Keratin degradation by bacteria and fungi isolated ...

This article was downloaded by: [Sultan Qaboos University] On: 22 April 2015, At: 22:36 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20

Keratin degradation by bacteria and fungi isolated from a poultry farm and plumage a

b

N. Sivakumar & S. Raveendran a

Department of Biology, College of Science, Sultan Qaboos University, Muscat, Oman

b

Bio Nano Electronics Research Center, Graduate School of Interdisciplinary New Science, Toyo University, Kawagoe, Japan Accepted author version posted online: 03 Jan 2015.Published online: 22 Apr 2015.

Click for updates To cite this article: N. Sivakumar & S. Raveendran (2015) Keratin degradation by bacteria and fungi isolated from a poultry farm and plumage, British Poultry Science, 56:2, 210-217, DOI: 10.1080/00071668.2014.996119 To link to this article: http://dx.doi.org/10.1080/00071668.2014.996119

PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions

British Poultry Science, 2015 Vol. 56, No. 2, 210–217, http://dx.doi.org/10.1080/00071668.2014.996119

Keratin degradation by bacteria and fungi isolated from a poultry farm and plumage N. SIVAKUMAR

AND

S. RAVEENDRAN1

Downloaded by [Sultan Qaboos University] at 22:36 22 April 2015

Department of Biology, College of Science, Sultan Qaboos University, Muscat, Oman, and 1Bio Nano Electronics Research Center, Graduate School of Interdisciplinary New Science, Toyo University, Kawagoe, Japan

Abstract 1. Poultry processing generates a large quantity of feather waste. Feathers are a rich source of keratin and could be used as a feather meal in the feed industry if the keratin is degraded using suitable micro-organisms. 2. In this study, keratin-degrading micro-organisms were isolated from a poultry farm. The predominant organisms were identified as Bacillus subtilis, Aspergillus flavus, A. fumigatus and Trychophyton sp. 3. The isolates were inoculated into feather medium and observed for keratin degradation by measuring the protein content, free amino acids and change in pH. 4. During feather degradation by B. subtilis, the concentration of soluble protein released to the medium increased gradually and reached the maximum (433 µg/ml) during d 7 of incubation and the pH increased from the initial 6.9 to 8.4 on d 9 of incubation. Similarly, the maximum protein content of 414 µg/ml and pH of 8.5 was observed for A. fumigatus on d 21 of incubation. 5. B. subtilis and A. fumigatus showed almost the same level of keratinase activity.

INTRODUCTION Birds are bearing one of the most complex epidermal derivatives called feathers which are the major habitat for many keratinophilic and keratinolytic micro-organisms. Feathers are unique in their structure and protect the birds from the external environment (Hudon, 2005). They are complex integumentary appendages in the birds, classified under keratins with β-confirmation. Usually keratins are non-vulnerable to the action of common proteases secreted by the resident flora of birds. But the feathers could be degraded easily once exposed to a certain environment. The poultry-processing industry generates several million tons of chicken feathers as a by-product and disposes of the feathers as a waste (Williams et al., 1991). Hence a great amount of useful proteins and amino acids are wasted. The feather keratin accumulates in the soil, either with the death of the bird or by disposal during poultry processing. The keratin is in the form of microfibrils which are a combination of 4 parallel protofibrils.

These are highly resistant to common proteolytic enzymes (Scott and Untereiner, 2004). Owing to poor digestibility, the feathers are not included in animal feed stuffs. Keratinolytic microbes play a vital role in the process of deriving nourishment out of these waste feathers, by converting the feather protein into an easily digestible form. Feather meal production from waste feathers available in the poultry farm would be an additional commercial benefit for the poultry industry. So the recycling of waste feathers by producing feather meal using keratinase-producing micro-organisms is a subject of interest (Gupta and Ramnani, 2006). Several micro-organisms such as fungi (Gradisar et al., 2005; Anbu et al., 2007), Bacillus species (Ramnani and Gupta, 2004; Werlang and Brandelli, 2005) and a few actinomycetes (Ivanko and Varbanets, 2004) were reported to degrade keratinaceous substrates by producing keratinase enzyme. Hence the search for efficient keratinase producing microbes is of significant importance. In this study, plumage collected from a large scale

Correspondence to: N. Sivakumar, Department of Biology, College of Science, Sultan Qaboos University, PC 123 Muscat, Oman. E-mail: [email protected] Accepted for publication 24 October 2014.

© 2015 British Poultry Science Ltd

MICROBIAL DEGRADATION OF KERATIN

poultry farm was used to isolate keratinolytic microorganisms. The isolates were studied for degradation of feather keratin substrate and their respective ability to release the constituent amino acids and peptides, thus investigating the possibility to include the keratin metabolites as feather meal in poultry feed.

MATERIALS AND METHODS Collection of feather samples


Feather samples were collected from the soil inside and outside a poultry farm and also from the bodies of birds. Collected feather samples were aseptically transferred to sterile plastic bags and taken to the laboratory for culturing the microbial population. Isolation and identification of microbes from the feather Appropriate dilutions of samples were cultured on nutrient agar, mannitol salt agar and De Mann Rogosa and Sharpe (MRS) medium to isolate different types of bacteria and on Sabourd dextrose agar to isolate fungi. The feather impregnation method was adopted to isolate the keratinophilic micro-organisms. After purification, individual colonies of the isolates were subjected to biochemical characterisation and identified according to Bergey’s manual of determinative bacteriology (1994). Carbohydrate utilisation tests were performed using API 50 strips (bioMerieux) and the results were analysed using APILAB software (API® - BioMerieux). Fungal isolates were stained using lactophenol cotton blue to study the morphological characteristics and identified according to Dugan (2006).

211

Fungal keratin degradation assay Feather samples (0.5 g) were added to 50 ml of sterile basal medium (K2HPO4: 1.5, MgSO4. 7H2O: 0.025, CaCl2: 0.025, FeSO4.7H2O: 0.015, ZnSO4.7H2O: 0.005) (Deshmukh and Agrawal, 1985). The flasks were inoculated with spores of isolated fungi. All the inoculated flasks and the uninoculated keratin control were incubated at 27°C for 4 weeks in a shaker at 300 rpm. During fermentation, 2 ml of the culture fluid was removed after every week and filtered by Whatman No.1 filter paper to remove the mycelial mat. The filtrate was used as a crude enzyme. The filtrate was assayed for release of proteins, total free amino acids and change in pH. Protein released was estimated by the method of Lowry et al. (1951).

Keratinase assay Keratinase activity was assayed according to the method of Letourneau et al. (1998) using keratin azure as substrate. The reaction mixture was 0.2 ml of the crude enzyme solution (supernatant), 0.8 ml 0.01 M Tris–HCl buffer (pH 8.5) and 4 mg of keratin azure. Control contained the boiled mixture of enzyme and substrate. The reaction mixture was incubated at 50°C for one hour. Then the substrate was removed by centrifugation at 24000 g for 10 min. The optical density of the supernatant was measured at 595 nm for the release of the azo dye, against the control. One unit of keratinase activity was defined as the amount of enzyme that increases the absorbance by 0.1 at 595 nm within one hour, under the conditions described.

RESULTS Bacterial keratin degradation assay Feather samples (0.5 g) were cut into small pieces, washed in sterile distilled water and added to 50 ml of the basal medium (composition g/l: NH4Cl: 0.5, NaCl: 0.5, K2HPO4: 0.3, KH2PO4: 0.4, MgCl2.6H2O: 0.1, yeast extract: 0.1, pH: 7.0) (Williams et al., 1990). After sterilisation, medium was inoculated with 24 h culture of the isolates and incubated at 37°C for 10 d in a shaker at 175 rpm. During fermentation, samples were collected aseptically at regular time intervals and centrifuged at 11000 g for 10 min to remove cells and insoluble residues. The obtained culture supernatant was used to analyse the pH, soluble proteins, total free amino acids, as well as used as a crude enzyme extract to analyse the keratinolytic activity. Total dry weight of feather remains was also measured.

Different types of bacteria and fungi were isolated from poultry feathers and poultry farm soil.These micro-organisms were identified with their respective cultural, microscopic and biochemical characteristics (Tables 1 and 2). Different species of bacteria such as B. subtilis, B. licheniformis, E. coli, Klebsiella sp., S. aureus, Pseudomonas sp., Salmonella sp. and fungi such as A. flavus, A. fumigatus, Trichophyton and yeast were identified. It was observed that one of the bacterial colonies, white, opaque, irregular, raised and endospore forming was predominant in the medium. Collective characteristic features indicated that this isolate was Bacillus subtilis. Among the fungi Aspergillus flavus, A. fumigatus and Trichophyton and yeast were identified with their cultural and microscopic characteristics. Identified B. subtilis and fungal cultures were

212

N. SIVAKUMAR AND S. RAVEENDRAN

Table 1. Micro-organisms isolated from poultry feathers S. no.

Samples

NA

MRS

MSA

SDA

1.

Feather from farm soil

E. coli Klebsiella Bacillus Staphylococcus Pseudomonas

Yeast Bacillus

Staphylococci

Yeast, A. niger, A. flavus Trichophyton

2.

Feather from outside farm soil

Bacillus E. coli Salmonella Klebsiella Staphylococci

Bacillus Yeast

Staphylococci Bacillus

A. flavus A.fumigatus A. niger Yeast

NA: Nutrient agar, MRS: De Mann Rogosa and Sharpe, MSA: Mannitol salt agar, SDA: Sabourd dextrose agar medium.


Table 2. S.no. 1 2 3 4 5 6 7 8

Organism B. subtilis B. licheniformis Lactobacillus sp. S. aureus E. coli K. pneumonia Salmonella sp. Pseudomonas sp.

Indole

MR

− − − − + − + −

− − − + + − − −

Biochemical characteristics of isolated bacterial species

VP Citrate + + + ± − ± + −

− − − − − + − +

Glu

Lac Xylose Starch

A AG AG A AG AG A A

− AG AG A AG AG − −

A AG − − − − − −

+ + − − − − − −

Anaerobic growth

Growth at 60°C

− + − − − − − −

+ + + − − − − −

Oxidase Catalase Motility − + − − − − − +

+ + + + + + + +

− + − − + − + −

SF (spore former): B. subtilis, B. licheniformis, Lactobacillus sp.; NSF (non spore former): S. aureus, E. coli, K. pneumoniae, Salmonella sp., Pseudomonas sp.; A: acid; AG: acid gas; +: positive; −: negative.

subjected to fermentation analysis in which feather keratin was added as prime substrate. Feather degradation B. subtilis was grown on feathers containing basal salt medium. Almost complete degradation of feathers was observed after 7 d of incubation at 37°C. Biodegradation was measured by the increase of free amino acids and soluble protein

in the growth medium. The concentration of soluble protein released to the medium increased gradually, reached a maximum on d 7 of incubation and declined thereafter. Similarly, free amino acids released in the medium increased up to 3 d then started to decrease (Figure 1). B. subtilis grown in feather medium secretes keratinase that hydrolyses the β-keratin network of the feathers thereby releasing oligopeptides

Figure 1. Protein concentration and total amount of free amino acids available in the medium during feather keratin degradation by B. subtilis.


and amino acids in the medium. Total dry weight of feathers before and after the fermentation showed a significant decrease in its weight. Using the Kjeltec 2100 distillation assembly, the total soluble protein available in the medium after the keratinolysis was estimated at 84.89% (Table 3). Another major factor analysed during the fermentation assay of B. subtilis was changes in the hydrogen ion concentration. It was observed that the pH of the medium increased to 8.44 from the initial pH of 6.8 by d 9 of B.

213

subtilis incubation (Figure 2). The range of pH during the keratinolytic process indicates the action of the exoenzyme keratinase, which releases soluble amino group to the medium by the process of deamination resulting in ammonia production. But any further increase in the pH was limited by the volatility of ammonia, which escapes from alkaline solutions in gaseous form. Keratinase activity of B. subtilis increased continuously until the maximum (35.3 U/ml) was reached on d 7 of incubation (Figure 3) correlating with

Table 3. Weight and protein content of feathers before and after keratin degradation


S.no. 1. 2. 3. 4.

Organism

Initial weight of feather (g)

B. subtlis A. flavus A. fumigatus Trichophyton sp.

0.5 0.5 0.5 0.5

Final weight of feather (g) 0.182 0.06 0.034 0.27

± ± ± ±

0.035 0.01 0.01 0.01

Initial protein (%) 20.0 19.68 19.68 19.68

Figure 2. Changes in pH during keratin degradation by B. subtilis.

Figure 3. Keratinase activity of B. subtilis at different time intervals.

± ± ± ±

0.29 0.03 0.02 0.05

Final protein (%) 84.89 14.01 9.24 13.66

± ± ± ±

0.33 0.018 0.03 0.06

214


an increase in protein concentration of the medium.


Fungal keratin degradation Spores of Aspergillus flavus, A. fumigatus and Trichophyton were inoculated into basal salt media with 0.5 g of feathers and maintained for 4 weeks in an incubator shaker. A matt growth was observed in all the three flasks with fungal cultures with cottony fungal mycelial growth around each and every fibre of the feather. This very well indicated the tendency of all three fungi to grow on keratin substrate as its sole source. Decreased protein content of the media was observed after feather fermentation (Table 3). But during fermentation, the most rapid release of protein was observed after three weeks of incubation in each case. The highest value of released protein, that is, 413 μg/ml was obtained after three weeks of feather degradation by A. fumigatus followed by

Trichophyton (375 μg/ml) and A. flavus (310 μg/ ml) (Figure 4), thus indicating a maximum keratinolysis by A. fumigatus followed by Trichophyton and A. flavus. Concentration of protein estimated by the Kjeltec 2100 distillation method suggested that all the three fungi were able to degrade a good amount of feather keratin as a substrate. A change in pH of the culture fluid during the experimental period was noted (Figure 5). A maximum pH around 8.5 was observed after three weeks of incubation in all cases indicating that the release of protein might have increased the alkalinity of the medium. Highest alkaline pH was observed for Trichophyton followed by A. flavus and A. fumigatus. The keratinase activity of all three fungi showed the same trend. All three fungi exhibited maximum keratinase activity during the third week of incubation. A. fumigatus showed higher activity (33.7 U/ml) than Trichophyton (29 U/ml) and A. flavus (25.7 U/ml) (Figure 6).

Figure 4. Amount of protein present in the medium at different time intervals during fungal keratin degradation.

Figure 5. Changes in pH during feather degradation by fungi.


215


Figure 6. Keratinase activity of three fungi isolated from a poultry farm.

DISCUSSION Feathers are highly resistant to the action of common proteolytic enzymes. Feathers are composed of more than 90% protein and are accumulated in large amounts as a waste from poultry processing. Dumping the feather waste will cause environmental pollution and a waste of feather protein (Gousterova et al., 2005). Traditional ways of feather degradation by alkali hydrolysis and steam pressure results in the destruction of amino acids and also consume large amounts of energy. Biodegradation of feathers by keratinase producing micro-organisms would be an alternative energy-conserving approach (Cai et al., 2008). Almost all keratinases are inducible enzymes and various keratin-containing materials such as feathers, hair and wool were used as substrates for keratinase production. Among them, feathers were the mostly utilised substrate (Gupta and Ramnani, 2006). In this study, B. subtilis bacteria isolated from feathers collected in a poultry farmyard were able to degrade feather keratin by using feathers as a primary source of energy, carbon and sulphur. One explanation for the presence of this species in a poultry farmyard may be the indigenous nature of this bacterium in the chicken gut. Hence, it is more likely to be present in the environment where poultry excreta are present. Hence, these bacteria have adapted to utilise feathers present in the poultry farm soil as their substrate (Kunert, 2000). When cells of B. subtilis were grown aerobically on feather medium, they released soluble peptides and sulphhydryl containing amino acids as degradation products of keratin in the fermentation broth. The increase in soluble peptides and free amino acids suggests that the bacterial isolate possess proteolytic enzymes which cleave the disulphide linkages in the

feather keratin. It was found that the total free amino acid concentration first increases and then gradually decreases. This could be due to the amino acid utilisation by B. subtilis, enhancing the conversion of amino acids into ammonia, which is volatile and escapes out immediately. Medium supplemented with 1% (w/v) feather was used in this study to induce maximum keratinase production. Similarly, Werlang and Brandelli (2005) reported that 1% (w/v) feather powder yielded the highest keratinase activity for Bacillus spp. It was also demonstrated that higher concentrations (3–5%) (w/v) of feather caused substrate inhibition or repression of keratinase production by Bacillus spp. FK46 (Suntornsuk and Suntornsuk, 2003). Maximum keratinase activity was achieved at d 7 of incubation by B. subtilis. Microbes with differing rates of keratin degradation were already reported. Similar to the B. subtilis of this study, Streptomyces lavendulae required 7 d for maximum keratinase production (Chitte et al., 1999) whereas B. licheniformis PWD-1 degraded intact feather completely at 50°C in 10 d (Williams et al., 1990). It was also reported that Streptomyces sp. MS-2 produced maximum keratinase activity after 72 h (Mabrouk, 2008). Keratinophilic fungi can grow on keratinaceous substrates in distilled water, but grow better in solutions of salts. Hence, the basal medium with feathers as a substrate was used for fungal keratin degradation. The degradation imparted by all the three fungi reached a maximum on d 21 of fermentation and they are also capable of releasing a considerable amount of keratinase enzyme. When spores of genus Aspergillus and Trichophyton were inoculated, the mycelium clustered forming a matt growth around the feather fibres after 4 d of incubation. After the establishment of growth in the feather fibres, the fungi


216


started secreting keratinase, which was evident from the increase in concentration of peptides and amino acids in the surrounding medium. This is probably because of the breaking of disulphide bridges in the preliminary steps of keratin degradation. Towards the end of the third week a noticeable amount of soluble peptides and amino acids were harvested from the culture fluid. Keratinophilic fungi were adapted to utilise these proteins and amino acids as the main or sole source of nutrition. This could be a possible reason for the low protein content in the feather medium inoculated with fungi. The fungi might have utilised the released proteins and amino acids in the medium more effectively than the bacteria (Kunert, 2000). It has been suggested that feathers subjected to a brief steam treatment followed by controlled biodegradation are an energy-efficient medium for production of digestible protein (Williams et al., 1990). The concentration of degradation products from protein hydrolysis may reach hundreds of micrograms per ml, but it is only weakly related to the rate of substrate degradation. A better indicator of keratinolysis is the rise in pH. It was observed that the pH of the medium increased to 8.44 from the initial neutral pH of 6.8 in the keratinolytic bacterial culture of B. subtilis after 7–11 d of incubation. Similarly, during keratin degradation by fungi the pH increased from neutral to 8.5. The increased pH reflects the utilisation of keratin proteins, deamination and ammonia production. All keratinolytic micro-organisms increase the pH to at least 8 (McQuade, 1964). Previous findings reported that either neutral or basic conditions were required for keratinase production by feather-degrading bacteria (Cheng et al., 1995; Riffel et al., 2003; Werlang and Brandelli, 2005). In cultures of keratinolytic fungi, pH usually peaks at 8–9; pH exceeding 10 were rarely reported (Kunert, 2000). The only waste from poultry processing is feather keratins while other wastes can be recycled in feedstuffs. By applying the appropriate microorganism for keratin fermentation, the feather keratin could be utilised effectively. From an industrial point of view it would be best to stop keratin fermentation when the protein and amino acid concentration is high in the fermentation medium. Extended fermentation results in the utilisation of these proteins and amino acids by the inoculated micro-organisms. The products of feather degradation could have a potential to be used as a feed supplement because of a high protein and amino acid content. This could further increase the profit of the poultry industry and utilise feathers instead of disposing them as waste.

REFERENCES ANBU, P., GOPINATH, S.C.B., HILDA, A., LAKSHMIPRIYA, T. & ANNADURAI, G. (2007) Optimization of extracellular keratinase production by poultry farm isolate Scopulariopsis brevicaulis. Bioresource Technology, 98: 1298–1303. doi:10.1016/j. biortech.2006.05.047 BERGEY, N.R.G. (1994) Bergey’s Manual of Determinative Bacteriology (Philadelphia, PA, Williams and Wilkins). CAI, C., LOU, B. & ZHENG, X. (2008) Keratinase production and keratin degradation by a mutant strain of Bacillus subtilis. Journal of Zhejiang University SCIENCE B, 9: 60–67. doi:10.1631/jzus.B061620 CHENG, S.W., HU, H., SHEN, S.W., TAKAGI, H., ASANO, M. & TSAI, Y.C. (1995) Production and characterization of keratinase of a feather-degrading Bacillus licheniformis PWD-1. Bioscience Biotechnology and Biochemistry, 59: 2239–2243. doi:10.1271/ bbb.59.2239 CHITTE, R.R., NALAWADE, V.K. & DEY, S. (1999) Keratinolytic activity from the broth of a feather-degrading thermophilic Streptomyces thermoviolaceus strain SD 8. Letters in Applied Microbiology, 28: 131–136. doi:10.1046/ j.1365-2672.1999.00484.x DESHMUKH, S.K. & AGRAWAL, S.C. (1985) Degradation of Human Hair by some dermatophytes and other keratinophilic fungi: Der abbau von menschlichem Haar durch einige dermatophyten und andere keratinophile pilze. Mycoses, 28: 463–466. doi:10.1111/j.1439-0507.1985. tb02160.x DUGAN, F.M. (2006) The Identification of Fungi: An Illustrated Introduction with Keys, Glossary, and Guide to Literature (St. Paul, MN, APS Press). GOUSTEROVA, A., BRAIKOVA, D., GOSHEV, I., CHRISTOV, P., TISHINOV, K., VASILEVA-TONKOVA, E., HAERTLE, T. & NEDKOV, P. (2005) Degradation of keratin and collagen containing wastes by newly isolated thermoactinomycetes or by alkaline hydrolysis. Letters in Applied Microbiology, 40: 335–340. doi:10.1111/j.1472-765X.2005.01692.x GRADISAR, H., FRIEDRICH, J., KRIZAJ, I. & JERALA, R. (2005) Similarities and specificities of fungal keratinolytic proteases: Comparison of keratinases of paecilomyces marquandii and doratomyces microsporus to some known proteases. Applied and Environmental Microbiology, 71: 3420– 3426. doi:10.1128/AEM.71.7.3420-3426.2005 GUPTA, R. & RAMNANI, P. (2006) Microbial keratinases and their prospective applications: An overview. Applied Microbiology and Biotechnology, 70: 21–33. doi:10.1007/ s00253-005-0239-8 HUDON, J. (2005) Considerations in the conservation of feathers and lair, particularly their pigments. Proceedings of the CAC/ACCR 31st Annual conference, Jasper, pp. 127–141. IVANKO, O.V. & VARBANETS, L.D. (2004) Keratinolytic activity of Streptomyces sp. 1382. Mikrobiol Z, 66: 3–9. KUNERT, J. (2000) Physiology of keratinophilic fungi, in: KUSHWAHA, R.K.S. & GUARRO, J. (Eds). Biology of Dermatophytes and Other Keratinophilic Fungi, pp. 77–85 (Bilbao, Spain, Revista Iberoamericana de Micologia). LETOURNEAU, F., SOUSSOTTE, V., BRESSOLLIER, P., BRANLAND, P. & VERNEUIL, B. (1998) Keratinolytic activity of streptomyces sp. SK1–02: A new isolated strain. Letters in Applied Microbiology, 26: 77–80. doi:10.1046/j.1472-765X.1998.00281.x LOWRY, O.H., ROSEBROUGH, N.J., FARR, A.L. & RANDALL, R.J. (1951) Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 193: 265–275. MABROUK, M.E.M. (2008) Feather degradation by a new keratinolytic Streptomyces sp. MS-2. World Journal of Microbiology and Biotechnology, 24: 2331–2338. doi:10.1007/s11274-008-9748-9 MCQUADE, A.B. (1964) Microbiological degradation of wool. Dermatology, 128: 249–266. doi:10.1159/000254758 RAMNANI, P. & GUPTA, R. (2004) Optimization of medium composition for keratinase production on feather by Bacillus licheniformis RG 1 using statistical methods involving



response surface methodology. Biotechnology and Applied Biochemistry, 49: 191–196. RIFFEL, A., LUCAS, F., HEEB, P. & BRANDELLI, A. (2003) Characterization of a new keratinolytic bacterium that completely degrades native feather keratin. Archives of Microbiology, 179: 258–265. SCOTT, J.A. & UNTEREINER, W.A. (2004) Determination of keratin degradation by fungi using keratin azure. Medical Mycology, 42: 239–246. doi:10.1080/13693780310001644680 SUNTORNSUK, W. & SUNTORNSUK, L. (2003) Feather degradation by Bacillus sp. FK 46 in submerged cultivation. Bioresource Technology, 86: 239–243. doi:10.1016/S0960-8524(02)00177-3

217

WERLANG, P.O. & BRANDELLI, A. (2005) Characterization of a novel featherdegrading Bacillus sp strain. Applied Biochemistry and Biotechnology, 120: 71–79. doi:10.1385/ ABAB:120:1:71 WILLIAMS, C.M., LEE, C.G., GARLICH, J.D. & SHIH, J.C.H. (1991) Evaluation of a bacterial feather fermentation product, feather-lysate, as a feed protein. Poultry Science, 70: 85–94. doi:10.3382/ps.0700085 WILLIAMS, C.M., RICHTER, C.S., MACKENZIL, J.M. & SHIH, J.C.H. (1990) Isolation, identification and characterization of a feather degrading bacterium. Applied and Environmental Microbiology, 56: 1509–1515.