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

Effect of different carbon sources and carbon concentrations on growth and feather degradation of Chryseobacterium indologenes K14 .... Effect of different nitrogen sources and nitrogen

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

Can Tho, 05/2013

APPROVAL

SUPERVISOR STUDENT

Can Tho, May , 2013 PRESIDENT OF EXAMINATION COMMITTEE

Dr DUONG THI HUONG GIANG

i

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 feather- degrading 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

ii

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

iii

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

1

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

2

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)

3

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

4

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 (108cells/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

5

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

6

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

7

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

(°C)

pH value

Kerainolytic activity (U/ml)

Extent of feather degradation (%)

Bacterial Growth (x 109CFU/ml)

25

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 12.02ghij 33.98cdef 4.5ghij

6 14.09efgh 34.03cdef 5.5efgh

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

9

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

10

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 feather degradati

on (%)

Bacterial Growth (x 109CFU/ml)

0 Control 15.567de 38.40g 12.2abc