effects of culture conditions on growth and feather degradation capability of chryseobacterium indologenes k14

MINISTRY OF EDUCATION & TRAINING
CAN THO UNIVERSITY
BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE

SUMMARY
BACHELOR OF SCIENCE THESIS
THE ADVANCED PROGRAM IN BIOTECHNOLOGY

EFFECTS OF CULTURE CONDITIONS ON
GROWTH AND FEATHER DEGRADATION
CAPABILITY OF CHRYSEOBACTERIUM
INDOLOGENES K14

SUPERVISOR

STUDENT

Dr. BUI THI MINH DIEU

DANG CHI THIEN
Student’s code: 4087890
Session: 34 (2008 - 2013)

Can Tho, 05/2013


APPROVAL

SUPERVISOR

Dr. BUI THI MINH DIEU

STUDENT

DANG CHI THIEN

Can Tho, May , 2013
PRESIDENT OF EXAMINATION COMMITTEE

Dr. DUONG THI HUONG GIANG


Abstract
The aim of this study was to investigate the optimal cultural
conditions affecting the growth and feather degradation
capability of Chryseobacterium indologenes K14, a featherdegrading mesophilic bacterium. The results showed that 30°C
and 8.0 as the optimum culture temperature and medium pH for
growth and keratinase production of this bacteria. Keratinolytic
activity of the bacterium were also depended on source and
concentration of carbon, nitrogen in culture medium. 2% (w/v)
corn starch and 0.1% (w/v) soy flour were determined as the best
carbon and nitrogen concentrations showing highly significant
values comparing to other treatments in keratinase activity (23.21
U/ml and 26.61 U/ml) and feather degradation capability
(46.33% and 48.26%), respectively. The amount of keratinolytic
enzyme production depended on feather concentrations and it was
found highly significant with 1% (w/v) supplement. Maximum
enzyme activity was found to be 23.63 (U/ml) on the fourth day of
culture period.
Key words: feather degradation, keratinolytic activity, optimal
conditions

i


CONTENTS

ABSTRACT………………………………………………...........i
CONTENTS..………………………..……………...……....…...ii
1. INTRODUCTION ..................................................................... 1
2. MATERIALS AND METHODS .............................................. 2
2.1. Materials ............................................................................. 2
2.2. Methods .............................................................................. 2
2.2.1. Method for testing keratinolytic activities ................... 2
2.2.2. Method for determinating of bacterial growth and
feather degradation ...................................................... 3
2.2.3. Effect of medium pH and cultural temperature on
growth and feather degradation of Chryseobacterium
indologenes K14.......................................................... 3
2.2.4. Effect of different carbon sources and carbon
concentrations on growth and feather degradation of
Chryseobacterium indologenes K14 ........................... 4
2.2.5. Effect of different nitrogen sources and nitrogen
concentrations on growth and feather degradation of
Chryseobacterium indologenes K14 ........................... 5
2.2.6. Effect of feather concentrations on growth and feather
degradation of Chryseobacterium indologenes K14 ... 5
2.2.7. Effect of time course on growth and feather
degradation of Chryseobacterium indologenes K14 ... 6
2.2.8. Data analysis ............................................................... 6
ii

3. RESULTS AND DISCUSSION ............................................... 7
3.1. Effect of temperature and pH on growth and feather
degradation by Chryseobacterium indologenes K14 ......... 7
3.2. Effect of different carbon sources and carbon
concentrations on growth and feather degradation of
Chryseobacterium indologenes K14 ................................ 10
3.3. Effect of different nitrogen sources and nitrogen
concentrations on growth and feather degradation of
Chryseobacterium indologenes K14 ................................ 12
3.4. Effect of feather concentrations on growth and feather
degradation of Chryseobacterium indologenes K14........ 14
3.5. Effect of time course on growth and feather degradation of
Chryseobacterium indologenes K14 ................................ 15
4. CONCLUSIONS AND SUGGESTIONS ............................... 18
4.1. Conclusions ...................................................................... 18
4.2. Suggestions ...................................................................... 18
REFERENCES ............................................................................ 19

iii


1. INTRODUCTION
Feathers are composed of over 90% protein and produced in
large amount as a by poultry processing worldwide, reaching
about 8.5 billion of tons per year with potential environmental
impact (Agrahari and Wadhwa, 2010). Accumulation of feathers
will lead to environmental pollution and feather protein wastage
(Gousterova et al., 2005). Therefore, their proper disposal may be

considered as a means of avoiding environmental pollution.
Traditional ways to degrade feathers such as physical and
chemical treatments may not only destroy the amino acids but
also consume large amounts of energy (Wang and Parson, 1997).
Biodegradation of feathers by keratinase from microorganisms
may provide a viable alternative.
Keratinases are the enzymes that can hydrolyze keratin
substrates produced by a large number of bacteria, actinomycetes,
and fungi. The keratinolytic microorganisms and technologies
developed for feather degradation not only remove the waste
feathers efficiently from the nature but also make the by-products
of the process as a valuable protein supplement.
Hence, biodegradation of feather keratin by microorganisms
represents an alternative method to improve the nutritional value
of feather waste and to prevent environment contamination.
Objectives
To optimize the culture conditions for growth and feather
degradation by Chryseobacterium indologenes K14.
1


2. MATERIALS AND METHODS
2.1. Materials
The strain Chryseobacterium indologenes K14 collected from
Molecular Biology Laboratory, Biotechnology Research &
Development Institute, Can Tho University.
Medium: The basal salts medium used contained the
following (g/l): 0.5 NH4Cl, 0.5 NaCl, 0.3 K2HPO4, 0.4 KH2PO4,
and 0.1 MgCl2.6H2O (Bo Xu et al., 2009)
Raw feathers were obtained from a poultry-processing plant.

They were washed extensively with tap water and dried at 80°C
for 48 h, and then kept at 4°C until used.
Chemicals and equipments in Molecular Biology laboratory.
2.2. Methods
2.2.1. Method for testing keratinolytic activities
This procedure tested the keratinolytic activity of keratinase
on azokeratin to begin the process, 5 mg of azokeratin was added
to a 1.5 ml centrifuge tube along with 0.8 ml of 50 mM potassium
phosphate buffer, pH 7.5. This mixture was agitated until the
azokeratin was completely suspended. A 0.2 ml aliquot of
supernatant of crude enzyme was added to the azokeratin, mixed
and incubated for 15 min at 50°C with shaking. The reaction was
terminated by adding 0.2 ml of 10% trichloroacetic acid (TCA).
The reaction mixture was filtered and analyzed for activity (Burtt
and Ichida, 1999).
2


The absorbance of the filtrate was measured at 450 nm with a
UV-160 spectrophotometer (LaboMed.Inc). A control sample was
prepared by adding the TCA to a reaction mixture before the
addition of enzyme solution. A unit of keratinase activity was
defined as a 0.01 unit increase in the absorbance at 450 nm as
compared to the control after 15 min of reaction (Burtt and Ichida,
1999).
2.2.2. Method for determinating of bacterial growth and
feather degradation
Bacterial growth was determined by total plate count on
nutrient agar. Feather in cultures was harvested by filtration with
the filter paper, washed twice with distilled water and dried at

80°C to constant weight. The percentage of feather degradation
was calculated from the differences in residual feather dry weight
between a control (feather without bacterial inoculation) and
treated sample.
2.2.3. Effect of medium pH and cultural temperature on
growth

and

feather

degradation

of

Chryseobacterium

indologenes K14
To investigate the optimum assay pH and temperature on
growth and feather degradation capability of C. indologenes K14.
This activity were carried out at different pH levels (5.0, 6.0,
7.0, 8.0, and 9.0) and different temperature levels (20, 30, 37, 40,
and 45ºC). The treatments were designed in triplicate randomly.
3


Procedure: Weigh 0.5 g feather meal that was washed and
dried. Add the feather meal into each of 250-ml Erlenmeyer
flasks containing 50 ml of salts medium. The pH was adjusted as
experimental design. They were sterilized at 121°C in 20 minutes.

Inoculate 1 ml of C. indologenes K14 suspension (108
cells/ml) grown in nutrient broth at 30°C for 48 h into Erlenmeyer
flasks which was sterilized before.
Cultivations were performed at 150 rpm for 7 days in a rotary
shaker with the different temperature levels as experimental
design.
Record the bacterial density, the feather degradation and the
keratinase activity. Compare the results and then select the
optimal medium pH and cultural temperature.
2.2.4. Effect of different carbon sources and carbon
concentrations on growth and feather degradation of
Chryseobacterium indologenes K14
This activity investigated the effect of carbon sources on
growth and feather degradation capability of C. indologenes K14.
Different carbon source like glucose, cornstarch and molasses
were added at different concentrations (1%, 2% and 3% w/v).
This activity was carried out in triplicate.
Procedure: performed similar to activity 2.2.3. Incubate flasks
at a suitable pH and temperature finding from results of activity
2.2.3. Add carbon concentrations as experimental design.

4


Record the bacterial density, the feather degradation and the
keratinase activity. Compare the results and then select
appropriate carbon source and carbon concentration.
2.2.5. Effect of different nitrogen sources and nitrogen
concentrations on growth and feather degradation of
Chryseobacterium indologenes K14

In order to find out a suitable nitrogen source on growth and
feather degradation capability of C. indologenes K14, the
following different nitrogen sources such as yeast extract, soy
flour, soybean residue, NH4Cl at different concentrations (0.1%,
0.5%, 1% w/v) were amended in the medium. This activity was
carried out in triplicate.
Procedure: performed similar to activity 2.2.3. Incubate flasks
at a suitable pH, temperature and carbon source finding from
results of the previous activities. Add nitrogen concentrations as
experimental design.
Record the bacterial density, the feather degradation and the
keratinase activity. Compare the results and then select
appropriate nitrogen source and nitrogen concentration.
2.2.6. Effect of feather concentrations on growth and feather
degradation of Chryseobacterium indologenes K14
This activity examined the growth and feather degradation
capability

of

C.

indologenes

K14

at

different

feather

concentrations (0.5%, 1%, 1.5%, 2%, 2.5%, 3% w/v). This
activity was carried out in triplicate.
5


Procedure: performed similar to activity 2.2.3. Incubate flasks
at a suitable pH, temperature, carbon source and nitrogen source
finding from results of the previous activities. Add feather
concentrations as experimental design.
Record the bacterial density, the feather degradation and the
keratinase activity. Compare the results and then select
appropriate feather concentration.
2.2.7. Effect of time course on growth and feather degradation
of Chryseobacterium indologenes K14
This activity examined the growth and feather degradation
capability of C. indologenes K14 on time course.
This experiment was studied at different time (day 1, day 2,
day 3, day 4, day 5, day 6, and day 7). This activity was carried
out in triplicate.
Procedure: performed similar to activity 2.2.3. Incubate flasks
at a suitable pH, temperature, carbon source, nitrogen source and
feather concentration finding from results of the previous
activities. Add substrate concentrations as experimental design.
Record the bacterial density, the feather degradation and the
keratinase activity with. Compare the results and then select
appropriate culture time.
2.2.8. Data analysis
The experiments were completely randomized design and

using Microsoft Excel version 2013 and Statgraphic version 15.1
for data analysis.
6


3. RESULTS AND DISCUSSION
3.1. Effect of temperature and pH on growth and feather
degradation by Chryseobacterium indologenes K14
Temperature and pH are important factors affecting the
growth and metabolites production by bacteria. Riffel et al.
(2003) reported the optimal temperature for growth and keratinase
production of Chryseobacterium sp. was between 25-40°C; the
maximum production enzyme was below 50°C and the pH range
from 6.0-8.0, while the maximum keratinolytic activity was
showed in this temperature range.
Table 5’. Effect of temperature and pH on keratinolytic
enzyme production, feather degradation and bacterial growth
of C. indologenes K14
Temperature

pH

(°C)

value

25

30

Kerainolytic
activity
(U/ml)

Extent of

Bacterial

feather

Growth

degradation

(x 109

(%)

CFU/ml)

5

10.33ijk

32.18fg

4.8fghi

6

11.71hijk

32.74efg

6.0defg

7

15.59bcde

33.87cdef

6.8cde

8

16.01bcde

35.11bcde

7.5bcd

9

14.19efgh

34.17cdef

6.7cde

5

14.6defg

34.83bcde

6.2def

7


37

40

45

6

15.22cdef

35.87abcd

7.4bcd

7

17.03bcd

36.33ab

8.0ab

8

20.9a

37.83a

9.5a

9

15.77bcde

36.20abc

8.1abc

5

12.02ghij

33.98cdef

4.5ghij

6

14.09efgh

34.03cdef

5.5efgh

7

18.07b

34.68bcde

6.4de

8

17.34bc

35.90abcd

8.0abc

9

16.24bcde

35.26bcde

7.2bcd

5

9.92jk

28.26i

3.4ijk

6

11.03ijk

29.65hi

3.7ijk

7

12.79fghi

30.83gh

4.6ghij

8

13.27efgh

33.29defg

4.0hijk

9

12.6fghij

32.30fg

3.8ijk

5

8.98k

22.51j

1.8l

6

10.63ijk

23.42j

3.4ijk

7

11.23ijk

24.75j

3.2jkl

8

11.4ijk

23.78j

3.4ijkl

9

9.99jk

24.46j

2.6kl

Note: the data that carrying different subscripts present
statistically difference at 95% confidence level.
The strain C. indologenes K14 was able to degrade feather
after 7 days of incubation. Table 5’ illustrated the results of pH
and temperature interaction which showing 30°C and 8.0 as the
8


optimum temperature and pH of cultural medium for keratinase
production and feather degradation of C. indologenes K14 strain.
At 25°C with pH in 7.0-8.0, the keratinolytic activity was rather

high, but it showed smaller than the one was compared at 30°C
with pH 8.0. It was found that the room temperature (about 37°C)
is relatively stable for the growth of bacteria, whereas the enzyme
production was not significant. With the range from 40 to 45°C,
the growth and the keratinolytic activity of strain C. indologenes
K14 were decreased dramatically. It seems that high temperature
inhibited the growth and keratinase production of the bacteria, so
the extent of feather degradation will be rather low. These results
are similar with that of other C. indologenes (Sangali and
Brandelli., 2000), which reported the temperature for the best
growth and keratinase production with the range from 20°C to
30°C. From the above results, the optimal temperature for the
growth and feather degradation capability of strain C. indologenes
K14 was 25°C to 37°C that more suitable for the typical climate
conditions in Mekong delta region.
Regarding effect of pH, the results in figure 5’ showing that
the growth and keratinase production of C. indologenes K14 were
inhibited at pH 5.0-6.0, therefore the feather degradation ability
was rarely low. It looks like the medium pH extremely effect to
the ability of the enzyme production by bacteria. The optimum
medium pH for keratinolytic activity and feather degradation was
from 7.0 to 9.0, and the maximum keratinase production were
recorded at pH 8.0. These results are similar with the report from
9


Sangali and Brandelli (2000); Riffel et al. (2003) in the study of
strains Vibrio kr2 and Chryseobacterium kr6.
3.2.

Effect

of

different

carbon

sources

and

carbon

concentrations on growth and feather degradation of
Chryseobacterium indologenes K14
To optimize the culture media for the growth and
keratinolytic enzyme production by C. indologenes K14, different
carbon sources were supplemented in experimental treatments.
The results in Table 6’ showed that the highest keratinolytic
activity and feather degradation were recorded with addition of
2% (w/v) corn starch. It may be explained that cornstarch not only
provides nutrients for the bactria to thrive but also promote the
increasing of keratinase production. These results are dissimilar
with the report from Mohammad Shahnoor Hossain et al. (2007)
that show 1% (w/v) molasses as the enhancement for keratinase
production by Bacillus licheniformis MZK-3. Furthermore, T.
Jayalakshmi et al. (2011) reported the supplement of 2% (w/v)
carbon source supplement in the medium increased effectively the
keratinolytic activity of Streptomyces sp. JRS19, but sucrose was

the suitable carbon source in this study.
Table 6’. Effect of carbon sources on feather degradation
and keratinolytic activity of C. indologenes K14
Extent of Bacterial
Concent
Keratinolytic
Carbon
feather
Growth
ration
activity
sources
degradati
(x 109
(%)
(U/ml)
on (%)
CFU/ml)
0

Control

15.567de
10

38.40g

12.2abc

1

2

3

Glucose

19.067b

43.93b

14.1a

Cornstarch

17.056cd

42.24cd

12abc

Molasses

16.044de

42.10cd

12.3abc

Glucose

14.850ef

41.44de

10.6cde

Cornstarch

46.33a

13.1ab

Molasses

23.211a
18.1bc

42.92bc

11.1cd

Glucose

12.444g

39.22fg

6.6f

Cornstarch

14.967ef

41.47cde

9.7de

Molasses

13.489fg

40.02ef

8ef

Note: the data that carrying different subscripts present
statistically difference at 95% confidence level.
At the supplement of 1% (w/v) carbon sources in culture
medium, the bacterial density and keratinase activity were highest
in the case of adding glucose, thus the feather degradation ability
would be increased significantly in this medium. However, the
feather degradation rate in case of adding 2% (w/v) corn starch
was higher than the addition of 1% (w/v) glucose, moreover the
price of corn starch that is also cheaper than glucose. The results
in Table 6’ further showed that supplement of 3% (w/v) glucose,
corn starch or molasses inhibited growth and keratinase
production. It seems that high carbon concentration supplement of
carbon may cause the repression in keratinase production.

11


3.3. Effect of different nitrogen sources and nitrogen
concentrations on growth and feather degradation of
Chryseobacterium indologenes K14
The effect of nitrogen sources on growth and keratinolytic
enzyme production is also shown in the results of Table 7’.
Feather medium supplemented with 0.1% (w/v) soy flour showed
maximum production of keratinase and feather degradation
capability by C. indologenes K14. It can be explained that using
organic nitrogen sources was more effective than using inorganic
nitrogen sources by bacteria during the keratinase enzyme
production. In the medium containing yeast extract as nitrogen
source, the bacterial

density was highest, whereas the

keratinolytic activity was not higher than in the medium which
supplemented 0.1% (w/v) soy flour. These results contrasted from
the case of Bacillus licheniformis MZK-3, in which NH4Cl (0.1%
w/v) encouraged bacterial growth and keratinase production by
increasing about 12% of keratinolytic activity (Mohammad
Shahnoor Hossain et al., 2007).
Table 7’. Effect of nitrogen sources on feather degradation
and keratinolytic activity of C. indologenes K14
Extent of
Keratinolytic
Concentr Nitrogen

feather
activity
ation (%)
sources
degradatio
(U/ml)
n (%)
0

Control

18.767e
12

42.07cdef

Bacterial
Growth
(x 109
CFU/ml)
9.8bc


Yeast
extract

22.889b

45.01b

14.3a

Soyflour

26.611a

48.26a

11.1b

Soybean
residue

21.156bc

42.78bcde

7.5cd

NH4Cl

21.522bc

44.10bc

10.9b

Yeast
extract

19.289de

40.41ef

9.4bcd

Soyflour

20.578cd

42.11cdef

7.7cd

Soybean
residue

22.094bc

43.71bcd

11.1b

NH4Cl

21.944bc

45.26b

9.0bcd

Yeast
extract

15.044f

35.98g

4.4e

Soyflour

14.233f

39.62fg

7.1d

Soybean
residue

14.950f

41.31def

7.4cd

NH4Cl

14.533f

40.02ef

8.0cd

0.1

0.5

1

Note: the data that carrying different subscripts present
statistically difference at 95% confidence level.
Feather medium supplemented with 0.5% (w/v) soybean
residue, the bacterial density and keratinolytic activity showed
significantly high, whereas the feather degradation ability was not
higher than the medium containing NH4Cl with the same
13


concentration. In the medium with supplying 1% (w/v) nitrogen
source, the keratinolytic activity was reduced significantly in all
nitrogen sources. Therefore, the high concentration of nitrogen in
the medium may inhibited the synthesis of keratinase enzyme by
C. indologenes K14. It seems that bacteria use the easier digested
nutrients instead of working hard in biosynthesize the enzymes to
degrade keratin containing substrate for growth. These results
share similarity the case of Bacillus sp. JB 99 in the study of
Kainoor and Naik (2010).
3.4. Effect of feather concentrations on growth and feather

degradation of Chryseobacterium indologenes K14
The effect of different feather concentrations on growth and
keratinolytic enzyme production is shown in Figure 11’. The
amount

of

keratinase

production

depended

on

feather

concentrations. Keratinolytic enzyme production increased as the
amount of feather increased (0.5-1.5% w/v); but when the
concentration was raised to 2.0% (w/v), enzyme production
slightly decreased. The maximum enzyme keratinase production
occurred with the supplemented of 1% (w/v) feather in the
medium. It looks like that high substrate concentration may cause
the repression of keratinase production. Hence, the feather
degradation took place inversely with increasing enzyme activity.
Cheng et al. (1995) reported that 1% (w/v) feather powder gave
the highest keratinase activity for B. licheniformis PWD-1.
Similar result was also reported for Bacillus sp. FK46
(Suntornsuk and Suntornsuk, 2003). The latter authors reported
14

that higher concentrations (3% and 5% w/v) may cause substrate
inhibition or repression in keratinase production.

Fig. 11. Effect of feather concentration on growth and feather
degradation capability of C. indologenes K14
The bacterial density was significantly influenced with
increasing the concentration of substrate (feather meal). High
feather concentration increased medium viscosity which possibly
resulting in oxygen limitation for bacterial growth. As a result, the
feather degradation capability was significantly decreased with
increasing the concentration of feather meal.
3.5. Effect of time course on growth and feather degradation
of Chryseobacterium indologenes K14
The time course of feather degradation and keratinolytic
activity of C. indologenes K14 culture grown in the medium
containing 2% (w/v) corn starch, 0.1% (w/v) soy flour, 1% (w/v)
feather concentration, medium pH 8.0 and cultural temperature at
30°C were shown in Figure 12’. The keratinase activity occurred
15


at the first day incubation with 10.68 U/ml, and the bacterial
density was about 1.3 x 109 CFU/ml. After the second day,
keratinolytic activity was still continued and started to increase
significantly at the third day. In this stage, the keratinase
production occurred strongly because of the adaptation of bacteria
in the medium and using the nutrients of the medium with the
biochemical activities for bacterial growth. The maximum

keratinolytic activity of C. indologenes K14 was about 23.63
U/ml after the fourth day cultivation. Maximum enzyme activity
was observed in the late logarithmic growth phase. On the other
hand, the feather degradation capability increased significantly
from the third day and continued to raise slowly to the seventh
day. These results are dissimilar with the report from Geun-Tae
Park and Hong-Joo Son (2009) that the maximum keratinolytic
activity of B. megaterium F7-1 occurred after 5 days of
cultivation.

16


Fig. 12. Effect of time course on growth and feather
degradation capability of C. indologenes K14
After 5 days, the keratinase production was slowly reduced
due to the bacteria use easier digested nutrients for growth instead
of working for production of enzyme. Thus, the longer incubation
time was, the worse keratinolytic activity would be. Nevertheless,
the keratinolytic activity of C. indologenes K14 still continued
and the maximum protein content was produced about 354.46
(µg/ml) at the sixth day. It provides the potential for poultry
processing from waste feather into livestock, and other probiotics.

17


4. CONCLUSIONS AND SUGGESTIONS
4.1. Conclusions
The elements of cultured medium as temperature, pH of

medium, carbon source, nitrogen source, feather concentration,
time for culturing effects to ability to degrade feather and
synthesize keratinase enzyme of C. indologenes K14.
The optimal cultural conditions for growth and feather
degradation by C. indologenes K14: temperature 30°C, pH 8.0,
2% (w/v) corn starch, 0.1% (w/v) soy flour, 1% (w/v) feather
concentration supplements and time course for 5 days of
incubation.
4.2. Suggestions
Studying the effect of the entire cultural conditions to the
biosynthesis keratinase capability by C. indologenes K14.
Continuing study the processes to develop the optimal
conditions for keratinase production to achieve economic
efficiency and usability.

18


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Vietnamese
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English
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Geun-Tae Park, Hong-Joo Son. 2009. Keratinolytic activity of Bacillus
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Microbiological Research. 164, 478-485.
Jayalakshmi, P. Krishnamoorthy, G. Ramesh kumar, P. Sivamani. 2011.
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19


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Naik,

G

R.

2010.

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Mozammel Hoq. 2007. Production and

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