Bio-utilization of keratinous waste biomass for the production of keratinolytic proteases by Chryseobactreium aquifrigidense isolated from poultry waste dumpsite
- Authors: Bokveld, Amahle
- Date: 2021-02
- Subjects: Keratin
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/20550 , vital:46120
- Description: Keratin is an insoluble and a fibrous protein that is mostly found in feathers, animal wool, and hair, making them mechanically stable. Avian feathers are the most ubiquitously generated keratinous waste biomass from the poultry processing plants. Keratinous waste biomass valorization could produce amino acids and bioactive peptides. Hence, the bio-recycling of keratin-rich wastes bears an advantage over the chemical and thermal means. In recent times, the microbial keratinases continues to gain traction due to the litany of their potential significance in green technology. Consequently, this study assessed chicken feather degrading and keratinase production potentials of bacteria isolated from a local poultry dumpsite. Soil samples were collected from the poultry dumpsite. Bacteria were isolated using basal salt medium and screened for keratinolytic activity. The identification of potent chicken feather degrading bacterial isolates was through 16S rRNA gene sequence analysis. Keratinase production efficiency of isolates on chicken feather constituted medium was optimized. Hydrolysate's amino acid compositions were quantified, and the keratinases produced was characterized. Out of 22 bacteria isolated from the soil samples, 12 showed a varying degree of proteolytic activity on skimmed milk agar. Four (4) isolates, coded as FPS-01, FPS-07, FPS-09, WDS-06, out of the 12 proteolytic strains further displayed remarkable degradation of the intact chicken feather; percentage degradation ranged from 69 percent for FPS-01 to 88 percent for FPS-09. The extracellular keratinase activity ranged from 610.91 ± 56.57 U/mL for WDS-06 and 834.55 ± 66.86 U/mL for FPS-07. Additionally, the sulfhydryl group concentration quantified from the fermentation broth was 2.22 ± 0.37 (mM), 0.22 ± 0.08 (mM), 2.27 ± 0.09 (mM), and 2.76 ± 0.79 (mM) for FPS-01, FPS-07, FPS-09 and WDS-06, respectively. Based on 16S rRNA gene sequencing and phylogenetic analyses, the isolates FPS-07 and FPS-09 were identified as Chryseobacterium aquifrigidense FANN1 and Chryseobacterium aquifrigidense FANN2. Following the optimization process, the improved fermentation conditions were pH 6, inoculum side (4 percent, v/v), temperature (30 oC), and chicken feather (0.5-1.5 percent, w/v) for FANN1. FANN2 optimal fermentation conditions were pH 6, inoculum (5 percent, v/v), temperature (30 oC), and chicken feather (1.5 percent, w/v). Both bacterial isolates showed the highest extracellular keratinase production after 72 h of the fermentation time. Analysis of the hydrolysates generated from the bacteria fermentation showed a high concentration of arginine, serine, glutamic acid, glycine, proline, valine, and leucine at a respective concentration of 1.13, 1.02, 0.83, 0.94, 0.85, 0.84, and 0.86 (g/100g sample) against FANN1. Similarly, FANN2 generated hydrolysate showed high concentrations of glutamic acid, arginine, serine, aspartic acid, and glycine at this respective abundance 2.52, 1.92, 2.12, 2.25 and 1.9 (g/100g sample). Keratinases from FANN1 and FANN2 showed optimal catalytic efficiency at pH 8 and temperature between 40-50 oC. The enzyme was considerably thermostable at 40 oC and 50 oC after 120 min of preheating. Both FANN1 and FANN2 showed variable residual activity in the presence of the different metal ions. Keratinase from FANN1 recorded the following residual activity of Fe3+ (120 ± 5.06 percent), Ca2+ (100 ± 10.33 percent), Na+ (122 ± 2.95 percent), Al3+ (106 ± 10.33 percent). Likewise, FANN2 keratinase showed remarkable stability against Na+ (108 ± 13.71 percent), Ba2+ (102 ± 0.86 percent), Al3+ (105 ± 2.57 percent), and Ca2+ (96 ± 2.99 percent). Keratinase from FANN1 was catalytically activated after 60 min of pre-treatment with the following detergents, Sunlight (129 percent), Ariel (116 percent), MAQ (151 percent), and Surf (143 percent) compared to the control. FANN2 keratinase showed less stability with laundry detergents after 60 min of preincubation. FANN1 keratinase showed remarkable stability in the presence of chemical agents tested, with residual activity of 90 ± 0.18 percent, 105 ± 7.55 percent, 108 ± 4.31 percent, 123 ± 1.44 percent, 132 ± 1.26 percent, 96 ± 7.19 percent, and 101 ± 3.06 percent for DTT, hydrogen peroxide, DMSO, acetonitrile, triton X-100, tween-80, and SDS, respectively. The enzyme activity was also considerably inhibited by PMSF and EDTA, which suggested a mixed type of protease. Furthermore, keratinase from FANN2 was inhibited by EDTA, and such inhibition pattern grouped it as a metallo-type of protease. The enzyme was also stable in the presence of other chemical agents tested. Therefore, the findings suggest the isolates and their enzymes' relevance to sustainable recycling of recalcitrant keratinous wastes into high-value products with immense application potentials. The remarkable stability shown by keratinases from FANN1 and FANN2, post detergent and chemical agents pre-treatment, indicates promise for the biotechnology and industrial sector. , Thesis(MSc) (Microbiology) -- University of Fort Hare, 2021
- Full Text:
- Authors: Bokveld, Amahle
- Date: 2021-02
- Subjects: Keratin
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/20550 , vital:46120
- Description: Keratin is an insoluble and a fibrous protein that is mostly found in feathers, animal wool, and hair, making them mechanically stable. Avian feathers are the most ubiquitously generated keratinous waste biomass from the poultry processing plants. Keratinous waste biomass valorization could produce amino acids and bioactive peptides. Hence, the bio-recycling of keratin-rich wastes bears an advantage over the chemical and thermal means. In recent times, the microbial keratinases continues to gain traction due to the litany of their potential significance in green technology. Consequently, this study assessed chicken feather degrading and keratinase production potentials of bacteria isolated from a local poultry dumpsite. Soil samples were collected from the poultry dumpsite. Bacteria were isolated using basal salt medium and screened for keratinolytic activity. The identification of potent chicken feather degrading bacterial isolates was through 16S rRNA gene sequence analysis. Keratinase production efficiency of isolates on chicken feather constituted medium was optimized. Hydrolysate's amino acid compositions were quantified, and the keratinases produced was characterized. Out of 22 bacteria isolated from the soil samples, 12 showed a varying degree of proteolytic activity on skimmed milk agar. Four (4) isolates, coded as FPS-01, FPS-07, FPS-09, WDS-06, out of the 12 proteolytic strains further displayed remarkable degradation of the intact chicken feather; percentage degradation ranged from 69 percent for FPS-01 to 88 percent for FPS-09. The extracellular keratinase activity ranged from 610.91 ± 56.57 U/mL for WDS-06 and 834.55 ± 66.86 U/mL for FPS-07. Additionally, the sulfhydryl group concentration quantified from the fermentation broth was 2.22 ± 0.37 (mM), 0.22 ± 0.08 (mM), 2.27 ± 0.09 (mM), and 2.76 ± 0.79 (mM) for FPS-01, FPS-07, FPS-09 and WDS-06, respectively. Based on 16S rRNA gene sequencing and phylogenetic analyses, the isolates FPS-07 and FPS-09 were identified as Chryseobacterium aquifrigidense FANN1 and Chryseobacterium aquifrigidense FANN2. Following the optimization process, the improved fermentation conditions were pH 6, inoculum side (4 percent, v/v), temperature (30 oC), and chicken feather (0.5-1.5 percent, w/v) for FANN1. FANN2 optimal fermentation conditions were pH 6, inoculum (5 percent, v/v), temperature (30 oC), and chicken feather (1.5 percent, w/v). Both bacterial isolates showed the highest extracellular keratinase production after 72 h of the fermentation time. Analysis of the hydrolysates generated from the bacteria fermentation showed a high concentration of arginine, serine, glutamic acid, glycine, proline, valine, and leucine at a respective concentration of 1.13, 1.02, 0.83, 0.94, 0.85, 0.84, and 0.86 (g/100g sample) against FANN1. Similarly, FANN2 generated hydrolysate showed high concentrations of glutamic acid, arginine, serine, aspartic acid, and glycine at this respective abundance 2.52, 1.92, 2.12, 2.25 and 1.9 (g/100g sample). Keratinases from FANN1 and FANN2 showed optimal catalytic efficiency at pH 8 and temperature between 40-50 oC. The enzyme was considerably thermostable at 40 oC and 50 oC after 120 min of preheating. Both FANN1 and FANN2 showed variable residual activity in the presence of the different metal ions. Keratinase from FANN1 recorded the following residual activity of Fe3+ (120 ± 5.06 percent), Ca2+ (100 ± 10.33 percent), Na+ (122 ± 2.95 percent), Al3+ (106 ± 10.33 percent). Likewise, FANN2 keratinase showed remarkable stability against Na+ (108 ± 13.71 percent), Ba2+ (102 ± 0.86 percent), Al3+ (105 ± 2.57 percent), and Ca2+ (96 ± 2.99 percent). Keratinase from FANN1 was catalytically activated after 60 min of pre-treatment with the following detergents, Sunlight (129 percent), Ariel (116 percent), MAQ (151 percent), and Surf (143 percent) compared to the control. FANN2 keratinase showed less stability with laundry detergents after 60 min of preincubation. FANN1 keratinase showed remarkable stability in the presence of chemical agents tested, with residual activity of 90 ± 0.18 percent, 105 ± 7.55 percent, 108 ± 4.31 percent, 123 ± 1.44 percent, 132 ± 1.26 percent, 96 ± 7.19 percent, and 101 ± 3.06 percent for DTT, hydrogen peroxide, DMSO, acetonitrile, triton X-100, tween-80, and SDS, respectively. The enzyme activity was also considerably inhibited by PMSF and EDTA, which suggested a mixed type of protease. Furthermore, keratinase from FANN2 was inhibited by EDTA, and such inhibition pattern grouped it as a metallo-type of protease. The enzyme was also stable in the presence of other chemical agents tested. Therefore, the findings suggest the isolates and their enzymes' relevance to sustainable recycling of recalcitrant keratinous wastes into high-value products with immense application potentials. The remarkable stability shown by keratinases from FANN1 and FANN2, post detergent and chemical agents pre-treatment, indicates promise for the biotechnology and industrial sector. , Thesis(MSc) (Microbiology) -- University of Fort Hare, 2021
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Keratinous poultry waste valorization through novel keratinases of C. cucumeris and S. multivorium isolated from poultry sludge
- Authors: Qaphela, Hendrick
- Date: 2021-02
- Subjects: Food--Biotechnology , Poultry , Poultry industry
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/20860 , vital:46640
- Description: Annually, about 55 percent of keratinous wastes are generated from various agro-industrial processing farms in South Africa. These wastes are difficult to handle due to structural integrity; hence, they constitute environmental issues due to the disposal means. Degradation of keratinous wastes using microbial-based technology has been deemed advantageous as it generates products with high-end values. Therefore, in this study, chicken feather and soil samples were collected from a local poultry farm, and bacteria were isolated using basal salt media supplemented with chicken feathers. The isolates were evaluated for proteolytic and keratinolytic potentials. The potent isolates were identified through 16S rDNA sequence and phylogenetic analysis. Fermentation media were optimized for enhanced keratinase production, and the amino acids liberated in the media during feather biodegradation were quantified. The biochemical properties of the keratinases produced were likewise determined. Ten (10) proteolytic bacteria were obtained from 20 isolates recovered from the samples with a diameter of halo on skimmed milk agar plate ranging from 15.5 ± 0.71 (mm) for isolate coded as PSW-15 to 28 ± 1.41 (mm) for isolate coded as PSW-08. The proteolytic bacteria showed variable keratinolytic potentials with percentage feather degradation that ranged from 29 percent for PSW-11 to 84 percent for PSW-14, and keratinase activity ranging from 99.99 U/mL for PSW-15 to 761.82 U/mL for PSW-14. The most potent isolates coded as PSW-14 and PSW-16 were identified as Chryseobacterium cucumeris FHN1 and Sphingobacterium multivorum HNFx. Their nucleotide sequences were submitted to the GenBank as MW16587 and MK82939, respectively. The optimization of fermentation conditions; C. cucumeris FHN1 showed improved activity at pH 5 - 6, inoculum size (4 percent, v/v), chicken feather concentration (1 percent, w/v), fermentation temperature (25o C). Similarly, S. multivorum HNFx showed optimal activity at pH 4.0, inoculum size (5 percent, v/v), chicken feather concentration (2.5 percent, w/v), and fermentation temperature (25-30 oC). C. cucumeris FHN1 and S. multivorum HNFx showed maximum keratinase production of 485.54 U/mL and 526.36 U/mL at 96 h and 72 h of incubation period respectively. Serine, aspartic acid, glutamic acid, and proline were the most abundant amino acids in the degraded chicken feathers, and upon quantitation, the following concentration was respectively obtained; 3.71, 3.4, 4.19 and 4.35 (g/100g sample) against C. cucumeris FHN1. While S. multivorum HNFx yielded aspartic acid (2.04 g/100g sample) and glutamic acid (2.0 g/100g sample) in high concentration. The keratinases showed optimal catalytic efficiency at pH and temperature of 8.0 and 90 oC, respectively. C. cucumeris FHN1 keratinase was inhibited by metal ion chelating agents; EDTA and 1,10-phenanthroline, suggesting a metallo-type of protease. The enzyme showed remarkable stability after pre-treatment with DTT, Fe2+, Fe3+ and Cu2+, with respective residual activity of 108 percent, 102 percent, 114 percent, and 104 percent. The S. multivorum HNFx keratinase; activity was not inhibited by serine- and metallo-protease inhibitors. It maintained the following residual activity against the following chemical agents; DTT (124 percent), hydrogen peroxide (152 percent), DMSO (161 percent), triton X-100 (152 percent), tween-80 (101 percent), and metal ions; Fe2+ (128 percent), Fe3+ (104 percent), K+ (117 percent), Ca2+ (104 percent), Na+ (103 percent), Ba2+ (115 percent), Al3+ (126 percent). The enzyme showed a substantial loss of catalytic efficiency after pre-incubation with various laundry detergents. The keratinases' remarkable stability in the presence of various chemical agents and metal ions tested suggests biotechnological and industrial application potentials. Consequently, the isolates portend industrial relevance for keratinous waste valorization and an excellent source of keratinases of industrial relevance. , Thesis (MSc) (Microbiology) -- University of Fort Hare, 2021
- Full Text:
- Authors: Qaphela, Hendrick
- Date: 2021-02
- Subjects: Food--Biotechnology , Poultry , Poultry industry
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/20860 , vital:46640
- Description: Annually, about 55 percent of keratinous wastes are generated from various agro-industrial processing farms in South Africa. These wastes are difficult to handle due to structural integrity; hence, they constitute environmental issues due to the disposal means. Degradation of keratinous wastes using microbial-based technology has been deemed advantageous as it generates products with high-end values. Therefore, in this study, chicken feather and soil samples were collected from a local poultry farm, and bacteria were isolated using basal salt media supplemented with chicken feathers. The isolates were evaluated for proteolytic and keratinolytic potentials. The potent isolates were identified through 16S rDNA sequence and phylogenetic analysis. Fermentation media were optimized for enhanced keratinase production, and the amino acids liberated in the media during feather biodegradation were quantified. The biochemical properties of the keratinases produced were likewise determined. Ten (10) proteolytic bacteria were obtained from 20 isolates recovered from the samples with a diameter of halo on skimmed milk agar plate ranging from 15.5 ± 0.71 (mm) for isolate coded as PSW-15 to 28 ± 1.41 (mm) for isolate coded as PSW-08. The proteolytic bacteria showed variable keratinolytic potentials with percentage feather degradation that ranged from 29 percent for PSW-11 to 84 percent for PSW-14, and keratinase activity ranging from 99.99 U/mL for PSW-15 to 761.82 U/mL for PSW-14. The most potent isolates coded as PSW-14 and PSW-16 were identified as Chryseobacterium cucumeris FHN1 and Sphingobacterium multivorum HNFx. Their nucleotide sequences were submitted to the GenBank as MW16587 and MK82939, respectively. The optimization of fermentation conditions; C. cucumeris FHN1 showed improved activity at pH 5 - 6, inoculum size (4 percent, v/v), chicken feather concentration (1 percent, w/v), fermentation temperature (25o C). Similarly, S. multivorum HNFx showed optimal activity at pH 4.0, inoculum size (5 percent, v/v), chicken feather concentration (2.5 percent, w/v), and fermentation temperature (25-30 oC). C. cucumeris FHN1 and S. multivorum HNFx showed maximum keratinase production of 485.54 U/mL and 526.36 U/mL at 96 h and 72 h of incubation period respectively. Serine, aspartic acid, glutamic acid, and proline were the most abundant amino acids in the degraded chicken feathers, and upon quantitation, the following concentration was respectively obtained; 3.71, 3.4, 4.19 and 4.35 (g/100g sample) against C. cucumeris FHN1. While S. multivorum HNFx yielded aspartic acid (2.04 g/100g sample) and glutamic acid (2.0 g/100g sample) in high concentration. The keratinases showed optimal catalytic efficiency at pH and temperature of 8.0 and 90 oC, respectively. C. cucumeris FHN1 keratinase was inhibited by metal ion chelating agents; EDTA and 1,10-phenanthroline, suggesting a metallo-type of protease. The enzyme showed remarkable stability after pre-treatment with DTT, Fe2+, Fe3+ and Cu2+, with respective residual activity of 108 percent, 102 percent, 114 percent, and 104 percent. The S. multivorum HNFx keratinase; activity was not inhibited by serine- and metallo-protease inhibitors. It maintained the following residual activity against the following chemical agents; DTT (124 percent), hydrogen peroxide (152 percent), DMSO (161 percent), triton X-100 (152 percent), tween-80 (101 percent), and metal ions; Fe2+ (128 percent), Fe3+ (104 percent), K+ (117 percent), Ca2+ (104 percent), Na+ (103 percent), Ba2+ (115 percent), Al3+ (126 percent). The enzyme showed a substantial loss of catalytic efficiency after pre-incubation with various laundry detergents. The keratinases' remarkable stability in the presence of various chemical agents and metal ions tested suggests biotechnological and industrial application potentials. Consequently, the isolates portend industrial relevance for keratinous waste valorization and an excellent source of keratinases of industrial relevance. , Thesis (MSc) (Microbiology) -- University of Fort Hare, 2021
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Critical analysis of anti-corruption strategies, policies, plans and practices- in the selected municipalities in the case of the Eastern Cape, Joe Gqabi District (2008-2011)
- Authors: Nonjola, Mcebisi Patrick
- Date: 2012-01
- Subjects: Local government -- South Africa -- Eastern Cape , Municipal services -- South Africa
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/25959 , vital:64637
- Description: Corruption is a virus that has infected all sectors of society not only in South Africa but also in the whole world. The various spheres of government are seemingly not immune against this scourge and its paralysing effect on service delivery. There is no universally acceptable definition of corruption. Its causes are diverse, ranging from administrative to socio-economic, and it manifests itself in various ways. The plethora of anti-corruption legislation and agencies has only served to minimise but not to eradicate corruption from the face of the public institutions. In order to scale up its fight against corruption, the Department of Public Service and Administration developed a strategy aimed at preventing, detecting and investigating corruption in government departments. Subsequently, the Department of Local Government and Traditional Affairs, now called Co-operative Governance and Traditional Affairs, developed a similar strategy for the municipalities. The strategy requires inter alia municipalities to develop minimum anti-corruption capacity which entail developing strategies, systems, policies, procedures and plans to prevent, detect and investigate fraud and corruption. The study sought to establish, in selected municipalities in Joe Gqabi District municipality, whether or not they have established the minimum capacity envisaged in the National anti-corruption strategy. The study found municipalities to have progressed at different stages of establishing and ensuring functioning of those systems, policies and plans but none of them had developed anti-corruption strategy. Consequently, the study recommends inter alia that the establishment of minimum anti-corruption capacity be made one of the national key performance indicators which must be incorporated in the performance agreements of the Senior management of municipalities. , Thesis (MPA) -- Faculty of Management and Commerce, 2012
- Full Text:
- Authors: Nonjola, Mcebisi Patrick
- Date: 2012-01
- Subjects: Local government -- South Africa -- Eastern Cape , Municipal services -- South Africa
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/25959 , vital:64637
- Description: Corruption is a virus that has infected all sectors of society not only in South Africa but also in the whole world. The various spheres of government are seemingly not immune against this scourge and its paralysing effect on service delivery. There is no universally acceptable definition of corruption. Its causes are diverse, ranging from administrative to socio-economic, and it manifests itself in various ways. The plethora of anti-corruption legislation and agencies has only served to minimise but not to eradicate corruption from the face of the public institutions. In order to scale up its fight against corruption, the Department of Public Service and Administration developed a strategy aimed at preventing, detecting and investigating corruption in government departments. Subsequently, the Department of Local Government and Traditional Affairs, now called Co-operative Governance and Traditional Affairs, developed a similar strategy for the municipalities. The strategy requires inter alia municipalities to develop minimum anti-corruption capacity which entail developing strategies, systems, policies, procedures and plans to prevent, detect and investigate fraud and corruption. The study sought to establish, in selected municipalities in Joe Gqabi District municipality, whether or not they have established the minimum capacity envisaged in the National anti-corruption strategy. The study found municipalities to have progressed at different stages of establishing and ensuring functioning of those systems, policies and plans but none of them had developed anti-corruption strategy. Consequently, the study recommends inter alia that the establishment of minimum anti-corruption capacity be made one of the national key performance indicators which must be incorporated in the performance agreements of the Senior management of municipalities. , Thesis (MPA) -- Faculty of Management and Commerce, 2012
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