Bioconversion of chicken feather into amino acids and keratinase production by mesophilic Chryseobacterium proteolyticum and Pseudomonas aeruginosa isolated from municipal waste dumpsites
- Giwu, Nonkonzo https://orcid.org/0000-0001-9416-7896
- Authors: Giwu, Nonkonzo https://orcid.org/0000-0001-9416-7896
- Date: 2021-02
- Subjects: Poultry -- Processing , Proteolytic enzymes
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/22732 , vital:52720
- Description: Chicken feathers are by-products of poultry processing which are generated in large amount because of the global growing demand for poultry meats. They have high contents of crude proteins in the form of keratin which could be valorized into digestible products. Keratinases are classified as a specific collection of proteolytic enzymes that have the ability for the degradation of recalcitrant keratinous substrates. Isolation and characterization of these enzymes from various microbial producers are gaining prominence in recent years due to their industrial and biotechnological application potentials. For this research, the collection of soil samples was done as well as the isolation of bacteria and the screening for keratinolytic activity. 16S rDNA sequencing and phylogenetic analysis were used to identify the isolates with efficient chicken feathers degrading capacity. Optimum conditions for the fermentation prcocess was enhanced for the production of keratinase. The fermentation broth was also analysed for various amino acids of protein, and the biochemical properties of the enzymes were likewise determined. Twenty two (22) bacteria were isolated from the soil samples, and 18 out of the 22 isolates showed proteolytic activity on solid media with diameters of halo zone that ranged from 5 ± 0.71 mm for isolate coded as PSS-03 to 25 ± 1.41 mm for isolate coded as PSS-06. Intact chicken feathers were degraded by proteolytic bacterial isolates in variable degree that ranged from 24percent for PSS-10 and 81percent for DSS-02. Extracellular keratinase production recorded for the isolates ranged from 63.63 ± 4.14 U/mL for PSS-10 to 693.63 ± 62.99 U/mL for DSS-02. Based on 16S rDNA sequence and phylogenetic analysis, the 2 isolates with remarkable keratinolytic activity coded as DSS-02 and PSS-14 were identified as Chryseobacterium proteolyticum FGNn and Pseudomonas aeruginosa GNFx. C. proteolyticum showed the maximum keratinase production of 756.36 U/mL after 72 h of incubation at optimized fermentation conditions which involved initial medium pH (4), incubation temperature (30 oC), inoculum size (2percent; v/v), and chicken feathers (1.5percent; w/v). Similarly, P. aeruginosa optimally produced keratinase (1055.45 U/mL) after 96 h of incubation at optimized fermentation conditions that involved initial medium pH (7-8), incubation temperature (30 oC), inoculum size (5percent; v/v), and chicken feathers (2.5percent; w/v). Furthermore, feather hydrolysate from C. proteolyticum FGNn had relatively higher abundance (>1.5g/100g sample) of arginine (1.85), serine (1.63), glycine (1.9) and lysine (1.62); while P. aeruginosa GNFx feather hydrolysate showed high abundance of arginine, serine, aspartic acid, glutamic acid, glycine, alanine, valine, and leucine with respective concentration of 2.06, 1.67, 2.39, 3.05, 1.87, 1.73, 1.56 and 1.65 (g/100g sample). The results showed that keratinases from the two bacterial isolates were optimally active at pH 8, and temperature of 50 oC for FGNn keratinase and 50-60 oC for GNFx keratinase. The enzymes displayed remarkable pH stability. Keratinase from C. proteolyticum was catalytically inhibited by EDTA and 1,10-phenanthroline but not affected by PMSF; while P. aeruginosa keratinase was not significantly affected by those class of protease inhibitors. Adiitionally, FGNn keratinase demonstrated high residual activity of 90percent, 103percent, 101percent, 110percent, 130, and 105percent in the presence of DTT, hydrogen peroxides, acetonitrile, triton X-100, tween-80 and SDS, respectively. Similarly, catalytic efficiency of GNFx keratinase was promoted in the presence of hydrogen peroxides (119percent), triton X-100 (140percent), tween-80 (150percent) and SDS (147percent) compared to the control. Furthermore, the keratinases from the both bacterial isolates exhibited catalytic efficiency enhancement and remarkable structural stability in the presence of laundry detergents tested. The findings from the study suggest the application potentials of the isolates for the bioconversion of recalcitrant keratinous wastes into digestible and quality protein hydrolysates. The properties of these microbial keratinases indicate that they may be exploited for various biotechnological and industrial processes especially in the formulation of detergents. , Thesis (MSc) -- Faculty of Science and Agriculture, 2021
- Full Text:
- Date Issued: 2021-02
- Authors: Giwu, Nonkonzo https://orcid.org/0000-0001-9416-7896
- Date: 2021-02
- Subjects: Poultry -- Processing , Proteolytic enzymes
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/22732 , vital:52720
- Description: Chicken feathers are by-products of poultry processing which are generated in large amount because of the global growing demand for poultry meats. They have high contents of crude proteins in the form of keratin which could be valorized into digestible products. Keratinases are classified as a specific collection of proteolytic enzymes that have the ability for the degradation of recalcitrant keratinous substrates. Isolation and characterization of these enzymes from various microbial producers are gaining prominence in recent years due to their industrial and biotechnological application potentials. For this research, the collection of soil samples was done as well as the isolation of bacteria and the screening for keratinolytic activity. 16S rDNA sequencing and phylogenetic analysis were used to identify the isolates with efficient chicken feathers degrading capacity. Optimum conditions for the fermentation prcocess was enhanced for the production of keratinase. The fermentation broth was also analysed for various amino acids of protein, and the biochemical properties of the enzymes were likewise determined. Twenty two (22) bacteria were isolated from the soil samples, and 18 out of the 22 isolates showed proteolytic activity on solid media with diameters of halo zone that ranged from 5 ± 0.71 mm for isolate coded as PSS-03 to 25 ± 1.41 mm for isolate coded as PSS-06. Intact chicken feathers were degraded by proteolytic bacterial isolates in variable degree that ranged from 24percent for PSS-10 and 81percent for DSS-02. Extracellular keratinase production recorded for the isolates ranged from 63.63 ± 4.14 U/mL for PSS-10 to 693.63 ± 62.99 U/mL for DSS-02. Based on 16S rDNA sequence and phylogenetic analysis, the 2 isolates with remarkable keratinolytic activity coded as DSS-02 and PSS-14 were identified as Chryseobacterium proteolyticum FGNn and Pseudomonas aeruginosa GNFx. C. proteolyticum showed the maximum keratinase production of 756.36 U/mL after 72 h of incubation at optimized fermentation conditions which involved initial medium pH (4), incubation temperature (30 oC), inoculum size (2percent; v/v), and chicken feathers (1.5percent; w/v). Similarly, P. aeruginosa optimally produced keratinase (1055.45 U/mL) after 96 h of incubation at optimized fermentation conditions that involved initial medium pH (7-8), incubation temperature (30 oC), inoculum size (5percent; v/v), and chicken feathers (2.5percent; w/v). Furthermore, feather hydrolysate from C. proteolyticum FGNn had relatively higher abundance (>1.5g/100g sample) of arginine (1.85), serine (1.63), glycine (1.9) and lysine (1.62); while P. aeruginosa GNFx feather hydrolysate showed high abundance of arginine, serine, aspartic acid, glutamic acid, glycine, alanine, valine, and leucine with respective concentration of 2.06, 1.67, 2.39, 3.05, 1.87, 1.73, 1.56 and 1.65 (g/100g sample). The results showed that keratinases from the two bacterial isolates were optimally active at pH 8, and temperature of 50 oC for FGNn keratinase and 50-60 oC for GNFx keratinase. The enzymes displayed remarkable pH stability. Keratinase from C. proteolyticum was catalytically inhibited by EDTA and 1,10-phenanthroline but not affected by PMSF; while P. aeruginosa keratinase was not significantly affected by those class of protease inhibitors. Adiitionally, FGNn keratinase demonstrated high residual activity of 90percent, 103percent, 101percent, 110percent, 130, and 105percent in the presence of DTT, hydrogen peroxides, acetonitrile, triton X-100, tween-80 and SDS, respectively. Similarly, catalytic efficiency of GNFx keratinase was promoted in the presence of hydrogen peroxides (119percent), triton X-100 (140percent), tween-80 (150percent) and SDS (147percent) compared to the control. Furthermore, the keratinases from the both bacterial isolates exhibited catalytic efficiency enhancement and remarkable structural stability in the presence of laundry detergents tested. The findings from the study suggest the application potentials of the isolates for the bioconversion of recalcitrant keratinous wastes into digestible and quality protein hydrolysates. The properties of these microbial keratinases indicate that they may be exploited for various biotechnological and industrial processes especially in the formulation of detergents. , Thesis (MSc) -- Faculty of Science and Agriculture, 2021
- Full Text:
- Date Issued: 2021-02
Chicken feather delipidation by lipolytic bacteria isolated from an aquatic environment
- Shiri, Tariro https://orcid.org/0000-0002-0290-9854
- Authors: Shiri, Tariro https://orcid.org/0000-0002-0290-9854
- Date: 2021-02
- Subjects: Keratin
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/21479 , vital:48693
- Description: Keratinous biomass contributes a significant proportion of agro-based wastes in the ecosystem with minimal potentials for valuable product recovery. The generation of huge quantities of chicken feather from poultry processing farms prompts the valorization attempt via diverse avenues. Chicken feathers are a rich source of valuable keratin, yet the overall value chain is rudimentary based on unsustainable recovery techniques involving corrosive chemicals and high energy input processes. Although attempts have been made to extract keratin using microbial techniques successfully, the pre-treatment stage remains dominated by chemical use. Chicken feathers are composed of approximately 91percent keratin, 1percent lipids, and 8percent water. Therefore, lipid removal is a critical step in the valorization process as they contribute to access hindrance of the keratinases and other sulfitolytic systems to keratin. Consequently, the study undertook to explore the environment for lipolytic bacteria capable of degrading chicken feathers' lipid components. Sediment samples were collected for bacteria isolation. The bacteria were evaluated for lipolytic activity, and the potent isolates were identified based on 16S rRNA gene sequencing. The fermentation conditions for the production of extracellular lipases were optimized, and the produced lipases were characterized. Lastly, chicken feather lipids were hydrolysed with lipolytic bacteria. Out of twenty bacteria isolated from the sediment samples, six isolates coded as ACT003, ACT004, ACT010, ACT013, ACT016, and ACT019 showed lipolytic activity on solid media with a respective diameter of 12 mm, 66 mm, 29 mm, 11 mm, 12 mm, and 10 mm. Based on 16S rRNA gene sequencing and phylogenetic analysis, the isolates coded as ACT004 and ACT010 were identified as Bacillus sp. TTs1 and Bacillus sp. TTs2; and the nucleotide sequences were submitted to GenBank (NCBI) with the accession numbers MW556206 and MW556207, respectively. Bacillus sp. TTs1 showed the maximum lipase production of 641.25 U/mL at 72 h, under optimized conditions that included initial pH (5), inoculum size (2percent, v/v), incubation temperature (45 oC), agitation speed (140 rpm), CaCl2 (0.01percent, w/v), yeast extract (1percent, w/v), and tween-80 (10percent, v/v). Similarly, the lipase production by Bacillus sp. TTs2 peaked at 96 h with enzyme activity of 618.8 U/mL in improved fermentation conditions consisting of initial pH (5), inoculum size (2-8percent, v/v), incubation temperature (25 oC), agitation speed (180 rpm), CaCl2 (0.01percent, w/v), yeast extract and peptone (1percent, w/v), and tween-80 (10percent, v/v). The evaluation of chicken feather concentrations on free fatty acid liberation showed that 6-8percent (w/v) chicken feather was adequate with free fatty acids contents of 0.58percent and 0.86percent for Bacillus sp. TTs1 and Bacillus sp. TTs2, respectively. Both isolates' lipases showed remarkable catalytic efficiency at pH and temperature of 7 and 40oC, respectively. The comparative analysis of residual lipids between pre-and post-fermentation indicated a 39.9 ± 7.8percent and 51.2 ± 20.2percent hydrolysis efficiency for Bacillus sp. TTs1 and Bacillus sp. TTs2, respectively. This study's findings indicated the lipolytic potentials of Bacillus spp. and suggest the possibility of a full bio-based approach for chicken feather lipid removal in the valorization of chicken feathers. , Thesis (MSc) -- Faculty of Science and Agriculture, 2021
- Full Text:
- Date Issued: 2021-02
- Authors: Shiri, Tariro https://orcid.org/0000-0002-0290-9854
- Date: 2021-02
- Subjects: Keratin
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/21479 , vital:48693
- Description: Keratinous biomass contributes a significant proportion of agro-based wastes in the ecosystem with minimal potentials for valuable product recovery. The generation of huge quantities of chicken feather from poultry processing farms prompts the valorization attempt via diverse avenues. Chicken feathers are a rich source of valuable keratin, yet the overall value chain is rudimentary based on unsustainable recovery techniques involving corrosive chemicals and high energy input processes. Although attempts have been made to extract keratin using microbial techniques successfully, the pre-treatment stage remains dominated by chemical use. Chicken feathers are composed of approximately 91percent keratin, 1percent lipids, and 8percent water. Therefore, lipid removal is a critical step in the valorization process as they contribute to access hindrance of the keratinases and other sulfitolytic systems to keratin. Consequently, the study undertook to explore the environment for lipolytic bacteria capable of degrading chicken feathers' lipid components. Sediment samples were collected for bacteria isolation. The bacteria were evaluated for lipolytic activity, and the potent isolates were identified based on 16S rRNA gene sequencing. The fermentation conditions for the production of extracellular lipases were optimized, and the produced lipases were characterized. Lastly, chicken feather lipids were hydrolysed with lipolytic bacteria. Out of twenty bacteria isolated from the sediment samples, six isolates coded as ACT003, ACT004, ACT010, ACT013, ACT016, and ACT019 showed lipolytic activity on solid media with a respective diameter of 12 mm, 66 mm, 29 mm, 11 mm, 12 mm, and 10 mm. Based on 16S rRNA gene sequencing and phylogenetic analysis, the isolates coded as ACT004 and ACT010 were identified as Bacillus sp. TTs1 and Bacillus sp. TTs2; and the nucleotide sequences were submitted to GenBank (NCBI) with the accession numbers MW556206 and MW556207, respectively. Bacillus sp. TTs1 showed the maximum lipase production of 641.25 U/mL at 72 h, under optimized conditions that included initial pH (5), inoculum size (2percent, v/v), incubation temperature (45 oC), agitation speed (140 rpm), CaCl2 (0.01percent, w/v), yeast extract (1percent, w/v), and tween-80 (10percent, v/v). Similarly, the lipase production by Bacillus sp. TTs2 peaked at 96 h with enzyme activity of 618.8 U/mL in improved fermentation conditions consisting of initial pH (5), inoculum size (2-8percent, v/v), incubation temperature (25 oC), agitation speed (180 rpm), CaCl2 (0.01percent, w/v), yeast extract and peptone (1percent, w/v), and tween-80 (10percent, v/v). The evaluation of chicken feather concentrations on free fatty acid liberation showed that 6-8percent (w/v) chicken feather was adequate with free fatty acids contents of 0.58percent and 0.86percent for Bacillus sp. TTs1 and Bacillus sp. TTs2, respectively. Both isolates' lipases showed remarkable catalytic efficiency at pH and temperature of 7 and 40oC, respectively. The comparative analysis of residual lipids between pre-and post-fermentation indicated a 39.9 ± 7.8percent and 51.2 ± 20.2percent hydrolysis efficiency for Bacillus sp. TTs1 and Bacillus sp. TTs2, respectively. This study's findings indicated the lipolytic potentials of Bacillus spp. and suggest the possibility of a full bio-based approach for chicken feather lipid removal in the valorization of chicken feathers. , Thesis (MSc) -- Faculty of Science and Agriculture, 2021
- Full Text:
- Date Issued: 2021-02
Keratinous poultry wastes valorization through novel keratinases of Chryseobacterium cucumeris and Sphingobacterium multivorum isolated from poultry sludge
- Hendrick, Qaphela https://orcid.org/0000-0001-7529-8129
- Authors: Hendrick, Qaphela https://orcid.org/0000-0001-7529-8129
- Date: 2021-02
- Subjects: Agricultural wastes , Factory and trade waste
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/21400 , vital:48537
- Description: Annually, about 55percent 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 29percent for PSW-11 to 84percent 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 (4percent, v/v), chicken feather concentration (1percent, w/v), fermentation temperature (25o C). Similarly, S. multivorum HNFx showed optimal activity at pH 4.0, inoculum size (5percent, v/v), chicken feather concentration (2.5percent, 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 108percent, 102percent, 114percent, and 104percent. 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 (124percent), hydrogen peroxide (152percent), DMSO (161percent), triton X-100 (152percent), tween-80 (101percent), and metal ions; Fe2+ (128percent), Fe3+ (104percent), K+ (117percent), Ca2+ (104percent), Na+ (103percent), Ba2+ (115percent), Al3+ (126percent). 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) -- Faculty of Science and Agriculture, 2021
- Full Text:
- Date Issued: 2021-02
- Authors: Hendrick, Qaphela https://orcid.org/0000-0001-7529-8129
- Date: 2021-02
- Subjects: Agricultural wastes , Factory and trade waste
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/21400 , vital:48537
- Description: Annually, about 55percent 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 29percent for PSW-11 to 84percent 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 (4percent, v/v), chicken feather concentration (1percent, w/v), fermentation temperature (25o C). Similarly, S. multivorum HNFx showed optimal activity at pH 4.0, inoculum size (5percent, v/v), chicken feather concentration (2.5percent, 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 108percent, 102percent, 114percent, and 104percent. 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 (124percent), hydrogen peroxide (152percent), DMSO (161percent), triton X-100 (152percent), tween-80 (101percent), and metal ions; Fe2+ (128percent), Fe3+ (104percent), K+ (117percent), Ca2+ (104percent), Na+ (103percent), Ba2+ (115percent), Al3+ (126percent). 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) -- Faculty of Science and Agriculture, 2021
- Full Text:
- Date Issued: 2021-02
Waste keratinous biomass valorization and characterization of keratinases produced by exiguobacteria species
- Authors: Dlume, Tutuka
- Date: 2021-02
- Subjects: Factory and trade waste -- Biodegradation , Bioremediation
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/20695 , vital:46438
- Description: Keratinous wastes are emanating in a million tons, as by-products, from various agro-industrial processing plants. Consequently, they create a serious solid waste problem in the environment due to poor handling. Microbial keratinases are proteolytic enzymes that effectively participate in keratin-rich biomass hydrolyses such as feathers, nail, hair, hooves, and horns. Therefore, proper management of these wastes via recycling into useful products is ecologically imperative. Biodegradation of keratin-rich biomass has been identified as an economical and environmentally friendly way of transforming these recalcitrant agro wastes into useful products, hence the motivation for this study. Feather degrading bacterial strains previously isolated from a municipal dumpsite and coded as SSB-02 and SSB-03 was identified through 16S rDNA sequencing and phylogenetic analysis. The fermentation conditions for keratinase production were optimized. The protein and amino acids constituents of the hydrolyzed chicken feather were analyzed. The biochemical properties of the keratinase produced were determined. Also, the effect of laundry detergents on the stability of the keratinase was studied. The isolates coded as SSB-02 and SSB-03 showed a high percentage of sequence homology with Exguobacterium spp., hence they were identified as Exiguobacterium sp. FBH5 and Exiguobacterium acetylicum FHBD, respectively. Exiguobacterium sp. FBH5 showed the highest extracellular keratinase production of 934.58 ± 27.27 U/mL at 72 h of incubation; in optimized fermentation conditions that included pH (5.0), temperature (30 oC), and chicken feather (0.5percent, w/v). Similarly, E. acetylicum FHBD displayed optimal keratinase production of 1023.64 ± 25.71 U/mL at 120 h of fermentation and improved fermentation conditions that involved pH (3.0), temperature (35 oC) and chicken feathers (0.5-1.5percent; w/v). The amino acid analysis showed that arginine, aspartic acid and glutamic acid were the most abundant amino acids cleaved from the degradation of chicken feathers by Exiguobacterium sp. FBH5 at a respective concentration of 1.16, 1.28 and 1.45 (g/100g sample). Additionally, hydrolysate that emanated from E. indicum FHBD degradation of feather showed high concentrations of arginine, serine, aspartic acid, glutamic acid, and glycine at the respective concentration (g/100g sample) of 1.2, 1.12, 1.34, 1.58 and 1.29. The keratinases were optimally active at pH and temperature of 8.0 and 50 oC, respectively. EDTA and PMSF did not highly repress keratinolytic proteases' activity, and this inhibitory pattern suggests that they may belong to a mixed protease family. Keratinase from E. acetylicum FHBD was highly stable in the presence of SDS, with 99percent residual activity and displayed variable stability in other chemical agents tested. A similar stability pattern was observed with keratinase from Exiguobacterium sp. FBH5, although the enzyme lost about 40percent of its original activity in the presence of SDS. Evaluation of metal ion stability indicated that E. acetylicum FHBD keratinase was remarkably stable in the presence of Fe3+, Mg2+, Cu2+, Zn2+, and Ba2+, with residual activity of 94percent, 88percent, 89percent, 90percent, and 97percent, respectively. Similarly, Exiguobacterium sp. FBH5 keratinase was considerably stable after treatment with Co2+, K+, and Zn2+ as it displayed a residual activity of 85percent, 84percent and 93percent, respectively. The study of the keratinases stability in laundry detergents showed that E. acetylicum FHBD keratinolytic proteases was activated in the presence of Omo, Surf, Sunlight, and Pro wash after 60 min of pre-incubation compared to 30 min, with residual activity of 94 ± 2.94percent, 91 ± 2.53percent, 95 ± 2.89percent and 87 ± 2.89percent respectively. Likewise, Exiguobacterium sp. FBH5 keratinase activity was promoted after 60 min of incubation compared to 30 min, with a residual enzyme activity of 79percent, 84percent, 101percent, 103percent and 105percent and 106percent for Ariel, Surf, Prowash, Freewave, Sky and Evaklin, respectively. Therefore Exiguobacterium spp., demonstrated excellent keratinolytic potentials that could be exploited for sustainable development of bio-innovative products. The study keratinases' properties suggest their industrial and biotechnological application potentials, especially as bio-additive in the formulation of laundry detergents. , Thesis (MSc) -- Faculty of Science and Agriculture, 2021
- Full Text:
- Date Issued: 2021-02
- Authors: Dlume, Tutuka
- Date: 2021-02
- Subjects: Factory and trade waste -- Biodegradation , Bioremediation
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/20695 , vital:46438
- Description: Keratinous wastes are emanating in a million tons, as by-products, from various agro-industrial processing plants. Consequently, they create a serious solid waste problem in the environment due to poor handling. Microbial keratinases are proteolytic enzymes that effectively participate in keratin-rich biomass hydrolyses such as feathers, nail, hair, hooves, and horns. Therefore, proper management of these wastes via recycling into useful products is ecologically imperative. Biodegradation of keratin-rich biomass has been identified as an economical and environmentally friendly way of transforming these recalcitrant agro wastes into useful products, hence the motivation for this study. Feather degrading bacterial strains previously isolated from a municipal dumpsite and coded as SSB-02 and SSB-03 was identified through 16S rDNA sequencing and phylogenetic analysis. The fermentation conditions for keratinase production were optimized. The protein and amino acids constituents of the hydrolyzed chicken feather were analyzed. The biochemical properties of the keratinase produced were determined. Also, the effect of laundry detergents on the stability of the keratinase was studied. The isolates coded as SSB-02 and SSB-03 showed a high percentage of sequence homology with Exguobacterium spp., hence they were identified as Exiguobacterium sp. FBH5 and Exiguobacterium acetylicum FHBD, respectively. Exiguobacterium sp. FBH5 showed the highest extracellular keratinase production of 934.58 ± 27.27 U/mL at 72 h of incubation; in optimized fermentation conditions that included pH (5.0), temperature (30 oC), and chicken feather (0.5percent, w/v). Similarly, E. acetylicum FHBD displayed optimal keratinase production of 1023.64 ± 25.71 U/mL at 120 h of fermentation and improved fermentation conditions that involved pH (3.0), temperature (35 oC) and chicken feathers (0.5-1.5percent; w/v). The amino acid analysis showed that arginine, aspartic acid and glutamic acid were the most abundant amino acids cleaved from the degradation of chicken feathers by Exiguobacterium sp. FBH5 at a respective concentration of 1.16, 1.28 and 1.45 (g/100g sample). Additionally, hydrolysate that emanated from E. indicum FHBD degradation of feather showed high concentrations of arginine, serine, aspartic acid, glutamic acid, and glycine at the respective concentration (g/100g sample) of 1.2, 1.12, 1.34, 1.58 and 1.29. The keratinases were optimally active at pH and temperature of 8.0 and 50 oC, respectively. EDTA and PMSF did not highly repress keratinolytic proteases' activity, and this inhibitory pattern suggests that they may belong to a mixed protease family. Keratinase from E. acetylicum FHBD was highly stable in the presence of SDS, with 99percent residual activity and displayed variable stability in other chemical agents tested. A similar stability pattern was observed with keratinase from Exiguobacterium sp. FBH5, although the enzyme lost about 40percent of its original activity in the presence of SDS. Evaluation of metal ion stability indicated that E. acetylicum FHBD keratinase was remarkably stable in the presence of Fe3+, Mg2+, Cu2+, Zn2+, and Ba2+, with residual activity of 94percent, 88percent, 89percent, 90percent, and 97percent, respectively. Similarly, Exiguobacterium sp. FBH5 keratinase was considerably stable after treatment with Co2+, K+, and Zn2+ as it displayed a residual activity of 85percent, 84percent and 93percent, respectively. The study of the keratinases stability in laundry detergents showed that E. acetylicum FHBD keratinolytic proteases was activated in the presence of Omo, Surf, Sunlight, and Pro wash after 60 min of pre-incubation compared to 30 min, with residual activity of 94 ± 2.94percent, 91 ± 2.53percent, 95 ± 2.89percent and 87 ± 2.89percent respectively. Likewise, Exiguobacterium sp. FBH5 keratinase activity was promoted after 60 min of incubation compared to 30 min, with a residual enzyme activity of 79percent, 84percent, 101percent, 103percent and 105percent and 106percent for Ariel, Surf, Prowash, Freewave, Sky and Evaklin, respectively. Therefore Exiguobacterium spp., demonstrated excellent keratinolytic potentials that could be exploited for sustainable development of bio-innovative products. The study keratinases' properties suggest their industrial and biotechnological application potentials, especially as bio-additive in the formulation of laundry detergents. , Thesis (MSc) -- Faculty of Science and Agriculture, 2021
- Full Text:
- Date Issued: 2021-02
Assessment of some bacteria species isolated from woodlands of Raymond Mhlaba Local Municipality for high activity laccase production
- Authors: Gogotya, Asemahle
- Date: 2019
- Subjects: Laccase
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: http://hdl.handle.net/10353/19098 , vital:39879
- Description: The function of enzymes in keeping the earth clean is enormous; being executed in the biodegradation of different natural pollutants and biocatalysis of different responses by substituting the ecologically risky and harmful concoction impetuses offering a situation inviting option, laccases is an example of an enzyme described as it doesn’t produce harmful byproducts. Laccases are employed in several industrial processes that play a key responsibility in transformation of life and making the environment a better place. Bacteria have been described as best producers of laccases with a potential in the industrial scale processes. Laccase was produced from different bacterial isolates identified and named as Bacillus sp. strain GFN1 isolated from soil sample, Bacillus sp. strain GLN and Streptomyces sp. strain LAO both isolated from decaying wood samples in Raymond Mhlaba local municipality with accession numbers MK290988 to MK290990 respectively, as identified by partial sequencing, these were the best producers some of which were positive for Napthol and guaiacol; even upon quantitative screening they were better laccase producers. For quantitative laccase screening, 2,2'-azino-bis(3-ethylbenzothiazoline-6- sulphonic acid) was utilised as the substrate for laccase assays. These laccase producing bacteria were subjected to optimization of growth conditions using submerged fermentation which increased the activity of the produced laccase in great amounts. In optimizing basal medium growth conditions where laccase was harvested after every 72 hours where optimal activity was 16 obtained, studying several factors such as pH which turned out to be pH 5 for all isolates, effect of supplemented carbon and nitrogen sources with the be best being lactose and urea respectively with their effective concentrations using lignin as the main carbon and nitrogen source. Copper sulfate was used as the main inducer and the species preferred guaiacol and ferullic acid and the Fe2+ asthe best supplemented metal ion. The time course was done investigating parameters such as cell growth which was determined by observing the optical density, laccase activity, protein concentration and pH and the presented results suggested that when there was an increase in cell growth, enzyme activity decreased pH had no much effect on the enzyme production as it was almost stable all the time with protein concentration exhibiting no direct effect on enzyme activity also. Characterization of the crude enzyme was done to check the stability of the enzyme produced in various parameters. The enzymes produced by the different strains were thermophilic as they were able to withstand elevated temperatures between 90 and 100 C, with pH stability within an extensive variety of alkaline pH, typical of most bacterial laccases. Various metal ions affected the stability of the enzyme with CuSO4 increasing the stability of two of the bacterial enzyme and appeared to slightly decrease the stability of one enzyme. The studied inhibitors only decreased the stability on the enzyme and couldn’t completely inhibit the enzyme, and the enzymes showed specificity towards varying substrates. The studied bacterial laccases exhibit tremendous characteristics which are of great significance in the industries and will add to the novelty of bacterial laccases and their stability amongst the most studied fungal laccases.
- Full Text:
- Date Issued: 2019
- Authors: Gogotya, Asemahle
- Date: 2019
- Subjects: Laccase
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: http://hdl.handle.net/10353/19098 , vital:39879
- Description: The function of enzymes in keeping the earth clean is enormous; being executed in the biodegradation of different natural pollutants and biocatalysis of different responses by substituting the ecologically risky and harmful concoction impetuses offering a situation inviting option, laccases is an example of an enzyme described as it doesn’t produce harmful byproducts. Laccases are employed in several industrial processes that play a key responsibility in transformation of life and making the environment a better place. Bacteria have been described as best producers of laccases with a potential in the industrial scale processes. Laccase was produced from different bacterial isolates identified and named as Bacillus sp. strain GFN1 isolated from soil sample, Bacillus sp. strain GLN and Streptomyces sp. strain LAO both isolated from decaying wood samples in Raymond Mhlaba local municipality with accession numbers MK290988 to MK290990 respectively, as identified by partial sequencing, these were the best producers some of which were positive for Napthol and guaiacol; even upon quantitative screening they were better laccase producers. For quantitative laccase screening, 2,2'-azino-bis(3-ethylbenzothiazoline-6- sulphonic acid) was utilised as the substrate for laccase assays. These laccase producing bacteria were subjected to optimization of growth conditions using submerged fermentation which increased the activity of the produced laccase in great amounts. In optimizing basal medium growth conditions where laccase was harvested after every 72 hours where optimal activity was 16 obtained, studying several factors such as pH which turned out to be pH 5 for all isolates, effect of supplemented carbon and nitrogen sources with the be best being lactose and urea respectively with their effective concentrations using lignin as the main carbon and nitrogen source. Copper sulfate was used as the main inducer and the species preferred guaiacol and ferullic acid and the Fe2+ asthe best supplemented metal ion. The time course was done investigating parameters such as cell growth which was determined by observing the optical density, laccase activity, protein concentration and pH and the presented results suggested that when there was an increase in cell growth, enzyme activity decreased pH had no much effect on the enzyme production as it was almost stable all the time with protein concentration exhibiting no direct effect on enzyme activity also. Characterization of the crude enzyme was done to check the stability of the enzyme produced in various parameters. The enzymes produced by the different strains were thermophilic as they were able to withstand elevated temperatures between 90 and 100 C, with pH stability within an extensive variety of alkaline pH, typical of most bacterial laccases. Various metal ions affected the stability of the enzyme with CuSO4 increasing the stability of two of the bacterial enzyme and appeared to slightly decrease the stability of one enzyme. The studied inhibitors only decreased the stability on the enzyme and couldn’t completely inhibit the enzyme, and the enzymes showed specificity towards varying substrates. The studied bacterial laccases exhibit tremendous characteristics which are of great significance in the industries and will add to the novelty of bacterial laccases and their stability amongst the most studied fungal laccases.
- Full Text:
- Date Issued: 2019
Distribution of virulence determinants in some members of the ESKAPE pathogens isolated from selected hospital wastewater effluents in the Eastern Cape, South Africa
- Authors: Mapipa, Qaqamba
- Date: 2019
- Subjects: Effluent quality Water -- Purification
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: http://hdl.handle.net/10353/14704 , vital:40061
- Description: Members of ESKAPE pathogens are grouped under the acronymn made up of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. ESKAPE pathogens, isolated from hospitals and communities, have just been characterized for new pathogenesis and transmission patterns and overcoming the usefulness of available drugs, this leads to infections that tend to increase the rate of disease in a population and death rate and cost our society massively. The distribution of virulence determinants and the antibiotic susceptibility patterns in some members of the ESKAPE pathogens isolated from hospital wastewater facilities that were selected within the Eastern Cape Province, South Africa was evaluated. Water samples from the hospital wastewater facilities were collected fortnightly over a period of three (3) months (February to April). Spread plate technique was employed for the enumeration and isolation of the members of ESKAPE pathogens. A total of 725 presumptive ESKAPE isolates were collected, screened and Polymerase Chain Reaction (PCR) amplification of target determinants was performed. Results showed that Klebsiella pneumoniae tested positive for ninety two (92=35.9%) samples, Enterobacter cloacae fifty seven (57=22.2%) samples tested positive, Pseudomonas aeruginosa fifty four (54=21.09%) isolates tested positive and fifty three (53=20.7%) isolates tested positive for Acinetobacter baumannii making a total of 256 isolates which tested positive for some members of ESKAPE pathogens. The detection of some members of the ESKAPE pathogens in these Hospitals is a cause of concern to public health. A total of 256 ESKAPE isolates that were obtained from the hospital wastewater facilities; were subjected to antibiotic susceptibility test in order to determine their antibiotic-resistant profiles. ESKAPE isolates recovered from the three hospital effluents showing resistance to antimicrobial agents in this study were further evaluated for resistance determinants namely, sulfonamides resistance determinants (sulI and sulII), β-lactams (ampC, blaTEM and blaZ), tetracyclines (tetA, tetB, tetC and tetM), 10 chloramphenicol’s (catI and catII) and aminoglycoside resistance determinants (aacC2, apHAI and apHAII). The resistant determinants targeted have been evaluated through their prevalence and the following distributions were obtained: [β-Lactams; ampC (80%), blaTEM (77%)]; [Tetracyclines; tetA (87%)]; [Sulfonamides; sulI (79%)]; [Aminoglycosides; aacC2 (12%), aphA2 (52%) and aadA (18%)]. With the worldwide increase in these resistant occurrences, the execution of strict infection control policies and treatment with different antimicrobials needs hasty detection. Comprehensive data are therefore needed so that the history of infection, identification of genes present in organisms and monitoring of changes in infection patterns can be traced correctly.
- Full Text:
- Date Issued: 2019
- Authors: Mapipa, Qaqamba
- Date: 2019
- Subjects: Effluent quality Water -- Purification
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: http://hdl.handle.net/10353/14704 , vital:40061
- Description: Members of ESKAPE pathogens are grouped under the acronymn made up of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. ESKAPE pathogens, isolated from hospitals and communities, have just been characterized for new pathogenesis and transmission patterns and overcoming the usefulness of available drugs, this leads to infections that tend to increase the rate of disease in a population and death rate and cost our society massively. The distribution of virulence determinants and the antibiotic susceptibility patterns in some members of the ESKAPE pathogens isolated from hospital wastewater facilities that were selected within the Eastern Cape Province, South Africa was evaluated. Water samples from the hospital wastewater facilities were collected fortnightly over a period of three (3) months (February to April). Spread plate technique was employed for the enumeration and isolation of the members of ESKAPE pathogens. A total of 725 presumptive ESKAPE isolates were collected, screened and Polymerase Chain Reaction (PCR) amplification of target determinants was performed. Results showed that Klebsiella pneumoniae tested positive for ninety two (92=35.9%) samples, Enterobacter cloacae fifty seven (57=22.2%) samples tested positive, Pseudomonas aeruginosa fifty four (54=21.09%) isolates tested positive and fifty three (53=20.7%) isolates tested positive for Acinetobacter baumannii making a total of 256 isolates which tested positive for some members of ESKAPE pathogens. The detection of some members of the ESKAPE pathogens in these Hospitals is a cause of concern to public health. A total of 256 ESKAPE isolates that were obtained from the hospital wastewater facilities; were subjected to antibiotic susceptibility test in order to determine their antibiotic-resistant profiles. ESKAPE isolates recovered from the three hospital effluents showing resistance to antimicrobial agents in this study were further evaluated for resistance determinants namely, sulfonamides resistance determinants (sulI and sulII), β-lactams (ampC, blaTEM and blaZ), tetracyclines (tetA, tetB, tetC and tetM), 10 chloramphenicol’s (catI and catII) and aminoglycoside resistance determinants (aacC2, apHAI and apHAII). The resistant determinants targeted have been evaluated through their prevalence and the following distributions were obtained: [β-Lactams; ampC (80%), blaTEM (77%)]; [Tetracyclines; tetA (87%)]; [Sulfonamides; sulI (79%)]; [Aminoglycosides; aacC2 (12%), aphA2 (52%) and aadA (18%)]. With the worldwide increase in these resistant occurrences, the execution of strict infection control policies and treatment with different antimicrobials needs hasty detection. Comprehensive data are therefore needed so that the history of infection, identification of genes present in organisms and monitoring of changes in infection patterns can be traced correctly.
- Full Text:
- Date Issued: 2019
Evaluation of some Eastern beach bacteria for high activity laccase production
- Authors: Ntlatywa, Nonkolisi
- Date: 2019
- Subjects: Laccase
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/${Handle} , vital:40025
- Description: Laccases are multi-copper oxidases endowed with huge applicability and versatility. They are found in nature, fungi, insects, archaea and bacteria; however, there is a dearth of information regarding bacterial laccases. Thus, the search for novel bacterial laccases becomes crucial. Therefore, the broad aim of the study was the assessment of some bacteria on marine water isolated from Eastern beach for the production of high activity laccases. In this study, marine water samples obtained from the Eastern beach in East London were investigated for bacterial laccase-producing isolates. The samples were analysed using different microbiological media and varied substrates via qualitative methods. The isolates were tested for laccase activity using ABTS (2, 2’- azinobis 3-ethyl-benzothiazoline-6-sulphonate), as a substrate. The present study also dealt with the optimization of culture conditions for laccase production and characterization of laccase. A total of forty-eight (48) bacterial isolates were obtained, which were identified based on their morphological and cultural characteristics. Many of the isolates (42) were categorized as Gram positive and a few (6) as Gram negative upon subjecting to microscopic examination. Result showed that on average, laccase activity ranged from 5.33 – 6.5 U/L as demonstrated by three bacterial isolates (Bacillus sp. NFN1, Bacillus sp. NLN and Escherichia coli LO). Laccase production was highly enhanced by the supplementation with vanillic acid by Bacillus sp. strain NLN with yield of 15.83 ± 0.7 U/L, Bacillus sp. strain NFN1, 12.72 ± 0.21 U/L. However, Escherichia coli LO demonstrated enhanced laccase production with guaiacol (12.84 ± 1.12 U/L) followed by ferrulic acid (12.8 ± 0.36 U/L). While optimization results revealed that the laccase yield was enhanced under the following conditions: 72 h of incubation, pH 5, 0.5mg/L lactose as a carbon source, 1. 5 mg/L yeast extract as a nitrogen source as well as in the presence of corn stover and mandarin peels as lignocellulosic substrates depending on the bacteria.The time course and stability of laccase against temperature, pH, metal ions and organic solvents were investigated using previously described methods. In the findings obtained for the time course, the laccase activity ranged from 22.2- 23.98 U/L. In general, the optimum parameters for the optimum laccase activities depended on the bacterial isolates and the time of incubation and included 80 °C and 70 °C, pH 3 and 9 while, the thermal stability was over 100%. Overall, NaCl, ZnSO4, NiCl and 20% acetone demonstrated positive effect on the residual activity of the laccases of all the bacterial isolates (Bacillus sp. NFN1, Bacillus v sp. NLN and Escherichia coli LO). In conclusion the findings of the study, suggest that marine water of the Eastern beach is a rich source for laccase producing bacteria with potential of high activity.
- Full Text:
- Date Issued: 2019
- Authors: Ntlatywa, Nonkolisi
- Date: 2019
- Subjects: Laccase
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/${Handle} , vital:40025
- Description: Laccases are multi-copper oxidases endowed with huge applicability and versatility. They are found in nature, fungi, insects, archaea and bacteria; however, there is a dearth of information regarding bacterial laccases. Thus, the search for novel bacterial laccases becomes crucial. Therefore, the broad aim of the study was the assessment of some bacteria on marine water isolated from Eastern beach for the production of high activity laccases. In this study, marine water samples obtained from the Eastern beach in East London were investigated for bacterial laccase-producing isolates. The samples were analysed using different microbiological media and varied substrates via qualitative methods. The isolates were tested for laccase activity using ABTS (2, 2’- azinobis 3-ethyl-benzothiazoline-6-sulphonate), as a substrate. The present study also dealt with the optimization of culture conditions for laccase production and characterization of laccase. A total of forty-eight (48) bacterial isolates were obtained, which were identified based on their morphological and cultural characteristics. Many of the isolates (42) were categorized as Gram positive and a few (6) as Gram negative upon subjecting to microscopic examination. Result showed that on average, laccase activity ranged from 5.33 – 6.5 U/L as demonstrated by three bacterial isolates (Bacillus sp. NFN1, Bacillus sp. NLN and Escherichia coli LO). Laccase production was highly enhanced by the supplementation with vanillic acid by Bacillus sp. strain NLN with yield of 15.83 ± 0.7 U/L, Bacillus sp. strain NFN1, 12.72 ± 0.21 U/L. However, Escherichia coli LO demonstrated enhanced laccase production with guaiacol (12.84 ± 1.12 U/L) followed by ferrulic acid (12.8 ± 0.36 U/L). While optimization results revealed that the laccase yield was enhanced under the following conditions: 72 h of incubation, pH 5, 0.5mg/L lactose as a carbon source, 1. 5 mg/L yeast extract as a nitrogen source as well as in the presence of corn stover and mandarin peels as lignocellulosic substrates depending on the bacteria.The time course and stability of laccase against temperature, pH, metal ions and organic solvents were investigated using previously described methods. In the findings obtained for the time course, the laccase activity ranged from 22.2- 23.98 U/L. In general, the optimum parameters for the optimum laccase activities depended on the bacterial isolates and the time of incubation and included 80 °C and 70 °C, pH 3 and 9 while, the thermal stability was over 100%. Overall, NaCl, ZnSO4, NiCl and 20% acetone demonstrated positive effect on the residual activity of the laccases of all the bacterial isolates (Bacillus sp. NFN1, Bacillus v sp. NLN and Escherichia coli LO). In conclusion the findings of the study, suggest that marine water of the Eastern beach is a rich source for laccase producing bacteria with potential of high activity.
- Full Text:
- Date Issued: 2019
Distribution of virulence determinants in Plesiomonas shigelloides and Vibrio species isolated from selected hospital wastewater effluents in the Eastern Cape Province, South Africa
- Authors: Mashudu, Mavhungu
- Date: 2018
- Subjects: Vibrio
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: http://hdl.handle.net/10353/14505 , vital:39996
- Description: Plesiomonas shigelloides and some Vibrio species are amongst the bacteria species classified into the emerging pathogenic bacteria grouping, and they portend increasing virulence and high resistance to some of the most potent antimicrobial agents used as most important and critical lines of defence against pathogenic infectious agents. Hospital wastewater has attracted significant attention as a pool for the regurgitation of pathogens into the environment which eventually becomes a problem to public health. It has been variously reported that hospital wastewater critically serves also as a puddle for microbial genetic interaction with outcomes including the development of resistance to antimicrobial agents among other pathogenic traits. And, in the same vein, exchanges and gene recombination may have been leading to the emergence of virulence determinants not previously known owing to the capacity of hospital wastewater to encompass a vast diversity of pathogenic bacteria and antimicrobial agents at the same material time. Hence, this research aimed at the evaluation of the prevalence of virulence and antibiotic resistance in Plesiomonas shigelloides and some human pathogenic Vibrio species isolated from selected hospital wastewater in the Eastern Cape Province of South Africa. Wastewater samples were collected at two weeks intervals throughout three months sampling duration from hospital wastewater facility in the Eastern Cape Province. The wastewater samples were from the secondary hospital and, tertiary hospital along with Limbede community wastewater facilities based in the Eastern Cape Province of South Africa. The wastewater samples were aseptically screened for the presence of presumptive Plesiomonas shigelloides and selected Vibrio species using the culture-based method. The identity of the presumptive Plesiomonas shigelloides and Vibrio species (Vibrio cholerae, V. fluvialis, V. parahaemolyticus, and V. vulnificus) isolates were confirmed using species specific primers. After that, the presence 14 of virulence determinants and the antimicrobial susceptibility profiles of the positive strains as well as the antimicrobial resistance factors were all determined. The distribution of the presumptive isolates was as follows; 322 Plesiomans shigelloides and 378 Vibrio species respectively. However, upon molecular confirmation studies, 85 out of 322; 26% of the total presumptive isolates, were confirmed as positive for Plesiomans shigelloides and, on the other hand, from the 378 presumptive Vibrio species, 71% (270) was as positive to Vibrio genus, and upon speciation of the positive isolates; 27% (73) was confirmed as Vibrio cholerae, 9% (24) as Vibrio parahaemolyticus, 4% (12) as Vibrio vulnificus and 3% (8) as Vibrio fluvialis. The remaining 57% (153) which was unaccounted for may belong to other Vibrio species not included in this studies. Various virulence determinants were shown to be present in the Vibrio species identified and the degree varied in line with the species. P. shigelloides showed high levels of resistance to impregnated antibiotics (18 different types). The antibiogram characteristics showed P. shigelloides to possess 100% resistance to Ciprofloxacin, Nalidixic acid, Trimethoprim, Ampicillin, Penicillin, Tetracycline, Doxycycline, Erythromycin, and Cephalothin. These findings are of crucial importance to public health and the science of infectology as it gives insight into the role of the hospital wastewater as a puddle for the recombination of antibiotic resistance determinants and virulence emergence in bacteria. On the strength of the finding of this research, it is essential to indicate that there is a need for an immediate review of hospital wastewater disposal process. Some form of treatment or pretreatment of the hospital wastewater is required before discharge into municipal wastewater treatment facilities. And, perhaps, continuous monitoring of hospital wastewater to reduce the risk posed to public health.
- Full Text:
- Date Issued: 2018
- Authors: Mashudu, Mavhungu
- Date: 2018
- Subjects: Vibrio
- Language: English
- Type: Thesis , Masters , MSc (Microbiology)
- Identifier: http://hdl.handle.net/10353/14505 , vital:39996
- Description: Plesiomonas shigelloides and some Vibrio species are amongst the bacteria species classified into the emerging pathogenic bacteria grouping, and they portend increasing virulence and high resistance to some of the most potent antimicrobial agents used as most important and critical lines of defence against pathogenic infectious agents. Hospital wastewater has attracted significant attention as a pool for the regurgitation of pathogens into the environment which eventually becomes a problem to public health. It has been variously reported that hospital wastewater critically serves also as a puddle for microbial genetic interaction with outcomes including the development of resistance to antimicrobial agents among other pathogenic traits. And, in the same vein, exchanges and gene recombination may have been leading to the emergence of virulence determinants not previously known owing to the capacity of hospital wastewater to encompass a vast diversity of pathogenic bacteria and antimicrobial agents at the same material time. Hence, this research aimed at the evaluation of the prevalence of virulence and antibiotic resistance in Plesiomonas shigelloides and some human pathogenic Vibrio species isolated from selected hospital wastewater in the Eastern Cape Province of South Africa. Wastewater samples were collected at two weeks intervals throughout three months sampling duration from hospital wastewater facility in the Eastern Cape Province. The wastewater samples were from the secondary hospital and, tertiary hospital along with Limbede community wastewater facilities based in the Eastern Cape Province of South Africa. The wastewater samples were aseptically screened for the presence of presumptive Plesiomonas shigelloides and selected Vibrio species using the culture-based method. The identity of the presumptive Plesiomonas shigelloides and Vibrio species (Vibrio cholerae, V. fluvialis, V. parahaemolyticus, and V. vulnificus) isolates were confirmed using species specific primers. After that, the presence 14 of virulence determinants and the antimicrobial susceptibility profiles of the positive strains as well as the antimicrobial resistance factors were all determined. The distribution of the presumptive isolates was as follows; 322 Plesiomans shigelloides and 378 Vibrio species respectively. However, upon molecular confirmation studies, 85 out of 322; 26% of the total presumptive isolates, were confirmed as positive for Plesiomans shigelloides and, on the other hand, from the 378 presumptive Vibrio species, 71% (270) was as positive to Vibrio genus, and upon speciation of the positive isolates; 27% (73) was confirmed as Vibrio cholerae, 9% (24) as Vibrio parahaemolyticus, 4% (12) as Vibrio vulnificus and 3% (8) as Vibrio fluvialis. The remaining 57% (153) which was unaccounted for may belong to other Vibrio species not included in this studies. Various virulence determinants were shown to be present in the Vibrio species identified and the degree varied in line with the species. P. shigelloides showed high levels of resistance to impregnated antibiotics (18 different types). The antibiogram characteristics showed P. shigelloides to possess 100% resistance to Ciprofloxacin, Nalidixic acid, Trimethoprim, Ampicillin, Penicillin, Tetracycline, Doxycycline, Erythromycin, and Cephalothin. These findings are of crucial importance to public health and the science of infectology as it gives insight into the role of the hospital wastewater as a puddle for the recombination of antibiotic resistance determinants and virulence emergence in bacteria. On the strength of the finding of this research, it is essential to indicate that there is a need for an immediate review of hospital wastewater disposal process. Some form of treatment or pretreatment of the hospital wastewater is required before discharge into municipal wastewater treatment facilities. And, perhaps, continuous monitoring of hospital wastewater to reduce the risk posed to public health.
- Full Text:
- Date Issued: 2018
Characterisation of antibacterial compounds produced by Pseudomonas spp. isolated from Hogsback wetlands, Eastern Cape, South Africa
- Authors: Obi, Akudo Odochi
- Date: 2017
- Subjects: Bacteriology -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10353/15688 , vital:40507
- Description: With microbial pathogens developing resistance against currently existing antimicrobials, there is an earnest need to discover and develop new therapeutic compounds with unique and/or precise modes of action. Natural products have provided the inspiration for most of the active ingredients in medicines and thus are regarded as a potential screening resource lead for discovery of new drugs. Pseudomonads are ubiquitous, Gram-negative, motile bacteria that flourish and inhabit a diversity of environments. Literature has vastly revealed that some members of the genus Pseudomonas are capable of producing a plethora of biologically active metabolites and thus are regarded as “attractive” bacteria for testing as potential sources for new therapeutic compounds. In this regard, this study was undertaken with an aim to isolate and characterize antibacterial compounds produced by Pseudomonas species isolated from aquatic milieu of the Hogsback wetlands, Eastern Cape, South Africa. A total of 12 water samples were aseptically collected from Hogsback wetlands. To ascertain antimicrobial potential of the isolates, 9 test bacterial (sensitive) strains, namely Escherichia coli ATCC 3695, Vibrio fluvialis, Salmonella typhimurium, Salmonella enteritidis, Pseudomonas aeruginosa ATCC 27853, Escherichia coli (2), Listeria ivanovii, Listeria monocytogenes and Enterococcus faecalis ATCC 19433 were used for antimicrobial screening. A total of 500 presumptive Pseudomonas spp. were isolated and screened for the production of antibacterial compound through the Agar-well diffusion method. About 8percent of the presumptive Pseudomonas spp. produced compound with activity against the test bacteria and based on the levels of activity shown, three isolates coded as A53, Y55 and Y138 were selected and identified by 16S rDNA sequence analysis and The Basic Local Alignment Search Tool analysis revealed that the three isolates (A53, Y55 and Y138) belonged to the Pseudomonas genus. Using MEGA6 software, a molecular phylogenetic tree was constructed by Maximum Likelihood Method. The phylogenetic analysis by this method showed that all three isolate codes (A53, Y55 and Y138) formed the same clade with Pseudomonas viridiflava strain P3 (Accession number: AY972186) and Pseudomonas plecoglossicida strain P9 (Accession number: AY972231) at a high bootstrap value of 100percent. Isolate code A53 and Y55 also grouped together at a bootstrap value of 96percent. Factors affecting antibacterial compound production by the three isolates were elucidated. The effect of pH on the three isolates revealed that isolate code A53 and Y55 could produce antibacterial compounds over all pH ranges (pH 4-9), however, antibacterial production was produced optimally at acidic conditions (pH 4) and a less acidic-neutral condition (pH 6) respectively. Isolate Y138 showed optimal production at pH 5. The effect of temperature on all three isolates was studied at different temperatures ranging between 25°C-40°C. Study on effect of temperature variations on antibacterial production revealed that all three isolates are capable of growing and producing antibacterial compounds over all the different temperature ranges, however, maximum/optimum production was observed at temperature of 30°C. In order to establish the role of plasmids in antibacterial production, two curing agents; Sodium dodecyl sulfate (SDS) and Ethidium bromide under different sub-lethal concentrations (1 mg/ml -11mg/ml and 2.5 μg/ml- 125 μg/ml respectively) were used to cure the selected isolates. Plasmid DNA samples of A53, Y55 and Y138, were separated by electrophoresis on a 0.7percent (w/v) agarose. Agarose gel electrophoresis revealed that not all the concentrations used were effective in curing the plasmid. A Quick Load 1 kb Extend DNA ladder marker was used for determining the size of the plasmids. Isolate code A53 and Y138 had a plasmid size of 48.5 kb while isolate Y55 had a plasmid size of approximately 20 kb. Antibacterial activity by agar-well diffusion method after the curing process revealed that antibacterial production was not lost or prevented, but rather increased. Thus, it is suggested that the genes responsible for antibiotic production in this study were chromosomal-encoded and not plasmid-encoded. This study revealed that Hogsback wetlands may represent a previously unexplored environment/source of discovering novel and potent antibacterial compounds.
- Full Text:
- Date Issued: 2017
- Authors: Obi, Akudo Odochi
- Date: 2017
- Subjects: Bacteriology -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10353/15688 , vital:40507
- Description: With microbial pathogens developing resistance against currently existing antimicrobials, there is an earnest need to discover and develop new therapeutic compounds with unique and/or precise modes of action. Natural products have provided the inspiration for most of the active ingredients in medicines and thus are regarded as a potential screening resource lead for discovery of new drugs. Pseudomonads are ubiquitous, Gram-negative, motile bacteria that flourish and inhabit a diversity of environments. Literature has vastly revealed that some members of the genus Pseudomonas are capable of producing a plethora of biologically active metabolites and thus are regarded as “attractive” bacteria for testing as potential sources for new therapeutic compounds. In this regard, this study was undertaken with an aim to isolate and characterize antibacterial compounds produced by Pseudomonas species isolated from aquatic milieu of the Hogsback wetlands, Eastern Cape, South Africa. A total of 12 water samples were aseptically collected from Hogsback wetlands. To ascertain antimicrobial potential of the isolates, 9 test bacterial (sensitive) strains, namely Escherichia coli ATCC 3695, Vibrio fluvialis, Salmonella typhimurium, Salmonella enteritidis, Pseudomonas aeruginosa ATCC 27853, Escherichia coli (2), Listeria ivanovii, Listeria monocytogenes and Enterococcus faecalis ATCC 19433 were used for antimicrobial screening. A total of 500 presumptive Pseudomonas spp. were isolated and screened for the production of antibacterial compound through the Agar-well diffusion method. About 8percent of the presumptive Pseudomonas spp. produced compound with activity against the test bacteria and based on the levels of activity shown, three isolates coded as A53, Y55 and Y138 were selected and identified by 16S rDNA sequence analysis and The Basic Local Alignment Search Tool analysis revealed that the three isolates (A53, Y55 and Y138) belonged to the Pseudomonas genus. Using MEGA6 software, a molecular phylogenetic tree was constructed by Maximum Likelihood Method. The phylogenetic analysis by this method showed that all three isolate codes (A53, Y55 and Y138) formed the same clade with Pseudomonas viridiflava strain P3 (Accession number: AY972186) and Pseudomonas plecoglossicida strain P9 (Accession number: AY972231) at a high bootstrap value of 100percent. Isolate code A53 and Y55 also grouped together at a bootstrap value of 96percent. Factors affecting antibacterial compound production by the three isolates were elucidated. The effect of pH on the three isolates revealed that isolate code A53 and Y55 could produce antibacterial compounds over all pH ranges (pH 4-9), however, antibacterial production was produced optimally at acidic conditions (pH 4) and a less acidic-neutral condition (pH 6) respectively. Isolate Y138 showed optimal production at pH 5. The effect of temperature on all three isolates was studied at different temperatures ranging between 25°C-40°C. Study on effect of temperature variations on antibacterial production revealed that all three isolates are capable of growing and producing antibacterial compounds over all the different temperature ranges, however, maximum/optimum production was observed at temperature of 30°C. In order to establish the role of plasmids in antibacterial production, two curing agents; Sodium dodecyl sulfate (SDS) and Ethidium bromide under different sub-lethal concentrations (1 mg/ml -11mg/ml and 2.5 μg/ml- 125 μg/ml respectively) were used to cure the selected isolates. Plasmid DNA samples of A53, Y55 and Y138, were separated by electrophoresis on a 0.7percent (w/v) agarose. Agarose gel electrophoresis revealed that not all the concentrations used were effective in curing the plasmid. A Quick Load 1 kb Extend DNA ladder marker was used for determining the size of the plasmids. Isolate code A53 and Y138 had a plasmid size of 48.5 kb while isolate Y55 had a plasmid size of approximately 20 kb. Antibacterial activity by agar-well diffusion method after the curing process revealed that antibacterial production was not lost or prevented, but rather increased. Thus, it is suggested that the genes responsible for antibiotic production in this study were chromosomal-encoded and not plasmid-encoded. This study revealed that Hogsback wetlands may represent a previously unexplored environment/source of discovering novel and potent antibacterial compounds.
- Full Text:
- Date Issued: 2017
Evaluation of some pseudomonas species isolated from Hogsback forest reserve for the production of antibacterial compounds
- Authors: Mkono, Yonela Pelokazi
- Date: 2017
- Subjects: Bacteriology -- South Africa -- Eastern Cape Pseudomonas
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10353/5961 , vital:29458
- Description: Pseudomonas species are Gram-negative bacteria most abundant in soil and water bodies, with the capacity to thrive in varied environments. They are largely associated with resistant pathogenic bacteria linked to human and plant diseases. Species such as Pseudomonas aeruginosa have been particularly targeted as case studies due to the extremity to which they pose a threat to human health. With more focus directed at using these species for biocontrol and bioremediation purposes, their role in bioactive compound production may be equally important. As the crisis on antimicrobial resistance still persists, the need for effective antimicrobial compounds is ever more urgent and solutions may possibly still be dormant in bacterial species whose potential has not been fully investigated. On a bid to source out potential antimicrobial compound producers, soil samples were collected from Hogback forest reserve in the province of the Eastern Cape, South Africa. For bacterial screening, M1 and R2A agar were used and the cultures grown at 37˚C for a period of seven days. After the presumed Pseudomonas species were identified, antimicrobial production was determined by submerged fermentation method using nutrient broth as media of choice. Active isolates were further studied to determine the optimum conditions which best facilitate for antimicrobial compound production, with parameters such as temperature (25˚C – 40˚C) and pH (4 – 9) considered. The role plasmids play in antimicrobial compound production was also investigated. Each isolate was grown in fermentation media containing Sodium dodecyl sulphate and Ethedium Bromide, at varying concentrations, to facilitate for plasmid curing. With each sample, distinct colonies were identified with varying pigmentations most dominant being a cream colour. The identity of the isolated strains was achieved through sequencing of 16S rDNA. Phylogenetic analysis showed that isolate A16 had 80 percent homology with Pseudomonas plecoglossicida strain P4 and share a close ancestor with isolates Y52 and Y81, also isolate Y89 showed a 90 percent homology with Pseudomonas sp. Co-11a. With the exception of isolate A16, the isolates which were active against Gram-negative bacteria lost activity as the screening processes continued. When looking at temperature variations, isolates Y81 and A16 were highly active with maximum activity observed at 35˚C while Y89 performed best at 25˚C and Y52 showed constant activity across all studied temperatures. The plasmids in all isolates were found to be 48.5 kb in size with the exception of isolate Y89 which was 20 kb. The plasmids were cured at concentrations of (1 mg/ml; 5 mg/ml; 7 mg/ml; 10 mg/ml; 11 mg/ml) SDS and (125 μg/ml; 6.5 μg/ml; 5μg/ml) EtBr. The curing process also showed changes in both the antimicrobial activity of the isolates as well as their physical characteristics. The isolates are the first reported Pseudomonas species from Hogsback forest reserve with the ability to produce antimicrobial compounds which are active against Gram-positive and Gram-negative bacteria. These mesophilic bacteria also show that plasmids do not pay any role in the production of antimicrobial compounds and that the biosynthesis genes are highly likely to be chromosomal borne meaning that the production cannot be linked to horizontal transfer of genes. Therefore, these isolated Pseudomonas species provide a potential reservoir of antimicrobial compounds which may play an important role in the antimicrobial resistance phenomenon.
- Full Text:
- Date Issued: 2017
- Authors: Mkono, Yonela Pelokazi
- Date: 2017
- Subjects: Bacteriology -- South Africa -- Eastern Cape Pseudomonas
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10353/5961 , vital:29458
- Description: Pseudomonas species are Gram-negative bacteria most abundant in soil and water bodies, with the capacity to thrive in varied environments. They are largely associated with resistant pathogenic bacteria linked to human and plant diseases. Species such as Pseudomonas aeruginosa have been particularly targeted as case studies due to the extremity to which they pose a threat to human health. With more focus directed at using these species for biocontrol and bioremediation purposes, their role in bioactive compound production may be equally important. As the crisis on antimicrobial resistance still persists, the need for effective antimicrobial compounds is ever more urgent and solutions may possibly still be dormant in bacterial species whose potential has not been fully investigated. On a bid to source out potential antimicrobial compound producers, soil samples were collected from Hogback forest reserve in the province of the Eastern Cape, South Africa. For bacterial screening, M1 and R2A agar were used and the cultures grown at 37˚C for a period of seven days. After the presumed Pseudomonas species were identified, antimicrobial production was determined by submerged fermentation method using nutrient broth as media of choice. Active isolates were further studied to determine the optimum conditions which best facilitate for antimicrobial compound production, with parameters such as temperature (25˚C – 40˚C) and pH (4 – 9) considered. The role plasmids play in antimicrobial compound production was also investigated. Each isolate was grown in fermentation media containing Sodium dodecyl sulphate and Ethedium Bromide, at varying concentrations, to facilitate for plasmid curing. With each sample, distinct colonies were identified with varying pigmentations most dominant being a cream colour. The identity of the isolated strains was achieved through sequencing of 16S rDNA. Phylogenetic analysis showed that isolate A16 had 80 percent homology with Pseudomonas plecoglossicida strain P4 and share a close ancestor with isolates Y52 and Y81, also isolate Y89 showed a 90 percent homology with Pseudomonas sp. Co-11a. With the exception of isolate A16, the isolates which were active against Gram-negative bacteria lost activity as the screening processes continued. When looking at temperature variations, isolates Y81 and A16 were highly active with maximum activity observed at 35˚C while Y89 performed best at 25˚C and Y52 showed constant activity across all studied temperatures. The plasmids in all isolates were found to be 48.5 kb in size with the exception of isolate Y89 which was 20 kb. The plasmids were cured at concentrations of (1 mg/ml; 5 mg/ml; 7 mg/ml; 10 mg/ml; 11 mg/ml) SDS and (125 μg/ml; 6.5 μg/ml; 5μg/ml) EtBr. The curing process also showed changes in both the antimicrobial activity of the isolates as well as their physical characteristics. The isolates are the first reported Pseudomonas species from Hogsback forest reserve with the ability to produce antimicrobial compounds which are active against Gram-positive and Gram-negative bacteria. These mesophilic bacteria also show that plasmids do not pay any role in the production of antimicrobial compounds and that the biosynthesis genes are highly likely to be chromosomal borne meaning that the production cannot be linked to horizontal transfer of genes. Therefore, these isolated Pseudomonas species provide a potential reservoir of antimicrobial compounds which may play an important role in the antimicrobial resistance phenomenon.
- Full Text:
- Date Issued: 2017
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