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

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 Dr. BUI THI MINH DIEU STUDENT TRAN LE BACH Can Tho, May , 2013 PRESIDENT OF EXAMINATION COMMITTEE 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 40oC 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 i 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 ii 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 iii 1. INTRODUCTION 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, 1 sodium tetrathionate these methods are not suitable for the large scale application. In order to overcome these limitations the use of microbial enzymes which improves the nutritional value of feather wastes have been implemented in recent years. Keratinase enzyme are among the most important industrial enzymes; they are primarily employed as detergent additives, accounting for 40-60% of the global enzyme market. Alkaline proteases are group of proteolytic enzymes that are able to hydrolyze insoluble hard proteins more effectively than other proteases such as trypsin, pepsin and papain. The genus Bacillus provides most of the keratinase enzyme with commercial value, offering the advantages of being easy to culture and maintain and they are potentially valuable in the bioconversion of keratinous wastes; in the detergent, fertilizer, biopolymer, pharmaceutical and animal feed industries; and in leather processing and hydrolysis of prion proteins as well. Therefore, such microorganisms and enzymes have been the focus in several studies and Bacillus alkaline proteases are reported to be among the most efficient keratin degraders. Against these backdrops, this study aim to optimize the culture conditions for growth and feather degradation by Bacillus pumilus K8. 2 2. MATERIALS AND METHODS 2.1. Materials Microorganism Bacillus pumilus K8 was collected from Molecular Biology Laboratory, Biotechnology Research & Development Institute, Can Tho University. 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. 3 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 4 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 (108 cells/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. 5 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. 6 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 7 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. 8 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. 9 Figure 5: Extent of feather degradation of B.pumilus K8 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 10 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 11 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 12 The highest levels of feather degradation were also obtained in the medium containing 1.5% of feathers. However, at lower or higher feather concentrations, keratinolytic activity, feather degradation and microorganisms population were reduce. In fact, it was demonstrated that high feather concentration may cause substrate inhibition or repression of keratinase production, resulting in a low percentage of feather degradation. On the other hand, high feather concentration increased medium viscosity which possibly resulting in oxygen limitation for bacterial growth. Although the substrate concentration at 1.5% bacterial biomass does not increase sharply as the concentration of 1%, but for the purpose of receiving the high activity of keratinase enzyme that 1.5% feather concentration were chosen for culturing bacteria. 3.5 Effect of time course on growth and feather degradation of Bacillus pumilus K8. Time course from 1 to 7 days was followed in shaking and static incubated flasks containing the optimized of Bacillus pumilus K8 (Fig.). Maximum keratinolytic activity was reached after 5 days shaking incubation period for Bacillus pumilus K8. It seems to be stable on the 4th and 5th days of the static incubation period having maximum enzyme production on the 5th. A gradual decrease in activity was noticed after 5th day incubation was recorded at 22.9 (U/ml). From the 5th after, the enzyme activity began to decrease due to keratinolytic activity is ending and the nutrients from the medium began to deplete. The study of 13 Teodoro và Martins (2000) and Riaz (2009) have also same result. Thus, from the results of this experiment, the incubation period for Bacillus pumilus K8 to have the optimal keratinolytic activity is 120 hours. 14 Figure 9: Effect of time course on keratinolytic activity, micro organism population and feather degradation of B.pumilus K8 15 4. CONCLUSIONS AND SUGGESTIONS 4.1. Conclusions The elements of cultured medium as temperature, pH of medium, carbon source, nitrogen source, feather concentration, time showed effect on feather degradation capacity and keratinase synthesis enzyme of Bacillus pumilus K8. The optimal conditions of cultured medium for Bacillus pumilus K8 to keratinase synthesis enzyme is: temperature 40°C, pH 6.0, 2% cornstarch concentration (w/v), 0.5% NH4Cl (w/v), 1.5% keratin (w/v) and time course for culturing should be 5 days. 4.2. Suggestions Studying the effect of the entire cultural conditions to the biosynthesis keratinase capability by Bacillus pumilus K8. Continuing study the processes to develop the optimal conditions for production to achieve economic efficiency and usability. 16 REFERENCES Vietnamese Cao Ngọc Điệp và Nguyễn Hữu Hiệp. 2008. Giáo trình thực hành Vi sinh vật học đại cương. Viện NC & PT Công Nghệ Sinh Học, Trường Đại Học Cần Thơ. Dương Thị Hương Giang. 2008. Giáo trình thực tập Protein và Enzyme học. Viện NC & PT Công Nghệ Sinh Học, Trường Đại Học Cần Thơ: Trang 11-14. Trần Đình Toại và Trần Thị Hồng. 2007. Tương lai ứng dụng Enzyme trong xử phế thải. Tạp chí Khoa học Đại học Quốc gia Hà Nội, Khoa học Tự nhiên và Công nghệ 23. 75-85. English Bo Xu, Qiaofang Zhong, Xianghua Tang, Yunjuan Yang and Zunxi Huang. 2009. Isolation and characterization of a new keratinolytic bacterium that exhibits significant feather-degrading capability. African Journal of Biotechnology Vol. 8 (18), pp. 4590-4596 Kim, J.M., W.J.Lim, H.J. Suh. 2001. Feather-degrading Bacillus species from poultry waste. Process Biochem., 37:287-291.Kreplak L, Doucet J, Dumas P, Briki F. 2004. New aspects of the α-helix to β-sheet transition in stretched hard α-keratin fibers. Biophys J 87:640–647 Papadopoulos MC. 1986. The effect of enzymatic treatment on amino acid content and nitrogen characteristics of feather meal. Anim Feed Sci Technol, 16:151–156 17 Riffel, A. and A. Brandelli, 2006. Keratinolytic bacteria isolated from feather waste. Brazilian Journal of Microbiology, ISSN 15178382. Rozs M, Mancziger L, Vagvolgyi C & Kevei F, Secrection of a trypsinlike thiol protease from by a new keratiolytic strain of Bacillus lichenformis, FEMS Microbiol Lett, 205 (2001) 221-224. Sangali, S, and A. Brandell, 1999. Isolation and characterization of a novel feather-degrading bacterial strain. Humana Press Inc, 02732289/00/87/0017 Shih JCH, 1993. Recent development in poultry waste digestion and feather utilization-a review. Poultry Sci, 72:1617–1620 Vidal L, Christen P & Coello M N, Feather degradation by Kocuria rosea in submerged culture, World J Microbiol Biotechnol, 16 (2000) 551-554. Williams, C.M, C.S. Richter, J.M. Mackenzie and J.C.H. Shih, 1990. Isolation, identification and characterization of a feather degrading bacterium. Appl. Environ. Microbiol., 56: 1509-1515. Webpage http://www.ncbi.nlm.nih.gov (November 20th, 2011) 18 [...]... 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 10 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... 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...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... proteins and cell wall components Figure 7: Effect of nitrogen sources on keratinolytic activity and feather degradation of B .pumilus K8 11 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... 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,... population and feather degradation of B .pumilus K8 12 The highest levels of feather degradation were also obtained in the medium containing 1.5% of feathers However, at lower or higher feather concentrations, keratinolytic activity, feather degradation and microorganisms population were reduce In fact, it was demonstrated that high feather concentration may cause substrate inhibition or repression of keratinase... results of the previous activities Add nitrogen concentrations as experimental design 6 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. .. 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... 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... results of this experiment, the incubation period for Bacillus pumilus K8 to have the optimal keratinolytic activity is 120 hours 14 Figure 9: Effect of time course on keratinolytic activity, micro organism population and feather degradation of B .pumilus K8 15 4 CONCLUSIONS AND SUGGESTIONS 4.1 Conclusions The elements of cultured medium as temperature, pH of medium, carbon source, nitrogen source, feather ... feather concentrations on growth and feather degradation by Bacillus pumilus K8 2.2.7 Effect of time course on growth and feather degradation by Bacillus pumilus K8 RESULTS AND DISCUSSION ... carbon sources on growth and feather degradation by Bacillus pumilus K8 2.2.5 Effect of different nitrogen sources on growth and feather degradation by Bacillus pumilus K8 2.2.6 Effect of feather. .. 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