effects of culture conditions on keratin degrading capability of bacillus subtilis k15

The bacterium grew with an optimum at pH 7.0–8.0 and 30–35 o C, where maximum bacterial growth, keratin degradation, keratinolytic activity and soluble protein concentration were also ob

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 KERATIN DEGRADING CAPABILITY OF

BACILLUS SUBTILIS K15

SUPERVISOR STUDENT

Dr BUI THI MINH DIEU TRUONG VAN THO Student’s code: 3082631 Session: 34 (2008 - 2013)

Can Tho, 5/2013

APPROVAL

SUPERVISOR STUDENT

Dr BUI THI MINH DIEU TRUONG VAN THO

Can Tho, May, 2013 PRESIDENT OF EXAMINATION COMMITTEE

Abstract

Microorganisms grow at different ranges of environmental conditions The aim of this study was to investigate the culture conditions affecting keratin degradation by bacillus subtilis k15,

a feather-degrading mesophilic bacterium The bacterium grew with an optimum at pH 7.0–8.0 and 30–35 o C, where maximum bacterial growth, keratin degradation, keratinolytic activity and soluble protein concentration were also observed The keratin degradation rate obtained 47% and 46% when the bacterium was cultivated in a chicken feather medium contains 0.5% (w/v) skim milk and yeast extract Additionally, corn meal had a positive influence on keratin degradation; the treatment degraded 48% chicken feather meal, and showed 36.8% higher than the control The amount of chicken feather degraded higher in a medium contains 1.2-1.8% (w/v) chicken feather meal Besides, the quantity of keratinolytic enzyme production depended on chicken feather meal concentrations Bacillus subtilis k15 degraded 30- 40% chicken feather meal after 5 days of incubation at 150 rpm; bacterial growth, keratinolytic enzyme and soluble protein concentration also got the highest value at that time The bacterium effectively degraded chicken feather meal and duck feather meal, whereas hair human and pig fur showed relatively low degradation rates In short, Bacillus subtilis k15 presented high keratinolytic activity and was very effective in keratin degradation, providing potential use for biotechnological processes of keratin degradation

Keywords: Bacillus subtilis, chicken feather meal, keratin

degradation, keratinolytic enzyme

ii

CONTENTS

Abstract i

CONTENTS ii

1 INTRODUCTION 1

2 MATERIALS AND METHODS 3

2.1 Materials 3

2.2 Methods 3

2.2.1 Effect of medium temperature on the keratin degradation of Bacillus subtilis k15 3

2.2.2 Effect of medium pH on the keratin degradation of Bacillus subtilis k15 4

2.2.3 Effect of different nitrogen sources on the keratin degradation of Bacillus subtilis k15 4

2.2.4 Effect of different carbon sources on the keratin degradation of Bacillus subtilis k15 5

2.3.5 Effect of different chicken feather concentration on the keratin degradation of Bacillus subtilis k15 5

2.2.6 Time course of keratin degradation 6

2.2.7 The keratin degradation capability of Bacillus subtilis k15 degraded the different keratin sources 6

2.2.8 Methods determined characteristic samples in the experiments 6

3 RESULTS AND DISCUSSION 10

3.1 Effect of temperature on keratin degradation 10

3.2 Effect of pH on keratin degradation 11

3.3 Effect of nitrogen sources on keratin degradation 13

3.4 Effect of carbon sources on keratin degradation 14

3.5 Effect of chicken feather concentrations on keratin

degradation 16

3.6 Time course of keratin degradation 18

3.7 The ability of Bacillus subtilis k15 degrades different keratin sources 19

4 CONCLUSIONS AND SUGGESTIONS 20

4.1 Conclusion 20

4.2 Suggestions 20

REFERENCES 21

1

1 INTRODUCTION

Environmental wastes are found in large quantities in many countries Feathers are largely produced as a waste byproduct at poultry plants, reaching millions of tons per year worldwide Additionally, the accumulation of some of these wastes in nature

is considered to be a serious source of pollution and health hazards Therefore, their proper disposal may be considered as a means of avoiding environmental pollution (Williams et al., 1991)

Feathers are insoluble structural proteins cross-linked by disulfide, hydrogen and hydrophobic bonds but could represent a rich protein resource because they contain over 90% (w/w) keratins Keratins cannot be degraded by the usual proteolytic enzymes such as pepsin, trypsin and papain (Kumar et al., 2007) The mechanical stability of keratin and its resistance to biochemical degradation depend on the tightly packed protein chains in α-helix (α-keratin) and β-sheet (β-keratin) structures In addition, these structures are cross-linking by disulfide bridges in cystines residues (Riffel et al., 2007)

Feathers are almost pure keratin protein, potential alternative to more expensive dietary ingredients for animal feedstuffs Generally, they become feather meal used as animal feed after undergoing physical and chemical treatments These processes require significant energy and also destroy certain amino acids (Papadoulos and Ketelaars, 1986) An alternative and attractive method for improving the digestibility of feathers is biodegradation by keratinolytic microorganisms A number of keratinolytic microorganisms can produce keratinases which are

capable of degrading keratin Various authors have reported that, among the keratinolytic microorganisms, some species of

bacillus, actinomycetes and fungi are able to produce these

keratinases and peptidases (Sangali and Brandelli, 1999)

Bacteria are dependent on their environment to provide for their basic needs There are several factors that influence the growth of bacteria: nutrition, oxygen, pH, temperature, and moisture Adverse conditions can alter their growth rate or kill them By understanding the factors affecting the growth of bacteria we can know how to create an optimum condition in which bacteria grow fastest and effectively degrade keratin

Objectives:

Screening of cultural conditions affecting on the growth

and keratin-degrading capability of Bacillus subtilis k15

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2 MATERIALS AND METHODS

2.1 Materials

− Bacillus subtilis k15 from the research of Nguyễn Thị

Hồng Thẩm (2012), Biotechnology Research and Development Institute, Can tho university

and 10 chicken feather meals The pH was adjusted to 7 before sterilization The agar medium added 20 agar and 0.1 yeast extract These samples were enriched in broth containing feather

(g/l), pH 7.5)

milled prior to being added to the medium

2.2 Methods

2.2.1 Effect of medium temperature on the keratin

degradation of Bacillus subtilis k15

This activity examined the keratin degrading capability of bacteria at different temperature levels (25, 30, 35, 40, 45, and 50ºC) The capacity of degradation of keratin substrates was tested on the feather medium Using 250 ml Erlenmeyer flasks added 50 ml medium, sterilized at 121ºC in 15 minutes,

cultivated for 5 days with constant shaking at 150 rpm After 5

keratinolytic activity and protein

2.2.2 Effect of medium pH on the keratin degradation of

Bacillus subtilis k15

This activity screened the keratin degrading capability of bacteria at different pH levels (4, 5, 6, 7, 8, 9, 10, 11) The capacity of degradation of keratin substrates was tested on the feather medium 50 ml of the medium was dispensed into each of 250-ml Erlenmeyer flasks followed by inoculation with 1ml of

Bacillus subtilis k15 culture (107CFU/ml) Flasks were sterilized

at 121ºC in 15 minutes before inoculating the bacteria Cultivations were performed at optimum temperature at experiment temperature and 150 rpm for 5days All samples were determined bacterial growth, keratin degradation, keratinolytic activity, protein

2.2.3 Effect of different nitrogen sources on the keratin

degradation of Bacillus subtilis k15

The feather medium was supplemented with 0.5% (w/v)

the effects of addition different nitrogen sources on keratin degradation The capacity of degradation of keratin substrates was tested on the feather medium; pH was adjusted to optimum pH

50 ml of the medium was dispensed into each of 250-ml

Erlenmeyer flasks followed by inoculation with 1ml of Bacillus

subtilis k15 culture (107CFU/ml) Flasks were sterilized at 121ºC

in 15 minutes before inoculating the bacteria Cultivations were performed at optimum temperature and 150 rpm for 5 days All samples were determined bacterial growth, keratin degradation, keratinolytic activity, protein

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2.2.4 Effect of different carbon sources on the keratin

degradation of Bacillus subtilis k15

The feather medium was supplemented with 1% (w/v) glucose, sucrose, starch, molasses and corn meal to study the effects of addition different carbon sources on keratin degradation The capacity of degradation of keratin substrates was tested on the feather medium; pH was adjusted to optimum pH before sterilization 50 ml of the medium was dispensed into each

of 250 ml Erlenmeyer flasks followed by inoculation with 1ml of

Bacillus subtilis k15 culture (107CFU/ml) Flasks were sterilized

at 121ºC in 15 minutes before inoculating the bacteria Cultivations were performed at optimum temperature and 150 rpm for 5days All samples were determined bacterial growth, keratin degradation, keratinolytic activity, protein

2.3.5 Effect of different chicken feather concentration on the

keratin degradation of Bacillus subtilis k15

The feather medium was supplemented with 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.8%, 2%(w/v) chicken feather powder to study the effects of addition different chicken feather concentration on keratin degradation The capacity of degradation

of keratin substrates was tested on the feather medium; pH was adjusted to optimum pH 50 ml of the medium was dispensed into each of 250ml Erlenmeyer flasks followed by inoculation with

sterilized at 121ºC in 15 minutes before inoculating the bacteria Cultivations were performed at optimum temperature and 150 rpm for 5 days All samples were determined bacterial growth, keratin degradation, keratinolytic activity, protein

2.2.6 Time course of keratin degradation

The capacity of degradation of keratin substrates was tested

on the feather medium; pH was adjusted to optimum pH before sterilization 50 ml of the medium was dispensed into each of 250-ml Erlenmeyer flasks followed by inoculation with 1ml of

Bacillus subtilis k15 culture (107CFU/ml) Flasks were sterilized

at 121ºC in 15 minutes before inoculating the bacteria Cultivations were performed at optimum temperature and 150 rpm from 1 to 7 days All samples were determined bacterial growth, keratin degradation, keratinolytic activity, protein every day

2.2.7 The keratin degradation capability of Bacillus subtilis

k15 degraded the different keratin sources

The feather medium was supplemented with 1%(w/v) duck feather, chicken feather, human hair, goat hair, pig fur to study the effects of addition different keratin sources on keratin degradation The capacity of degradation of keratin substrates was tested on the feather medium; pH was adjusted to optimum pH

50 ml of the medium was dispensed into each of 250-ml

Erlenmeyer flasks followed by inoculation with 1ml of Bacillus

subtilis k15 culture (107CFU/ml) Flasks were sterilized at 121ºC

in 15 minutes before inoculating the bacteria Cultivations were performed at optimum temperature and 150 rpm for 5 days All samples were determined bacterial growth, keratin degradation, keratinolytic activity, protein

2.2.8 Methods determined characteristic samples in the experiments

b Determination of keratin degradation

Feather in cultures was harvested by filtration with filter

constant weight The percentage of keratin degradation was calculated from the differences in residual feather dry weight between a control (feather without bacterial inoculation) and treated sample (bacteria were inoculated)

c Determination of kerinolytic activity

Azokeratin (Joshi el at., 2007):

Azokeratin was prepared by a similar method Ball-milled feather powder was prepared 1 g portion of the chicken feather powder (the keratin source) was placed in a 100-ml round bottomed reaction flask with 20 ml of sterilized water The suspension was mixed with a magnetic stirrer Two ml of 10%

separate 10-ml tube, 174 mg of sulfanilic acid were dissolved in 5

the tube and dissolved The solution was acidified with 0.4 ml of

5 N HCl, mixed for 2 min and neutralized by adding in 0.4ml of 5

N NaOH This solution was added to the feather keratin suspension and mixed for 10 min The reaction mixture was filtered and the insoluble azo-keratin was rinsed thoroughly with deionized water The azo-keratin was suspended in water and shaken at 50°C, for 2 hr and filtered again This wash cycle was

repeated until the pH of the filtrate reached 6.0-7.0 and the spectrophotometric absorbance of the washing at 450 nm was less than 0.01 Finally, the wash cycles were repeated at least twice using 50mM potassium phosphate buffer, pH 7.5 The azokeratin was washed once again with water and dried in vacuum overnight

at 50°C

Extraction of Enzyme (Park and Son, 2009)

Bacillus subtilis k15 incubated in feather medium at

different culture conditions

Filtration: The culture medium was filtered through filter paper to remove undegraded residues

Centrifugation: The filtrate was then subjected to centrifugation at 12,000 rpm for 5 min to remove bacterial residue It was used as the crude enzyme keratinase

This procedure tested the keratinolytic activity

5 mg of azokeratin was added to a 1.5 ml centrifuge tube along with 0.8ml of 50 mM potassium phosphate buffer, pH 7.5 This mixture was agitated until the azo-keratin was completely suspended A 0.2ml 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) Control sample was prepared by adding the TCA to a reaction mixture before the addition of enzyme The absorbance of the filtrate was measured at 450 enzyme solution 1 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

d Protein determination

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Protein concentration was measured by the method of Bradford (1976), using bovine serum albumin (BSA) as standard