Research Article

Received: 12 November 2015 Revised: 1 February 2016 Accepted article published: 5 February 2016 Published online in Wiley Online Library: 4 March 2016

(wileyonlinelibrary.com) DOI 10.1002/jctb.4912

Transformation of raw feather waste

into digestible peptides and amino acids
Hana Stiborova,a* Barbora Branska,b Tereza Vesela,a Petra Lovecka,a
Milena Stranska,c Jana Hajslova,c Monika Jiru,c Petra Patakovab and
Katerina Demnerovaa
ABSTRACT
BACKGROUND: Millions of tons of feather waste from the poultry industry are disposed of annually despite containing a
high level of keratin. The aim of this study was to compare the hydrolysis of non-treated feather waste using three different
approaches (whole cell microbial digestion, enzymatic and chemical cleavage) and to test the use of hydrolysates as peptone
substitutes in a culture medium.

RESULTS: Among bacterial isolates, Pseudomonas sp. P5 exhibited the highest keratinolytic activity and efficiency to hydrolyse
raw feather material. The hydrolysates contained up to 301 mg L−1 of free amino acids and 6.2 g L−1 of peptides. Hydrolysates
obtained by digestion using semi-purified keratinase from Pseudomonas sp. P5 were richer in amino acids (1191 mg L−1 , 56%
essential ones) but peptides were present in lower amounts (up to 3.3 g L−1 ). The third approach was feather treatment under
mild alkaline conditions. This provided the highest amount of peptides (17.2 g L−1 ) but a significantly lower level of amino acids,
especially the essential ones.

CONCLUSIONS: All approaches tested could convert raw feather waste into products of commercial value with proven use in a
cultivation medium. The level of peptides, their molecular size and amino acid composition was dependent on the method used.
© 2016 Society of Chemical Industry

Supporting information may be found in the online version of this article.

Keywords: feather waste; amino acids; peptides; microbial hydrolysis; alkaline hydrolysis; substrate

INTRODUCTION During the last decades, several methods of feather processing

While the products of the poultry industry provide a major source
of protein in the human diet, the industry also produces a vast
amount of waste that must be dealt with. One of the major waste
products is feathers, which account for 5–10% of the live weight of
an adult chicken. The amount of chicken feather generated by the
poultry industry is approximately 5 million tons annually.1 Classical
methods of feather disposal, such as incineration or landfilling,
have already been restricted or banned in some countries due to
the pollution they create.2 Thus, there is an increasing need for new
technologies that enable environmentally friendly processing of
feather waste.
Ideally, such technologies should also create valuable
by-products. Feathers contain more than 90% protein in the
form of keratin, which, being rich in cysteine, arginine, threo-
nine and hydrophobic amino acids, has high nutrient potential.3
Feather keratin could be used as an alternative to the more
expensive dietary ingredients used in animal feeds, fertilizers or
feed supplements. However, due to keratin’s rigid structure, in
which protein chains are tightly packed and stabilized through
hydrophobic interactions and disulphide bonds, degradation by
common proteolytic enzymes such as pepsin, papain or trypsin, is
unachievable.4 Unhydrolysed milled feather powder has only low
nutritional potential when converted into feed supplements, and
have been considered, but there are issues associated with each
of them. For example, feather hydrolysis with strong alkali or acid
not only leads to environmental pollution, but also affects the
amino acid composition of the products such that hydrolysates
have limited use in food applications. More recently, novel phys-
ical feather treatments have been described. Enhanced hydroly-
sis by microwave irradiation yielded high levels of amino acids
due to their low degradation when processed under such mild
conditions.5 Gao et al.6 used feather pyrolysis in supercritical
carbon dioxide and obtained two products: (i) carbon micro-
spheres that displayed super-hydrophobicity for use as a fab-
ric coating material; and (ii) ammonium bicarbonate that can be
∗ Correspondence to: H Stiborova, UCT Prague, Faculty of Food and Biochemical
Technology, Department of Biochemistry and Microbiology, Technická 3, 16628
Prague 6, Czech Republic. E-mail: hana.stiborova@vscht.cz
a UCT Prague, Faculty of Food and Biochemical Technology, Department of
Biochemistry and Microbiology, Technická 3, 16628, Prague 6, Czech Republic
b UCT Prague, Faculty of Food and Biochemical Technology, Department of
Biotechnology, Technická 5, 16628, Prague 6, Czech Republic
c UCT Prague, Faculty of Food and Biochemical Technology, Department of Food
1629

has low biodegradability when used in agriculture. Analysis and Nutrition, Technická 3, 16628, Prague 6, Czech Republic

J Chem Technol Biotechnol 2016; 91: 1629–1637 www.soci.org © 2016 Society of Chemical Industry
 www.soci.org
utilized as a fertilizer. However, a temperature as high as 600 ∘ C was
required otherwise little or no product was generated.
Other approaches for dealing with feather waste involve the use
of feather degrading microorganisms or their keratinases. Williams
et al.7 first isolated and characterized the feather degrading bac-
terium Bacillus licheniformis PWD-1. Since then, diverse groups
of bacteria and fungi, and their extracellular keratinases, have
attracted attention due to their ability to hydrolyse feathers into
products with high nutritional potential.2,3,8 – 10
Although the products prepared by the above methods contain
amino acids and peptides suitable for industrial applications,11 – 13
the feathers have to be washed, ground or milled, each of which
increases preparation costs. To investigate a lower-cost alternative,
we utilized raw feathers obtained directly from the poultry indus-
try. In addition, we directed our study to a comparison of yields and
the composition of amino acids and peptides in hydrolysates pre-
pared by three different methods: (i) microbial degradation using
Pseudomonas sp. P5 isolated from poultry bedding; (ii) keratin
hydrolysing enzymes obtained by semi-purification from Pseu-
domonas sp. P5; and (iii) weak alkaline conditions. Use of the
hydrolysates as a sole source of carbon and nitrogen in a cultiva-
tion medium was subsequently tested.
MATERIAL AND METHODS
Chicken feathers were supplied by the RABBIT s.r.o. poultry pro-
cessing plant, Czech Republic, and raw feathers were used for all
hydrolysis and cultivation experiments. Prior to all experiments the
raw feathers were sterilized by autoclaving for 20 min at 121 ∘ C.
The effect of autoclaving on feather hydrolysis was verified by
detection of free amino acids and peptides in solution.
Isolation and identification of bacteria with keratinolytic
activity
Bacteria with keratinolytic activity were isolated from poultry
bedding using enrichment cultivation on mineral medium with
feather material as sole source of carbon and energy. The mineral
medium was used according to Williams et al.7 except for no addi-
tion of yeast extract. The amount of raw wet feather material used
in cultivations varied from 30 to 90 g L−1 (10.7 to 31.9 g dry weight
L−1 ). All cultivations were carried out at 28 ∘ C or 37 ∘ C with shak-
ing at 130 rpm. The isolated bacteria were screened for prote-
olytic activity on milk agar medium14 and positive isolates were
subsequently tested for their ability to hydrolyse feathers and
for keratinolytic activity. The bacterial isolates were identified by
MALDI-TOF/TOF mass spectrometry. For the preparation of bac-
terial samples, direct transfer was used as previously described.15
Briefly, bacterial material from a single colony was smeared directly
on the MALDI target in two parallel strips, and air dried. Each
sample was then overlaid with 1 μL of freshly prepared HCCA
matrix (10 mg per mL 𝛼-cyano-4-hydroxy-cinnamic acid diluted
in 50% acetonitrile and 2.5% trifluoroacetic acid) and the mix-
ture was air dried again. Bacterial identification was performed
on the Autoflex speed MALDI-TOF/TOF instrument and spectra
were recorded in positive linear mode within the mass range
2–20 kDa at the maximum laser frequency. For each sample, sin-
gle spectra gained from 2000 laser shots in 200-shot steps from
10 randomized positions on the target spot were summarized.
The spectra were externally calibrated by a Bruker bacterial test
standard (Bruker Daltonics GmbH, Bremen, Germany). Automated
1630
analysis of raw spectra was performed by MALDI Biotyper 3.1
wileyonlinelibrary.com/jctb © 2016 Society of Chemical Industry
H Stiborova et al.
software (Bruker Daltonics GmbH, Bremen, Germany) equipped
with the 5627 MSP library (released in December 2013) using the
MALDI Biotyper pre-processing standard and the MALDI Biotyper
MSP identification standard methods.
Keratinase assay and protein determination
Keratinolytic activity was determined by a modified method pre-
viously described by Letourneau et al.16 5 mg of keratin azure
(Sigma- Aldrich) were used as substrate in the reaction prepared
by mixing 1 mL of 50 mmol L−1 Tris/HCl and 1 mL of supernatant.
The mixture was incubated for 1 h at 50 ∘ C, 130 rpm and stopped
by cooling on ice for 10 min. Excess substrate was removed by cen-
trifugation (10 000 g, 5 min) and the released azo dye was mea-
sured at 595 nm. One unit was defined as the amount of enzyme
causing an increase in absorbance of 0.01 compared with controls.
Proteins were determined using the method of Lowry.17
Keratinase production and feather hydrolysis by bacterial
isolate
The bacterial isolate with the highest keratinolytic activity and
efficacy of feather hydrolysis was chosen for subsequent experi-
ments. Production of extracellular keratinase was monitored dur-
ing 7 days of cultivation with different amounts of added wet
feathers, and ranged from 30 to 90 g L−1 (10.7 to 35.1 g dry
weight L−1 ). Feather hydrolysis was determined gravimetrically.
After 5 days of cultivation, the residual feather material was dried
at 105 ∘ C for 48 h and the percentage of degraded mass was
calculated.
Semi-purification of keratinase
Strain Pseudomonas sp. P5 was cultivated in 300 mL of mineral
medium supplemented with 70 g L−1 of raw wet feathers at 28 ∘ C,
130 rpm for 5 days. The supernatant was collected by centrifu-
gation at 15000 g for 10 min and extracellular proteins were pre-
cipitated at 4 ∘ C by addition of 80% (w/v) ammonium sulphate.
The precipitated proteins were harvested by centrifugation (15
000 g, 10 min) and then resuspended in 15 mL Tris/HCl buffer
(50 mmoL−1 , pH 7.5).
Proteins were concentrated using ultrafiltration by an Amicon
system (Millipore), cut-off 15 000 Da, and then desalted using
PD 10 (GE Healthcare) according to the gravity protocol. The
active fractions were pooled and keratinolytic activity and pro-
tein concentration were determined. The purified keratinase was
stored at 4 ∘ C and its stability was monitored over a period of
31 days.
Alkaline hydrolysis
Raw feathers were sterilized in an autoclave (121 ∘ C, 20 min) and
dried at 105 ∘ C to constant weight. Dry feathers, 2.49 g and 3.19 g
(corresponding to 7 g and 9 g of wet material) were placed into
250 mL Erlenmeyer flasks with 100 mL of 0.6% KOH and incubated
for 24 h at 70 ∘ C and 130 rpm. Unhydrolysed feather material was
removed by centrifugation at 8400 g, washed with distilled water
and dried at 105 ∘ C to determine the degree of hydrolysis. For
subsequent cultivation experiments, the pH of the supernatant
was adjusted to 7.2 ± 0.2 using 10% HCl. Acid hydrolysis was also
performed at the same conditions used for the alkali hydrolysis
with similar molarity. However, the hydrolysis efficiency was very
low (Fig. S1, Supplementary material), therefore all experiments
were conducted only for alkali hydrolysate.
J Chem Technol Biotechnol 2016; 91: 1629–1637
Transformation of raw feather waste into digestible peptides and amino acids
Amino acid analysis in feather hydrolysates
Hydrolysates were microfiltered (0.22 μm), diluted in acetoni-
trile:water (85:15, v/v) and directly analysed. Five microliters
of sample were injected into the UHPLC-OrbitrapMS system.
UHPLC (Ultra high-performance liquid chromatography) was
performed using a Waters ACQUITY UPLCTM SYSTEM (Waters,
USA), equipped with a binary solvent delivery system and an
autosampler. Chromatographic separations were performed on a
Waters Atlantis HILIC silica column (100 × 2.1 mm, 3 μm particle
size) (Waters, USA). The mobile phase was composed of solvent A
(water, 0.2% formic acid) and solvent B (acetonitrile) with a gradi-
ent elution: 0–6 min, 25 − 50% A; 6–8 min, 50% A; 8.1 − 12 min,
25% A. The flow rate of the mobile phase was 0.6 mL min−1 , and
the column temperature was maintained at 40 ∘ C. High resolution
mass spectrometric detection was performed on the ExactiveTM
Orbitrap MS (Thermo Scientific, USA), which was equipped with
a heated electrospray interface, and was operated in the positive
mode, scanning ions in m/z range 50–1000. The resolving power
was set to 100 000 full with half maximum resulting in a scan time
of 1 s. The interface parameters were as follows: spray voltage
3.5 kV, capillary voltage 25 V, capillary temperature 280 ∘ C, sheath
and auxiliary gas flow 35. Data were recorded using Xcalibur soft-
ware version 2.2 (Thermo Scientific). Twenty-three amino acids
(Ala, Arg, Asp, Asn, Cys, Cystine, Gln, Glu, Gly, His, H-Lys, H-Pro, Ile,
Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val) obtained from
Sigma Aldrich (USA) were used as external calibration standards.
Peptide concentrations in feather hydrolysates
Hydrolysates were microfiltered (0.22 μm) and further diluted in
distilled water and directly analysed. Two microliters of sample
were injected into the HPLC-DAD system. HPLC (high-performance
liquid chromatography) was performed using a Hewlett Packard
HPLC system, series 1100 (Hewlett Packard, USA) equipped with
a binary solvent delivery system and an autosampler. Chromato-
graphic separations were performed on a Yarra™ 3u SEC-2000
column, 300 × 4.6 mm, 3 μm particle size (Phenomenex, USA).
The mobile phase comprised 50 mmol L−1 phosphate buffer with
300 mmol L−1 sodium chloride at pH 6.8, elution was isocratic and
the analytical time was 20 min. The flow rate of the mobile phase
was 0.35 mL min−1 , and the column temperature was maintained
at 25 ∘ C. UV-DAD detection was achieved at a wavelength of
220 nm, which is optimum for peptide bonds. As an external cal-
ibration standard, the SEC200 reference protein standard (Waters,
USA) was used.
Determination of peptide size distribution using
MALDI-TOF/TOF mass spectrometry
Before mass spectrometric analysis, feather hydrolysates were
desalted and concentrated by ZipTip C18 pipette tips (Millipore,
USA) according to the manufacturer’s instructions. Then, 1 μL of
each sample was mixed directly on the MALDI target with 1 μL
of DHB matrix solution (20 mg mL−1 2,5-dihydroxybenzoic acid
diluted in 70:30 (v/v) ACN:0.1% TFA). Peptide Calibration Standard
II (Bruker Daltonics, Germany), used as an external calibration stan-
dard, was prepared in the same way as samples. Analysis was
performed on the Autoflex speed MALDI-TOF/TOF mass spec-
trometer (Bruker Daltonics, Germany), equipped with UV nitrogen
lasers (337 nm). The spectra were collected in the positive reflector
mode in the range 600 to 4000 m/z, where the final spectrum was
obtained by the accumulation of individual spectra measured by
500 laser shots from at least 10 different positions on the sample.
J Chem Technol Biotechnol 2016; 91: 1629–1637 © 2016 Society of Chemical Industry
www.soci.org
Signals in the spectra were then manually labelled in the mMass
version 5.4.1 software.18
Sugar content analysis
HPLC analysis (Agilent Series 1200 HPLC; Agilent, Spain) with
refractive index detection (Agilent Series 1200 Refractive Index
Detector; Agilent, Spain) was used to determine the presence of
glucose; separation conditions were as follows: IEX H+ polymer
column (Watrex, Czech Republic), 5 mmol L−1 H2 SO4 with flow rate
of 0.5 mL min−1 and column temperature 60 ∘ C.
Exploitation of feather hydrolysates – bioscreen analysis
An inoculum of Escherichia coli (DSM 6897) was prepared in 100 mL
of LB medium and cultivated for 15 h at 37 ∘ C and 130 rpm. All
types of feather hydrolysates prepared by different methods were
sterilized by filtration (0.45 μm, nitrate cellulose). Then, 100 μL
of filtered hydrolysate was inoculated with 10 μL of E. coli cell
suspension and cultivation tests were carried out at 37 ∘ C for 48 h
on a Bioscreen C micro-cultivation device (Labsystems, Finland).
Culture growth was quantified by absorbance at 600 nm.
RESULTS
Screening and identification of strains with proteolytic
and keratinolytic activities
A total of 50 bacterial strains isolated from poultry bedding were
tested. Isolates were preliminarily tested for production of extra-
cellular proteolytic enzymes by the appearance of a clearing zone
around the bacterial colony due to hydrolysis of casein in the milk
agar. From these, 11 strains exhibited both proteolytic activity
determined on milk agar as well as keratinolytic activity. For
these isolates, proteins in the MW range from 2000 to 20 000 Da
were used as phylogenetic markers and MALDI-TOF/TOF MS was
used for taxonomic identification using the Bruker Daltonics
MALDI BioTyper database. These strains belonged to the gen-
era Microbacterium, Pseudomonas, Bacillus, Cellulosimicrobium
and Filifactor. Isolates, belonging to the genera Microbacterium,
Cellulosimicrobium and Filifactor exhibited only poor hydrolytic
abilities (less than 25% feather degradation). Strains belonging
to Bacillus genera were able to moderately hydrolyse feathers
(40–60% of degradation), while two isolates belonging to the
Pseudomonas genera showed the highest feather hydrolysis.
For further experiments, Pseudomonas sp. strain P5 was chosen
based on the highest hydrolysis of feather as well as the highest
keratinolytic activity detected among the bacterial isolates tested.
Feather hydrolysis by Pseudomonas sp. P5
The overall hydrolysis of feather material by Pseudomonas sp. P5
ranged from 70 to 93% after 5 days of cultivation. The degree
of feather hydrolysis was dependent on the initial weight of
feathers tested, in concentrations from 30 to 90 g of wet raw
feather L−1 (10.7 to 35.1 g dry weight L−1 ). The highest degree
of hydrolysis was determined when 30 g L−1 of wet raw feathers
were used. On the other hand, when 70 and 90 g of raw wet
feathers L−1 were used, Pseudomonas sp. P5 biomass increased
by more than 40%, which also led to increased production of
extracellular enzymes exhibiting keratinolytic activity. Activity in
the supernatant increased from 8.5 ± 2.1 U mL−1 to a maximum
of 36.2 ± 8.4 U mL−1 when cultivated on 70 g L−1 of feathers.
Elevated enzyme activity with increased feather substrate was
1631
also documented in other studies.19,20 However, in these studies,
wileyonlinelibrary.com/jctb
 www.soci.org H Stiborova et al.

predominantly formed by feather digestion using Pseudomonas

Table 1. The content of amino acids and peptides in feather
hydrolysates sp. P5 (Fig. 1). The highest proportion of peptides comprised peaks
with m/z of 655 and 1335. Although we also observed some ions
Type of hydrolysis Amino acids (mg L−1 ) Peptides (g L−1 ) with m/z up to 2116, their relative distribution was low.
Pseudomonas sp. P5 (A) 301.2 ± 31.2 6.2 ± 0.2
Pseudomonas sp. P5 (B) 274.8 ± 6.6 4.6 ± 0.1
Feather hydrolysis by semi-purified keratinase from
Keratinase (A) 1047.6 ± 14.6 3.2 ± 0.2 Pseudomonas sp. P5
Keratinase (B) 1090.7 ± 47.6 3.3 ± 0.2
The production of extracellular keratinase by Pseudomonas sp. P5
Alkali (A) 326.9 ± 45.4 17.2 ± 2.6
was monitored over the course of 170 h. In general, a higher pro-
Alkali (B) 377.8 ± 17.0 14.3 ± 0.1
duction of keratinase occurred in late exponential and stationary
Control (A) 63.3 ± 1.6 0.7 ± 0.0
phases of microbial growth20 and, except for anaerobic bacteria,23
Control (B) 60.7 ± 7.1 0.7 ± 0.1
aeration usually increased enzyme production.19 To maintain good
(A) The initial amount of wet raw feather material was 90 g L−1 aeration, the Erlenmeyer flasks were agitated at 130 rpm and their
(31.9 g L−1 of dry feather). volume was always 5 times larger than the volume of the medium.
(B) The initial amount of wet raw feather material was 70 g L−1 The highest keratinolytic activity, 36.2 ± 8.4 U mL−1 , was detected
(24.9 g L−1 of dry feather).
after 120 h of cultivation in supernatant when 70 g L−1 of wet
raw feathers were used. Keratinase produced by Pseudomonas sp.
P5 was semi-purified by ammonium sulphate precipitation and
beyond a certain feather concentration, a negative impact on desalted. Specific activities of keratinase in the post-cultivation
activity was observed,21,22 probably caused by substrate inhibition supernatant and in the semi-purified concentrated product were
or by limited access of oxygen due to viscosity of the medium. 3.16 U mg−1 and 585.7 U mg−1 , respectively. The keratinase activity
A similar trend was observed for Pseudomonas sp. P5. optima, which were tested at ranges of pH from 7 to 9 and tem-
The levels of free amino acids and peptides in hydrolysates pre- peratures from 37–70 ∘ C, were 50 ∘ C and pH 7.5. The stability of
pared by cultivation of Pseudomonas sp. P5 on 70 and 90 g L−1 prepared enzyme, stored at 4 ∘ C, was tested over the course of 1
of raw wet feathers are summarized in Table 1. The relative dis- month. The activity of keratinase was unchanged during the first
tributions of individual amino acids in hydrolysates are described week, after 11 days activity decreased to 80%, but after 31 days
in Table 2. The essential amino acid content was more than 47%. only 18% of keratinolytic activity remained.
Among these, lysine, valine and a mixture of isoleucine and leucine Hydrolysis of 70 g and 90 g of raw wet feathers (24.9 and 31.9 g
each accounted for more than 10%. The molecular mass profiles of dry feathers) L−1 was carried out at 50 ∘ C, Tris/HCl buffer pH 7.5
of keratin peptides were analysed by MALDI-TOF/TOF MS. Low for 24 and 48 h. The degree of hydrolysis and the content of amino
molecular weight peptides in the range from 655 to 1430 m/z were acids and peptides were tested in ranges of keratinase activity from

Table 2. Relative distribution of amino acids in hydrolysates

Pseudomonas sp. P5 (A) Pseudomonas sp. P5 (B) Keratinase (A) Keratinase (B) Alkali (A) Alkali (B)

Asn 0.4 ± 0.2 0.2 ± 0.0 2.5 ± 0.1 2.5 ± 0.0 3.4 ± 0.2 2.8 ± 0.1
Asp 4.0 ± 0.2 3.9 ± 0.4 8.0 ± 0.1 7.8 ± 0.1 2.8 ± 0.0 2.7 ± 0.1
Phe 5.6 ± 0.3 5.8 ± 0.5 7.1 ± 0.1 7.1 ± 0.0 2.3 ± 0.1 2.3 ± 0.1
Tyr 3.8 ± 0.3 4.1 ± 0.2 6.2 ± 0.1 6.1 ± 0.0 2.4 ± 0.1 2.2 ± 0.1
Ile + Leu 11.8 ± 0.3 11.9 ± 0.9 21.9 ± 0.0 21.6 ± 0.2 3.7 ± 0.3 3.6 ± 0.2
Met 3.0 ± 0.4 3.3 ± 0.1 1.9 ± 0.1 1.9 ± 0.0 0.4 ± 0.0 0.4 ± 0.1
Glu 11.3 ± 2.0 13.7 ± 3.7 10.0 ± 0.1 10.0 ± 0.1 9.2 ± 0.3 8.2 ± 0.4
Val 11.9 ± 1.8 10.0 ± 0.2 11.1 ± 0.2 11.0 ± 0.5 1.3 ± 0.1 1.2 ± 0.1
Gln 1.1 ± 0.1 1.0 ± 0.0 1.1 ± 0.1 1.2 ± 0.1 7.9 ± 0.1 8.2 ± 0.1
Ala 5.1 ± 0.2 5.9 ± 0.2 1.8 ± 0.0 1.8 ± 0.1 4.2 ± 0.2 4.0 ± 0.2
Ser 1.7 ± 0.3 1.2 ± 0.0 1.6 ± 0.0 1.5 ± 0.0 36.7 ± 0.3 38.4 ± 0.9
Gly 0.5 ± 0.1 1.3 ± 0.0 1.6 ± 0.1 1.5 ± 0.1 14.8 ± 0.3 15.0 ± 0.5
Pro 0.4 ± 0.2 0.3 ± 0.1 2.5 ± 0.0 2.5 ± 0.1 4.3 ± 0.4 4.3 ± 0.1
Thr 2.4 ± 0.02 2.1 ± 0.1 1.1 ± 0.0 1.2 ± 0.2 3.3 ± 0.1 3.2 ± 0.1
His 2.5 ± 0.3 2.7 ± 0.1 6.0 ± 0.1 6.1 ± 0.1 0.8 ± 0.1 0.8 ± 0.2
Lys 11.0 ± 0.5 11.5 ± 0.5 5.8 ± 0.1 5.9 ± 0.1 1.0 ± 0.1 1.0 ± 0.1
Arg 8.3 ± 0.6 8.8 ± 0.7 7.4 ± 0.3 7.4 ± 0.3 1.2 ± 0.2 1.1 ± 0.0
cystin 4.3 ± 0.7 0.9 ± 0.2 2.6 ± 0.1 2.9 ± 0.2 n.d. 0.3 ± 0.2
H-Pro 11.0 ± 0.5 11.5 ± 0.5 n.d. n.d. 0.3 ± 0.0 0.4 ± 0.1
Essential 48.2 47.2 54.9 54.8 12.8 12.5

n.d. not detected

Amino acids tryptophan, H-lysine and cysteine were not detected in any hydrolysate. The amino acids marked in bold belong into the group of
essential ones. (A) The initial amount of wet raw feather was 90 g L−1 (31.9 g L−1 of dry feather ). (B) The initial amount of wet raw feather was 70 g L−1
(24.9 g L−1 of dry feather).
1632

wileyonlinelibrary.com/jctb © 2016 Society of Chemical Industry J Chem Technol Biotechnol 2016; 91: 1629–1637
Transformation of raw feather waste into digestible peptides and amino acids www.soci.org

Figure 1. MALDI-TOF MS analysis of peptides in hydrolysates prepared from raw feather using digestion by Pseudomonas sp. P5, keratinase and under
1633

mild alkali conditions.

J Chem Technol Biotechnol 2016; 91: 1629–1637 © 2016 Society of Chemical Industry wileyonlinelibrary.com/jctb
 www.soci.org
1 U to 2 U mg−1 dry feather. The yields of amino acids and pep-
tides in hydrolysates were not significantly affected by prolonged
digestion times from 24 to 48 h, by 1 U or 2 U of keratinase applied
to 1 mg of dry feather material. The amino acid content was higher
in comparison with other types of hydrolysis (Table 1). In contrast,
the yield of peptides was lower. Essential amino acids accounted
for more than 54% of total amino acids, from which isoleucine and
leucine represented more than 21%. Although both approaches:
(i) direct feather digestion by Pseudomonas sp. P5; and (ii) conver-
sion using semi-purified keratinase, were dependent on the same
extracellular enzymes, the amino acid distribution differed among
them (Table 2). This variability could be caused by subsequent use
of the amino acids by Pseudomonas sp. P5 for growth. On the other
hand, the molecular mass distribution of peptides was similar and
peptides were formed in the lower size range from 727 to 2116 m/z
(Fig. 1). The highest proportion comprised peaks corresponding to
peptides with m/z of 1025 and 1378.
Feather hydrolysis by weak alkali
Alkaline hydrolysis was carried out for similar feather concentra-
tions as in the previous experiments with keratinolytic strains and
isolated keratinase. Hydrolytic conditions were set with regard to
microbial strain requirements, in particular to keep salinity at a
physiological level while achieving a reasonable degree of feather
transformation. More than 80% hydrolysis was achieved at 70 ∘ C
with 0.6% KOH for 24 h on a rotary shaker at 130 rpm, maintaining
a maximum salt concentration of 8 g L−1 after neutralization with
HCl. Therefore, the hydrolysate was used directly for cultivation
without subsequent desalting. This approach is environmentally
friendly and low cost.
Experiments with 70 g and 90 g of wet untreated feather per
litre achieved 85.9 ± 0.5% and 82.9 ± 0.2% conversion, respec-
tively. Although the levels of free amino acids obtained were con-
siderably lower than with enzymatic hydrolysis, the soluble pep-
tides concentration was increased at least 4 times (Table 1). The
molecular mass distribution of peptides for an alkaline hydrolysate
was higher compared with both methods (direct digestion by
A B
0.9
0.8
0.7
0.6
OD (600 nm)
0.5
0.4
0.3
0.2
0.1
0 0
0 10 20 30 40 50
Time (h)
Figure 2. Escherichia coli growth curves on different types of hydrolysates after alkali (black diamonds), enzymatic (grey squares) and micro-
bial – Pseudomonas sp. P5 (circle) transformation together with control media (triangles) and LB media (asterix); (A) initial feather concentration 70 g L−1 ,
1634
(B) 90 g L−1 .
wileyonlinelibrary.com/jctb © 2016 Society of Chemical Industry
H Stiborova et al.
Pseudomonas sp. P5 and enzymatic hydrolysis) with the most
abundant fragments being 1971 m/z and 2058 m/z (Fig. 1).
Exploitation of feather hydrolysates as a source of nutrients
for bacterial growth
All feather hydrolysates were subjected to a cultivation test per-
formed using a Bioscreen microcultivation device. Escherichia coli
was chosen as a model microorganism owing to its rapid prolifera-
tion and ability to utilize amino acids and peptides as a sole source
of carbon and nitrogen without the need for glucose addition.
Detailed analysis of feather peptone performed by revealed pres-
ence of sugar,37 therefore HPLC analysis was performed to deter-
mine potential glucose content in alkali hydrolysate, nevertheless,
no glucose was detected.
Growth curves obtained for all hydrolysates (Fig. 2) verified their
potential in microbial growth medium as a substitute for expen-
sive culture media such as Luria-Bertani broth. Exponential growth
with a minimal lag period was recorded for enzymatic and alkaline
hydrolysates containing free amino acids. The high biomass con-
centrations achieved for both alkaline hydrolysates corresponded
with the several times higher concentration of soluble peptides.
In culture medium prepared using Pseudomonas sp. keratinolytic
activity showed a prolonged lag phase that lasted for approxi-
mately 31 h for both initial feather concentrations of 70 g L−1 and
90 g L−1 . This could be caused by inhibitory secondary metabolites
produced by Pseudomonas sp. P5 when degrading the feather ker-
atin. As a negative control, a buffer solution containing peptides
released by autoclaving (no enzyme addition) the same concen-
trations of feathers was used and incubated under identical con-
ditions. In the contol samples no E. coli proliferation was observed
within 48 h.
The generation time of E. coli was higher on LB medium and
thus the stationary phase was reached faster (after 14 h), the final
biomass concentration was only slightly higher in comparison with
growing on hydrolysates prepared by mild alkali conditions (Fig. 2).
This is promising as the composition of feather hydrolysates were
not further optimized by addition of other substances, e. g. by
0.9
0.8
0.7
0.6
OD (600 nm)
0.5
0.4
0.3
0.2
0.1
0 10 20 30 40 50
Time (h)
J Chem Technol Biotechnol 2016; 91: 1629–1637
Transformation of raw feather waste into digestible peptides and amino acids
addition of yeast extract or by combination with other peptone
hydrolysates, which is common in complex cultivation media.
DISCUSSION
Due to the fact that feathers consist mainly of the protein keratin,
feather hydrolysates containing free amino acids and digestible
peptides have potential applications in agriculture,24 as well as
in cosmetics12 or as food supplements.2 Up to now, many meth-
ods have been applied to hydrolyse feathers. However, prior to
hydrolysis, pre-treatments such as washing to remove fat, cut-
ting into small pieces, or grinding into feather powder were often
used.12,25,26 But these additional steps increased the cost of final
products. The goal of feather hydrolysis was to use conditions
that are economically favourable for industrial applications (low
cost demanding) and are environmentally friendly. In order to
simplify the subsequent technological processing, three aspects
were maintained: (i) feather hydrolyses were conducted with raw
feather waste produced by the poultry industry (Fig. S1); (ii) no
additional nutrients, such as yeast extract or carbon source (such
as sugars) were added when feathers were used for microbial cul-
tivations; and (iii) alkaline hydrolysis was carry out under mild
conditions (0.6% KOH and 70 ∘ C). In this study, we compared the
composition of hydrolysates prepared by three different methods
and after that, the use of hydrolysates as substrates for bacterial
cultivation.
Microorganisms with keratinolytic activity were isolated from
diverse environmental samples.4,7,26 We used poultry bedding and
isolated 11 strains with both proteolytic and keratinolytic activities.
Among them, Pseudomonas sp. P5 exhibited the highest kerati-
nolytic activity as well as the greatest degree of feather hydrol-
ysis. Although there are disadvantages in using direct bacterial
degradation due to partial consumption of amino acids and pep-
tides by the bacteria,27 the hydrolysates contained peptides in
concentrations up to 6.2 g L−1 . These peptides had a low molec-
ular mass with the highest distribution between 655 and 1430 m/z
(Fig. 1). A similar pattern was also described for feather hydrolysis
by Bacillus subtilis,12 whereas some bacteria produced peptides in
higher molecular mass ranges up to 10 000 m/z.28 Lower molecu-
lar mass peptides were proved to have a better ability to penetrate
into hair fibres and thus positively influence hair structure.12 There-
fore they have a potential usage in cosmetics.
The mechanism of feather keratin degradation by microor-
ganisms is not fully documented4 but it is known that bacte-
ria enzymatically decompose keratin by reduction of disulphide
bonds. Yamamura et al.29 described two types of extracellular pro-
teins produced by Stenotrophomonas sp. D-1 that promote ker-
atin degradation; a serine protease and a disulfite reductase-like
protein. Neither of these enzymes exhibited keratinolytic activ-
ity alone and keratin degradation relied on the presence of
both enzymes. Similarly, strains Pseudomonas aeruginosa KS-1
and Pseudomonas stutzeri K4 produced a mixture of extracellu-
lar keratinases that differed in molecular weight while growing
on feathers.30 – 32 Therefore, we did not aim to purify kerati-
nase to high purity, but rather employed simple precipitation
by ammonium sulphate (80%), followed by desalting and subse-
quent concentration. Keratinase, applied at 1 U mg−1 dry feather,
converted feather keratin into hydrolysates containing up to
1091 mg L−1 amino acids. As the essential amino acids isoleucine
and leucine, valine, phenylalanine, lysine, histidine, threonine and
methionine, represented more than 54% of total amino acids,
these hydrolysates provided better nutritional ingredients for
J Chem Technol Biotechnol 2016; 91: 1629–1637 © 2016 Society of Chemical Industry
www.soci.org
animal feed supplements than hydrolysis by strong alkali, acids
or steam-pressure processing.2,33 Peptides were detected in the
lower size range up to 3.3 g L−1 compared with direct degradation
with Pseudomonas sp. P5 or alkaline hydrolysis (Table 1). Oligopep-
tides also had a lower molecular mass (Fig. 1).
Alkaline hydrolysis under mild conditions was the third method
applied to convert feather into a protein hydrolysate. Employing
mild alkaline conditions for the preparation of soluble peptides
from the recalcitrant keratin structure present in chicken feathers
has already been described,34,35 however, in those studies alkaline
hydrolysis served as a pre-treatment step prior to enzymatic cleav-
age. Nevertheless, such a strategy combined the disadvantages of
both enzymatic and alkaline hydrolysis, particularly the high price
of enzymes and increased salinity after neutralization. We have
shown that even very low hydroxide concentrations achieved effi-
cient hydrolysis of feathers, providing high bioavailability of break-
down products.
As peptone is the most expensive ingredient in a culture
medium, the utilization of feather hydrolysates offers a cheap
variant for microbial cultivation. The hydrolysates prepared by
digestion with strong acids (6 N) have already been success-
fully tested as growth substrates for microorganisms.36,37 In our
study, we tested hydrolysates prepared by all three approaches
as a medium for subsequent E. coli growth. This model organ-
ism has often been used in recombinant DNA technology38,39
and its growth characteristics are well known. Escherichia coli is
also an important member of the normal mammalian intestinal
microflora, although different E. coli strains can also cause diverse
intestinal and extra-intestinal diseases.40 Therefore, rapid growth
of E. coli is important in clinical microbiology.
Both keratinase and alkaline hydrolysates provided a satis-
factory medium for E. coli growth without the need for any
feather pre-treatment step (up-stream) or additional processing
of hydrolysates (down-stream) except pH adjustment. The high-
est E. coli biomass production was observed when grown on
hydrolysates prepared under mild alkaline conditions, which cor-
responds with the highest concentration of soluble peptides
(Fig. 2, Table 1).
Surprisingly, increasing initial feather concentrations did not
lead to higher biomass concentrations in the case of alkaline or
enzymatic hydrolysis. Such findings have also been described
previously,37 and imply a lower efficiency of hydrolysis with
increasing feather load. Both cultures showed a negligible lag
period followed by exponential growth, interrupted by the diauxic
behaviour of E. coli growing in medium prepared using alkaline
but not keratinase hydrolysates. This phenomenon may be caused
by higher molecular weight protein fragments originating from
alkaline conditions (Fig. 2) and lower amounts of amino acids
which are often added into culture media (e.g. in the form of yeast
extract).
Usually, the peptones are produced industrially by controlled
enzymatic digestion of selected material such as animal tissues,
plants, protein (casein) or yeast. The production often includes
ultrafiltration with a cut-off 10 kDa to remove the endotoxins and
therefore the peptides in peptones have common size distribution
in the range 500 to >10 000. Some producers use the cut-off
up to 50 kDa so as not to lose the higher molecular weight
peptides.41 In our case, the feather hydrolysates obtained by alkali
hydrolysis contained peptides with size distribution from 617 m/z
to 2990 m/z. For example, the peptides size distribution in meat
peptones varied, with average molecular weight from 985 to
1635
3081 Da and the content of free amino acids was in the range 2.8
wileyonlinelibrary.com/jctb
 www.soci.org
to 12.9 g per 100 g of peptone powder (www.organotechnie.com).
With the typical use of 5 to 10 g L−1 of peptones in culture media
the final concentration of free amino acids is in a similar range as
was detected in hydrolysates prepared by alkaline conditions.
We have shown that such hydrolysates offer a cheap substrate
variant for microbial growth and eventually could be effectively
used as a culture medium for industrial fermentation processes
such as for the production of antibiotics, enzymes, organic acids
or biofuels.
CONCLUSION
Raw untreated feathers were converted into hydrolysates by all
three approaches tested, but the amount and molecular size of
peptide products as well as free amino acid compositions differed.
The highest transformation of feathers into soluble peptides and
amino acids (almost 60%) was achieved by alkaline hydrolysis. This
hydrolysate also supported the highest biomass growth of E. coli
when used as a peptone substitute in the cultivation medium.
In contrast, the highest amino acid production (including the
proportion of essential amino acids) was achieved by keratinase
hydrolysis. Therefore, the application of any particular method
of feather hydrolysis will depend on the subsequent use of the
hydrolysate, such as in organic fertilizers, peptone substitutes in
culture media, low cost supplements or in cosmetics.
ACKNOWLEDGEMENTS
This study was funded by the Technology Agency of the Czech
Republic Grant No. TE01020080. The authors gratefully thank Petra
Junkova for the mass spectra analysis using MALDI-TOF/TOF MS
and Livie Kralikova from UCT, Prague for isolating the strains with
keratinolytic activity.
Supporting Information
Supporting information may be found in the online version of this
article.
REFERENCES
1 Poole AJ, Church JS and Huson MG, Environmentally sustainable fibers
from regenerated protein. Biomacromolecules 10:1–8 (2009).
2 Brandelli A, Sala L, Kalil SJ. Microbial enzymes for bioconversion of
poultry waste into added-value products. Food Res Int 73:3–12
(2015).
3 Tiwary E and Gupta R, Medium optimization for a novel 58 kDa dimeric
keratinase from Bacillus licheniformis ER-15: biochemical charac-
terization and application in feather degradation and dehairing of
hides. Bioresource Technol 101:6103–6110 (2010).
4 Kornillowicz-Kowalska T and Bohacz J, Biodegradation of keratin
waste: theory and practical aspects. Waste Manage 31:1689–1701
(2011).
5 Chen J, Ding S, Ji Y, Ding J, Yang X, Zou M et al., Microwave-enhanced
hydrolysis of poultry feather to produce amino acid. Chem Eng
Process: Process Intensification 87:104–109 (2015).
6 Gao L, Hu H, Sui X, Chen C and Chen Q. One for two: conversion of
waste chicken feathers to carbon microspheres and (NH4)HCO3.
Environ Sci Technol 48:6500–6507 (2014).
7 Williams CM, Richter CS, MacKenzie Jr JM and Shih JCH, Isolation, iden-
tification, and characterization of a feather-degrading bacterium.
Appl Environ Microbiol 56:1509–1515 (1990).
8 Habbeche A, Saoudi B, Jaouadi B, Haberra S, Kerouaz B, Boudelaa
M et al., Purification and biochemical characterization of a
detergent-stable keratinase from a newly thermophilic actino-
mycete Actinomadura keratinilytica strain Cpt29 isolated from
1636
poultry compost. J Biosci Bioeng 117:413–421 (2014).
wileyonlinelibrary.com/jctb © 2016 Society of Chemical Industry
H Stiborova et al.
9 Gupta R and Ramnani P, Microbial keratinases and their prospec-
tive applications: an overview. Appl Microbiol Biotechnol 70:21–33
(2006).
10 Błyskal B, Fungi utilizing keratinous substrates. Int Biodeter Biodegrad
63:631–653 (2009).
11 Darah I, Nur-Diyana A, Nurul-Husna S, Jain K and Lim SH, Microsporum
fulvum IBRL SD3: as novel isolate for chicken feathers degradation.
Appl Biochem Biotechnol 171:1900–1910 (2013).
12 Villa ALV, Aragão MRS, dos Santos EP, Mazotto AM, Zingali RB, de Souza
EP et al., Feather keratin hydrolysates obtained from microbial ker-
atinases: effect on hair fiber. BMC Biotechnol 13:15 (2013).
13 Bach E, Cannavan FS, Duarte FRS, Taffarel JAS, Tsai SM and Brandelli A,
Characterization of feather-degrading bacteria from Brazilian soils.
Int Biodeterior Biodegrad 65:102–107 (2011).
14 Riffel A and Brandelli A, Keratinolytic bacteria isolated from feather
waste. Brazilian J Microbiol 37:395–399 (2006).
15 Uhlík O, Strejček M, Junková P, Šanda M, Hroudová M, Vlček Č et
al., Matrix-Assisted Laser Desorption Ionization (MALDI)-Time of
Flight Mass Spectrometry- and MALDI Biotyper-based identifica-
tion of cultured biphenyl-metabolizing bacteria from contaminated
horseradish rhizosphere soil. Appl Environ Microbiol 77:6858–6866
(2011).
16 Letourneau F, Soussotte V, Bressollier P, Branland P and Verneuil B,
Keratinolytic activity of Streptomyces sp SK1-02: a new isolated
strain. Lett Appl Microbiol 26:77–80 (1998).
17 Lowry OH, Rosebrough NJ, Farr AL and Randall RJ, Protein mea-
surement with the folin phenol reagent. J Biol Chem 193:265–275
(1951).
18 Strohalm M, Kavan D, Novák P, Volný M and Havlíček V, MMass 3: a
cross-platform software environment for precise analysis of mass
spectrometric data. Analyt Chem 82:4648–4651 (2010).
19 Jeevana Lakshmi P, Kumari Chitturi CM and Lakshmi VV, Efficient
degradation of feather by keratinase producing Bacillus sp. Int J
Microbiol 2013:1–7.
20 Daroit DJ, Correa APF and Brandelli A, Production of keratinolytic
proteases through bioconversion of feather meal by the Amazonian
bacterium Bacillus sp P45. Int Biodeterior Biodegrad 65:45–51 (2011).
21 Park GT and Son HJ, Keratinolytic activity of Bacillus megaterium
F7-1, a feather-degrading mesophilic bacterium. Microbiol Res
164:478–485 (2009).
22 Suntornsuk W and Suntornsuk L, Feather degradation by Bacillus sp.
FK 46 in submerged cultivation. Bioresource Technol 86:239–243
(2003).
23 Riessen S and Antranikian G, Isolation of Thermoanaerobacter ker-
atinophilus sp. nov., a novel thermophilic, anaerobic bacterium with
keratinolytic activity. Extremophiles 5:399–408 (2001).
24 Kucinska JK, Magnucka EG, Oksinska MP and Pietr SJ, Bioefficacy of
hen feather keratin hydrolysate and compost on vegetable plant
growth. Compost Sci Utilization 22:179–187 (2014).
25 Grazziotin A, Pimentel FA, Sangali S, de Jong EV and Brandelli A,
Production of feather protein hydrolysate by keratinolytic bac-
terium Vibrio sp. kr2. Bioresource Technol 98:3172–3175 (2007).
26 Khardenavis AA, Kapley A and Purohit HJ, Processing of poultry feath-
ers by alkaline keratin hydrolyzing enzyme from Serratia sp. HPC
1383. Waste Manage 29:1409–1415 (2009).
27 Matsui T, Yamada Y, Mitsuya H, Shigeri Y, Yoshida Y, Saito Y et al.,
Sustainable and practical degradation of intact chicken feathers by
cultivating a newly isolated thermophilic Meiothermus ruber H328.
Appl Microbiol Biotechnol 82:941–950 (2009).
28 Paul T, Das A, Mandal A, Halder SK, DasMohapatra PK, Pati BR et al.,
Valorization of chicken feather waste for concomitant production
of Keratinase, Oligopeptides and essential amino acids under sub-
merged fermentation by Paenibacillus woosongensis TKB2. Waste
Biomass Valorization 5:575–584 (2014).
29 Yamamura S, Morita Y, Hasan Q, Rao SR, Murakami Y, Yokoyama K et al.,
Characterization of a new keratin-degrading bacterium isolated
from deer fur. J Biosci Bioeng 93:595–600 (2002).
30 Sharma R, Verma VV and Gupta R, Functional characterization of
an extracellular keratinolytic protease, Ker AP from Pseudomonas
aeruginosa KS-1: a putative aminopeptidase with PA domain. J Mol
Catal B: Enzymatic 91:8–16 (2013).
31 Sharma R and Gupta R, Extracellular expression of keratinase
Ker P from Pseudomonas aeruginosa in E. coli. Biotechnol Lett
32:1863–1868 (2010).
32 Chaturvedi V, Bhange K, Bhatt R and Verma P, Production of kertinases
using chicken feathers as substrate by a novel multifunctional
J Chem Technol Biotechnol 2016; 91: 1629–1637
Transformation of raw feather waste into digestible peptides and amino acids
strain of Pseudomonas stutzeri and its dehairing application. Biocatal
Agricult Biotechnol 3:167–174 (2014).
33 Dalev P, Ivanov I and Liubomirova A, Enzymic modification of feather
keratin hydrolysates with lysine aimed at increasing the biological
value. J Sci Food Agric 73:242–244 (1997).
34 Dalev P, An enzyme-alkaline hydrolysis of feather keratin for obtaining
a protein concentrate for fodder. Biotechnol Lett 12:71–22 (1990).
35 Mokrejs P, Svoboda P, Hrncirik J, Janacova D and Vasek V, Processing
poultry feathers into keratin hydrolysate through alkaline-
enzymatic hydrolysis. Waste Manage Res 29:260–267 (2011).
36 Taskin M, A new strategy for improved glutathione production from
Saccharomyces cerevisiae: use of cysteine- and glycine-rich chicken
feather protein hydrolysate as a new cheap substrate. J Sci Food
Agricult 93:535–541 (2013).
J Chem Technol Biotechnol 2016; 91: 1629–1637 © 2016 Society of Chemical Industry
www.soci.org
37 Taskin M and Kurbanoglu EB, Evaluation of waste chicken feath-
ers as peptone source for bacterial growth. J Appl Microbiol
111(4):826–834 (2011).
38 Lee SY, Mattanovich D and Villaverde A, Systems metabolic engineer-
ing, industrial biotechnology and microbial cell factories. Microb Cell
Fact 11:1–3 (2012).
39 Johnson IS, Human insulin from recombinant DNA technology. Science
219:632–637 (1983).
40 Kaper JB, Nataro JP and Mobley HLT, Pathogenic Escherichia coli. Nat
Rev Micro 2:123–140 (2004).
41 Pasupuleti VK and Braun S, State of the art manufacturing of protein
hydrolysates. Protein Hydrolysates Biotechnol 11–32 (2010).
1637
wileyonlinelibrary.com/jctb