Isolation and selection the thermophilic keratin degradation bacteria in dong thap province

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MINISTRY OF EDUCATION & TRAINING CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE SUMMARY BACHELOR OF SCIENCE THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY ISOLATION AND SELECTION THE THERMOPHILIC KERATIN DEGRADATION BACTERIA IN DONG THAP PROVINCE SUPERVISOR STUDENT Dr. BUI THI MINH DIEU PHAM MINH TRIET Student’s code: 3082639 Session: 34 (2008- 2013) Can Tho, 05/2013 APPROVAL SUPERVISOR Dr. BUI THI MINH DIEU STUDENT PHAM MINH TRIET Can Tho, May , 2013 PRESIDENT OF EXAMINATION COMMITTEE Abstract Using of microorganisms, especially bacteria to process keratin-containing waste source has demonstrated their potential and advantages in many areas of life: food processing for cattle and poultry, bio-fertilizer production with lower cost and environmental friendly ... The temperatures in the waste graves can raise highly and inhibit almost activities of the useful bacteria. Therefore, the thesis was carried out for isolation and selection the thermophilic keratinolytic bacteria strains which have high degradation capability of keratin-containing wastes. Total of 18 aerobic keratinolytic bacteria strains were isolated of five soil and two wastewater samples from animal and poultry processing plants. The result showed that all the isolated strains were capable of growth and degradation keratin at 45°C; in which ten strains can grow at 50°C; and two strains can o survive at 55 C. All isolated strains expressed protease activity with the halo zone creating on the skim milk medium after 24 hours incubation. The halo diameter ranged from 4.7mm to 10.3mm. The results of feathers degradation study was recorded that 18 strains were capable of reducing 14.59% to 37.76% poultry feather; and 25.12% to 42.18 goat hair. In which, V1, V2 and V9 strains showed the ability of keratin degradation effectively than the others. The results of sequencing analysis of partial 16S rRNA gene showed that the gene sequences of V1 shared 99% similarity with Bacillus megaterium species and V2 shared 99% similarity with Bacillus sp. P014. Key words: Bacillus, Bacillus megaterium, keratinase, keratin degradation, thermophilic bacteria i CONTENTS ABSTRACT………………………………………………...........i CONTENTS..………………………..……………...……....…...ii 1. INTRODUCTION ................................................................. 1 2. MATERIALS AND METHODS ............................................ 3 2.1. Materials ............................................................................ 3 2.2. Methods ............................................................................. 3 2.2.1. Collection and treatment samples method ................... 3 2.2.2. Method of isolation ..................................................... 3 2.2.3. Survey morphology and mobility of bacteria ............... 4 2.2.4. Gram staining ............................................................. 4 2.2.5. Study the biochemical properties ................................ 5 2.2.6. Count for bacteria cell density..................................... 5 2.2.7. Study protease activity of bacteria strains .................... 5 2.2.8. Study the poultry feather degradation of bacteria strains ............................................................................................ 6 2.2.9. Study the goat feather degradation of bacteria strains .. 6 2.2.10. Identification of the thermophilic bacteria with high capable of keratin degradation by molecular technique ............... 7 3. RESULTS AND DISCUSSION ............................................ 8 3.1. Isolation and thermotolerance condition study .................. 8 3.2. Morphology of isolated bacteria strains............................. 8 3.3. Study of biochemical properties ...................................... 10 3.4. Study protease activity of bacteria strains ......................100 3.5. Study the poultry feather degradation of bacteria strains . 12 3.6. Study the goat feather degradation of bacteria strains ...... 14 ii 3.7. Identification of high keratin degradation thermophilic bacteria strains ......................................................................... 15 4. CONCLUSIONS AND SUGGESTIONS ............................170 4.1. Conclusions ...................................................................190 4.2. Suggestions ...................................................................191 REFERENCES .......................................................................212 iii 1. INTRODUCTION Today, the environmental problem is set many big challenges for human. Using of microorganisms to treat environmental pollution were showed their potential and benefits. In which keratin degradation bacteria group are interested in many scientists. Keratin is virtual resistant to the degradation of general proteases as trypsin, pepsin…due to their stable structure. This protein is major component of poultry feather and cattle feather (up to 90%) (Gousterova et al., 2005.) The dismission of poultry feather and cattle feather directly to the environment not only polluted the environment but also wasted the protein sources. Therefore, there are many researches to process this waste source. However, almost of physical methods and chemical methods were consumed big energy and environmental unfriendly; and also reduces the nutritional value of the product due to loss of several amino acids (Wang and Parsons, 1997). Use of the thermophilic keratinolytic bacteria strains is the new way to handle the problem above. This novel method of treatment is potential and wide application. It helps to enrich the nutritional value of feather products and environment friendly (Lee et al., 1991). The temperatures in the waste graves can rise highly and inhibit almost activities of the useful bacteria. For that reason, the thesis "Isolation and selection thermophilic keratin degradation bacteria in Dong Thap province" was conducted to select the thermophilic keratinolytic bacteria strains which have high degradation capability of keratin-containing wastes. 1 Objectives Isolation and selection of thermophilic bacteria strains with high keratin degradation capability. 2 2. MATERIALS AND METHODS 2.1. Materials Five soil and two wastewater samples were collected from Cao Lanh district, Lai Vung district and Cao Lanh city, Dong Thap province. These samples were stored at Molecular Biotechnology laboratory, Can Tho University. Medium: skim milk medium (peptone 5g/l, yeast extract 3g/l, non-fat milk 100ml/l, agar 12g/l) (Riffel và Brandelli, 2006), medium with poultry feather addition (NaCl 0.5g/l, KH2PO4 0.4g/l, K2HPO4 0.3g/l, NH4Cl 0.5g/l, Agar 20g/l, poultry feather powder 10g/l) (Daniel et al., 2008), Luria Bertani medium (Bacto tryptone 10g/l, Bacto yeast extract 5g/l, Nacl 10g/l). Chemicals and equipments in Molecular Biotechnology laboratory. 2.2. Methods 2.2.1. Collection and treatment samples method - Soil samples: took soil 20cm deep from the surface at poultry or cattle farm and slaughter places. - Water samples: collected directly at wastewater sources. Samples were stored at laboratory until the experiments are carried out. 2.2.2. Method of isolation Add 1ml (water sample) or 1g (soil sample) of sample to erlen 250ml containing 100ml medium (NaCl 0.5g/l, KH2PO4 0.4g/l, K2HPO4 0.3g/l, NH4Cl 0.5g/l, poultry feather powder 3 10g/l) sterilized at 121ºC in 10 minutes. Growing bacteria at 37°C, 120rpm for 24h to 48h. Dilute from 10-6 to 10-8 times and streak on skim milk medium. Incubate at 45ºC for 24h. After that select the colonies with variant morphologies and halo zone creating to grow on medium with feather powder addition for 24h at 45ºC until they get pure strains. Study the thermotolerance of bacteria strains by growing them at 50ºC, 55ºC, 60ºC… Record their development in 24h to 48h. 2.2.3. Survey morphology and mobility of bacteria Prepare bacteria samples as: Drop 5  l of sterilized distilled water on lame  take a little of colony and stretch out regularly on water drop  put lamen on the lame and observe their morphology and mobility under x400 microscope. 2.2.4. Gram staining Drop 20  l sterilized distilled water on lame take a little of colony and stretch out regularly on water drop  place the lame on fire (not over heat)  put one or two drops of Crystal Violet for two minutes wash with sterilized distilled water, dried slightly slower  Instil one or two drops of iodine solution for a minute Wash with sterile distilled water, dried slightly slower  Instil one or two drops Fusin for a minute  Wash with sterilized distilled water. Observe sample in optical microscope at magnification x1000 and record by Gram bacteria. Bacteria is Gram-positive if sample has blue purple color of 4 Crystal violet, Gram-negative if sample is red color of Fusin (Cao Ngoc Diep and Nguyen Huu Hiep, 2008). 2.2.5. Study the biochemical properties Biochemical properties was performed for identification the bacteria. Study catalase activity: Add one to three drops of H2O2 3% to Petri dish containing bacteria. Record the results. Study methyl red test: Add five drops of methyl red to 1ml of bacteria suspension. Record the results. Study amylase activity: Inoculate 1ml of bacteria solution to 1ml of salt solution 0.1%. After that add 1ml of starch solution, incubate at 30°C for 30 minutes. Record the results. 2.2.6. Count for bacteria cell density Add 100  l of bacteria suspension to eppendorf containing 900  l sterilized distilled water to have diluted sample 10-1. Dilute the sample to 10-10. Take 5  l of bacteria suspension and drop on feather powder addition medium. Record the results after 24h to 48h at conditions of study. 2.2.7. Study protease activity of bacteria strains Protease activity can determine by the ability of creating halo zone on milk medium. Drop 5  l of bacteria suspension on skim milk medium. Record the results after 24h at different conditions of study. The degrading diameter was calculated by: Degrading diameter = Halo diameter – Diameter of bacteria growing drop 5 2.2.8. Study the poultry feather degradation of bacteria strains Prepare a Erlenmeyer flask 250ml contain 100ml medium (NaCl 0.5 g/l, KH2PO4 0.4 g/l, K2HPO4 0.3 g/l, NH4Cl 0.5 g/l and 1g of poultry feather powder) sterilize at 121°C for 10 minutes. Inoculate 5ml of bacteria suspension to Erlenmeyer flask; incubate at 120rpm at study temperature for one week. Filter medium by filter paper and dry at 75°C until weight is constant. Record the weight. The percentage of poultry feather degradation A(%) are calculated by: A (%) = (mBĐ - mC) x 100/ mBĐ mBĐ: weight at beginning mC: weight after experiment * Study the degradation of whole chicken feather Put whole chicken feather (same size and weight) to test tube containing 20ml of (NaCl 0.5 g/l, KH2PO4 0.4 g/l, K2HPO4 0.3 g/l, NH4Cl 0.5 g/l), sterilized at 121°C for 10 minutes. Inoculate 4ml of bacteria suspension and incubate at different temperature of study, 120rpm for 10 days. Record the results. 2.2.9. Study the goat feather degradation of bacteria strains Prepare an Erlenmeyer flask 250ml contain 100ml medium (NaCl 0.5 g/l, KH2PO4 0.4 g/l, K2HPO4 0.3 g/l, NH4Cl 0.5 g/l and 1g of goat feather powder) sterilize at 121°C for 10 minutes. Inoculate 5ml of bacteria suspension to Erlenmeyer flask; incubate at 120rpm at study temperature for one week. Filter medium by filter paper and dry at 75°C until weight is constant. 6 Record the weight. The percentage of goat feather degradation A(%) are calculated as the form of experiment 2.2.8. 2.2.10. Identification of the thermophilic bacteria with high capable of keratin degradation by molecular technique The bacterial isolate was identified by sequence analysis of partial 16S rRNA gene. A couple of primer including forward primer 27F: 5’-AGAGTTTGATCCTGGCTC-3’and reverse primer 1492R: 5’-TACGGTTACCTTGTTACGACT-3’ were employed to amplify the target gene through PCR technique. PCR product was sequenced and analyzed at Molecular Biology laboratory, Research and Development Biotechnology Institute, Can Tho University. 7 3. RESULTS AND DISCUSSION 3.1. Isolation of thermolytic keratinolytic bacteria Total 18 aerobic keratinolytic bacteria strains were isolated from five soil and two wastewater samples using poultry feather supplemented medium. The bacteria strains were named Vx with x stand for ordinal number of the isolated bacteria strains. The results of thermotolerance condition study showed that all of 18 strains can grow and degrade keratin at 45°C (V1 to V18); 10 strains can grow at 50°C (V1, V10, V11, V12, V13, V14, V15, V16, V17, V18) and two strains can survive at 55°C (V17, V18). 3.2. Morphology of isolated bacteria strains Colony of 18 isolated strains has circle form, white color. Colony diameter ranged from 0.5mm to 6.0mm. The results of Gram staining at x1000 microscope showed that the cell of 14 bacteria strains were Gram negative (4 strains were Gram positive: V1, V2, V5 and V9). Almost bacteria strains were rod shaped (except strain V3 was sphere) (Table 8’). Table 3’: Colony properties of bacteria strains Colony properties Name Form Elevation Margin Color Diameter (mm) V1 Circular Raised Entire Light pink V2 Circular Crateriform Entire Opaque white 8 1.5 6 V3 Circular Raised Entire Opaque white 2 V4 Circular Raised Entire Light yellow 1.5 V5 Circular Raised Entire Light green 4 V6 Circular Raised Entire Opaque white 0.8 V7 Circular Crateriform Entire Opaque white 2.5 V8 Circular Raised Entire Opaque white 3 V9 Circular Crateriform Waxy Light yellow 5 V10 Circular Crateriform Waxy Opaque white 1.2 V11 Circular Raised Entire Opaque white 0.7 V12 Circular Crateriform Waxy Opaque white 0.8 V13 Circular Raised Entire Light yellow 1 V14 Circular Raised Entire Opaque white 1.1 V15 Circular Raised Entire Opaque white 0.9 V16 Circular Raised Entire Opaque white 0.5 V17 Circular Crateriform Entire Opaque white 3 V18 Circular Raised Entire Light yellow 0.5 9 3.3. Study of biochemical properties 3.3.1. Catalase activity The results of experiment showed that 14 strains had catalase activity (bubble grew up, about 77.8%); four strains were negative (including V1, V7, V10, V11). 3.3.2. Methyl red test Four strains were considered as high acid creation as ++ value of three scale of value: 0, +, ++ (V3, V7, V13, V14) and 14 strains were low acid production as + value. 3.3.2. Amylase activity Almost bacteria strains, except strain V16 showed amylase activity with the ability to discolor blue color of mixed iodine and starch. 3.4. Study protease activity of bacteria strains. All isolated strains expressed protease activity with the halo zone creating on the skim milk medium after 24h incubation. Halo diameter ranged from 4.7mm to 10.3mm. In which, the strains V1, V2, V9, V16 had halo circular diameter more than 9mm (difference was statistically significant at the 5% level compared with other strains in each group of thermotolerant study). Degrading halo diameters in this research were higher than the research of Le Dinh Quyen and Tran Thi Lan Phuong (2001) with 9.5mm degrading halo diameter creation by Bacillus strain. 10 Halo diameter (mm) 12.0 10.3 10.2 10.0 7.3 8.0 6.3 6.0 5.0 5.3 v3 v4 6.0 4.7 4.0 2.0 0.0 v2 v5 v6 v7 v8 v9 Strain Figure 7: Degrading halo diameter of bacteria strains on skim milk medium after 24h incubation at 45°C The result of Figure 7 showed that strains V2 and V9 could create degrading halo diameter more than 9mm (difference was statistically significant at the 5% level compared with the others). The others also expressed protease activity. However, the result Halo diameter (mm) was not high. 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 9.3 8.3 9.3 8.7 7.7 7.7 6.0 4.7 v1 v10 v11 v12 v13 v14 v15 v16 Strain Figure 8: Degrading halo diameter of bacteria strains on skim milk medium after 24h incubation at 50°C Figure 8 represented the result of degrading halo diameter creation on skim milk medium after 24h incubation at 50°C. In general, the result of this group was higher than the previous 11 group. However, the maximum halo diameter creation was lower (9.3mm of V1 compared with 10.3mm of V2). The results of study on degrading halo diameter creation of bacteria strains at 55°C showed that this group could create degrading diameter. However, the results were not important. 3.5. Study the poultry feather degradation of bacteria strains After one week inoculation, the results demonstrated that total 18 strains have keratin degradation capability by degradation poultry feather powder (ranged from 14.59% to 37.76%). Difference was statistically significant at 5% level compared with the control treatment. The study group at 45°C gave better results. Strain V9 showed the best degradation result than other strains (37.76%). These results were lower than the research of Nguyen Huy Hoang et al. (2010) with degradation results reached from 52.40% to 98.45% after one week inoculation. Optimum the condition as temperature and medium components can investigate degrading rate. The results were showed in Figure 10, Figure 11, Degrading percentage (%) and Figure 12. 37.76 40.00 32.29 35.00 30.00 24.79 22.96 25.00 20.00 14.59 15.88 16.96 18.44 v5 v6 v7 15.00 10.00 5.00 2.52 0.00 C v2 v3 v4 v8 v9 Strains Figure 9: Degradation of poultry feather powder by bacteria strains after one week inoculation at 45°C C: control treatment 12 Total eight strains of this group could degrade poultry feather powder. Difference was statistically significant at 5% level compared with the control treatment. In which V2 and V9 strains expressed degradation capability more effectively than the Degrading percentage (%) others (32.29% of V2 and 37.76% of V9). 40.00 35.64 35.00 30.67 30.68 30.00 28.28 29.82 26.44 28.81 28.96 v15 v16 25.00 20.00 15.00 10.00 5.00 2.29 0.00 C v1 v10 v11 v12 v13 v14 Strains Figure 10: Degradation of poultry feather powder by bacteria strains after one week inoculation at 50°C C: control treatment Figure 11 represented the result of poultry feather powder degradation at 50°C. All strains of this group degraded feather powder more than 26% after one week inoculation. The strain V1 showed the best result (35.64%). The result of degradation capability of bacteria strains at 55°C was statistically significant difference at the 5% level compared with control treatment (27.75% of V17 and 26.75% of V18 compared with 2.38% of control treatment). However, the maximum degrading percentage was lower than two previous groups. 13 * Study chicken shaft degradation Total 18 isolated bacteria strains were showed the capable of chicken feather degradation in time study, except the feather shaft. The study group at 45°C gave better result than other group. Strain V2 and V9 were broken down whole chicken feather after 10 days inoculation. Strain V13 and V1 degraded well with more than 70% broken up of chicken feather. The others also degraded feather. However, the rate was low. 3.6. Study degradation of goat hair powder by bacteria strains The aim of this experiment was selection bacteria strain with high keratin degradation capability. The results of each group were similar to poultry feather degradation test. The best result was recorded by strain V9 with 42.18% degrading feather weight. Strain V2 showed good result with 40.85%. Remainder strains showed the degradation capability ranged from 25% to 40.47%. Figure 14, Figure 15, Figure 16 represented the goat hair Degrading percentage (%) degradation capability. 45.00 40.00 35.00 30.00 25.00 20.00 15.00 10.00 5.00 0.00 42.18 40.85 36.95 34.50 29.91 31.15 30.32 v5 v6 v7 25.12 2.38 C v2 v3 v4 v8 v9 Strains Figure 12: Degradation of goat hair powder by bacteria strains after one week inoculation at 45°C C: control treatment 14 Figure 14 showed the result of goat hair powder degradation of bacteria strains at 45°C. Almost strains could degrade goat hair powder more than 30%. Strains V2 and V9 Degrading percentage (%) gave effective results with 40.85% of V2 and 42.18% of V9. 45.00 40.00 35.00 30.00 25.00 20.00 15.00 10.00 5.00 0.00 40.48 31.65 31.07 30.65 34.29 37.32 33.53 28.19 2.18 C v1 v10 v11 v12 v13 v14 v15 v16 Strains Figure 13: Degradation of goat hair powder by bacteria strains after one week inoculation at 50°C C: control treatment Total eight strains of this group showed goat hair powder degradation capability with degrading percentage ranged from 28.19% to 40.48%. Strain V1 showed the best result with 40.48% goat hair powder degradation. 3.7. Identification of the high thermophilic keratinolytic bacteria strains Based on the study of keratin degradation, tow strains V1 and V2 were selected to sequence 16S rRNA gene and BLAST on NCBI website. The result showed that the gene sequences of isolated V1 shared 99% similarity with Bacillus megaterium; isolated V2 shared 99% similarity with Bacillus sp. P014. The result of 16S rRNA gene sequencing of strain V1 15
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