Bioconversion of chicken feather into amino acids and keratinase production by mesophilic Chryseobacterium proteolyticum and Pseudomonas aeruginosa isolated from municipal waste dumpsites
- 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
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Chicken feather delipidation by lipolytic bacteria isolated from an aquatic environment
- 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
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Keratinous poultry wastes valorization through novel keratinases of Chryseobacterium cucumeris and Sphingobacterium multivorum isolated from poultry sludge
- 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
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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
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