International Biodeterioration & Biodegradation 103 (2015) 134e140

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An optimization approach to scale up keratinase production

by Streptomyces sp. 2M21 by utilizing chicken feather
Tug €

çe Demir, E. Esin Hameş*, Suphi S. Oncel, Fazilet Vardar-Sukan
_
Department of Bioengineering, Faculty of Engineering, Ege University, 35100, Bornova-Izmir, Turkey

a r t i c l e i n f o a b s t r a c t

Article history: The aim of this study was to optimize the culture conditions for keratinase production by a new isolate,
Received 6 April 2015 Streptomyces sp. 2M21 utilizing chicken feather as an alternative low-cost substrate. Contribution of
Received in revised form different independent fermentation variables on keratinase activity was investigated by Plackett-Burman
25 April 2015
design. Parameters that have the highest contribution were selected for the optimization experiments
Accepted 25 April 2015
which were carried out by using response surface methodology (RSM). Relative and mutual effects of
Available online
fermentation variables on keratinase activity were analyzed with “23 Full Factorial Central Composite
Design”. Cultivation processes were implemented in 250 ml Erlenmeyer flasks containing 50 ml medium.
Keywords:
Fermentation
Maximum keratinase activity was obtained under optimal conditions of 28
C, 5.0 g l1 chicken feather
Environmental biotechnology and 5.5 days of process time. As a result of the optimization studies, a 15 fold increase in keratinase
Keratinase activity was reached compared to the unoptimized conditions. Furthermore, similar results were ob-
Feather tained in the 2 l bioreactor under optimized conditions.
Experimental design © 2015 Elsevier Ltd. All rights reserved.
Optimization

Introduction Keratinases, have potential usage in leather industry for de-

Organic wastes with their high pollution loads exhibit a threat
for sustainability demanding researchers to seek alternative
methods of valorization, leading to zero waste processes within a
biorefinery concept. Utilization of organic wastes as substrates for
the production of high value products is a common approach.
Chicken feather which is almost pure keratin is accumulated
from poultry processes and is produced in millions of tons annually.
It has a great potential as a raw material for industrial applications
(Riffel et al., 2003; Werlang and Brandelli, 2005; De Azeredo et al.,
2006). Feather is currently converted into feather meal by chemical
or thermal processes for utilization as feed in poultry industry.
However these processes decrease the nutrient quality of feather
and need high energy consumption (Papadopoulos et al., 1986;
Grazziotin et al., 2006). Feather meal production by the alterna-
tive microbial hydrolysis method, provides higher nutritional
values as well as improving the quality (Onifade et al., 1998;
Werlang and Brandelli, 2005). Feather hydrolysates produced can
also be used as soil fertilizers due to their high nitrogen content
(Brandelli, 2008).
* Corresponding author. Tel./fax: þ90 232 3884955.
E-mail addresses: esin.hames@ege.edu.tr, esinhames@gmail.com (E.E. Hameş).
hairing process that improves leather quality. They can also be
used in textile industry for wool and silk cleaning and modification
of fibers. Capability of attaching on hard surfaces and removing
keratinous blemish also makes keratinases attractive for detergent
market. Some of the other potential applications are production of
biodegradable films, coatings and glues, bioactive peptides as well
as prion hydrolysis, drug delivery and waste water treatment
(Gupta and Ramnani, 2006; Brandelli, 2008; Sharma and Gupta,
2012).
Keratinous substrates contain disulfide bonds, hydrogen bonds
and hydrophobic interactions in addition to cross-linking of protein
chains by cysteine bridges, which are responsible for their recalci-
trant nature, cannot be degraded by common proteolytic enzymes
(Werlang and Brandelli, 2005; Karthikeyan et al., 2007; Brandelli
et al., 2010). Some microorganisms belong to fungi and bacteria
have the ability to degrade these substrates (Onifade et al., 1998;
Werlang and Brandelli, 2005). Streptomyces sp. is a promising
extracellular enzyme producer with keratinolytic activity in addi-
tion to their capabilities for the production of valuable secondary
metabolites such as antibiotics and antitumor agents (Deshpande
et al., 1988; Çetinel-Aksoy et al., 2012; Demir et al., 2013).
Understanding the interactions of process parameters on pro-
ductivity enables optimization which is a helpful tool to show the
correlations between the process parameters (Demir et al., 2013).

http://dx.doi.org/10.1016/j.ibiod.2015.04.025
0964-8305/© 2015 Elsevier Ltd. All rights reserved.
 T. Demir et al. / International Biodeterioration & Biodegradation 103 (2015) 134e140 135

Several reports are available presenting statistical optimization of
keratinase production by using chicken feather. However optimi-
zation has not been used as a tool for scale-up for keratinase pro-
duction (Ramnani and Gupta, 2004; Thys et al., 2004; Anbu et al.,
2005; Bernal et al., 2006; Casarin et al., 2008; Rai et al., 2009; Rai
and Mukherjee, 2011). The aim of this study is to focus on the
optimization of keratinase production by a new isolate, Strepto-
myces sp. utilizing chicken feather, as a raw material and use this
approach for scale-up.
Materials and methods
Microorganisms
In this study, alkaliphilic actinomycetes strains (180 strains)
from Actinomycetes Culture Collection (ACTINOCC) founded by
Bioengineering Department of Ege University, registered to World
Federation of Culture Collections No. 952 were used.
Chicken feathers
Feathers were obtained from a local poultry processing plant

lu Co., Manisa, Turkey) were washed with warm distilled
(Keskinog
water and dried at 45
C in an incubator for 2 days. Dried feathers
were autoclaved at 121
C for 45 min and stored at room temperature.
Screening and selection of the strain
Preliminary screening experiments were implemented using
agar plates technique for the 180 alkaliphilic actinomycetes strains.
In this step protease activity of the strains were evaluated by single
point inoculation of actinomycetes onto agar plates (containing
g l1: starch 10, yeast extract 4, peptone 2, skim milk 10 and agar 15;
pH 10). The plates were then incubated at 28
C for 3 days and 10
promising protease producers were selected according to the ratio of
clear zone diameter around the colony to the diameter of the colony.
Selected strains were then investigated for their feather degra-
dation ability using chicken feather as the sole carbon and nitrogen
source. For this purpose these strains were cultivated in 250 ml
Erlenmeyer flasks with 50 ml of mineral salt medium (g l1: CaCO3
5, NaCl 5, K2HPO4 3, feather 5; at pH 8.0, 28
C, 150 rpm for 6 days
inoculated with 108 spores ml1). Produced mycelial biomass and
residual solid substrate were removed by centrifugation (Hettich
Universal, 32) at 5000 rpm for 3 min. Cell free fermentation broth
was used for extracellular keratinase activity assay.
Keratinase activity assay
Keratinase activity was measured according to Delmar et al.
(1979). Suc-Ala-Ala-Pro-Phe-NA (BaChem) was dissolved in
mixture of 0.05 M TriseHCl (pH 8) buffer and dimethyl sulfoxide
(DMSO) (Merck Chemicals) [1:99 (v v1)] and used as substrate
solution. The reaction mixture (140 ml of 0.05 TriseHCl buffer, 50 ml
of 2 mM substrate solution and 10 ml of cell free fermentation broth)
was incubated at 37
C for 10 min. Amount of released pNA (p-
nitroanilide) was measured by using spectrophotometer (Versa-
Max, USA) at 405 nm against the blank which consisted of 150 ml of
named as Streptomyces sp. 2M21. 16S rDNA sequence was compared
with the data accessible through NCBI (National Centre for
Biotechnology Information) using BLASTN. DNA sequence of the
isolate 2M21 was deposited to GenBank under Accession number
JX000224.
Design of experiments and statistical analysis
Selection of the key fermentation parameters for optimization of
keratinase production through keratinase activity was performed
according to Plackett-Burman design. Following the selection step,
optimization experiments were carried out by using response
surface methodology (RSM) with “23 Full Factorial Central Com-
posite Design (CCD)”.
Design Expert Software 7.0.0 (Stat-Ease, Inc., Minneapolis, USA)
was used for experimental designs, regression and variance anal-
ysis of models and calculation of polynomial model equations.
Analysis of variance (ANOVA) was used to evaluate the interaction
of variables with the predicted response.
Plackett-Burman experimental design
Nine fermentation variables; time, temperature, pH, inoculum
size, amount of feather, concentrations of CaCO3, NaCl, K2HPO4,
MgSO4$7H2O were used in the design. Each parameter was tested,
in 250 ml Erlenmeyer flasks (50 ml medium) with continuous
mixing (150 rpm), at two levels (maximum: “þ” and minimum:
“”) (Table 1) in the defined experimental design matrix (Table 2).
The values of the tested parameters were determined according to
the preliminary experiments considering their effects on keratinase
production. Two dummy factors in the experimental design were
added and statistical analysis was performed with 12 experiments.
The first order polynomial is used to predict the response of
variables:
X
Y ¼bþ b i xi
where Y is the predicted response, b is the model intercept is the
linear coefficient and is the level of the independent variable.
Response surface methodology for optimizing the selected
fermentation conditions
RSM was used for the optimization of fermentation conditions
(in 250 ml Erlenmeyer flasks with 50 ml mineral salt medium,
continuous mixing at 150 rpm, utilizing 8  108 spores ml1) tar-
geting maximum keratinase activity. The experiments were
designed by CCD with three key factors, namely temperature, time
and the amount of feather, which were selected according to their
impact on the keratinase activity through Plackett-Burman design.
The design matrix was consisted of 20 experimental runs at five
different levels including 8 factorial points, 6 axial points and 6
Table 1
Minimum and maximum levels of variables used in Plackett-Burman.
Variable Symbol Minimum Maximum
values () values (þ)

TriseHCl buffer and 50 ml of substrate solution. One unit of kera- Time x1 1 day 4 days
tinase activity was defined as the amount of enzyme required to Temperature x2 20
C 30
C
liberate 1 mmol pNA under standard conditions. pH x3 5.0 9.0
Feather x4 5 g l1 20 g l1
Inoculum size x5 4*108 spores ml1 8*108 spores ml1
Identification of the strain CaCO3 x6 0 g l1 5 g l1
NaCl x7 0 g l1 5 g l1
The strain selected with regards to the highest keratinase ac- K2HPO4 x8 0 g l1 1 g l1
MgSO4$7H2O x9 0 g l1 1 g l1
tivity was identified according to 16S rDNA sequence analysis,
136 T. Demir et al. / International Biodeterioration & Biodegradation 103 (2015) 134e140

Table 2
Plackett-Burman experimental design matrix and experimental results.

Run Variable Time Temp. pH Feather Inoculum CaCO3 NaCl K2HPO4 MgSO4$7H2O Dummy Keratinase
variables activity (U ml1)

Symbol x1 x2 x3 x4 x5 x6 x7 x8 x9 x10 x11

Effect (%) 29.62 40.80 1.12 10.25 0.016 7.93 2.40 3.735E-003 7.01

1 þ þ e þ þ þ e e e þ e 55.26
2 e þ þ e þ þ þ e e e þ 25.53
3 þ e þ þ e þ þ þ e e e 35.34
4 e þ e þ þ e þ þ þ e e 17.54
5 e e þ e þ þ e þ þ þ e 3.21
6 e e e þ e þ þ e þ þ þ 3.09
7 þ e e e þ e þ þ e þ þ 8.97
8 þ þ e e e þ e þ þ e þ 35.99
9 þ þ þ e e e þ e þ þ e 27.09
10 e þ þ þ e e e þ e þ þ 30.43
11 þ e þ þ þ e e e þ e þ 20.36
12 e e e e e e e e e e e 1.31

replicates at the center point. The actual values of the variables at
different levels were shown in Table 3.
Second order polynomial is used to predict the response of
variables:
X X X Results and discussion
Y ¼ b0 þ bi xi þ bii x2i þ bij xi xj
where Y is the predicted response, b0 offset term, bi linear effect, bii
squared effect, bij interaction effect.
Production in 2 l bioreactor
Scale up was carried out by using a 2 l stirred tank bioreactor
(Sartorious Biostat A, Germany) batch wise (in triplicate) with a
working volume of 1.5 l under the optimized conditions (28
C,
5 g l1 feather in mineral salt medium, pH 8.0, 5.5 days). Mixing was
provided with 150 rpm agitation and 1.25 vvm aeration rate.
Inoculum was prepared in 250 ml Erlenmeyer flasks with
50 ml glucose-yeast extract (GY) broth (containing g l1: glucose
10, yeast extract 2, peptone 25, K2HPO4 0.1 and pH 8.0) with
continuous mixing at 150 rpm, utilizing 8  108 spores ml1 for
36 h. Produced mycelial biomass in GY broth was collected by
centrifugation at 5000 rpm for 5 min and suspended in distilled
water prior to inoculation to the bioreactor. At the end of the
production cell free fermentation broth was achieved for kerati-
nase assay.
The key in the selection of the process variables was based on
the nature of keratinase production. Feather was used as the sole
carbon and nitrogen source, and a culture medium was defined
with the help of the other studies, with feather and mineral salts
(Letourneau et al., 1998; Mohamedin, 1999; Syed et al., 2009;
Vasileva-Tonkova et al., 2009). Utilization of this simple me-
dium has fewer variables to analyze for successful optimization.
This also supported the aim of the study providing an approach
for scale-up with cost effectiveness, using an alternative by-
product.
Screening of keratinase producers and identification of isolate 2M21
After the screening stage, isolate 2M21 was selected as the
highest keratinase producer with a keratinase activity of 32 U ml1.
The 16S rDNA gene sequences shares 99% homology with that of
Streptomyces genus.

Table 3
CCD matrix with predicted and experimental responses.

Run Independent variables Response; keratinase activity (U ml1)

1
A; temperature ( C) B; feather (g l ) C; time (day) Experimental Predicted

1 20 5.4 3 40.153 18.69

2 35 5.4 3 333.063 292.93
3 20 12.6 3 41.613 27.67
4 35 12.6 3 400.720 380.65
5 20 5.4 6 392.523 372.35
6 35 5.4 6 346.937 320.61
7 20 12.6 6 340.721 340.59
8 35 12.6 6 311.622 330.21
9 15 9 4.5 8.036 43.924
10 40 9 4.5 276.306 297.267
11 27.50 3 4.5 281.171 348.272
12 27.50 15 4.5 405.496 395.334
13 27.50 9 2 41.117 100.732
14 27.50 9 7 389.910 387.177
15 27.50 9 4.5 364.775 366.88
16 27.50 9 4.5 402.342 366.88
17 27.50 9 4.5 381.712 366.88
18 27.50 9 4.5 370.281 366.88
19 27.50 9 4.5 301.264 366.88
20 27.50 9 4.5 390.678 366.88
 T. Demir et al. / International Biodeterioration & Biodegradation 103 (2015) 134e140 137

Table 4
ANOVA results for Plackett-Burman design.

Source Sum of squares Degrees of Mean square F-value P-value Prob > F
freedom

Model 2895.09 9 321.68 25.64 0.0381

x1 864.93 1 864.93 68.94 0.0142
x2 1191.30 1 1191.30 94.95 0.0104
x3 32.72 1 32.72 2.61 0.2477
x4 299.18 1 299.18 23.85 0.0395
x5 0.47 1 0.47 0.038 0.8642
x6 231.62 1 231.62 18.46 0.0501
x7 70.03 1 70.03 5.58 0.1420
x8 0.11 1 0.11 8.693E-003 0.9342
x9 204.75 1 204.75 16.32 0.0562
Residual 25.09 2 12.55
Cor Total 2920.18 11

Optimization of keratinase production
Plackett-Burman design
Based on the statistical analysis of the model, effects of the var-
iables are represented in Table 2. The ANOVA results for the Plackett-
Burman design are presented in Table 4 giving detailed information
about the model terms. The Model-F value (25.64) showed that the
model is significant (Table 4). There is only a 3.81% chance that a
“Model-F value” could occur due to noise. The determination coef-
ficient (R2) that explains the consistence of predicted and actual
responses with a value of 0.9914 validated the model. Adequate
precision value which measures the signal to noise ratio was
determined as 17.097 which is greater than desirable ratio (4)
indicating that this model can be used to navigate the design space.
According to the statistical analysis, time, temperature, amount of
feather are significant (p < 0.05) and their contribution percentages
showed that highest contribution was with temperature (40.80%),
followed by time (29.62%) and feather amount (10.25%) (Table 2).
pH, inoculum size, concentrations of NaCl, CaCO3, MgSO4$7H2O and
K2HPO4 are insignificant terms (Table 4). The influence of time,
temperature, amount of feather, concentration of CaCO3 are positive,
the variables including inoculum size, NaCl, K2HPO4 and
MgSO4$7H2O have negative influence on keratinase production at
their high levels. Previous reports related to optimization of kera-
tinase from Bacillus species using Plackett-Burman and RSM also
showed that mineral salts had some effect or were even ineffective
in the keratinase production (Ramnani and Gupta, 2004; Tatineni
et al., 2007; Fakhfakh-Zouari et al., 2010).
Response surface methodology
Plackett-Burman design showed that the major influencing pa-
rameters were time, temperature and amount of feather. Even if
other parameters especially the mineral salts such as CaCO3 (7.93%),
MgSO4$7H2O (7.01%) and NaCl (2.4%) had some effect on the pro-
duction monitored by the activity of keratinase, their influence was
weak compared to three major parameters reaching a total impact of
80.67% (Table 2). This comparison was the main goal for the selection
of the first 3 parameters to be used as the building block of the CCD.
Effects of the medium components including NaCl, CaCO3,
MgSO4$7H2O and K2HPO4 were insignificant but Streptomyces sp.
2M21 required these components for growth and keratinase pro-
duction. Therefore, concentrations of these components were used
at standard amounts in the mineral salt medium (pH 8.0) and
inoculum size (8.108 spores ml1).
In order to determine the optimum values of the selected vari-
ables and to investigate their mutual interaction on keratinase
production, analysis was performed at five different levels by CCD.
The design matrix calculated from regression model with respect to
experimental and predicted responses is presented in Table 3.
Experimental results were correlated as a second order polynomial
model. The empirical relationship between predicted keratinase
activity and variables in coded units regardless of their significance
was expressed as following quadratic equation:
Y ¼ 366:88 þ 75:31A þ 13:99B þ 85:15C þ 10:34AB  90:84AC
 19:53BC  69:38A2 þ 1:74B2  43:45C 2
where Y is the predicted keratinase activity, A, B, C are coded values
of temperature, amount of feather and time, respectively.
Statistical significance of the model was analyzed using Fischer's
F test and the analysis of variance of the model is shown in Table 5.
According to the statistical analysis, the P value of the quadratic
regression model was found to be much smaller (<0.0001) than the

Table 5
ANOVA results for CCD.

Source Sum of squares Degrees of Mean square F-value P-value Prob > F
freedom

Model 3.400e þ 005 9 37,775.72 16.15 <0.0001

A 77,457.76 1 77,457.76 33.12 0.0002
B 2673.71 1 2673.71 1.14 0.3101
C 99,013.93 1 99,013.93 42.34 <0.0001
AB 854.60 1 854.60 0.37 0.5590
AC 66,012.09 1 66,012.09 28.23 0.0003
BC 3051.14 1 3051.14 1.30 0.2800
A2 69,372.77 1 69,372.77 29.66 0.0003
B2 43.64 1 43.64 0.019 0.8941
C2 27,208.32 1 27,208.32 11.63 0.0066
Residual 23,387.72 10 2338.77
Lack of Fit 17,038.27 5 3407.65 2.68 0.1513
Pure Error 6349.45 5 1269.89
Cor Total 3.634E þ 005 19
138 T. Demir et al. / International Biodeterioration & Biodegradation 103 (2015) 134e140

desired significance level (0.05) which is a proof of a high signifi-

cancy. Also, values of ‘p’ less than 0.05 indicate that model terms are
significant. In this model, A, C, AC, A2, C2 are significant model terms.
Values greater than 0.1000 indicate model terms are insignificant.
The optimum values of fermentation variables for maximum ker-
atinase activity were determined from the quadratic model and the
response surface curves as A ¼ 28
C, B ¼ 5 g l-1and C ¼ 5.5 days.
According to the quadratic regression model, the predicted kera-
tinase activity for these conditions was 412 U ml1.
The R2 value of 0.9356 which indicates the relevancy of the fit
showed that the actual values were close to the predicted values for
the selected fermentation variables. The model explains 93.56% of
the total variations. The “Lack of Fit F-value” of 2.68 implies the lack
of fit is not significant relative to the pure error suggesting it is a
good fit for this model. The adequate precision value of 12.107
shows that the model can be used to navigate the design space.
The interaction between time, temperature and amount of
feather with respect to keratinase activity in 3D plots shows the
effect of these variables (Fig. 1). Specifically, as the saddle-like
formation implies (Fig. 1a and b), the effect of feather amount on
keratinase activity is minor compared to time and temperature.
This effect could also be observed in Run 6 and 8 of CCD experi-
ments in which, approximately two fold increase in the amount of
feather caused 10% increase in keratinase activity at 35
C for 6
days. The minor effect of feather could be explained by the change
in the rheology and the shift to the solid state appearance of
culture medium as the amount of feather increases. The increased
feather amount also limited aeration, caused an insufficiency for
growth and resulted in a decrease in enzyme productivity. This
effect was supported by the observations with Bacillus lichen-
iformis RGI utilizing chicken feather and Streptomyces sp7 using
feather meal for keratinase production (Ramnani and Gupta, 2004;
Tatineni et al., 2007).
The interaction between time and temperature exhibited in
Fig. 1c clarify their major effect on keratinase activity. The lowest
keratinase activity (Run 9: 8.03 U ml1) was with the lowest tem-
perature condition (15
C) which was actually outside the isolate's
growth temperature range (25e37
C) (Whitman et al., 2012). On
the other hand, the highest temperature (Run 10:40
C) resulted in
a higher activity (276.3 U ml1) but this result was less than the
highest activity reached at 27.5
C (405.5 U ml1), which was within
the growth temperature range. Similar observations were reported
previously with other bacteria (Thys et al., 2004; Anbu et al., 2008;
Casarin et al., 2008) describing of the effect of temperature on the
keratinase production. Also highest activities with thermo-tolerant
strains of Streptomyces were reported at temperatures of 45
C and
even 50
C, showing that keratinase production is a temperature
dependent process (Mohamedin, 1999; Gupta and Ramnani, 2006;
Cai et al., 2008; Syed et al., 2009). With regards to time interval, the Fig. 1. The 3D plot showing the interaction between (a) time and amount of feather on
keratinase activity, (b) temperature and amount of feather on keratinase activity, (c)
CCD was between days 2 and 7 with a shift from the 1e4 days in time and temperature on keratinase activity.
Plackett-Burman design. This actually depended on the laboratory
observations of Plackett-Burman studies, in which the feathers in
Predicted results obtained from the quadratic regression model
the Erlenmeyers were stable at day 1 and nearly vanished at day 4.
were confirmed by the experiments under predicted optimal
Based on the activity results, the production was expected to in-
fermentation conditions. The experimental response (28
C, 5 g l1
crease until day 4. Therefore, the process was extended up to 7 days
feather, 5.5 days) reached 487 U ml1 which was close to the pre-
in order to observe the decrease. This was also parallel to the
dicted response (412 U ml1). These results between the observed
studies related to keratinase production using a range of microor-
and predicted responses indicated the reliability of the model for
ganisms from actinomycetes, bacteria to fungi, with cultivation
keratinase activity.
periods from 24 h to several days (up to 10 days) (Gupta and
Ramnani, 2006; Anbu et al., 2008; Casarin et al., 2008; Rai et al.,
2009). The optimum duration for high keratinase activity was Production in 2 l bioreactor
around days 4e6 for Streptomyces in this study and this was in good
agreement with the results of Streptomyces gulbargensis DAS 131 In order to investigate the potential of optimization as a tool for
(Syed et al., 2009) and Streptomyces thermonitrificance scale up in keratinase production utilizing chicken feather, opti-
(Mohamedin, 1999) with values of 120 h and 96 h, respectively. mum process conditions of the Erlenmeyer experiments were used
 T. Demir et al. / International Biodeterioration & Biodegradation 103 (2015) 134e140
in 2 l bioreactor. An important observation when feather is used as
the sole substrate was that the feathers floated in the medium in
the beginning of the process (Fig. 2a). During the fermentation, the
rheology of the production medium was affected by the decrease in
the particle sizes, increase in the mycelia biomass during the pro-
cess. It was difficult to maintain a homogeneous environment in the
bioreactor, due to this ever-changing situation of the process
especially in the first 48 h (Fig. 2b and c).
Similar keratinase activities with respect to time were observed
in bioreactor experiments (Fig. 3) and Erlenmeyer studies (Fig. 1a).
A maximum keratinase activity of 400 ± 10 U ml1 obtained in the
bioreactor was close to the predicted value (412 U ml1) but lower
than the experimental value in Erlenmeyer scale (487 U ml1).
Reasons for the lower keratinase activity obtained in the 2 l
bioreactor scale compared to Erlenmeyer scale can be attributed to
the different conditions inside the vessel, specifically on the me-
chanical agitation and direct aeration as well as the consumption of
an insoluble, floating substrate like feather (Fig. 2a).
Change in environmental parameters (nutrient availability, pH,
temperature, shear, etc.) are mainly affected by agitation and
aeration conditions dominating the success of industrial produc-
tion (Garcia-Ochoa and Gomez, 2009). Since high agitation rates
may cause disruption of cells in the bioreactor by shear forces and
lower agitation rates may result in poor mass transfer for oxygen
and substrate, it is important to optimize aeration and agitation
rates for maximizing the yield (Large et al., 1998). Although the
batch production in bioreactor showed encouraging results with
respect to scale up of keratinase production by Streptomyces sp.
2M21 using chicken feather, the lower activity indicated the need of
statistical optimization to maximize the productivity of keratinase
in the batch culture.
Conclusions
The present study was the first in a series of studies empha-
sizing a scale up approach for keratinase production by Strepto-
myces sp. 2M21 utilizing chicken feather as a potential renewable
Fig. 2. (a) the insoluble, floating structure of feather (b) the fermentation medium in
2 l bioreactor at the end of 24 h (c) the fermentation medium in 2 l bioreactor at the
end of 48 h.
139
Fig. 3. Keratinase activity with respect to time in 2 l bioreactor.
raw material without a requirement for carbon or nitrogen sup-
plements. Obtaining a standard production yield from all batches
under the same conditions was not easy due to filamentous
structure of Streptomyces strain and the insoluble nature of the
substrate. However, optimization experiments were successfully
performed with a 15 fold increase in keratinase activity. On the
other hand, based on the encouraging results from the Erlenmeyer
and bioreactors, further studies are still under investigation to
fulfill the ultimate aim of maximizing the keratinase production in
higher scales (up to 30 l) with an optimization approach as a tool for
scale-up.
Acknowledgments
This work was supported by The Research Fund of Ege Univer-
sity (Grant number 11.MUH.005) and supported in part by TUBITAK
(The Scientific and Technical Research Council of Turkey) (Grant
number MAG-112M250).
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