This document discusses the isolation and characterization of a feather-degrading bacterium from poultry waste sites in Thiruvananthapuram, India. Soil samples were collected and plated on feather agar to isolate bacteria. The isolate showing highest keratinase activity was selected for further study. It was identified as Bacillus thuringiensis based on cultural, biochemical and molecular analysis. Microscopic analysis revealed spore formation. This feather-degrading bacterium could help reduce waste and environmental pollution from poultry industry.

1. V.P. Potty
Principal Scientist
CEPC
Kollam
G.M. Nair
Prof. & Head
Department of Plant Sciences
Central University of Kerala
Resmi Raj L
Research Scholar
Department of Botany
University of Kerala

2. Introduction
Feather as poultry waste.
Disposal of waste feathers.
Protein wastage and environmental
pollution.

3.  Feathers represent over 90% protein.
 β-keratin
 Structural rigidity due to disulfide, hydrogen and
hydrophobic bonds.
 Resistance to degradation by common microbial
proteases.
 Degradation by inducible enzyme - keratinase.

4.  Only a minor quantity of waste feathers is being
used, even though wide industrial applications are
possible.
 Pretreatment methods hydrolyze feather to
corresponding amino acids and small peptides.
 Drawbacks of the pretreatment methods gave a way
to use microbial keratinases.
 Thus there is a requirement of isolation of
keratinases from natural sources to meet the
industrial and environmental demand.

5. Objectives
 Isolation of feather degrading bacteria from
various poultry-waste dumping sites of
Thiruvananthapuram.
 Selection by screening of the maximum feather
degrading strain from among the isolates.
 Characterization of the selected strain by cultural
and biochemical characters; molecular and
microscopic techniques.

6. Materials & Methods
 Collection of soil samples from different poultry-waste
dumping sites.
 Plating of the bacterial suspension on feather agar
plates.
 Incubation of the plates at 37º C for 24 to 48 hrs.
 Selection of single colonies and maintenance at 4ºC for
further work.

7. The basic medium used for isolation and fermentation of
feather degrading micro organisms contained:
 NaCl - 0.5 g/L
 KH2PO4 - 0.7 g/L
 K2HPO4 - 1.4 g/L
 MgSO4 - 0.1 g/L
 Feather - 10 g/L
 1ml of 24hr grown culture (4.25×105 CFU/ml) in LB
broth was used as inoculum.

8. Keratinolytic activity determination
 0.5 % (w/v) soluble keratin was used as substrate for
keratinolytic activity determination (Wawrzkiewicz et
al., 1987).
 Crude enzyme was obtained from inoculated medium
after 96 hrs of cultivation and done enzyme assay
according to Cai et al., 2008 with uninoculated medium
as control.
 One unit of enzyme activity (U/ml) was defined as an
increase of corrected absorbance at 280 nm with that of
the control for 0.01/minute.

9. Taxonomic studies and identification
of the microorganism with potential
keratinolytic activity
 The cultural, morphological and biochemical characteristics of
the selected bacterial strain were compared with the data from
Bergey’s Manual of Determinative Bacteriology (Robert S. Breed
et al., 1957).
 Molecular characterization of 16S rRNA gene was carried out.
Bacterial 16S rDNA was amplified by using the universal primers:
 Forward primer: 8F 5’ AGAGTTTGATCCTGGCTCAG 3’
 Reverse primer: 1492R 5’CGGTTACCTTGTTACGACTT 3’

10.  Sequence obtained was aligned using BioEdit
sequence alignment software and verified for
correctness using the BLAST option available at:
http://www.ncbi.nlm.nih.gov/
 The 16S rRNA sequence of the strain was then
submitted to GenBank and obtained the
accession number. A phylogenetic tree was
constructed using TREECONW 3.1 software
package.

11. (a)Light microscopy
Sporulated culture was stained and examined in a
light microscope (Olympus BX51 Image Analyser)
to study the crystal morphology. The slide was
then heat fixed and stained (0.133% CBB R-250
stain in 50% acetic acid), rinsed in distilled water,
dried and observed under light microscope using
a 100x oil immersion objective.
Microscopic studies

12. (b) Phase Contrast microscopy
 Spore-crystal mixture was prepared in sterile distilled
water after heat treatment for 1hr and a wet mount of the
sample was observed in phase contrast microscope (Zeiss
Observer A1, AX10) under 100x oil immersion objective.
(c) Scanning Electron microscopy
 A thin smear of the spore-crystal suspension was
prepared on glass mounts and then coated with gold in
JEOL-JFC 1200 sputtering unit and photographed in
JEOL-JSM 5600LV scanning electron microscope
operated at a voltage of 20 kV.

13. Results & Discussion
Isolation of keratinolytic microorganisms
 Six morphologically different bacteria were
isolated from the collected soil samples. All the
strains were maintained in Nutrient Agar for
elucidating their keratinolytic activity as well as
feather degrading capacity. S3KUBOT showed
maximum activity as per the keratinolytic activity
determining assay.

14. Fig.1 Growth of the isolate S3KUBOT on (a) nutrient agar medium (b) feather agar medium
Fig.2 (a) uninoculated control medium (b) inoculated with S3KUBOT after 48hrs.

15. Keratinolytic activity determination
All the six strains isolated were
able to grow and produce
extracellular keratinase in the
medium in which whole
chicken feather served as the
sole carbon and nitrogen
sources. The maximum enzyme
activity was observed on the
4th day of inoculation for all
the isolates (Fig.3). The strain 3
designated as S3KUBOT was
selected which was found to
exhibit maximum enzyme
activity (31.92 U/ml) on the
basis of the assay. All the strains
except S4 showed the
morphological and cultural
characteristics of Bacillus
species.
0
5
10
15
20
25
30
35
S1 S2 S3 S4 S5 S6
Enzymeactivity(U/ml)
Isolates
24hrs
48hrs
72hrs
96hrs
120hrs
Fig. 3

16. Taxonomic studies and identification
•Phylogenetic tree was inferred from Tajma and Nei (1984)
distances using the neighbour-joining method and
constructed using the software TREECONW 3.1. (Fig.4)
•The branching pattern was checked by 100 bootstrap
replicates. The 1514 bp sequence obtained was submitted
to GenBank and obtained the GenBank accession number,
HQ832565.
•The cultural, morphological and biochemical features
agreed with the description of Bacillus species in
Bergey’s Manual of Systematic Bacteriology.

17. Morphological characteristics Inference
Form
Gram stain
Spore
Rod
Positive
Spore forming
Cultural characteristics
Feather Agar and Nutrient Agar Creamy white colonies
Biochemical Test Inference
Catalase
Oxidase
O-F
MR
VP
Citrate
Indole
Phenyl alanine
Nitrate reduction
Gelatin liquefaction
Gelatinase
Amylase
Lecithinase
Motility
Positive
Positive
Positive
Positive
Negative
Positive
Negative
Negative
Positive
Rapid liquefaction
Positive
Positive
Positive
Positive

18. Fig. 4

19. Microscopic studies
(a) Light microscopy
Fig. 5(a) Fig. 5(b)

20. (b) Phase Contrast microscopy
Fig. 6

21. (c) Scanning Electron microscopy
Fig.7

22. Conclusion
The strain S3KUBOT has been reported to produce
keratinases during vegetative phase which makes the
degradation of feather possible within 96 hrs of
incubation.
The inefficiency of the 16S rRNA gene analysis may be
the prime reason that a lot much of isolates belonging to
the Bacillus cereus group are still been identified as
Bacillus species within the GenBank.
The isolate S3KUBOT was confirmed as Bacillus
thuringiensis by electron microscopy to possess a
parasporal body.

23. References
•Cheng-gang Cai, Bing-gan Lou, Xiao-dong Zheng. (2008)
Keratinase production and keratin degradation by a mutant strain
of Bacillus subtilis. J Zhejiang Univ Sci B 9(1): 60-67.
•Robert, S. Breed, E. G. D. Murray and Nathan, R. Smith. (1957)
Bergey’s Manual of Determinative Bacteriology. Seventh Edition,
Baltimore. The Williams & Wilkins Company.
•Wawrzkiewicz, K., Lobarewski, J. and Wolski, T. (1987)
Intracellular keratinases of Trichophyton gallinae. J. Med. Vet.
Mycol. 25: 261–268.

24. Thank You