important organic acids has been successfully achieved via solid-state fermentation. Such organic acids include citric acid, lactic acid, acetic acid,

gluconic acid, glutamic acid, linolenic acid, and other fatty acids, and phenolics such as ferulic acid. In addition, Hesseltine (C. W Hesseltine et al. 1972) used rice as the substrate to produce aflatoxin by Aspergillus flavus and Aspergillus parasiticus via solid-state fermentation.
The best potential application of solid-state fermentation, however, is the production of various industrial enzymes such as amylases, proteases, cellulases, pectinases, rennet, invertase, lactase, peroxidase, and so on. The production of cellulase, a-amylase and β glucosidase from agricultural by-products by solid-state fermentation was investigated by
Zheng et al. 1972, 1993. Glucoamylases were produced by solid-state fermentation from cassava starch using Rhizopus species or corn flour and wheat bran. A thermostable a amylase was produced by thermophilic Bacillus coagulans in solid-state fermentation. A thermostable a-L-arabinofuranosidase was produced by solid-state fermentation with
Thermoascus aurantiacus on sugar beet pulp. a-Galactosidase was formed by As pergillus niger on wheat and rice bran-based solid-state medium. A novel carbohydrase complex was produced from solid-state fermentation by the aerobic fungus Penicillium capsulatum . A protease was produced from wheat bran by Rhizopus oryzae, with a maximum enzyme activity of 341 units per gram of wheat bran. A lipase was produced by solid-state fermentation using gingelly oil cake as the substrate from A. niger, and the enzyme activity of
363.6 units per gram of dry substrate was obtained under optimal conditions (N. R. Kamini, J.
G. S. Mala, and R. Puvanakrishnan et al. 1998).
Numerous products were produced by solid-state fermentation from different fruit processing wastes, and they will be discussed below in this article.
2.3.1 Ethanol
In general, ethanol is produced from a liquid substrate by a culture of Saccharomyces cerevisiae. Because of its physical nature, apple pomace is not readily amenable to

submerged yeast fermentation. A solid-state fermentation system for the production of ethanol from apple pomace with a strain of S. cerevisiae was devised. The yield of ethanol varied from about 29 g to more than 40 g per kilogram of apple pomace fermented at 3O
0
C in
24 h, depending on the samples fermented; the fermentation efficiency of this process was approximately 89%. The spent pomace resulting from the separation of alcohol should be a better animal feed supplement, because its protein content was enriched by the yeast biomass.
A novel process for concomitant production of ethanol and animal feed from apple pomace by solid-state fermentation was developed. Ethanol production from other fruit processing wastes such as corn fiber, carob, and banana waste has also been reported. Gupta et al. 1990 further studied the effect of nutrition variables on solid-state alcoholic fermentation of apple pomace by several strains of yeasts and improved the fermentation efficiency by adding various phosphates, nitrogen sources, or trace elements. Ngadi and Correia 1992 studied the effect of factors such as moisture and bioreactor mixing speed on the ethanol production from apple pomace. They even proposed a mathematical model to describe the kinetics of solidstate ethanol fermentation from apple pomace and suggested a logistic function of cell growth and ethanol production during solid-state fermentation.
2.3.1.1 Properties Of Ethanol
Molecular formula
Molar mass
Exact mass
Appearance
Density
Properties
C
2
H
6
O
46.07 g mol
−1
46.041864814 g mol
−1
Colorless liquid
0.789 g/cm
3

Melting point
Boiling point
−114 °C, 159 K, -173 °F
78 °C, 351 K, 172 °F
5.95 kPa (at 20 °C)
Vapor pressure
Acidity (p K a
) 15.9
-1.9 Basicity (p K b
)
Refractive index ( n
D
) 1.36
0.0012 Pa s (at 20 °C)
Viscosity
Dipole moment 1.69 D
2.3.1.2 Yeast Biology
Characteristics of Saccharomyces

Eukaryote: possesses a membrane bound nucleus

Reproduces by budding

Grows vegetatively as haploid (1N) or diploid (2N)

Capable of conjugation (1N to 2N) and sporulation (2N to 1N)

Non-motile
Saccharomyces is a Eukaryote
Saccharomyces is a member of the kingdom of fungi. Fungi possess plant-like cell walls, but have other features more in common with animals. A significant amount of information is known about Saccharomyces due to the utility of this organism as an experimental system.
Many of the fundamentals of genetic inheritance in eukaryotic cells were initially identified and studied in this yeast. The fungi are eukaryotic organisms meaning that they possess a membrane bound nucleus . The nucleus has a double membrane structure. The outer membrane is contiguous with an organelle known as the endoplasmic reticulum. The

endoplasmic reticulum (ER) is involved in secretion of extracellular proteins and in de novo biosynthesis of the plasma membrane.
Microscopic Morphology of Saccharomyces cerevisiae
2.3.1.3 Glycolytic Pathway
The universal biochemical pathway by which sugars are degraded in an energyyielding process to the three carbon compound pyruvate is called glycolysis. This pathway is found throughout the plant, animal, fungal, bacterial and archae kingdoms. Energy is generated in the form of ATP via a process called substrate level phosphorylation.
Glycolysis
The set of biochemical reactions converting hexose (6 carbon) sugars to two 3 carbon pyruvate molecules, during which energy is released and recaptured in the form of ATP. We can think of glycolysis as a process rearranging the energy in the bonds of a sugar molecule, so that a high-energy bond is formed that can then transfer that energy in a conservative manner to ADP generating ATP, the universal energy source. The energy in the ATP bond can be used to drive energetically unfavorable reactions.
Glycolysis
Glucose + 2ATP + 2NAD + + 2ADP + 2Pi
2Pyruvate + 4 ATP + 2NADH + heat

This process requires the cofactor NAD+ that is converted to the reduced form NADH. Heat is also given off as an end product of glycolysis. One sugar molecule plus two ATP and
2ADP molecules are converted into 2 pyruvate and 4 ATP molecules. Early steps in the glycolytic pathway consume ATP. The first reaction is a phosphorylation of glucose (or fructose) at the six position.
2.3.1.4 Where does ethanol come from?
The end products of glycolysis are pyruvate and 2 molecules of the reduced co-factor NADH.
Yeast cells regenerate NAD+ by transferring the hydrogen molecule (electron) to an organic molecule: acetaldehyde.
In the alcoholic fermentation, pyruvate is decarboxylated to acetaldehyde, which is then reduced to ethanol with concomitant formation of NADH.
Ethanol Formation
CH
3
-CO-COOH Pyruvate
CO
2
CH
3
-CHO Acetaldehyde
NADH
NAD +
CH
3
-CH
2
OH Ethanol
Carbon dioxide is also an end product of glycolytic metabolism. Thus, the 6-carbon sugars are converted into 2 one-carbon molecules of carbon dioxide and 2 of the twocarbon molecule ethanol. Other organisms use other means to regenerate NAD+. Some reduce pyruvate directly to lactic acid.

2.3.1.5 Ethanol Yield:
Ethanol Yield
1 M Glucose (Fructose)
2M CO2 + 2 M Ethanol
Theoratical Maximum:
180g 2(46g) = 92/180 = 51.1% w/w
= 63.9% v/w = ~0.6 Original Brix value
2.3.1.6
Uses of Ethanol
Ethanol is used:
 in the manufacture of alcoholic drinks, e.g. Vodka, etc.,
 as a widely used solvent for paint, varnish and drugs,
 in the manufacture of ethanal, (i.e. acetaldehyde), and ethanoic acid, (i.e. acetic acid),
 as a fuel (e.g. in Gasahol),
 as the fluid in thermometers, and
 in preserving biological specimens
2.3.2 Animal Feed
Apple pomace was directly used as a feed for cows and sheep, but its value as an animal feed is less than $7 per ton (W. J. Jewell and R. J. Cummings et al. 1984). Apple pomace is a poor animal feed supplement because of its low protein content. The nutritional value of apple pomace was improved by solid-state fermentation with a food yeast, Candida utilis . Yeast fermentation resulted in a 2.5-fold increase in protein, 3.4-fold increase in niacin, 10-fold

increase in pantothenic acid, 1.5-fold increase in riboflavin, and 1.2-fold increase in thiamine.
An improved stock feed was produced from apple pomace by solid-state fermentation with
KLoeckera apiculate and C. utilis .
A solid-state fermentation of orange processing wastes with Aspergillus niger and Rhizopus species enriched the protein content by 300%, and the fermented product could be readily sold as animal feed. Banana wastes were also used for protein enrichment and protein and biomass production by solid-state fermentation using A. niger , yeast Pichia spartinae , and
Saccharomyces uvarum (O. Enwefa et al. 1991).
2.3.3 Cellulase
Cellulase refers to a class of enzymes produced chiefly by fungi, bacteria, and protozoans that catalyze cellulolysis (i.e. the hydrolysis of cellulose). However, there are also cellulases produced by a few other types of organisms, such as some termites and the microbial intestinal symbionts of other termites. Several different kinds of cellulases are known, which differ structurally and mechanistically. The EC number for this group of enzymes is EC
3.2.1.4.
2.3.3.1 Reaction
Hydrolysis of 1,4-beta-D-glycosidic linkages in cellulose, lichenin and cereal beta-D-glucans.
2.3.3.2 Types and action
Five general types of cellulases based on the type of reaction catalyzed:
 Endocellulase breaks internal bonds to disrupt the crystalline structure of cellulose and expose individual cellulose polysaccharide chains
 Exocellulase cleaves two to four units from the ends of the exposed chains produced by endocellulase, resulting in the tetrasaccharides or disaccharides, such as cellobiose.

There are two main types of exocellulases [or cellobiohydrolases (CBH)] - CBHI works processively from the reducing end, and CBHII works processively from the nonreducing end of cellulose.
 Cellobiase or beta-glucosidase hydrolyses the exocellulase product into individual monosaccharides.
 Oxidative cellulases depolymerize cellulose by radical reactions, as for instance cellobiose dehydrogenase (acceptor).
 Cellulose phosphorylases depolymerize cellulose using phosphates instead of water.
2.3.3.3 Mechanism of cellulolysis
2.3.3.4 Role of Cellulase in various industries
Cellulases have a wide range of enormous potential applications in biotechnology and many thermo stable endoglucanase appeared to have a great potentiality for industrial use
(Karmakar and Ray, 2010a). In most of the cases they are used with hemicelluloses, pectinases, ligninase and allied enzymes. Some of the most important applications of cellulases are in food, brewery and wine, animal feed, textile and laundry, pulp and paper

industries, as well as in agriculture and for research purposes. Details of most promising applications are discussed below.

Cellulase in food processing industries:
Enzyme infusion has the potential of producing fruit and vegetable juices which is very important from commercial standpoint. The production of fruit and vegetable juices requires methods for extraction, clarification and stabilization. During early
1930s, when fruit industries began to produce juice, the yields were low and many difficulties were encountered in filtering the juice to an acceptable clarity (Uhlig,
1998). Subsequently, research on industrially suitable macerating enzymes from foodgrade micro-organisms ( Aspergillus niger and Trichoderma sp.), together with increased knowledge on fruit components, helped to overcome these difficulties.
During the production of juice from fruits such as apples and pears, the whole fruits were crushed to pulp mash, which, after mechanical processing (pressing, centrifuging and filtering), resulted into a clear fruit juice and a solid phase called pomace. Application of macerating enzymes could increase both production and process performance without additional capital investment. Macerating enzymes are generally used after crushing, to macerate the fruit pulp for partial or complete liquifaction, which increases the juice yield, reduces the processing time and improves the extraction of valuable fruit components. Thus, the macerating enzymes, composed of mainly cellulase and pectinase play a key role in food biotechnology and their demand will likely increase for extraction of juice from a wide range of fruits and vegetables including olive oil extraction, that has attracted the world market because of its numerous health claims. In addition, the infusion of pectinases and bglucosidases increases the aroma and volatile characteristics of specific fruits and vegetables (Humpf and Schrier, 1991; Marlatt et al ., 1992; Pabst et al ., 1991).


Cellulase in pharmaceutical industries:
Since, humans poorly digest cellulose fiber, taking a digestive enzyme product, like
Digestin, that contains cellulase enzymes is not only necessary, but also vital for healthy cells.

Cellulase in brewery and wine industries:
Bioconversion of cellulosic materials to bioalcohol involves a multistep process which first uses cellulolytic enzymes for hydrolysis of polymers to pentose/hexose sugars and fermentation followed by distillation of these sugars into ethanol. In the beer wort production, Pajunen (1986) opined that the enzyme preparation from
Trichoderma was the best as judged by its cost /performance ratio.

Cellulase in textile industries:
The textile industries take advantage of both complete and individual cellulase components to achieve partial cellulose hydrolysis and improve fabric properties, where the cellulase would act upon the fibre to reduce the cell wall thickness and would make the fibre more flexible and collapsible.

Cellulase in detergent industries:
Use of cellulase along with protease and lipase is a more recent innovation (Singh et al ., 2007). Removal of oil from interfibre space by selective contraction of fibres by the alkaline cellulase increases the cleansing capacity of a detergent. Nowadays, liquid laundry detergent containing anionic or nonionic surfactant, citric acid or a water-soluble salt, proteolytic enzyme, cellulase and a mixture of 1,2 propane diol and boric acid or its derivative. The compositions are prepared by adding the diol and boric acid before adding the citric acid/salt to the composition. This order of addition improves the stability of the cellulase. As most of the cellulose fibres in the modern textile industry enzymes are used increasingly in the finishing of fabrics and clothes

are arranged as long, straight chains some small fibres can protrude from the yarn or fabric. The correct application of a cellulase enzyme can remove these rough protuberances giving a smoother, glossier brighter colored fabric. This technique has become known biopolishing and results in not only a softer fabric but also improved color brightness. This process of washing has been adapted and included in some laundry detergents.

Application of cellulase in animal feed:
Cellulases have potential application in animal feed industry consumed by poultry, pigs, ruminants as well as pet and fish farming. In todays world there is a great deal of interest in using enzyme preparations containing high levels of cellulase and hemicellulase activities for improving the feed utilization , milk yield and body weight gain by ruminants. Nevertheless, the successful use of these enzymes in animal diet is to: eliminate Anti-Nutritional Factors (ANF) present in grains or vegetables; degrade certain cereal components in order to improve the nutritional value of feed; and/or to supplement animals’ own digestive enzymes (e.g., proteases, amylases and glucanases). Moreover, Cellulases and hemicellulases are responsible for partial hydrolysis of lignocellulosic materials, dehulling of cereal grains, hydrolysis of b-glucans and better emulsification and flexibility of feed materials which results in the improvement in the nutritional quality of animal feed.
Cellulases, hemicellulases and pectinases can cause partial hydrolysis of plant cell wall during silage and fodder preservation. They are responsible for the expression of preferred genes in ruminant and monogastric animals for high feed conversion efficiency. These commercially important enzymes can produce and preserve high quality fodder for ruminants; improving the quality of grass silage (Ali et al ., 1995;
Hall et al ., 1993; Selmer-Olsen et al ., 1993).


Application of cellulases in research and development:
Mixture of different cellulase along with hemi-cellulase and pectinase have immense potential and application in research and development area for controlling plant diseases and enhancing plant growth. A cocktail of different cellullases,hemicellulases and pectinases results in the solubilisation of fungal or plant cell wall to produce protoplast. Cellulases and related enzymes are used in the biocontrol of plant pathogens and different plant diseases by inhibiting the germination of spores of the plant pathogens. Even the cellobiohydrolase promoters of
Tricoderma is used for the expression of the different proteins, enzymes, antibodies in large amount. These are some of the main cellulase application in the research area
(Penttila, 1998).

Application in waste utilization:
Cellulose is the major part of plant biomass. Therefore, the wastes generated from forests, agricultural fields and agro industries contain a large amount of unutilized or underutilized cellulose. Agricultural and industrial wastes are among the causes of environmental pollution) These wastes generally accumulate in the environment causing pollution problem
Nowadays, these so called wastes are judiciously converted into valuable products such as enzymes Sugar, biofuels, chemicals, cheap energy sources for fermentation, improved animal feeds and human nutrients (Howard et al ., 2003), which is accomplished by cellulase. Therefore, the discarded biomass and agrowastes are successfully utilized for the production of enzymes, sugar and alcohols (Karmakar and Ray, 2010b; Omosajola et al ., 2008).

2.3.4 Pectin Esterase
Pectinesterase (PE) (EC 3.1.1.11) is a ubiquitous cell-wall-associated enzyme that presents several isoforms that facilitate plant cell wall modification and subsequent breakdown. It is found in all higher plants as well as in some bacteria and fungi. Pectinesterase functions primarily by altering the localised pH of the cell wall resulting in alterations in cell wall integrity.
Pectinesterase catalyses the de-esterification of pectin into pectate and methanol. Pectin is one of the main components of the plant cell wall. In plants, pectinesterase plays an important role in cell wall metabolism during fruit ripening. In plant bacterial pathogens such as
Erwinia carotovora and in fungal pathogens such as Aspergillus niger , pectinesterase is involved in maceration and soft-rotting of plant tissue. Plant pectinesterases are regulated by pectinesterase inhibitors, which are ineffective against microbial enzymes (Giovane A,
Tsernoglou D, Camardella L, Di Matteo A, Raiola A, Bonivento D, De Lorenzo G, Cervone
F, Bellincampi D (2005).
Function
Application
Production using gene technology
Labelling
Splitting of pectins
Fruit juice, preparation of vegetables and fruits widespread no
2.3.4.1 Function
Pectinesterases belong to the enzyme group of pectinases. They "crack" a very specific bond in molecules of pectin, which is a structural substance in the cell walls of plants.

2.3.4.2 Application
Similarly to pectinases, pectin esterases also are used primarily in the preparation of fruit and vegetables:
 in fruit juice: increasing the juice yield and clarifying juices through the elimination of lees
 in the production of concentrates from fruits and vegetables (carefully heated masses of uncooked plants or their parts), for example with tomatoes, onions, carrots, paprika or celery and also with plums, buckthorn and rosehips. These concentrates are used as an ingredient of various products
 also in the production of colouring extracts and food colour from plant-based raw materials
 feed additives

• Petri dishes,
• Glass slides,
•
Glass beakers,
•
Cover slips,
• Media bottles,
•
Conical flasks,
•
Pipette,
• Media bottles,
•
Test tubes,
•
Distillation unit
•
Alcohol,
•
Distilled water,
• Lacto phenol blue
•
Ethanol
•
DNSA
• Sodium sulphite
•
Concentric sulfuric acid
•
Potassium dichromate
• Potassium iodide
•
Sodium thiosulphate
•
Starch

•
Acetate buffer
• Ninhydrin reagent
• BSA(bovine serum albumin)
•
Brad ford reagent

All the instruments which were used are mentioned in the following table with their respective manufacturing company.
Table-3 Laboratory Instruments and Manufacturers
SN.
1.
2.
3.
INSTRUMENT
Weighing Balance pH meter
Autoclave
6.
7.
4.
5.
Hot Air oven
Laminar Air Flow
Incubator
Shaker Water Bath
8. Orbital Shaking incubator
9. Magnetic stirrer
10 Microscope
MANUFACTURED BY
Atco
Controlled Dynamics
Medica
Meca instrument
Micro-Filt [INDIA]
Meca instrument
Quality
Remi instruments
Remi equipments
Lebomed
11. UV visible spectophoto meter Milton Roy Company
12. Refrigerator LG,samsung
13. Centrifuge Remi

3.2.1 Substrate
Waste apples collected from local market or fruit processing centrewere used for ethanol.
Apples were cutinto slices in order to ensure rapid extraction of juice in a juice press. Apple juice was filtered and pretreated with HCl and H2SO4 to maintain the pH, and steamed at
90oC for 20 minutes in water bath (Campos et al ., 2002). Juice sample was filled in jars
(capacity 1.5 liters) andwas preserved at 4°C to prevent any possible degradation or spoilage during storage.
3.2.2 Microorganism
The Saccharomyces cerevisiae was used in the experiments because of its ability to ferment sugar. The commercial bakery yeast ( Saccharomyces cerevisiae ) was obtained from the laboratory,Mitcon. It was incubated in GYE-broth (50 ml) and incubated at 30ºC for 24h for refreshment. After that it served as the starter culture for ethanol production. The cultures were screened for maximum production of ethanol.
PREPARATION OF INOCULUM
Inoculum were used in order to obtain maximum production of ethanol and best inoculum was used for further studies. Inoculum was prepared by using 3days old slants that were transferred to
250mL flasks, containing 50 mL of a medium composed of GYE broth medium. The flasks were incubated in shaker at 32°C for 24h at 120 rpm.

3.2.4
Apple fruit (sorted for defects)
Dilute 0.05 HCL
Washing pressing
POMACE
In a Grater
Apple juice
4 days, 25+o C
Ammonium sulphate (1.0%)
Yeast Culture @ 5%
Sachharomyce Cerevisiae
Vaccum distillation (70 o C)
Left over Fermented Apple Pomace
Packaging (polythene bag)
Ethanol

3.2.5 Ethanol recovery by vacuum distillation method
Hot water at 50 ºC was added to the fermented apple pomace of the ratio (1:1). The material was allowed to stand for 30 min and was transferred to vacuum distilation flask which was kept on a hot plate. The temperature was maintained constantly at 70 ºC.
3.2.6 Drying of apple pomace residue
The fermented apple pomace was dried in a mechanical dehydrator at 60+-1 ºC for 8h and thereafter,ground into powder. It was packed in polythene bags. The dried apple pomace was analysed for different parameters. VIZ.proteins (cruide and soluble) and total sugars.
3.2.7 ANALYTICAL METHOD:
The estimation of the ethanol present in the fermentative media was performed by potassiumdichromate method. An accurately measured amount of potassium dichromate in
H2SO4 is added to the solution of alcohol. Reacting the sample with an excess of potassium dichromate, all ethanol is oxidized to acetic acid:
C2H5OH + [O] → CH3COOH
When alcohol vapor makes contact with the orange potassium dichromate solution, the color changes from orange to green. The degree of the color change is directly related to the level of alcohol in the sample.

Standard solution of 10 mg/ml prepared then different concentrations of Ethanol of
10-40 mg/tube (1-4 ml) and make up the volume to 5ml with distilled water.

Then 1 ml of Potassiumdichromate reagent was added. All the test tubes were kept in dark condition for 30 minutes.

Then add 4 ml of 20% KI in each of the flask and mix its contents well.

Titrate the librated iodine with 0.1N Na2S2O3 solution.


When most of the iodine has reacted as indicated by the solution acquiring a yellowish green color, add few drops of 1% starch solution, mix it well, the color should change to blue.

Continue the addition of thiosulfate solution dropwise and swirling the solution constantly, until 1 drop changes the color from greenish blue to light green.

Note the amount of Na2S2O3 requured to bring the color change consider aliquote as blank(B) while othe as experiment(E).

Calculate B-E for each aliquote.

Plot this on standard graph and find out the concentration in our unknown sample.
3.2.8 Physico-chemical analysis of dried apple pomace
In order to quantify different nutrition value after solid state fermentation of apple pomace, following experiments were conducted:
Reducing Sugars Content (DNSA METHOD)
1.
Reducing sugars analysis was conducted by using 2 ml of sample which was added to
3 ml of DNS and boiled for 15 min.
2.
After cooling, 6 ml of Distilled water was added to stabilize the color.
3.
The absorbance was recorded at 540 nm using a spectrophotometer against the blank of distilled water.
3.2.8.2 Total protein content (BRADFORD METHOD)
5mg albumin/ml was used as protein standard
Reagents used : Badford reagent (Annexure III)

Firstly standard curve with different concentrations of bovine albumin serum (BSA) namely-1-4 was made.

For analysis of total protein, set 5 ml volume with distilled water.


Then 1ml of bradford reagent was added to all tubes.

The tubes were then mix properly and absorbance was noted at 595 nm.
3.2.8.3 Total ash content
Material:

Oven

Scale

Desiccator

Crudible(platinumor silica)

2 ‐ 4g of the groundair driedmaterialweighedaccuratelyin a prviouslyingnitedand taredcrucible

Ingnitethe materialby graduallyincreasingthe heat500 ‐ 600oC untilitiswhite

Cool in a desiccatorand weigh
3.2.8.4 Amino acid
Reagents required

Standard amino acid stock solution (150 micrograms of Standard amino acid stock solution (100µg/ml).

0.2M Acetate buffer (pH=5.5).

O.8% w/v of Ninhydrin reagent [ Preparation: weigh 8g of ninhydrin and dissolve in
100ml of acetone].

50% v/v ethanol.

Distilled water.
Apparatus and Glasswares required:

Test / Boiling tubes.

Pipettes [glass / micropipette].

Waterbath.


Visible UV spectrophotometer
Quantitative estimation of amino acid- The Experiment:
1.
Pipette out different volumes(0.1ml-1ml) of standard amino acid solution to the respective labelled test tubes.
2.
Add distilled water in all the test tubes to make up the volume to 4ml.
3.
Add 4ml of distilled water to the test tube labelled Blank.
4.
Now add 1ml of ninhydrin reagent to all the test tubes including the test tubes labelled
'blank' and 'unknown'.
5.
Mix the contents of the tubes by vortexing /shaking the tubes.
6.
Put a few marble chips in each tube.
7.
Cover the mouth of the tubes with aluminium foil.
8.
Place all the test tubes in boiling water bath for 15 minutes.
9.
Cool the test tubes in cold water and add 1ml of ethanol to each test tube and mix well.
10.
Now record the absorbance at 570 nm of each solution using a colorimeter.
3.3 METHODS OF PRODUCING CELLULASE (ORANGE PEELS)
The process of producing cellulase from the orange peel waste can be explained in three parts.
They are
1.Pretreatment
2.Saccharification and fermentation
3.Estimation

3.3.1 Pretreatment
The pretreatment has been the most important part in the fermentation of the orange peels.
The main obstacle to fermentation of orange peel is the presence of peel oil (more than 95%
D-limonene, hereafter called limonene), a component that is extremely toxic to fermenting microorganisms. The antimicrobial effect of limonene was reported even at very low concentrations such as 0.01% (w/v), and resulted in complete failure of fermentations at higher concentrations. The removal of the limonene can be done in many ways. One is the heat treatment, where the orange peels are treated at 150°C (70 psi) by injecting high pressure steam. In this work we removed limonene by distillation. The peels were crushed to juice by the addition of little amount of water. Now this juice had been transferred into a round bottomed flask, which was connected to the distillation unit later. The heating mantel was placed below the round bottomed flask and a heat of 100°C was provided for approximately an hour. This resulted in the removal of the limonene from the orange peels.
Nutrition in orange peels:
Sr.No. Constituents Amount
1. Total fat 0.6%
2.
3.
Sodium
Protein
0.3%
6.2%
4. Calcium 32.2%
5. Iron 9.2%
3.3.2 Saccharification
The Saccharification is the process of converting the substrate into simpler sugars. The orange peels consist of saturated and unsaturated fats, zinc, and phytosterols. So cellulase,

Pectinase along with pseudomonas aureus was used. Pseudomonas aureus was a rapid starch degrading organism, which in the process of co-metabolism breaks the starch in glucose and so orgnisms directly used the substrate to grow. The chemicals cellulase and Pectinase were used as 1.0gm/100ml with continuous agitation for 2-4 hours and the inoculums of 10ml.
This media was incubated at 45°C for 24hours.
3.3.3 Microorganisms:
3.3.3.4 PROCEDURE
Soil from the local place was 1gm weighed and dissolved in 10ml srerile distilled water and name it as stock. Take 3 test tubes and add 10ml sterile distilled water in 1 st
tube and fill the rest with 9ml of saline water. Take 1ml stock solution and add into the first tube and mix thoroughly this makes the dilution rate of 10
-1
, now continue transferring 1ml from previous
10ml into next test tube until the dilution rate of 10 -3 was achieved at 3 rd tube. PDA media was prepared, autoclaved, poured into Petri plates and allowed to solidify. 0.1ml from 10
-3 was spread on PDA plate and incubated at 27 o c for 48 hours.
3.3.3.5 PREPARATION OF PURE CULTURE
Microorganisms can grow in almost any environment. In order to culture them they are to be inoculated in PDA media. When transferred aseptically the pure cultures are obtained. The microorganism will utilize the carbon source mainly carbohydrates, mineral and nutrients from the media in which they are inoculated. The organism only grows under favorable conditions that are mainly when incubated at 27 o
C in an incubator.
PROCEDURE
PDA media was prepared, autoclaved, poured and allowed to solidify. The organism was streaked on the plates using the inoculum loop and incubated at 27 o
C for 48 hours.

3.3.3.6 Preparation of inoculums:
Inoculum was prepared by suspending and scraping the spores of 4 day-old PDA slant of trichoderma viride with 3 ml sterile distilled water.
3.3.4 Production of cellulase under solid state fermentation
1.
Cellulase production experiments were carried out in 250 mL flasks containing 20 g of piece of orange peel moistened with 20ml of nutrient solution composed of 0.1%
Ammonium sulphate and 0.1% Magnesium sulfate were added to a moisture level of
50%.
2.
All flasks were sterilized at 121°C for 30 min.
3.
Then inoculated (10
8
spores/flask), 2 ml and then incubated at 30°C for 120 h.
4.
The samples were withdrawn at regular intervals to determine enzyme activities.
5.
To investigate the effect of temperature on cellulase production, different culture temperature (25°C, 30 o
C, 35°C and 40 o
C) was compared to investigate the effect of temperature on cellulase production.
6.
The effect of pH on enzyme production was studied in buffer over a wide range of pH
3-9 (sodium citrate for pH 3.0–6.0, sodium phosphate for pH 6.0–8.0, Tris–HCl for pH 8.0–9.0.) The production media were incubated over atemperture range of 20-
50
0
C for 48hrs.
3.3.5 Termination (preparation of crude enzyme):
1) After 5-6 days, terminate fermentation by taking 5 gm of fermented cake and mix it with 100 ml of sterile distilled water in sterile flask.
2) Add 0.1% tween 80 in it. Shake it vigourously for 3 min.
3) Centrifuge at 1500 rpm for 30 min.
4) Collect supernatent and used for enzyme assay.

3.4.5 Purification of Enzyme
Acetone precipitation method :
The proteins in the crude preparation were precipitated by the addition of equal volume of
Crysteline Acetone (99.9%). The precipitate was allowed to form at 4°C for 24 h, and was collected by centrifugation at 4,000 g in a cold centrifuge at 4°C for 30 min. The precipitate was redissolved in 10 ml of 0.2 M sodium acetate buffer, pH 5.5.
3.4.6 Measurement of Cellulase enzyme activity:
1.
Take 2 test tube and label them as 0.5 ml and 1.0 ml.
2.
Take 0.5 ml of enzyme sample in the test tube labelled as0.5 ml. In to that add 1 ml of citrate buffer and level it up to 2 ml.
3.
Now, into both tubes, add 1 ml substrate (1% Carboxy methyl cellulose) and level up to 3 ml.
4.
Now, incubate both tubes at 50 o
C for 30 min.
5.
After incubation, add 2 ml of DNSA to both tubes.
6.
Keep both tubes in boiling waterbath for 10 min.
7.
Cool the tubes and add 6 ml of distilled water and read tha absorbance at 540 nm against blank.
8.
In blank tubes instead of add substrate, add 1 ml citrate buffer in both tubes.
9.
One unit of enzyme activity was defined as the amount of enzyme that released 1 μM of glucose per min.
3.4.7 Analytical method
Determination of protein concentration:
The protein concentration of the crude as well as that of the purified enzyme was determined by the method of Bradford et al. (1951) using bovine serum albumin (BSA) as standard.

3.4 METHODS OF PRODUCING PECTIN ESTERASE (EC 3.1.1.11) [PINEPPLE
WASTE]
3.4.1 Microorganism:
The strain Aspergillus niger was obtained from the laboratory and employed to conduct fermentation. Inoculum is prepared by suspending one loop of inoculating needle (30 mm diameter full of spores of 5 day-old culture) in 2 mL of distilled water.
3.4.2 Substrate:
Pinepple waste: Fresh green-yellow peels are washed, cut into species of 1 mm. The moisture content is a range of 75 ÷ 80%. Using immediately after preparation.
3.4.3 Production of pectin esterase under solid state fermentation:
Pinepple Waste
Drying at 60 o C
Dried pineaaple peels
Grinding
Adjust pH, Add nitrogen source [Ammonium Sulphate (1% w/v)] in the fermentation medium
Sterilization of media (121 o C for 20 min.)
Aspergillus niger in PDA slant
Spore suspention in distilled water (one loop full of 30 mm diameter added to 2 ml distilled water for 100 gm medium (w/v)
Addition of inoculums to sterilized medium
Incubation at different temperature And pH

Fermented pineapple peels
Enzyme Extraction (Crude Enzyme Extract)
Purification (using Acetone precipitation method)
Partially purified Enzyme
3.4.4 Termination (preparation of crude enzyme):
5) After 5-6 days, terminate fermentation by taking 5 gm of fermented cake and mix it with
100 ml of sterile distilled water in sterile flask.
6) Add 0.1% tween 80 in it. Shake it vigourously for 3 min.
7) Centrifuge at 1500 rpm for 30 min.
8) Collect supernatent and used for enzyme assay.
3.4.5 Purification of Enzyme
Acetone precipitation :
The proteins in the crude preparation were precipitated by the addition of equal volume of
Crysteline Acetone (99.9%). The precipitate was allowed to form at 4°C for 24 h, and was collected by centrifugation at 4,000 g in a cold centrifuge at 4°C for 30 min. The precipitate was redissolved in 10 ml of 0.2 M sodium acetate buffer, pH 5.5 .
3.4.6 Measurement of pectin esterase enzyme activity:
1.
Add 2 ml of 1.5 M Nacl to 10 ml of 1% pectin solution.
2.
Mix by bubbling in carron dioxide free air (to make the air free of carbon dioxide, pass the air through NAOH solution).

3.
Add a few drops of flutions indicator and titrate to pH 7.5 with 0.02N NAOH.
4.
Transfer to a constant temperature water bath maintained at 30 o C.
5.
When the protein solution has attained the temperature of the bath, add enzyme and water to adjust volume to 20 ml.
6.
Immediately record the time and volume of alkali required to maintain the pH at the constant volume.
7.
Adjust the concentration of enzyme so as to require about 1 to 3ml of 0.02N alkali in
10 mins.
8.
Pectin methyl esterase units (U/ml):
V
(in mL) of 0.02 M NaOH consumed·1 min·3.1·dilution
V (in mL) of enzyme preparation··total time of determination in min
9.
The effect of pH on enzyme production was studied in buffer over a wide range of pH
3-6 (sodium citrate for pH 3.0–6.0, sodium phosphate for pH 6.0–8.0, Tris–HCl for pH 8.0–9.0.) The production media were incubated over a temperture range of 25 -
35
0
C for 48hrs.
3.4.7 Analytical method
Determination of protein concentration:
The protein concentration of the crude as well as that of the purified enzyme was determined by the method of Bradford et al. (1951) using bovine serum albumin (BSA) as standard.

1,5
1
0,5
0
3
2,5
2
4,5
4
3,5
4.0 Results and Discussion
4.1 Bioethanol and animal feed production
The use of apple pomace as a substrate contain enough amount of sugar, so that saccharomyces cervisiae were able to produce good yield of ethanol and increased nutritional value of apple pomace which use as a animal feed.
4.1.1 Bioethanol production under different culture conditions
4.1.1.1 Effect of temperature
The ethanol yield increase due to the increase temperature from 25 to 30°C with the incubation of sacharomyces cervisiae . Beyond this level the ethanol content decreases significantly. The maximum ethanol yield at 30°C was 4.2% from 120h of fermentation time
(Fig. 1). At low temperatures, a metabolic function was reduced and it slows the rate of conversion of substrate into ethanol (Hossain and Fazliny, 2010). A reason behind significant lower production of ethanol at high temperature is denature of cells at higher temperature
(Periyasamy et al ., 2009).
3,1
3,3
4,2
1,5
25 27
Temperature ( o C)
30 35

4.1.1.2 Effect of pH
The pH of media was adjusted to 4.5, 5.5, 6.5, 7.5 with HCl and NaOH. The ethanol yield increased significantly from 4.5 to 6.5 beyond this level there is decrease in ethanol yield.
The maximum ethanol yield of 4.0 % was obtained at pH 6.5 (Fig.2 ). Control of pH during ethanol fermentation is important for two reasons: yeast grows well in acidic conditions and the growth of harmful bacteria is retarded by acidic solution (Mathewson, 1980). In basic environment production of acid is more than ethanol (Kourkoutas et al ., 2004).
4,5
4
3,5
3
2,5
2
1,5
1
0,5
0
3
3,4
4
2
4,5 5,5 6,5 7,5 pH
4.1.2 Animal Feed production
After SSF of apple pomace, removal of ethanol and drying of fermented apple pomace was carried out. There was a considerable enhancement of nutritive value of fermented apple pomace comparative to before fermentation of apple pomace.(Fig 1)

Fig.1 (a) Non-Fermented apple pomace Fig.1 (b) Fermented Apple pomace (Animal feed)
Table 2: Comparison of physic-chemical characteristics of apple pomace powder before and after fermentation with s.cerevisiae
Parameters (%) Before fermentation After fermentation
Total sugar
Soluble protein
Total Ash
Aminoacids:
Aspartate
Glutamate
20.00
1.02
2.18
0.0013
0.0010
8.00
1.80
4.17
0.0054
0.0045

4.2 Cellulase Production
The use of orange peel (Figure 1) as a substrate contain enough amount of sugar, so that
Trichoderma viride were able to produce good yield of cellulose.
(Figure 2. Use of orange peels as substrate for the Cellulase production)
4.2.1 Microorganism
4.2.1.1 OBSERVATION
After incubation, different growth formation was observed in the PDA Petri plates (Fig.2)
Fig.2 Mix Growth of micorganisms on PDA plate
Trichoderma Viride

4.2.1.2 Pure culture
Observation
Growth of the microorganism was observed in the test tubes containing PDA slant after incubation. (Fig.3)
Fig.3 Growth of Trichoderma viride on PDA slant
4.2.1 Cellulase production under different culture conditions
4.2.1.1 Effect of temperature on cellulase activity
The effect of temperature on the activity of the enzymes is shown in Figure 4. The enzymes were activated at 25° to 35°C after which the activity began to drop. The maximum production of cellulase was 0.171 IU (µ mole/min) at temperature for enzyme activity was
35°C.

0,2
0,15
0,1
0,05
0
0,134
0,148
0,171
0,129
25 30
Temperature ( o C)
35 40
Figure 4 Effect of Different temperature on cellulase activity
4.2.1.2 Effect of pH on cellulase activity
The optimum pH on the enzyme activity is shown in Figure 5. The maximum production of cellulase enzyme was 0.166 IU (µ mole/min) at pH 3.5. The activity of cellulase decrease after pH 3.5.
0,2
0,15
0,1
0,05
0
0,162 0,166
0,137
0,129
3 3,5 4 4,5 pH
Figure 5. Effect of Different pH on cellulase activity
4.2.1.3 Effect of Time Duration on cellulase activity
The effect of time duration on the activity of the enzymes is shown in Figure 6. The maximum production of cellulase was 0.171 IU (µ mole/min) at after 96 hrs then after decrease the activity.

0,18
0,16
0,14
0,12
0,1
0,08
0,06
0,04
0,02
0
0,111
0,171
0,133
48 96
Time (hrs)
120
Figure 6. Effect of Different Time Duration on cellulase activity
4.2.1.4 Protein determination
The concentration of protein was increase after purification of cruide enzyme by Acetone precipitation method which shown in figure 7. The protein production of crude enzyme was
7.8% which increase to 11.2% after purification.
12
10
8
6
4
2
0
Crude Enzyme After Purification
Type of enzyme
Figure 7. Comparative study for the production of protein between crude and purified enzyme

4.3 PECTIN ESTERASE PRODUCTION
4.3.1 Effect of temperature on pectin esterase activity
The effect of temperature on the activity of the enzymes is shown in Figure 8. The maximum production of pectin esterase 0.42 U/ml was at temperature for enzyme activity was 25°C.
Then continuously decrease the activity with increase the temperature.
0,5
0,4
0,3
0,2
0,1
0
0,42
0,31
0,19
25 30
Temperature ( o C)
35
Figure 8. Effect of Different temperature on pectin esterase production activity
4.3.2 Effect of pH on pectin esterase activity
The optimum pH on the enzyme activity is shown in Figure 9. The maximum production of pectin esterase enzyme was 0.31 U/ml at pH 3.5. The activity of pectin esterase decrease continuously after pH 3.5 with increase the pH.
0,35
0,3
0,25
0,2
0,15
0,1
0,05
0
0,31
0,19
0,17
3,5 4 pH
5,2
Figure 9. Effect of Different pH on pectin esterase activity production

2
1
0
4
3
7
6
5
4.3.3 Protein determination
The concentration of protein was increase after purification of cruide enzyme by Acetone precipitation method which shown in figure 10. The protein production of crude enzyme was
4.7% which increase to 6.1% after purification.
Crude Enzyme Pure Enzyme
Fugure 10. Comparative study for the production of protein between crude and purified enzyme

5. Conclusions
Studies have been conducted on production of different value added products from the
Agricultural waste in system SSF. An overview of the various aspects shows that the SSF of agricultural waste has a large economic potential for conversion into several value added products. SSF of fruit processing waste is a simple, high yielding and economically feasible process. It is now technologically and economically feasible to produce low bulk, high value products like pectinases, cellulases, ethanol and animal feed. In future, SSF could become a potential tool for solid waste management of food processing plant and to prevent environment pollution as well. Inspite of these, there a few limitations of SSF in comparision to SmF which have to be solved to make the process workable at industrial scale. Some of the limitations of SSF are: more heat build up, occational bacterial and fungal contamination, limitation of microbial types that can be used, need for use of indirect methods for biomass estimation, need for production of large scale inoculums, non-availability of well defined scale up criteria and problem in product recovery and harvest.
More in-depth research on various problems could help in commercializing the various technologies. However, studies carried over in this direction reflect the magnitude of benefit which could be reaped from bioprocessing of waste from food industry.

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7.0 APPENDIX-I CULTURE MEDIUM:
7.1 GYE - Broth Medium
Ingredient
Yeast Extract
Peptone
Amount (g/l)
5.0 g
10.0 g
Glucose 10.0 g
Distilled Water 1000 ml pH was kept to 7.2
Add all components into Distilled Water. Bring volume to 1000 ml. Mix thoroughly and adjust the pH. Autoclave it at 121 o
C and 15 psi pressure for 30 minutes.
7.2 PDA (Potato Dextrose Agar) medium
Ingredient
Potatoes (Sliced washed unpeeled)
Dextrose
Amount (g/l)
300 g
20 g
Agar powder
Distilled Water pH was kept to 3.5
20 g
1000 ml
Potato infusion can be made by boiling 300 grams of sliced (washed but unpeeled) potatoes in ~ 1 litre water for 30 mins and then decanting the broth through cheesecloth. Distilled water is added such that the total volume of the suspension is 1 litre. 20 grams dextrose and 20 grams agar powder is then added and the medium is sterilized by autoclaving at 15 pounds per square inch for 15 minutes.

8.0 APPENDIX-II STAINS AND REAGENTS
 All the chemicals and reagents which were used are of Hi-Media Laboratories and all were of Laboratory grade.
1.
0.2 M Acetate Buffer
Add 27.2 gm of Sodium Acetate trihydrate in to 1 liter volumetric flask (1 lit
Deionized water) abd then properly mixed and adjust pH with 1 N HCl or 1 N NaOH to 5.5.
2.
10 N H
2
SO
4
Commercially available H
2
SO
4 is 36 N.
Dilute 10 ml of this acid to 26 ml Distilled water.
3.
0.2 N K
2
CR
2
O
7
in 10 N H
2
SO
4
Dissolve 9.8 gm K
2
CR
2
O
7
in 1000 ml of 10 N H
2
SO
4
(Equivalent at of K
2
CR
2
O
7
=
49.033).
4.
0.1 N NA
2
S
2
O
3
Solution
Dissolve 24.82 gm NA
2
S
2
O
3
(equivalent weight of NA
2
S
2
O
3
= 248.2) in 1000 ml of freshly boiled distilled water. Prepared fresh on the day of use.
5.
Citrate Buffer (0.05 M, pH 4.8)

0.05 M Solution of citric acid (C
6
H
8
O
7
.H
2
O)
Dissolved 10.51 gm Citric acid in 1000 ml distilled water

0.05 M Solition of sodium Citrate (C
6
H
5
Na
3
O
7
.2H
2
O)
Dissolved 14.71 gm of sodium Citrate in 1000 ml water

Adjust the pH of the 0.05 M Citrate solution to 4.8 with the 0.05 M Citric acid solution.