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:
- 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:
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:
- 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:
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:
- 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:
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