Journal of Research in Biology
Isolation and characterization of feather degrading
bacteria from poultry waste
Keywords:
Feather, keratin, feather degrading bacterium, poultry waste, keratinase, keratinolytic activity.
ABSTRACT:
The aim of this study was to characterize keratinolytic bacteria isolated from feather waste. Feather waste is generated in large amounts as a by-product of commercial poultry processing. This residue is almost pure keratin, which is not easily degradable by common proteolytic enzymes. The crude protein from feather has of high nutrient value and could be used as animal feed for livestock and fish feed in aquaculture. Feather constitutes over 90% protein, the main component being beta-keratin, a fibrous and insoluble structural protein extensively cross linked by disulfide bonds. This renders them resistant to digestion by animals, insects and proteases leading to serious disposal problems. It is degraded only by keratinase enzyme. These enzymes were produced by some species of Bacillus. In the present study, B. licheniformis was used for degrading keratin substrate such as feathers. Based on morphology and biochemical analysis, the isolates were identified as Bacillus spp. Fermentation using feather as a substrate was carried out on minimal salt media for seven days which resulted in almost complete degradation of feather. The optimum conditions for keratinase production were temperature 37°C, pH 7.0 and initial substrate concentration 1%. Maximum enzyme activity was found to be 100 U/L with the protein concentration of 4 μg/ml.
676-682 | JRB | 2012 | Vol 2 | No 7
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Authors:
Arun Kumar JM1, Lakshmi A2, Sangeetha Rani V2, Sailaja B2.
Institution:
2. B.E. Scholar, Dept of Biotechnology, Ballari Institute of Technology and Managment, Bellary.
Corresponding author:
Arun Kumar JM.
Web Address:
http://jresearchbiology.com/documents/RA0239.pdf.
Dates:
Received: 07 May 2012 Accepted: 26 May 2012 Published: 01 Nov 2012
Article Citation:
Arun Kumar JM, Lakshmi A, Sangeetha Rani V, Sailaja B.
Isolation and characterization of feather degrading bacteria from poultry waste.
Journal of Research in Biology (2012) 2(7): 676-682
Journal of Research in Biology
Original Research
An International Scientific Research Journal
INTRODUCTION
Feathers, which are almost pure keratin proteins, are produced in large amounts as a waste by-product at poultry-processing plant. A total of 5-7% weight of mature chicken comprises of feathers (Avinash et al., 2011). Feather waste constitutes beta-keratin, a insolvable protein (Swetlana and Jain, 2011). In addition to this, feather waste is produced at the rate of 22 million kg per year (US alone) (Savitha et al., 2007). A group of proteolytic enzymes which are able to hydrolyze insoluble keratins more efficiently than other proteases are called keratinizes produced by some microorganisms. These bacterial strains produce enzymes which selectively degrade the beta-keratin found in feathers. These enzymes make it possible for the bacteria to obtain carbon, sulphur and energy for their growth and maintenance from the degradation of beta keratin (Muthusamy et al., 2011). Keratins are the most abundant proteins in epithelial cells of vertebrates and represent the major constituents of skin and its appendages such as nail, hair, feather, and wool. Keratins are grouped into hard keratins (feather, hair, hoof and nail) and soft keratins (skin and callus) according to sulphur content (Scott and Untereiner 2004). The protein chains are packed tightly either in α-helix (α-keratins) or in β-sheet (β-keratins) structures, which fold into final 3-dimensional form (Veslava et al., 2009)
Keratinases which are produced by these keratinolytic organisms could be used to degrade feather waste and further the digested products could be a excellent material for producing animal feed, fertilizers or natural gas (Tamilmani et al., 2008). Use of keratinolytic microorganisms for feather degradation is an economical, environmental friendly alternative. Keratinolytic proteases offer considerable opportunities for a low energy consuming technology for bioconversion of poultry feathers from a potent pollutant to a nutritionally upgraded protein feed for live stock (Jahan et al., 2010; Vigneshwaran et al., 2010). Most feather waste is land filled or burned which involves expense and can cause contamination of air, soil and water. Bacterial strains are known which are capable of degrading feathers (Savitha et al., 2007). Traditional ways to degrade feathers such as alkali hydrolysis and steam pressure cooking may not only destroy the amino acids (methionine, lysine and histidine) but also consume large amounts of energy. Utilizing poultry feathers as a fermentation substrate in conjunction with keratin-degrading microorganism or enzymatic biodegradation may be a better alternative to improve nutritional value of poultry feathers and reduce environmental waste (Veslava et al., 2009). These feathers constitute a sizable waste disposal problem. Several different approaches have been used for disposing of feather waste, including land filling, burning, natural gas production and treatment for animal feed. Most feather waste is land filled or burned which involves expense and can cause contamination of air, soil and water (Rai Sapna et al., 2011; Savitha et al., 2007). Feathers hydrolysed by mechanical or chemical treatment can be converted to feedstuffs, fertilizers, glues and foils or used for the production of amino acids and peptides (Jahan et al., 2010; Andrea et al., 1996). An alternative to decrease this pollution is the utilisation of feather constitutes that can be used as animal feed, preventing accumulation in the environment and the development of some types of pathogens (Veslava et al., 2009).
MATERIALS AND METHODS
Enrichment
1 g of poultry waste was serially diluted in order to reduce the initial number of micro organisms. This dilution was then inoculated on minimal feather agar media. Feathers were washed, dried and hammer milled prior to being added to the medium. The medium was sterilized by autoclaving. All incubations were done at 37°C.
Screening
Skim milk agar (Himedia) was prepared and the above dilutions were streaked on milk agar plates for testing the caseinolytic activity of the organism. Bacteria were inoculated onto plates and incubated at 37°C for 24 h. Strains that produced clear zones in this medium were selected.
Subculturing
The organism screened with Keratin agar plates was subcultured by continuously growing the bacterium in minimal broth medium (3 days at 37°C, 120 rpm) and subsequently streaking on minimal agar medium (1.5% agar, 2 days 37°C).
Identification of Isolated feather degrading bacteria
Gram Stain, Spore staining, Motility test and Catalase Test.
Characterization of the isolate using Biochemical assays
IMViC Test, Hydrogen Sulfide Test, Urease Test, Lipid hydrolysis, Carbohydrate Fermentation, Starch hydrolysis and Gelatin liquefaction.
Production of keratinolytic enzyme
The bacterial isolate was cultivated in 100 ml minimal feather media. The samples were withdrawn and centrifuged at 6000 rpm for 10min. The supernatants were preserved at 4°C and assayed for protein.
Determination of keratinase activity
Azocasein hydrolysis was used as an alternative to the azokeratin hydrolysis. Keratinolytic protease activity was determined with azocasein (Sigma Co. St. Louis. Mo.) as a substrate by azocasein solution in 0.05 M Tris -HCI buffer at pH 8.5, which was incubated with 400 μl crude enzyme solution for 30 min at 37°C in a water bath with shaking. The reaction was terminated by addition of 1.4 ml of 10% trichloroacetic acid (TCA). After 15 min at 4°C, the reaction mixture was centrifuged at 10,000xg for 10 min. One ml supernatant was mixed with 1 ml of 0.5 N NaOH and the absorbance was read at 440 nm. The control was treated in the same way, except TCA was added before the addition of crude enzyme. One unit of caseinolytic activity was determined as the amount of enzyme that produces an increase in absorbance of 0.01 per min under the assay conditions. The soluble protein concentration in the culture supernatant was estimated according to the Bradford method (Bradford, 1976).
Enzyme characterization
Taking Temperature, pH, Substrate concentration, Activator and Inhibitor as parameters, characterization of enzyme was done.
Feather degradation
100 ml of Nutrient broth is prepared; 1 gm of feather is added to the media and sterilized. A loop full of inoculum is inoculated into the media and incubated for seven days at 37°C. Residual feathers were harvested from the fermentation media by filtering it over whatman filter paper No: 3. The harvested feathers were kept in hot air oven at 50°C until weight stabilized to constant value. The difference between the weight of residual feather obtained from the control and that of inoculated media has been used as measure of feather degradation. Degradation was expressed in percentage.
RESULTS
A screening program was employed to obtain bacterial isolates capable of producing feather degrading extracellular keratinase enzyme using feather (keratin) as sole carbon substrate. The potential isolate was then characterized and identified to its genus level.
Identification of bacteria was based on morphological, cultural and biochemical tests comparing the data with standard species (Hoq et al., 2010). Morphological and physiological characteristics of the bacteria were compared with the Bergey's Manual of Systemic Bacteriology. The isolate was Gram positive, rod shaped and spore-former and were able to utilize both glucose and sucrose but not lactose. They were also catalase positive, oxidase negative. MR test is positive and VP test negative for these organisms. The organisms were unable to utilize citrate and all were able to reduce nitrate to nitrite. The isolate showed typical characteristics of Bacillus licheniformis.
Characterization of Enzyme
Characterization was done by determining the effect of pH, Temperature, Activator, Inhibitor and Substrate.
Effect of Temperature and pH on Keratinase enzyme
The activity of the enzyme at various temperatures and pH was studied and graphs are plotted. The optimum temperature and pH is 37°C and 7 respectively (Fig 1 and 2).
Effect of Activator and Inhibitor on Keratinase enzyme
The enzyme samples were checked for the effect of activator and Inhibitor. Zinc Chloride is used as activator and EDTA is used as an inhibitor. It was observed that as the concentration of the activator increased, activity of the enzyme increased where as the concentration of the inhibitor increases the activity of the enzyme decreases (Fig 3 and 4).
Effect of Substrate on Keratinase enzyme
The activity of the enzyme at different concentration was carried out and the graph is plotted (Fig 5).
Rate of degradation
Bacillus sp was able to grow and produce keratinase in nutrient medium in which feather meal served as an additional carbon and nitrogen source and acted as enzyme inducer, resulted in 85% of feather degradation in seven days at 37°C. Kerotinolytic activity was measured in the absorbance at 440 nm by the standard enzyme assay method.
DISCUSSION
A bacterium isolated from poultry waste has been shown to degrade feather keratin. The identification of the keratinolytic bacteria was based on cell morphology, colony morphology, and several other methods. These results suggested that the strain belong to genus Bacillus (Saritha Agrahari and Neeraj Wadhwa 2010). The most studied keratinolytic bacteria are Bacillus spp., which have been described to possess feather-degrading activity (Kim et al., 2001; Lin et al., 1999). Through the strategy of isolation of keratinolytic
microorganisms utilized in this work, bacteria presenting high keratinolytic activity were selected. Considering that feather protein has been showed to be an excellent source of metabolizable protein (Klemersrud et al., 1998), and that microbial keratinases enhance the digestibility of feather keratin (Lee et al., 1991; Odetallah et al., 2003), these keratinolytic strains could be used to produce animal feed protein. The enzyme was stable at the pH range of 6-8 (Cheng et al., 2007). The activity decreased at pH 3.0 and 8.0. The isolated bacterium showed Optimum keratinolytic activity at 37°C and pH 7.0. The enzyme also showed to be stable at 60°C and pH 9.0 (Kurt Cotanch and Grant 2007). In the present study, it has been recorded that the Bacillus licheniformis degraded the feather at a rate of 85% at 37°C in seven days where as Williams et al., (1990) reported that Bacillus licheniformis PWD-1 degraded chicken feather completely at 50°C in 10 days. (Bockle et al.,1995) demonstrated that Streptomyces pactum DSM40530 partially degraded native chicken feather at 50°C, the maximum feather degrading activity was at 50°C.
On increasing the feather concentration the extent of feather degradation decreases because of a decrease in keratinase activity. It indicates that at higher substrate concentration repression of keratinase production can take place. This observation is similar to previous studies which concluded that a low concentration of substrate is optimum for yielding maximum enzyme activity (Avinash et al., 2011).
CONCLUSION
In the present study, we isolated the Bacillus sp. capable of producing keratinase from habitats where keratin containing substrate is decomposed under natural conditions. The isolate exhibited highest keratinase activity is the most potential isolate which degraded feather at a rate of 85% after seven days at 37°C. The keratinolytic microorganisms isolated in this study therefore could play an important role in the production of animal feed protein in addition to the biodegradation of poultry wastes. The degradation of feathers with keratinolyticmicro organisms is the best eco friendly approach in the poultry feather waste management. This novel keratinolytic isolate could be a potential candidate for the degradation of feather keratin and also in de-haring process at leather industry.
REFERENCES
Andrea B. Friedrich and Garabed Antranikian. 1996. Keratin Degradation by Fervidobacterium pennavorans, a Novel Thermophilic Anaerobic Species of the Order Thermotogales, Applied and Environmental Microbiology, 62:2875-2882.
Avinash Srivastava, Anshul Sharma and VuppuSuneetha. 2011. Feather Waste biodegradation as a source of Amino acids, European Journal of Experimental Biology, 1 (2):56-63.
Bockle V galunhki and muller R. 1995. ”Characterization of a keratinolytic serine trotease from strectomycespactm DSM40530. APPL environ microbial., 61:3705-3710.
Cheng-gang CA1, Bing-gan LOU and Xiao-dong ZHENG. 2007. Keratinase production and keratin degradation by a mutant strain of Bacillus subtilis, Journal of Zhejiang University SCIENCE B, 9(1):60-67.
Williams CM. 1990. Isolation, Identification, and Characterization of a Feather-Degrading Bacterium, Appl. Environ. Microbiol., 56(6):1509.
Vigneshwaran C, Shanmuga S and Sathish Kumar T. 2010. Screening and Characterization of Keratinase from Bacillus Licheniformis Isolated from Namakkal poultry farm, 2(4).
Scott JA and Untereiner WA. 2004. Determination of keratin degradation by fungi using keratin azure, Medical Mycology, 42:239-246.
Kim JM, Lim WJ, Suh HJ. 2001. Feather-degrading Bacillus species from poultry waste, Process Biochem., 37:287-291.
Klemersrud MJ, Klopfenstein TJ, Lewis AJ. 1998. Complementary responses between feather meal and poultry by-product meal with or without rumminally protected methionine and lysine in growing calves. J. Anim. Sci., 76:1970-1975.
Kurt Cotanch and Rick Grant. 2007. Analysis of Nutrient Composition of Feather Meal and Feather Meal with Blood, Projact thesis.
Lee CG, Ferket PR, Shih JCH. 1991. Improvement of feather digestibility by bacterial keratinase as a feed additive. FASEB J., 59:1312.
Lin X, Inglis G, Yanke L and Cheng KJ. 1999. Selection and characterization of feather-degrading bacteria from canola meal compost, J. Ind. Microbiol. Biotechnol., 23:149-153.
Muthusamy Govarthanan Selvankumar T and Arunprakash S. 2011. Production Of Keratinolytic Enzyme By A Newly Isolated Feather Degrading Bacillus Sp. from Chick Feather Waste, International Journal of Pharma and Bio Sciences, 2(3).
Odetallah NH, Wang JJ, Garlich JD, Shih JCH. 2003. Keratinase in starter diets improves growth of broiler chicks. Poultry Sci., 82:664-670.
Tamilmani P, Umamaheswari A, Vinayagam A and Prakash B. 2008. Production of an Extra Cellular Feather Degrading Enzyme by Bacillus licheniformis Isolated from Poultry Farm Soil in Namakkal District (Tamilnadu), International Journal of Poultry Science, 7(2):184-188.
Rai Sapna and Vishwakarma Yamini. 2011. Study Of Keratin Degradation By Some Potential Bacterial Isolates From Soil, Electronic Journal of Biotechnology 1(1):01-03.
Sarita Agrahari and Neeraj Wadhwa. 2010. Degradation of Chicken Feather a Poultry Waste Product by Keratinolytic Bacteria Isolated from Dumping Site at Ghazipur Poultry Processing Plan, International Journal of Poultry Science, 9(5):482-489.
Savitha G. Joshi, Tejashwini MM, Revati N, Sridevi R and Roma D. 2007. Isolation, Identification and Characterization of a Feather Degrading Bacterium, International Journal of Poultry Science, 6(9):689-693.
Swetlana Nagal and Jain PC. 2011. Feather Degradation By Strains Of Bacillus Isolated From Decomposing Feathers, Brazilian Journal of Microbiology, 41:196-200.
Veslava Matikevičienė, DanutėMasiliūnienė and SauliusGrigiškis. 2009. Degradation Of Keratin Containing Wastes By Bacteria With Keratinolytic Activity, Environment Technology Resources Proceedings of the 7th International Scientific and Practical Conference. Volume 1.
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Kumar et al., 2012
677 Journal of Research in Biology (2012) 2(7): 676-682
Kumar et al., 2012
Journal of Research in Biology (2012) 2(7): 676-682 678
Sl. No |
Bio-Chemical Test |
Bacillus sp |
1. |
Indole production Test |
Positive |
2. |
Methyl Red Test |
Positive |
3. |
Voges proskaeur Test |
Negative |
4. |
Citrate Utilization Positive |
Positive |
5. |
Carbohydrate fermentation Test |
Positive/Presence of Air Bubbles |
6. |
Catalase |
Positive |
7. |
Starch hydrolysis |
Positive |
8. |
Urease |
Positive |
Table 1: Biochemical Characterization.
Kumar et al., 2012
Time in weeks |
Concentration of feather in gms |
Rate of degradation in % |
1 |
1 |
25 |
2 |
1 |
79 |
3 |
1 |
85 |
Table 2 : Rate of feather degradation
679 Journal of Research in Biology (2012) 2(7): 676-682
Fig 1: Effect of Temperature
Fig 2: Effect of pH
Activity (U/L)
Activity (U/L)
Temperature (°C)
pH
Kumar et al., 2012
Journal of Research in Biology (2012) 2(7): 676-682 680
Fig 4: Effect of Inhibitor
Activity (U/L)
Concentration of Inhibitor (ml)
Fig 3: Effect of Activator
Activity (U/L)
Concentration of Activator (ml)
Fig 5: Effect of Substrate
Activity (U/L)
Concentration of Substrate (µl)
Kumar et al., 2012
681 Journal of Research in Biology (2012) 2(7): 676-682
Kumar et al., 2012
Journal of Research in Biology (2012) 2(7): 676-682 682
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