CHAPTER ONE: INTRODUCTION

Transcription

1 CHAPTER ONE: INTRODUCTION

2 1.1 World population and waste generation: Inflation of the global population: The world population has crossed 7 billion in the year 2011 (Anonymous, ). As of May 28, 2014 the world's human population has crossed billion by the United States Census Bureau (Anonymous, ). The United Nations has predicted a 15% increase in the world population by 2025, which means an estimated 8 billion (from 7 billion today). By 2083, the world population is predicted to be 10 billion (Anonymous, n.d.). According to the website, World Population Clock (Anonymous, Worldometers-Realtime world statistics, ), after 2025, it is expected that Asia will contain more than 65% of the world s population Solid waste as a by-product of population expansion: Obviously, with the expansion of human population, waste disposal is and will be one of the major concerns faced by the human race. Solid waste is a striking by- product of human civilization. Only a part of the discarded materials are recycled or subjected to compost; most are landfilled or incinerated. Since 1900, waste generation has increased 10 times, and by 2025, it will double (Hoornweg D et al., 2013). Solid waste generation will accelerate and reach a peak with the rising standard of living and urbanization. As quoted by an article published by Nature (Hoornweg D et al., 2013); 220 million urban residents (13% of the population) produced fewer than 300,000 tonnes of rubbish (such as broken household items, ash, food waste and packaging) per day in the year By 2000, the 2.9 billion urban residents; (49% of the world's population) were creating more than 3 million tonnes of solid waste per day. By 2025, solid waste production will increase to more than 6 million. One of the greatest expenses managed by the municipal budgets is solid-waste management. A large amount of waste that keeps on generating is like a man-made burden on the earth s land, water and air, thus limiting the earth s ability to restore itself. One such waste that poses a major threat is the waste generated from the poultry industry (Hoornweg D and Bhada-Tata P, 2012). School of Sciences, SVKM S NMIMS (Deemed-to-be) University 2

3 1.2 Poultry consumption in the world: Global poultry industry (Agribusiness Handbook for Poultry Meat & Eggs, 2010): In the last decade, the poultry industry has been the most dynamic by showing maximum growth among all the meat sectors. The dynamic growth of the poultry sector is favored by a steady increase in the demand world over. Certain factors which have resulted in the growth of the poultry market include- The reduced supply of pork meat in Eastern Asia in 2007 has resulted in an increase in more poultry consumption than pork; The growth of income in certain countries significantly favors the consumption of meat in general; Reduced consumption of pork in the countries of the Middle East. In Asia itself, the poultry consumption has hiked up to 40% of the world total, which is 42.5 million tons this year as compared to 35.4 million tons in the year All over the world, total poultry production has hiked from 94 million tons in 2008 to 106 million tons. This growth was achieved in spite of the restrictions associated with diseases linked to poultry animals, such as avian influenza (Agribusiness Handbook for Poultry Meat & Eggs, 2010). India is the fourth largest Broiler (poultry meat) producer in the world after China, U.S. and Brazil. The southern states of the country contribute to 60-70% total output of the poultry industry. Even egg production is represented more in the southern states especially Andhra Pradesh, Tamil Nadu and Maharashtra producing nearly 70% of the country s total egg production. The Indian poultry production is considered to be the cheapest in the world (Kotaiah T, 26 th July 2013; Terry Evans, ; Anonymous, Indian Poultry Industry, ) Consumption patterns- World and India: The steady growth in the economy promotes protein demand in developing countries especially for low-priced, high protein foods such as poultry meat and eggs. Poultry meat is not only low- priced as compared to other meat products, but it is also School of Sciences, SVKM S NMIMS (Deemed-to-be) University 3

4 considered as lean and easy to cook meat, thus complying with consumer demands. More than 50% population of the world depends on a non-vegetarian diet, and poultry meat and eggs comprise majorly of this non vegetarian diet. It is expected that by 2050 the demand for poultry is expected to double (Terry Evans, ). Asia, which includes populous countries like China, India, Indonesia and Pakistan and comprising about 60% of the world s population (Anonymous, ), has a growing demand for animal proteins. Chicken consumption in the Asian subcontinent is already 40% of the overall world production and is still increasing. China, which has consumed nearly 18 million tons of poultry meat in the year 2008, has surpasses United States and become the world s highest poultry consuming country in the world. India s population has reached 1.2 billion and is still growing. While the population is expanding, the Indian economy has been steadily increasing. Furthermore, the food requirements of the country are growing. As the growing Indian economy is promoting increased purchasing power, the demand and consumption of non- vegetarian food is increasing. India s per capita poultry consumption has increased by 16%, in 2014 from Some major factors which have contributed for increasing consumption of poultry include- an expansion of middle-class, better employment levels and incomes, growing demand for ready meals and products, increasing availability of quick service restaurants and a preference for poultry meat over other meats products due to low prices and cultural and religious restrictions for pork and beef consumption (International Egg and Poultry Review, 2013). 1.3 Poultry processing as a major contributor of solid waste: According to the EHS (Environment, Health and Safety) guidelines for Poultry Processing (2007), poultry by- products can be categorized as: Solid organic wastes and by-products, wastewater and emissions to air Solid organic wastes and by-products (EHS guidelines-poultry Processing, 2007): Slaughtering and rendering activities may generate significant quantities of organic waste. On an average, 75% of the live bird weight is retained as carcass. Solid School of Sciences, SVKM S NMIMS (Deemed-to-be) University 4

5 waste generated during poultry production contains waste feed, animal waste, carcasses, and sediments and sludge from on-site wastewater treatment facilities. Other wastes include various kinds of packaging (e.g. for feed and pesticides), used ventilation filters, unused / spoilt medications, and used cleaning materials. Composition, characterization and reprocessing of slaughterhouse solids (Williams CM, Poultry Development Review, 2010): Out of 70 to 75 percent of the live bird weight which is retained as carcass, the quantity of potentially sellable solid waste depends on the efficiency of the processing methods and the health of the birds prior to processing. Blood which is approximately 2 percent of the live bird weight, is obtained as a highly proteinecous by-product when filtered and dried to produce blood meal. Processed blood meal can be used in animal and fish feed as well as fertilizer. Feathers which form approximately 7-10 percent of the live bird weight are a rich source of the protein keratin. However, the utilization of feathers as a source of protein or an animal feed depends on further processing methods (e.g., high-pressure cooking at > 100 o C or enzymatic treatment) to improve digestibility. Processed feathers have several applications i.e. they can be used for bedding, clothing, feed and other sellable items for humans. Remaining slaughterhouse solids include head, feet and inedible viscera. These solids are further treated at high temperatures and pressures to be converted into useful products like protein-rich meals and fats. If proper biosecurity measures are fulfilled during these treatments, further processing of these solids may not be required. For example, high-quality inedible viscera wastes which are required for intensive fish culture can be simply grinded and mixed with a binder prior to use. Slaughterhouse solids generated from poultry operations contains low-risk materials originating from healthy birds and high risk materials which originates from dead or diseased birds which are unfit for human consumption. Thus, slaughterhouse solids need to be sorted and processed well Waste water (EHS guidelines-poultry Processing, 2007): A large amount of effluents are generated from poultry operations from various sources like runoff from poultry housing, feeding, and watering; from waste storage and School of Sciences, SVKM S NMIMS (Deemed-to-be) University 5

6 management facilities. Waste management measures like manuring may generate nonpoint source effluents due to runoff. Waste water generated from poultry processing can potentially lead to contamination of surface water and groundwater with nutrients, ammonia, sediment, pesticides, pathogens, feed additives, heavy metals, hormones, and antibiotics. Effluents from poultry operations are known to have a high content of organic material and therefore have a high biological oxygen demand (BOD) and chemical oxygen demand (COD), as well as suspended solids (TSS). Therefore, effluents generated must be adequately treated before being discharged Air Emissions (EHS guidelines-poultry Processing, 2007): Air emissions originate from odour, exhaust gases from utility operations, and particulate matter from smoking operations. Process odour comes from processes like scalding, live bird handling, wastewater treatment and rendering. Odour also arises from poultry material like by-products, blood collection tanks, manure piles, and fat traps. Boilers and generators produce exhaust. These poultry wastes are of major concern since they affect air, water and soil quality. The reported negative impacts that poultry waste has on the environment include contamination of nearby surface and/or groundwater. Impact on the air quality includes emission of ammonia, hydrogen sulfide, dust particles and volatile organic compounds Feather waste as a part of solid organic waste generated from poultry processing: About 89% of the poultry by-products comprise of in-edible waste. Of all the inedible wastes generated due to poultry consumption, feather wastes are of critical concern due to their rigid and chemically inert nature. Considering that feathers comprise about 7-10% of the weight of a chicken, and with above 400 million chickens being sacrificed each day, the feather waste generated will exceed 5 million tons. Feathers form the exoskeleton of birds and they fulfill the function of locomotion, insulation, and protection to these animals (Pettingill, 1970). Being biochemically rigid and unreactive, feathers therefore tend to accumulate. As a result, they pose environmental problems. Considering the increasing demand for poultry animals in the food sector in the coming years, the amount of feather waste generation would only keep on increasing, further adding to the environmental problems. School of Sciences, SVKM S NMIMS (Deemed-to-be) University 6

7 1.4 Traditional methods for disposal of feather waste and their disadvantages: Poultry wastes which are majorly responsible for environmental pollution include- poultry feathers, poultry offal, manure and litter (Thyagarajan D et al., 2013). Solid organic waste generated from poultry processing should be considered as both valuable resources as well as harmful environmental pollutants. The fate of these poultry wastes depends on their processing and treatment. Each year, billions of kilograms of waste feathers are produced by commercial poultry processing plants creating a serious solid waste management problem in many countries. Due to the high keratin content of the feathers, they are insoluble and poorly digested by proteolytic enzymes. As keratin is biochemically inert, feather wastes pose an environmental threat, as they tend to accumulate. Also, due to the presence of pathogenic microflora, efficient and immediate treatment of feather waste becomes necessary (Williams CM, Poultry Development Review, 2010). Disposal of feather waste is quite challenging. Considering the huge quantity generated, the methods that are adopted traditionally include drastic methods such as incineration, landfilling, composting or conversion to feather meal Incineration: Incineration as a method for feather waste disposal, involves combustion of solid wastes. Incineration plant temperatures are above 850 o C and mostly the waste is converted to carbon dioxide and water. Due to the requirement of high temperature, the operating costs are high, thus making incineration an expensive method for disposal. Although the method can bring down the original volume of the combustible by 85% to 90%, air pollution control as well as the cost factor remains a major problem in the implementation of incineration as a disposal method for feather waste (Knox A, 2005) Landfilling: Another method traditionally used for feather waste disposal is landfilling. Landfilling which involves disposal of waste materials by burial and is one of the oldest form of solid waste treatment. Historically, landfills have been the most popular methods of organized waste disposal and continue to remain in several places around the world (Remigios MV, 2010). Developed countries are well equipped with proper landfills with proper construction and maintenance of the same (Sudhakar Yedla, 2005). However, in School of Sciences, SVKM S NMIMS (Deemed-to-be) University 7

8 developing countries such as India, well designed and properly maintained landfills are rare. The waste is often disposed off in open dumps, which not only leads to severe environmental degradation but also results in loss of natural resources (Yedla and Parikh, 2001). The other adverse effects of landfilling include pollution of the local environment (such as contamination of groundwater and/or aquifers) by leakage or sinkholes and residual soil contamination during landfill usage. So, landfilling although being a lesser expensive way of disposal of feathers is not an efficient method, since it results in incomplete utilization of resources and pollution Composting: A cheap and easy way of feather waste management is Composting with manure. Composting is considered as one of the most economical viable and environmentally safe methods for recycling feather wastes (Ichida et al., 2001). Over the last three decades, studies are being carried out in order to achieve better recycling of animal wastes, including poultry wastes, via composting (Tiquia SM, 2002). Basically, the process of composting simply involves making a heap of wetted organic matter or waste known as green waste (leaves, food waste) and waiting for the materials to degrade into humus after a certain period of weeks or months. The compost thus generated can be used as a soil fertilizer or conditioner (Thyagarajan D et al., 2013) Production of Feather Meal: Feather meal is a by-product of processing poultry waste; it is made by partially hydrolyzing poultry feathers by treating them under elevated heat and pressure, and then grinding and drying. Typically, the process involves subjecting the feathers to a continuous feather hydrolyzing system, with a capacity of 20,000 pounds of raw material. The feathers are subjected to steam and high temperature treatment into the hydrolyzer at 142 o C, at approximately 380 kpa, for 30 to 40 minutes. The hydrolyzed feathers are then fed into the dryers where the temperature reaches to about 200 F at the dryer exit after which the product is subjected to more than of 300 F for approximately one hour. This treatment partially hydrolyzes the proteins, as a result of which denaturation occurs. The material is then dried, cooled and ground into a powder which can be used as a nitrogen source for animal feed (mostly ruminants) or as an organic soil enhancer. Sometimes, the finished product is required to be tested each month by an independent lab for Salmonella School of Sciences, SVKM S NMIMS (Deemed-to-be) University 8

9 as well as fecal count. Table 1.1 represents the composition of a typical feather meal (Handbook of Poultry feed from Waste: Processing and Use, 2000; Product Specification Sheet, WCRL, 2011). Table 1.1: Typical Analysis of Feather Meal (Ewing, 1997) Dry Matter 90% Crude Protein 82% Digestibility 75% min. Fat 6% Ash 4% Crude Fibre 0.6% Available Lysine 1.8% Available Lysine 1.8% Methionine + Cysteine 4.9% TME n (True metabolizable energy) 3.07 Kcal/g (12.8 MJ/kg) Containing up to 15% nitrogen, in the form of non-soluble keratin, feather meal is a source of slow-release, organic, high-nitrogen fertilizer for organic gardens (Haddas and Kautsky, 1994). Being a non-soluble nitrogen source, it is a slow release fertilizer, allowing plants to consume/utilize the nitrogen at their own pace. It is most suitable for plants like corn, leafy vegetables, and others which require a slow but constant release of fertilizers. There are certain disadvantages of the methods that are employed for the conversion of feather waste to feather meal. The employment of high temperature under pressure, with acids or alkali results in the loss of several valuable amino acids (Papadopoulus, 1989, Latshaw et al., 1994 and Wang and Parsons, 1997). Considering the disadvantages of the above methods employed for the management of poultry feathers, microbial degradation of the poultry feathers appears to be a viable alternative and it is therefore attracting the attention of scientists. School of Sciences, SVKM S NMIMS (Deemed-to-be) University 9

10 1.5 Feather Keratin (Martinez- Hernandez and Velasco-Santos, 2012): Keratin as a component of exo-skeleton: Keratin is a biofiber which is found in several natural sources such as hair, feather, skin, horns, nails etc. Keratin can be found in the exo-skeleton of several varied organisms such as human, horses, snakes and birds. This biopolymer has specific role in each of these living beings, depending upon the tissue where it is found. It is a structural protein and the principal component of the exo-skeleton and related appendages such as hair, horn, nails, and feathers. Keratins have been classified into two types- α- keratins, which occur in mammals, or β- keratins, which occur in birds and reptiles. Keratin fibers comprise 90% of the feather content, thus forming a considerable proportion. Keratin fibers from chicken feather are non- abrasive, eco-friendly, insoluble in organic solvents have good mechanical properties, low- density, hydrophobic behavior and also low cost Evolution of feather and keratin: Fibril proteins like keratin have been produced with a specific conformation, which is undoubtedly caused by several adaptation measures in response to environmental conditions for the purpose of survival. The creation of feather keratin can therefore be called an evolution process that involved a complex chain of mutations. Feather structure is a complex hierarchical arrangement of three level branched structure that comprises of rachis (primary shaft), barbs and barbules (secondary and tertiary branches respectively). According to the proposed developmental theories, the evolution of feathers occurred through the stages as illustrated in Figure 1.1. School of Sciences, SVKM S NMIMS (Deemed-to-be) University 10

11 Figure 1.1: Stages of feather evolution: 1) elongation of scales 2) appearance of the central shaft 3) differentiation of vanes into barbs 4) appearance of barbules and barbicel. First the feather originates as a hollow tube (phase 1), that changes into a series of barbs (phase 2). After this, the barbs self-organize along a rachis (phase 3). In phase 4, the origin of barbules takes place and in this stage, the organizational structure looks like a completely evolved feather. Figure 1.1: Phases of feather evolution Depending upon the structure and function, feathers can be classified into five main types: down feathers, contour feathers, semiplumes, filoplues and bristles. All these feather types have a common structural organization: a central hollow tube called calamus or rachis, the secondary structures called barbs, joined to the rachis and microscopic structures called barbules. These structures are illustrated in the following figure (Figure 1.2). School of Sciences, SVKM S NMIMS (Deemed-to-be) University 11

12 Figure 1.2: Images of feather structure: a) a semi plume feather b) optical micrograph c) and d) scanning electron micrograph 1.6 Feather Keratin Structure and Properties (Voet and Voet, 2011): Keratin: Keratin contains polypeptide chains containing amino acids linked by peptide bonds. According to Walker and Rogers, 1976, the polypeptide chain of feather keratin contains amino terminal and carboxy terminal regions which are rich in half-cysteine, and a large internal region devoid of half-cysteine and rich in hydrophobic amino acids. Depending upon the secondary structure i.e. the arrangement of the amino acid chain, keratin can be divided into two types: α- keratin and β- keratin. While both the types of keratins fulfill similar roles, they vary slightly in their structure, composition and properties. α- keratins are slightly basic and they form a right handed helix, while β- keratins are slightly acidic and they form a left handed helix. School of Sciences, SVKM S NMIMS (Deemed-to-be) University 12

13 The α-keratin structure is described as a coiled-coil structure. α- keratin exhibits a 5.1- o A pitch rather than 5.4 o A pitch as for a regular α- Helix. This happens when two keratin α-helices twist around one other thus forming a left handed coil. The normal 5.4 o A repeat distance in a regular α-helix is thereby tilted relative to the axis of the assembly, forming the observed 5.1- o A. This assembly is therefore called as coiled-coil structure. This structure is a result of keratin s primary structure which is an amino acid chain of approximately 300 residues, comprising a 7 residue pseudo repeat a-b-c-d-e-f-g, a and d predominantly being non-polar residues. Since the α- helix contains 3.6 residues per turn, the a and d residues get arranged along one side of the helix. The hydrophobic residue of one helix associates with that of the other helix. Since the 3.5 residue repeat in α-keratin is slightly smaller as compared to that of a standard α-helix, the two keratin α- helices are inclined 18 o relative to each other forming a coiled coil structure. This conformation allows the contacting side chains to inter-digitate. The below figure (Figure 1.3) illustrates the coiled- coil structure of α- keratin. School of Sciences, SVKM S NMIMS (Deemed-to-be) University 13

14 Figure 1.3: Coiled-coil structure of α- keratin: a) The helices have the pseudorepeating sequence a-b-c-d-e-f-g and a -b -c -d -e -f -g (as shwon in the image)in which residues a and d (and a and d ) are non-polar. The hydrophobic residues arrange along one side of each helix. b) Polypeptide backbone: skeletal form (left) and space-filling form(right). The contacting side chains (shown as red spheres in the space-filling model) interlock. The higher order structure of keratin is not well studied. The N-and C-terminal regions of each polypeptide arrange to form coiled coils (dimers) into protofilaments. Two such protofilaments constitute a protofibril. Four protofibrils arrange to form a microfibril, which associates with other microfibrils to form a macrofibril. The below figure (Figure 1.4) depicts the tertiary and quaternary structure of keratin. School of Sciences, SVKM S NMIMS (Deemed-to-be) University 14

15 Figure 1.4: Higher order keratin structure: a) Arrangment of two keratin polypeptides to form a coiled coil. b) Formation of Protofilament form coiled-coil: Two staggered rows of head-to tail association of coiled-coils. c) Protofibril: Protofilaments dimerize to form a protofibril, four of which form a microfibril. The structures of the latter assemblies are poorly characterized. School of Sciences, SVKM S NMIMS (Deemed-to-be) University 15

16 1.6.2 Various types of keratin secondary structures found in feathers: Chicken feathers contain up to 91% protein, predominantly keratin, 8% water and 1% fats. As mentioned earlier, biochemically, the amino acid content of feathers depends upon breed, food, and environmental conditions. Chicken feather keratin mainly includes cysteine, glutamine, valine, proline, serine and negligible amount of glutamic acid, lysine, glycine, histidine and tryptophan. Also, feather keratin possesses hydrophobic amino acids such as Leucine, Isoleucine, cysteine, Alanine, Phenylalanine and methionine, which would contribute to the hydrophobic nature of the feather keratin (Walker and Rogers, 1976; Saravanan K, 2012). According to several authors, feather keratin possesses β sheets, turn and random coils, whereas others consider α helix, or helical array of β sheets. Thus, secondary structure of feather keratin has been a topic of discussion, and there is no definite conclusion on it. According to Saravanan K, 2012, the feather barb section contains more α helical sections then β sheets, and its melting temperature is 240 o C. The feather quill possesses more β sheeted structures as compared to α helices and has a melting temperature of 230 o C. Thus, chicken feather fiber contains both- α helices and β sheets. The β sheets contain more cysteine residues, which result in more disulfide linkages that connect adjacent keratin fibers conferring more structural stability. According to several reports, as mentioned, the nature of keratin s secondary structure is varied. Below table (Table 1.2) represents the keratin structure types in feathers. School of Sciences, SVKM S NMIMS (Deemed-to-be) University 16

17 Table 1.2: Keratin secondary structures found in feathers of different species (Martinez-Hernandez and Velasco- Santos, 2012) Feather Part Avian Species Proposed Secondary structure Barbule Cockatoo β- Sheet Barbs Fowl 30% β- Sheet, turn and random coils King Penguin, β- keratin Wood stork, American Crow Chicken α- keratin type Quill Seagull β- type, feather keratin structure Pigeon β Sheet as dominant conformation with presence of minimal α- helix Rachis Non specified α Helix and pleated sheet layers Fowl Seagull Feather keratin structure, resembled α- keratin Chicken 78% β sheet, 18% αhelical from twisted sheet Remaining of turn and other random structures Calamus Goose β configuration and α protein School of Sciences, SVKM S NMIMS (Deemed-to-be) University 17

18 1.6.3 Hard and Soft keratin: (Ward WH and Lundgren HP, 1954): Based on the physical characters and the chemical composition, keratins can be classified into hard and soft keratins. The outer layer of the epidermis, the skin is the most common example of soft keratin. Soft keratin is distinguished to have a sulfur content of approximately 1% and a lipid content of 4%. The sulfur content is evenly distributed between cysteine and methionine. Also, there is an equimolar proportion of lysine and arginine. Hard keratins have much higher sulfur content, up to 5%, and predominatly in the form of cysteine. Non- protein components such as lipids and glycogen are negligible. The proportion of lysine and arginine is not appreciable i.e. arginine content is much higher as compared to lysine. Horn, nail, hair, feather, claw and wool are examples of hard keratin Bonds in keratin structure (Feughelman M, 1997): Besides the covalent bonds between two Carbon atoms and between Carbon and Nitrogen, the following bonds contribute to the structural properties of keratin: a) Hydrophobic Bonds: In keratin structure, hydrophobic bonds have a specialized role to play, in terms of binding the helical chains together to form double helical ropes, which form the organized structure in microfibrils, also known as the intermediate filaments. Hydrophobic bonds become effective in the presence of water molecules, and they result from the association between molecular groups that do not interact with water. These groups, associate with each other and reduce their interaction with the water molecules. Hydrophobic bonds therefore have a stabilizing role in the mechanical structure of keratin. b) Disulfide bridges: these are covalent bonds between the cysteine residues present on different parts of the protein. Since keratin has large number of cysteine residues, disulfide bonds result, which confers extra strength and rigidity by permanent, thermally stable crosslinking. c) Hydrogen Bonds: Typical hydrogen bonds (Figure 1.5) involve the amide proton (the hydrogen atom bonded to the nitrogen of the peptide bond) and the carbonyl oxygen (the oxygen atom bonded to the carbon atom in the amide plane). Hydrogen School of Sciences, SVKM S NMIMS (Deemed-to-be) University 18

19 bonds in keratin structure are of special interest since they are inter-turn bonds, thus stabilizing the repeating structures in keratin i.e. helices. Figure 1.5: Hydrogen bonding in a protein helix 1.7 Potential Uses of Feather Waste: About 90% of the feather content is proteins (remaining being fat, ash and water), out of which, keratin forms the major protein. Considering that keratin forms such a high proportion of feathers, which contains several nutritionally valuable amino acids, feather waste, can have several value-added applications. Thus, recycling of feather waste becomes interesting approach for managing huge quantities of feather waste. However, it is the complex structure of the feather keratin that interferes with most of the potential applications of the feather waste. Native keratin is highly inert, water-insoluble protein and undegraded by most proteolytic enzymes: trypsin, pepsin, papain etc. (Onifade et al., 1998). Some potential applications are to obtain nutritionally upgraded animal feedstuff or production of biofertilizer. Considering these applications and also the limitations due to keratin s compact structure; so far, the most common approach for recycling feather waste is conversion to feather meal. This is achieved by subjecting feathers to a School of Sciences, SVKM S NMIMS (Deemed-to-be) University 19

20 hydrothermal treatment resulting in a readily digestible feather meal. Basically, this treatment involves subjecting feather waste to high temperature and pressure, thus making the keratin less complex and easy to digest. While the advantage of high temperature being increasing the digestibility of feathers, it destroys several valuable amino acids, such as lysine, methionine and tryptophan that are originally present in the keratin and also results in the generation of non-nutritive amino acids such as lysinoalanine and lanthionine. Thus, hydrothermal treatment does not sufficiently enhance the nutritional content of the feather meal (Papadopoulus 1989, Latshaw et al., 1994 and Wang and Parsons 1997). A reasonable alternative would be microbial degradation for obtaining a feather meal that would be nutritionally upgraded with essential amino acids. This biotechnological approach, which would involve microbes and microbial enzymes, would not only transform the indigestible feather waste to a readily digestible feather meal, but it may not necessarily harm the valuable amino acids previously present in the keratin (Elmayergi and Smith 1971, Mohammed EI-Akied, 1987; Williams and Shih, 1989, Onifade et al., 1998). Thus, the feather meal obtained after such microbial treatment would not require any additives. Also, the technology would have less production cost since it would not require hydrothermal treatment. Moreover it involves usage of feather waste which would be a cheap raw material. The process is environmentally compatible as it would result in effective and safe disposal of feather waste as against landfilling or incineration. Thus, use of microbial technology to obtain feather meal from feathers would result in safe disposal of feather waste, lower the use of hydrothermal processing, substitute conventional methods of disposal and would generate valuable amino acids thus producing a complete meal and may potentially replace other costly animal feedstuffs such as soyabean meal and fish meal. Moreover it would benefit the environment and therefore mankind. 1.8 Microbial bio-degradation as an ecologically safe and alternative method: Considering the potent polluting implications and also the thermo-energetic cost of the above approaches for treatment of feather waste, an alternative which is ecologically safe, cost- effective and having prospects of nutritional enhancement is required; School of Sciences, SVKM S NMIMS (Deemed-to-be) University 20

21 microbial degradation is one such approach. Keratin (feathers) although being unusually stable does not accumulate in nature, which indicates that natural decomposition of keratin, does occur. This provides evidence of the existence of organisms that are capable of utilizing the keratin containing substances, thus preventing the accumulation of such rigid structures. This keratin degrading ability of micro-organisms provides an ecologically compatible way of disposal of keratin containing wastes which otherwise becomes an environmental hazard. Thus, on employing this ability of keratinolytic microorganisms on an industrial scale as a biotechnology, the environmental impacts of incineration and landfilling can be reduced to a great extent. A melange of micro-organisms exists in nature which is capable of carrying out keratin degradation. These microbes are natural decomposers or utilizers of keratins. Several bacteria and fungi have been reported and identified as keratin degraders. More than bacteria, keratin degrading fungi have been widely studied, since many pathogenic fungi are dermatophytes (Wawrzkiewicz et al., 1987, 1991; Apodaca and McKerrow, 1989; Hanel et al., 1991; Porro et al., 1997). In addition to dermatophytic fungi, nondermatophytic keratinolytic fungi have also been reported (Malviya et al., 1992, Dozie et al., 1994). Besides fungi, keratinolytic microorganisms include Actinomycetes spp. and bacteria belonging to Bacillus spp., Micrococcus spp., Clostridium sp., etc. Keratinolytic micro-organisms inhabit varied ecological and environmental conditions each possessing different capacities to solubilize keratin- containing substrates and other proteinaecious substrates (Lin et al,. 1999; Suh and Lee, 2001; Lucas et al., 2003, Thys et al., 2004; Bernal et al., 2006a). There have been studies involving attempts to carry out degradation of coloured feathers. A study carried out by Goldstein et al., 2004, involving comparative degradation of black and white chicken feathers indicated that melanized chicken feathers resisted degradation by B. licheniformis. Another study involving degradation of colored parrot feather by B. licheniformis (Burtt et al., 2010) indicated that the feathers with red psittacofulvins degraded at equal rate as those with melanin and more slowly than white feathers, which lack pigments. Several feather degrading bacteria, both Gram negative and Gram positive have been characterized from different sources. Most of these microorganisms have been School of Sciences, SVKM S NMIMS (Deemed-to-be) University 21

22 reported to be isolated from varied ecosystems such as soil- mostly those soil samples having abundant presence of keratinous material, poultry waste, hot springs, etc. Table 1.3 lists the source of various keratinolytic bacteria. Table 1.3: Origin of some feather degrading bacterial isolates Bacterial Isolate Origin Reference Gram Positive Bacillus licheniformis PWD-1 Poultry waste Williams et al., 1990 Bacillus subtilis S14 Soil Macedo et al.,2005 Bacillus pumilus, Bacillus Poultry waste Kim et al.,2001 licheniformis and Bacillus cereus Bacillus pseudofirmus Alkaline soda lake Gessesse et al., 2003 Streptomyces pactum DSM Collection culture Bockle et al., Streptomyces albidoflavus Hen house soil Bressolier et al., 1999 Streptomyces thermovioaceus Soil Chitte et al.,1999 Frevidobacterium spp. Hot springs Friedrich and Antarnikian 1996 Microbacterium spp. Decomposing Thys et al., 2004 feathers Microbispora aerate and Antarctic soil Gushterova et al., 2005 Streptomyces flavus Kocuria rosea Soil Bernal et al., 2005 Gram Negative Vibrio spp. kr2 Poultry soil Sangali et Brandelli 2006 Chryseobacterium spp. kr6 Decomposing Riffel et al., 2003 feathers Alcaligenes faecalis Dry meadow soil Lucas et al., 2003 School of Sciences, SVKM S NMIMS (Deemed-to-be) University 22

23 1.9 The major enzymes involved in feather degradation: Keratinases Proteases are a major group of commercial enzymes. They are a class of enzymes which have ample importance at industrial level majorly in the detergent industry and also in the leather industry (Kumar and Takagi 1999; Gupta et al., 2002). One such type of protease is the keratinase group of protease. Keratinases are proteolytic enzymes having the unique ability to digest the insoluble keratin substrates. Having this ability, keratinases have several value added applications and this category of protease is rapidly gaining added importance. Considering the resistance of keratinous substrates to other proteolytic enzymes such as trypsin, pepsin, papain etc., keratinases would play an essential role in bringing about efficient waste management of a significant amount of keratin containing by-products/wastes obtained from poultry industry, agro-industrial processing etc. Hydrolysis of such keratin containing wastes by means of microbial keratinases not only brings up an eco-friendly approach of their disposal but also recycles this waste to obtain value-added by-products. The enzymatic digestion of keratin containing substrates results in the conversion of insoluble keratin to a more solubilized and readily digestible material, which is supplemented with proteins and amino-acids (Papadopoulos et al., 1986; Onifade et al., 1998; Gupta and Ramnani 2006). Keratinases that have been reported so far mostly belong to the Serine protease group, while few others belong to the metalloprotease group. Keratinases from Gram positive isolates have been reported to be Serine proteases, while keratinases from Gram negative isolates belong to the category of metalloproteases (Brandelli, 2008). So far, the most well characterized keratinase is produced by B. licheniformis PWD-1 and is a serine- type protease. The gene ker A which codes for this keratinase, is expressed exclusively for feather degradation, thus making the enzyme inducible (Lin et al., 1995). The presence of feather keratin as a sole source of carbon and nitrogen is essential for this gene to be expressed and the enzyme to be produced. The biochemical properties of reported keratinases have been mentioned in Table 1.4. School of Sciences, SVKM S NMIMS (Deemed-to-be) University 23

24 Table 1.4: Biochemical properties of certain keratinases Producer Bacteria Catalytic Molecular Optimum Reference type mass (kda) ph B. licheniformis PWD-1 Serine Lin et al., 1992 B. subtilis KS-1 Serine Suh and Lee 2001 B. pseudofirmus FA 30- Serine Kojima et al., S. pactum DSM Serine Bockle et al., 1995 S. albidoflavus K1-02 Serine Bressolier et al., 1999 F. pennavorans Serine Friedrich and Antranikian 1996 X. maltophilia Serine 36 8 De Toni et al., 2002 Vibrio spp. Kr2 Serine 30 8 Sangali and Brandelli 2000 Chryseobacterium sp Metallo Riffel et al., 2007 Kr6 Microbacterium sp kr. 10 Metallo Thys and Brandelli 2006 Kocuria rosea LBP- 3 Serine Bernal et al., 2006 Although keratinases, both serine proteases and metalloproteases have their own catalytic mechanisms, a consortium of microorganisms may hydrolyse native keratin much efficiently as compared to a pure culture (Ichida et al., 2001). The exact mechanism of keratin hydrolysis is not yet known, however, the reduction of disulfide bridges must be a crucial step to allow further breakdown, and thus sufficient hydrolysis to occur. Keratinases are basically endo-proteases which have a broad range of activity (Lin et al., 1992; Gradisar et al., 2000; Brandelli, 2005), and their catalytic activity on School of Sciences, SVKM S NMIMS (Deemed-to-be) University 24

25 native keratin is linked to the cooperative action of multiple enzymes (Yamamura et al., 2002; Giongo et al., 2007). This happens due to the initial attack by keratinases and disulphide reductases, after which other less specific proteases act, resulting in keratin hydrolysis. The present study explores the potential of feather degrading bacteria for the purpose of effective waste management of poultry feather waste, such that it results in the transformation of the undigestible feather waste to a hydrolysate that is easily digestible and enriched with amino acids. School of Sciences, SVKM S NMIMS (Deemed-to-be) University 25