effects of culture conditions on growth and feather degradation capability of bacillus pumilus k8

0.5% ammonium chlorite was a good concentration of nitrogen source for the production of keratinolytic activity and feather degradation by Bacillus pumilus K8.. Effect of medium pH and c

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 BACILLUS PUMILUS K8

SUPERVISOR STUDENT

Dr BUI THI MINH DIEU TRAN LE BACH

Student’s code: 3083473 Session: 34 (2008- 2013)

Can Tho, 5/2013

APPROVAL

SUPERVISOR STUDENT

Dr BUI THI MINH DIEU TRAN LE BACH

Can Tho, May , 2013 PRESIDENT OF EXAMINATION COMMITTEE

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Abstract

An inducible keratinase is produced by Bacillus pumilus K8 in

a feather medium Under submerged fermentation condition with continuous agitation (180 rpm), high level of keratinase production occurred at 40 o C after 120 h at pH 6.0.Cornstarch supported the highest level of keratinolytic production at 2% concentration compare to other carbon sources 0.5% ammonium chlorite was a good concentration of nitrogen source for the production of keratinolytic activity and feather degradation by Bacillus pumilus K8 Present results indicate that Bacillus pumilus K8 can be a highly useful organism for feather meal production as animal feed staffs, biofertilizer and in leather industry

Keywords: Bacillus pumilus, feather degradation, feather

meal, keratin, keratinase

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CONTENTS

ABSTRACT……… i

CONTENTS ……… ……… …… … ii

Abstract i

1 INTRODUCTION 1

2 MATERIALS AND METHODS 3

2.1 Materials 3

2.2 Methods 4

2.2.1 Method for testing enzyme activities 4

2.2.2 Method for determinating of bacterial growth and feather degradation 4

2.2.3 Effect of medium pH and cultural temperature on growth and feather degradation by Bacillus pumilus K8 5

2.2.4 Effect of different carbon sources on growth and feather degradation by Bacillus pumilus K8 6

2.2.5 Effect of different nitrogen sources on growth and feather degradation by Bacillus pumilus K8 6

2.2.6 Effect of feather concentrations on growth and feather degradation by Bacillus pumilus K8 7

2.2.7 Effect of time course on growth and feather degradation by Bacillus pumilus K8 7

3 RESULTS AND DISCUSSION 9

3.1 Effect of medium pH and cultural temperature on growth and feather degradation by Bacillus pumilus K8 9

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3.2 Effect of different carbon sources on growth and feather

degradation by Bacillus pumilus K8 10

3.3 Effect of different nitrogen sources on growth and feather degradation by Bacillus pumilus K8 11

3.4 Effect of feather concentrations on growth and feather degradation by Bacillus pumilus K8 12

3.5 Effect of time course on growth and feather degradation by Bacillus pumilus K8 13

4 CONCLUSIONS AND SUGGESTIONS 15

4.1 Conclusions 16

4.2 Suggestions 16

REFERENCES 17

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Feathers represent 5-7% (w/w) of the total weight of mature chickens and are generated in large amounts as a waste product in commercial poultry-processing plants, reaching millions of tons per year worldwide Poultry feathers were discharged into the environment after the processing which leads to the environmental pollution Discarded feathers cause various human ailments including chlorosis, mycoplasmosis and fowl cholera Although the feathers are considered as wastages, it contains large amount of protein and this protein can be converted into animal feedstuffs which helps to reduce the protein shortage and cost effects Poultry feed production plays

an important role in protein supply and in agricultural economy The protein shortage for food and feed leads us to look for a new protein sources from wastage products like feather wastes Feathers are a significant source of protein for livestock because

of their high protein content (>85% CP) Feathers contain large amounts of cysteine, glycine, arginine and phenylalanine Raw feathers, however, are very poorly digested by non-ruminant animals because they contain a high proportion of keratin protein that has cysteine disulfide bonds The indigestible structure of raw feather must be hydrolyzed to be used as a feed ingredient for non-ruminant species Though keratin can be completely dissolved by reducing agents like copper sulphate, mercapto acetate, iodoacetic acid, amino, sodium sulphite,

Against these backdrops, this study aim to optimize the

culture conditions for growth and feather degradation by Bacillus pumilus K8

Production of seed culture

A single colony from a freshly subcultured nutrient agar plate was transferred into 250-ml conical flask containing 50 ml of nutrient broth The flask was incubated for 6-10 h at 37°C and 200 rpm in an orbital shaker until it reached to an absorbance of 0.5-0.8

at 600 nm

Fermentation and separation of culture filtrates

The seed culture (5 ml) was transferred to 95 ml of feather mill medium in a 500-ml Erlenmeyer flask Feather mill medium contained 0.075% NaCl, 0.21% K2HPO4, 0.105% KH2PO4, 0.015% MgCl2.6H2O, 0.009% CaCl2, 0.15% molasses and 0.75% feather mill (initial pH 7.5) The inoculated flasks were placed in a thermostated orbital shaker for 48 h, at 37°C and 150 rpm Samples were withdrawn at regular intervals and centrifuged at 5,000 rpm for 20 min The cell free supernatant was preserved at 4°C and used for enzyme assay and protein estimation (in duplicate)

Chemicals and equipments in Molecular Biology Laboratory

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2.2 Methods

2.2.1 Method for testing keratinolytic activity

This procedure tested the keratinolytic activity of keratinase

on azokeratin to begin the process, 5 mg of azokeratin was added

to a 1.5ml centrifuge tube along with 0.8ml of 50 mM potassium phosphate buffer, pH 7.5 This mixture was agitated until the azokeratin 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) The reaction mixture was filtered and analyzed for activity (Burtt and Ichida, 1999)

The absorbance of the filtrate was measured at 450nm 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

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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 Bacillus pumilus K8

To investigate the optimum assay pH and temperature on

growth and feather degradation capability of Bacillus pumilus K8

This activity were carried out at different pH levels (4.0, 5.0, 6.0, 7.0, and 8.0) and different temperature levels (20, 30, 37, 40, and 45ºC) The treatments were designed randomly with repeat 3 times

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

Inoculate 1ml of Bacillus pumilus K8 suspension (108cells/ml) grown in nutrient broth at 30°C for 48h 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 layout

Record the bacterial density, the feather degradation and the keratinase activity Compare the results and then select the optimal medium pH and cultural temperature

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2.2.4 Effect of different carbon sources on growth and

feather degradation of Bacillus pumilus K8

This activity investigated the effect of carbon sources on

growth and feather degradation capability of Bacillus pumilus K8

Different carbon source like glucose, cornstarch and molasses were added at different concentrations (1%, 2% and 3% w/v) This activity was carried out with triplicate

Procedure: performed similar to activity 2.2.3 Incubate flasks

at a suitable pH and temperature finding from results of activity 2.2.2 Add carbon concentrations as experimental design

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 on growth and

feather degradation of Bacillus pumilus K8

In order to find out a suitable nitrogen source on growth and

feather degradation capability of Bacillus pumilus K8, 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 with 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

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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 Bacillus pumilus K8

This activity examined the growth and feather degradation

capability of Bacillus pumilus K8 at different feather

concentration (0.5%, 1%, 1.5%, 2%, 2.5%, 3% w/v) This activity was carried out with triplicate

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 Bacillus pumilus K8

This activity examined the growth and feather degradation

capability of Bacillus pumilus K8 on time course

This experiment was studied at different levels (day 1, day 2, day 3, day 4, day 5, day 6, day 7) This activity was carried out with triplicate

Procedure: performed similar to activity 2.2.3 Incubate flasks

at a suitable pH, temperature, carbon source, nitrogen source and

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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

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3 RESULTS AND DISCUSSION

3.1 Effect of medium pH and cultural temperature on

growth and feather degradation of Bacillus pumilus K8

The effects of pH and temperature on keratinase activity were determined using azokeratin as substrate The optimal

temperatures of keratinolytic activity from other Bacillus were

between 30 and 40oC According to figure 1 and the result of statistical table (Table 2, Appendix 2.1) shown that the interaction

of pH and temperature effects on keratinolytic activity of Bacillus pumilus K8 In particular, the activity of keratinase reached the

highest value at 40oC, and also has the degradation of feather at highest value (38.04%) was described in Table 6 (Appendix 2.1)

In fact, it was demonstrated that if the temperature in the medium was too high may cause substrate inhibition or repression of keratinase production, resulting in a low percentage of feather degradation The result are in line with those reported by Daniel J

et al (2008) using the strain Bacillus pumilus A1

Figure 5: Extent of feather degradation of B.pumilus K8

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The effects of initial pH of feather medium on feather degradation are shown in Fig.5 The highest feather degradation

in B.pumilus K8 was obtainted with a starting pH of 6.0 at the

temperature is 40oC A different result is reported in

B.lincheniformis strain K-508 by A.Ganesh Kumar et al, (2006)

3.2 Effect of different carbon sources on growth and

feather degradation of Bacillus pumilus K8

Of all the tested carbon sources that add to culture medium (Fig.6) cornstarch supported the highest level of production for

Bacillus pumilus K8 The activities of respiratory enzymes in

cells grown on different carbon sources explained the differences

of the oxidative ability of cells grown acrobically on glucose

Figure 6: Effect of carbon sources on growth and feather

degradation of B.pumilus K8

Fig 6 shows that the concentrations of support the highest

production of keratinase by Bacillus pumilus K8 Futher, increase

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of the concentration cause gradual decrease in the keratinase production

3.3 Effect of different nitrogen sources on growth and

feather degradation of Bacillus pumilus K8

Ammonium chloride was a good nitrogen source for the

production of keratinase by Bacillus pumilus K8, in the medium

supplemented with 2% cornstarch (Fig.) None of the tested inorganic nitrogen salts supported the production of keratinase by

Bacillus pumilus K8 Delection of the organic nitrogen source

was more preferable as medium protein concenentration was increased In the most microorganisms, both inorganic and organic forms of nitrogen are metabolized to produce amino acids, nucleic acids, proteins and cell wall components

Figure 7: Effect of nitrogen sources on keratinolytic

activity and feather degradation of B.pumilus K8

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Fig.6 shows that of ammonium chloride was the best

concentration for optimal keratinase production by Bacillus pumilus K8 Further increase of ammonium chloride concentration resulted in a proportional decrease of keratinase production

3.4 Effect of feather concentrations on growth and feather

degradation of Bacillus pumilus K8

The microorganism Bacillus pumilus K8 was grown in

mineral medium containing different amounts of raw feathers as the sole carbon and nitrogen source The strain exhibited the highest enzyme production () in culture medium containing 1.5%

of feather concentration

Figure 8: Effect of feather concentration on keratinolytic activity, microorganism population and feather degradation

of B.pumilus K8