1. INTRODUCTION. Exploration of Keratinolytic Actinobacteria for the Bioconversion of Poultry Feather. Mr. SUBHASISH SAHA 1

Transcription

1 1. INTRODUCTION Feather waste is generated in large quantities as a by-product of commercial poultry processing. Feather consists of pure keratin protein. Keratin in its native state is not degradable by common proteolytic enzymes and that is the reason why a single feather naturally gets degraded in five to seven years. Due to the strange characteristics of feather, it gained enough consideration from the environmental scientist towards its control measurement because these large quantities of feather waste generates environmental pollution and also can leads towards various human ailments. Feather pollution and feather waste management received the healthy attention of scientists from more than last two decades, but unfortunately no solution was turned up strongly for feather waste management till date. Biological degradation of feather waste is more efficient than physical and chemical degradation, yielding more useful and toxic free by-product. In this context biological feather degradation has received the consideration from scientific community in recent days World population and feather The population of the world has exceeded 7 billion on March 12 th, 2012 and it is a well surveyed data suggested that more than half of this population are falling under the non-vegetarian food category. In this context, chickens are the most acceptable non vegetarian item around the globe for their meat and eggs to fulfil a huge demand of food habit for the non-vegetarian population. Around 24 billion chickens, per year are killed across the world to fulfil non-vegetarian food demand and as an outcome a huge feather waste are generating around the world annually. Feathers are one of the epidermal growths that form the distinctive outer covering on birds and they aid in flight, thermal insulation, water proofing, coloration that helps in communication and protection (Pettingill, 1970). Waste feathers from poultry were not a major concern for the environment or for the plants and poultry Exploration of Keratinolytic Actinobacteria for the Bioconversion of Poultry Feather. Mr. SUBHASISH SAHA 1

2 farms in the earlier days. But with the increasing production of poultry which might be accompanied with an increase in feather wastes Feather waste Feathers represent 4 to 7% of the total weight of mature chickens and are generated in huge quantities as a waste by-product at commercial poultry processing plants (Mohamedin, 1999). Worldwide, around 8.5 billion tons of poultry feather is generated annually, of which India's contribution alone is 350 million tons. The poultry feathers are either dumped, which pollute the soil or which again pollutes the air while burning. This mammoth size of discarded feather, apart from polluting the soil or air, also causes various human ailments including chlorosis, mycoplasmosis and fowl cholera Keratin Feathers are among the most complex integumentary appendages found in vertebrates and are formed in tiny follicles in the epidermis or outer skin layer, that produce keratin proteins. There are two main forms of keratin, α-keratin and β-keratin. α-keratin is seen in humans and other mammals; β-keratin is present in birds and reptiles. β-keratin is harder than α-keratin. Structurally α-keratin has α-helical coiled structure while β-keratin has twisted β-sheet structure. In the case of β-sheets, this allows sterically-unhindered hydrogen bonding between the amino and carboxyl groups of peptide bonds on adjacent protein chains, facilitating their close alignment which are then further twisted and cross linked by disulfide bridges into structures even tougher than the α-keratins of mammalian hair, horns and hoof. Fibrous keratin molecules can twist around each other to form helical intermediate filaments. Due to their extremely rigid structures, keratins are insoluble in nature and hard to degrade (Esawy, 2007) Use of feather waste Feathers are both soft and excellent at trapping heat; thus, they are sometimes used in high-class bedding, especially pillows, blankets and mattresses. They are also used as filling for winter clothing, such as quilted coats and sleeping bags (Bonser and Dawson, 1999). Bird feathers have long been used for fletching arrows. Colourful Exploration of Keratinolytic Actinobacteria for the Bioconversion of Poultry Feather. Mr. SUBHASISH SAHA 2

3 feathers such as those belonging to pheasants have been used to decorate fishing lures. Feathers of large birds have been and are used to make quill pens. But considering the huge feather waste generated worldwide, uses of feathers are very less and therefore the alternative uses of feather waste is very necessary to prevent the feather pollution. Thus scientist community has put more effort to find out suitable solution for feather waste management and their research came up successfully with various solutions such as feather waste can be converted into fertilizer, biodegradable plastic, biofuel; feather can be used for fuel storage, electrical device, but considerably the best option is conversion of feather waste into feather meal Feather meal Feather meal is a by-product of processing poultry feather waste. It is made from poultry feathers by partially hydrolyzing them under elevated heat and pressure, followed by grinding and drying. Feather meal is used for formulating animal feed. Hydrolyzed feather meal is a good source of natural protein for most animal diets and it can be used to replace a significant portion of other protein sources in livestock (Table 1). The addition of feather meal can increase lean percent in broilers and swine, provide important by-pass protein for ruminants. Table 1. Typical analysis of feather meal (Cotanch et al., 2007) Dry matter (DM), % 93.3 Crude protein, % of DM 87.8 Ether extract, % of DM 10.0 TMEn kcal/kg Ash, % of dry DM 1.9 Ca, % 0.54 P, % 0.34 Mg, % 0.03 Cl ion, % 0.17 K, % 0.13 Na, % S, % 0.11 Cu, ppm 11 Fe, ppm 239 Mn, ppm 7 Zn, ppm 87 Exploration of Keratinolytic Actinobacteria for the Bioconversion of Poultry Feather. Mr. SUBHASISH SAHA 3

4 1.6. Bioconversion of feather into feather meal Traditional methods such as steam, pressure and strong alkali or acid usually used in processing feathers required significant energy and resulted in destruction of some essential amino acids (Wang and Parsons, 1997). The most important factor affecting the quality of hydrolyzed poultry feathers is the extent of hydrolyzation. If less than 75 % of the crude protein content is digestible by the pepsin digestibility method, then hydrolyzation was incomplete and protein quality is reduced. Physical or chemical treatments of feather processes require significant energy and also destroy certain amino acids. Biodegradation by microorganisms possessing keratinolytic activity represents an alternative method to improve the nutritional value of feather waste (Williams et al., 1990). The nutritional upgrade of feather meal with the treatment of microbial keratinase might significantly increase amino acid availability of feather keratin (Odetallah et al., 2003). Keratinolytic enzymes offer opportunities for a low energy consuming technology for bioconversion of poultry feathers from a potent pollutant to a nutritionally upgraded protein rich feedstuff for livestock Keratinolytic enzymes Due to the strength and stability of keratin, very few microorganisms are able to break the feather keratin down and utilize it. A small number of microorganisms are capable of keratin decomposition into peptides and amino acids, further metabolized as a carbon and nitrogen source. Two steps in microbial keratinolysis have been reported: sulfitolysis or reduction in disulfide bonds and proteolysis. Specific proteases (keratinases), together with less specific proteinases are involved in the process. Microbial keratinases are predominantly extracellular inducible enzymes; a few cellbound and intracellular keratinases have also been reported (Gupta and Ramnani, 2006). Participation of intracellular disulfide reductases or reducing agents (sulfite, thiosulfate, dithiothreitol, mercaptoethanol, glutathione, cysteine, thioglycolate) in cleaving disulfide bridges of cystine is also suggested, which act in cooperation with keratinolytic proteases to bring about complete degradation of keratin (Onifade et al., 1998; Yamamura et al., 2002; Ramnani et al., 2005; Gupta and Ramnani, 2006; Brandelli, 2008). Exploration of Keratinolytic Actinobacteria for the Bioconversion of Poultry Feather. Mr. SUBHASISH SAHA 4

5 Keratinase properties depend upon its producers. It is usually a serine protease (Gradisar et al., 2005; Cortezi et al., 2008). Occasionally, it has been found to be a serine protease with a cysteine protease (Sangali and Brandelli, 2000a; Cortezi et al., 2008) and a metallo protease (Lee et al., 2002; Cortezi et al., 2008). Keratinolytic enzymes have important utilities in biotechnological processes involving keratin containing wastes from poultry and leather industries, through the development of nonpolluting processes. Keratinases have been reported from several species of bacteria, fungi and actinobacteria Keratinolytic bacteria Among the bacteria, feather degradation is mostly confined to Gram-positives, including Bacillus, Lysobacter, Nesternokia, Kocurica and Microbacterium. However, a few strains of Gram-negative bacteria, Vibrio, Xanthomonas, Stenotrophomonas and Chryseobacterium (Sangali and Brandelli 2000b; De Toni et al., 2002; Yamamura et al., 2002; Lucas et al., 2003), have also been recently reported. In addition, a few thermophiles and extremophiles belonging to the genera Fervidobacterium, Thermoanaerobacter, Bacillus and Nesternokia have also been described (Friedrich and Antranikian, 1996; Rissen and Antranikian, 2001; Nam et al., 2002; Gassesse et al., 2003) Keratinolytic fungi The most keratinolytic group among fungi belongs to fungi imperfecti including the following genera: Chrysosporium, Aspergillus, Alternaria, Trichurus, Curvularia, Cladosporium, Fusarium, Geomyces, Gleomastis, Monodictys, Myrothecium, Paecilomyces, Stachybotrys, Urocladium, Scopulariopsis, Sepedonium, Penicillium, Doratomyces. However, they do not have much commercial value as most of them are categorized as dermatophytes (Gradisar et al., 2000) Keratinolytic actinobacteria Most thermo active keratinases from mesophilic bacteria and dermatophytes producing keratinases are mostly pathogenic and they are undesirable for application (Gradisar et al., 2005) and on the other side, property of keratinolysis is widespread in Exploration of Keratinolytic Actinobacteria for the Bioconversion of Poultry Feather. Mr. SUBHASISH SAHA 5

6 the microbial world, but only a few have reached commercial exploitation. Hence, depending on need of keratinases that emphasizes their biotechnological potential with respect to feather meal production, the present study planned to explore the keratinolytic properties of soil actinobacteria. Actinobacteria are saprophytic bacteria capable of degrading many complex chemical substances and are crucial in the mineralization of organic matter in improving soil crumb structure (Miyadoh, 1997; Ryckeboer et al., 2003). Thermophilic actinobacteria have some advantages in comparison with mesophilic strains, such as accelerated accumulation of biomass and hydrolytic enzymes. Also, they can play a major role in the biodegradation of keratin-containing wastes (Edwards, 1993). Different species of Streptomyces like S. fradiae (Noval and Nickerson, 1959), S. pactum (Bockle et al., 1995), S. albidoflavus (Letourneau et al., 1998), S. thermoviolaceus SD8 (Chitte et al., 1999), S. graminofaciens (Szabo et al., 2000), Streptomyces sp. (Daniel and Maria, 2008), Streptomyces sp. strain 16 (Fuhong et al., 2010), Streptomyces sp. (Jayalakshmi et al., 2011; Ramakrishnan et al., 2011) and the Thermoactinomyces group, T. candidus (Ignatova et al., 1999) and another Thermoactinomyces sp. (Gousterova et al., 2005) is commonly described as keratin degraders with an ability to act on a wide variety of keratin substrates including hair, wool and feather. Some other genera of actinobacteria such as Saccharomonospora, Amycolatopsi, Terrabacter, Actinomadura keratinilytica sp. (Al-Zarban et al., 2002; Al-Musallam et al., 2003; Montero-Barrientos et al., 2005; Aaron et al., 2009), Nocardiopsis (Mitsuiki et al., 2004), Microbacterium (Thys et al., 2004) has also been reported with keratinolytic activity. Though the ample amount of research has been carried out on actinobacterial feather degradation, still in the context of feather meal formulation using thermoalkaliphilic actinobacteria and effect of feather meal on broiler chicken growth, findings are inadequate. Biodegradation of keratin by keratinolytic actinobacteria from the feather waste and produce feather meal as feed, which can achieve a good solution for the feather waste management. Tiruchirappalli and Namakkal district in Tamil Nadu, India are the hub for poultry industry and considering this fact, the present Exploration of Keratinolytic Actinobacteria for the Bioconversion of Poultry Feather. Mr. SUBHASISH SAHA 6

7 research is planned to explore keratinolytic actinobacteria from the Tiruchirappalli and Namakkal region soil samples and convert the feather waste into amino acid rich feed supplement. The outcome of this can be helped to prevent the environmental pollution implications as well as feather meal can come up as a good commercial benefit and also can develop an alternative economical support to the poultry industry Objectives of the present study In order to achieve the goal, the present study has been planned with following objectives: I. To isolate and screen the potential keratinolytic actinobacteria from the soil samples of feather waste dumping areas in and around Tiruchirappalli and various poultry farms at Namakkal, Tamil Nadu, India. II. To characterize and identify the potential keratinolytic actinobacteria by cultural, biochemical and molecular characterization based on 16S rrna analysis. III. To optimize the activity of keratinolytic enzymes under various physico chemical conditions. IV. To detect the keratinolytic enzymes by non denaturing polyacrylamide gel electrophoresis (ND-PAGE), zomography, MALDI TOF, Mascot and BLAST analysis. V. To formulate the amino acid rich feed supplement from feather waste using keratinolytic actinobacteria and study the effect of feed supplement on broiler chicken. Exploration of Keratinolytic Actinobacteria for the Bioconversion of Poultry Feather. Mr. SUBHASISH SAHA 7