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 REFERENCES Vietnamese 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. English Agrahari, S. and N. Wadhwa. 2010. Degradation of chicken feather a poultry waste product by keratinolytic bacteria isolated from dumping site at Ghazipur Poultry Processing Plant. International Journal of Poultry Science, 9 (5):482-489. 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. Burtt E.H. and Ichida M. 1999. Occurrence of feather degrading Bacilli in the Plumage of birds. Auk, 116:364-372. Cheng, S.W., H.M. Hu, S.W.Shen, H. Takagi, M. Asano, Y.C. Tsai. 1995. Production and characterization of keratinase of a featherdegrading Bacillus licheniformis PWD-1. Biosci. Biotechnol. Biochem., 59:2239-2243. Geun-Tae Park, Hong-Joo Son. 2009. Keratinolytic activity of Bacillus megaterium F7-1, a feather-degrading mesophilic bacterium. Microbiological Research. 164, 478-485. Jayalakshmi, P. Krishnamoorthy, G. Ramesh kumar, P. Sivamani. 2011. Optimization of culture conditions for Keratinase production in 19 Streptomyces sp. JRS19 for Chick feather wastes degradation. J. Chem. Pharm. Res., 3(4):498-503. Kainoor, Pushpalata S; Naik, G R. 2010. Production and characterization of feather degrading keratinase from Bacillus sp. JB 99. Indian Journal of Biotechnology. Vol 9, pp 384-390. Mohammad Shahnoor Hossain, Abul Kalam Azad, S.M. Abu Sayeni, Golam Mostafa and Mozammel Hoq. 2007. Production and Partial Characterization of Feather-degrading Keratinolytic Serine Protease from Bacillus licheniformis MZK-3. Journal of Biological Sciences 7. (4): 599-606, 2007. Riffle, A., F.S. Lucas, P. Heeb, A. Brandelli. 2003. Characterization of a new keratinolytic bacterium that completely degrades native feather. Arch. Microbiol., 179(4):258-265. Sangali, S, and A. Brandell. 2000. Feather keratin hydrolysis by a Vibrio sp. Kr2 strain. J. Appl. Microbiol., 89:735-743. Shih JCH. 1993. Recent development in poultry waste digestion and feather utilization-a review. Poultry Sci, 72:1617–162 Suntornsuk W, Suntornsuk L. 2003. Feather degradation by Bacillus sp. FK46 in submerged cultivation. Biores Technol; 86:239–43. Zeikus, J.G., Ng, T.K., Ben-Bassat, and Arie Lamed, R.J., 1983. Use of co-cultures in the production of ethanol by the fermentation of biomass. Journal of Free Patents Online. Wang, X. and C.M. Parsons. 1997. Effect of processing systems on protein quality of feather meal and hair meals. Poultry Sci, 76:491–496. Webpage 20 http://uv_vietnam.com.vn/SpecNewsDetail.aspx?newsId=947 (November 18th, 2012) www.nsl.hcmus.edu.vn/greenstone/collect/thesiskh/archives/HASH869 1.dir/6.PDF (November 18th, 2012) http://www.huaf.edu.vn/diendan/viewtopic.php?f=186&t=5220 (November 18th, 2012) 21 [...]... 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... 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. .. 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,... (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... 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... 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. .. 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. .. 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... 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 REFERENCES Vietnamese... 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,