SphereZyme (TM) technology for enhanced enzyme immobilisation application in biosensors
- Authors: Molawa, Letshego Gloria
- Date: 2011
- Subjects: Immobilized enzymes , Hydrolases , Hydrolysis , SphereZyme , Biosensors , Proteolytic enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3989 , http://hdl.handle.net/10962/d1004048 , Immobilized enzymes , Hydrolases , Hydrolysis , SphereZyme , Biosensors , Proteolytic enzymes
- Description: Self-immobilisation enzyme technologies, such as SphereZyme™, suffer from the lack of applicability to hydrolyse large substrates. Solid support immobilisation is usually a method of choice, to produce a stable biocatalyst for large substrates hydrolysis in the industry. In order to investigate this limitation, a commercial protease called Alcalase® was chosen as a model enzyme due to its natural activity (hydrolysis of large substrates-proteins). Prior to immobilising through the SphereZyme™ technology, Alcalase® was partially purified through dialysis followed by CM Sepharose™ FF cation exchanger. Sample contaminants, such as salts and stabilisers can inhibit protein crosslinking by reacting with glutaraldehyde. Alcalase® was successfully separated into 3 proteases with the major peak correlating to a positive control run on native PAGE, indicating that it was likely subtilisin Carlsberg. A 16% alkaline protease activity for azo-casein hydrolysis was retained when 5% v/v PEI: 25% v/v glutaraldehyde solution was used as a crosslinking agent in Alcalase® SphereZyme™ production. An increase in activity was also observed for monomeric substrates (PNPA) where the highest was 55%. The highest % activities maintained when 0.33 M EDA: 25% v/v glutaraldehyde solution was initially used as crosslinking agent were 4.5% and 1.6% for monomeric and polymeric substrates, respectively. PEI is a hydrophilic branched polymer with an abundance of amine groups compared to EDA. A comparison study of immobilisation efficiencies of SphereZyme™, Eupergit® and Dendrispheres was also performed for large substrate biocatalysis. The two latter technologies are solid-support immobilisation methods. Dendrispheres reached its maximum loading capacity in the first 5 minute of the one hour binding time. Twenty minutes was chosen as a maximum binding time since there was constant protein maintained on the solid support and no enzyme loss was observed during the 1 hour binding time. PEI at pH 11.5, its native pH, gave the highest immobilisation yield and specific activity over the PEI pH range of 11.5 to 7. SphereZyme™ had the highest ratio for azocasein hydrolysis followed by Dendrispheres and Eupergit®. The SphereZyme™ was also shown to be applicable to biosensors for phenol detection. Different modifications of glassy carbon electrode (GCE) were evaluated as a benchmark for the fabrication of SphereZyme™ modified phenol biosensor. GCE modified with laccase SphereZyme™ entrapped in cellulose membrane was the best modification due to the broad catechol range (<0.950 mM), high correlation coefficient (R2, 0.995) and relative high sensitivity factor (0.305 μA.mM-1). This type of biosensor was also shown to be electroactive at pH 7.0 for which its control, free laccase, lacked electroactivity. From the catalytic constants calculated, GCE modified with laccase SphereZyme™ entrapped in cellulose membrane also gave the highest effectiveness factor (Imax/Km app) of 1.84 μA.mM-1. The modified GCE with Alcalase® SphereZyme™ was relatively more sensitive than GCE modified with free Alcalase®.
- Full Text:
- Authors: Molawa, Letshego Gloria
- Date: 2011
- Subjects: Immobilized enzymes , Hydrolases , Hydrolysis , SphereZyme , Biosensors , Proteolytic enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3989 , http://hdl.handle.net/10962/d1004048 , Immobilized enzymes , Hydrolases , Hydrolysis , SphereZyme , Biosensors , Proteolytic enzymes
- Description: Self-immobilisation enzyme technologies, such as SphereZyme™, suffer from the lack of applicability to hydrolyse large substrates. Solid support immobilisation is usually a method of choice, to produce a stable biocatalyst for large substrates hydrolysis in the industry. In order to investigate this limitation, a commercial protease called Alcalase® was chosen as a model enzyme due to its natural activity (hydrolysis of large substrates-proteins). Prior to immobilising through the SphereZyme™ technology, Alcalase® was partially purified through dialysis followed by CM Sepharose™ FF cation exchanger. Sample contaminants, such as salts and stabilisers can inhibit protein crosslinking by reacting with glutaraldehyde. Alcalase® was successfully separated into 3 proteases with the major peak correlating to a positive control run on native PAGE, indicating that it was likely subtilisin Carlsberg. A 16% alkaline protease activity for azo-casein hydrolysis was retained when 5% v/v PEI: 25% v/v glutaraldehyde solution was used as a crosslinking agent in Alcalase® SphereZyme™ production. An increase in activity was also observed for monomeric substrates (PNPA) where the highest was 55%. The highest % activities maintained when 0.33 M EDA: 25% v/v glutaraldehyde solution was initially used as crosslinking agent were 4.5% and 1.6% for monomeric and polymeric substrates, respectively. PEI is a hydrophilic branched polymer with an abundance of amine groups compared to EDA. A comparison study of immobilisation efficiencies of SphereZyme™, Eupergit® and Dendrispheres was also performed for large substrate biocatalysis. The two latter technologies are solid-support immobilisation methods. Dendrispheres reached its maximum loading capacity in the first 5 minute of the one hour binding time. Twenty minutes was chosen as a maximum binding time since there was constant protein maintained on the solid support and no enzyme loss was observed during the 1 hour binding time. PEI at pH 11.5, its native pH, gave the highest immobilisation yield and specific activity over the PEI pH range of 11.5 to 7. SphereZyme™ had the highest ratio for azocasein hydrolysis followed by Dendrispheres and Eupergit®. The SphereZyme™ was also shown to be applicable to biosensors for phenol detection. Different modifications of glassy carbon electrode (GCE) were evaluated as a benchmark for the fabrication of SphereZyme™ modified phenol biosensor. GCE modified with laccase SphereZyme™ entrapped in cellulose membrane was the best modification due to the broad catechol range (<0.950 mM), high correlation coefficient (R2, 0.995) and relative high sensitivity factor (0.305 μA.mM-1). This type of biosensor was also shown to be electroactive at pH 7.0 for which its control, free laccase, lacked electroactivity. From the catalytic constants calculated, GCE modified with laccase SphereZyme™ entrapped in cellulose membrane also gave the highest effectiveness factor (Imax/Km app) of 1.84 μA.mM-1. The modified GCE with Alcalase® SphereZyme™ was relatively more sensitive than GCE modified with free Alcalase®.
- Full Text:
Towards a sustainable bioprocess for the remediation of acid mine drainage
- Authors: Mambo, Mutsa Prudence
- Date: 2011
- Subjects: Acid mine drainage , Algae culture , Reduction (Chemistry) , Hydrolysis , ASPAM model (Acid mine drainage) , Water -- Purification
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5955 , http://hdl.handle.net/10962/d1006167 , Acid mine drainage , Algae culture , Reduction (Chemistry) , Hydrolysis , ASPAM model (Acid mine drainage) , Water -- Purification
- Description: Acid mine drainage is of growing concern for both developing and developed economies. Thus there is increasing pressure to develop alternative remediation strategies. Biological sulphidogenic mechanisms have long since been studied but, very few have been implemented on a large scale. Limitations are due to the inability to acquire a suitable, low cost, environmentally friendly, renewable carbon source. The present study investigated the use of an algae biomass generated by the HRAOP of an IAPS as a carbon source for the EBRU 00AB/06 SRB consortium. The algae biomass and consortium were utilized together to remediate simulated AMD. Remediation involved decreasing the sulphate and metal concentrations in solution and decreasing the acidity of a simulated AMD. Experiments were carried out to investigate the capability of the EBRU 00AB/06 SRB consortium for sulphate reduction and sulphide generation. The consortium produced colonies when grown under anaerobic conditions in Petri dishes containing modified lactate SRB medium. The SRB consortium reduced the sulphate concentration of modified Postgates medium B and generated sulphide. Further analysis of the EBRU 00AB/06 SRB consortium revealed that the consortium was minimally impacted at pH 5 and by sulphate and iron at 3 g.L-1 and 0.5 g.L-1 respectively. The EBRU 00AB/06 SRB consortium was exposed to Actinomycin D and Ethidium Bromide to determine whether transcription and translation of proteins was required for sulphate reduction. Results indicated that sulphide generation and sulphate reduction were inducible. Analysis of the algae biomass used in this study revealed the empirical formula C1.0H1.91N0.084S0.003O0.36 indicating a carbon source rich in the nutrients required to sustain microbial development. Light microscopy revealed that algae cell walls and in particular those of Pediastrum were susceptible to acid hydrolysis. Dinitrosalicylic acid, Nile red, Bradford and Ninhydrin assays were used to determine the reducing sugar, lipid, protein and amino acid content respectively, of the mixed algae biomass. Results showed that upon exposure of the biomass to simulated AMD at pH 1 and pH 3, the concentration of reducing sugars and amino acids in solution increased. Whereas levels of lipids remained unchanged while the protein concentration decreased, indicating that, upon exposure of algae biomass to AMD, simulated or otherwise, cells ruptured, proteins were hydrolyzed and polysaccharides were broken down to sugars which are immediately available for SRB utilization. Exposure of biomass to simulated AMD revealed further that the presence of algae biomass increased the pH of simulated AMD (pH 3) to pH 7.67 after 4 d. Likewise, the pH of simulated AMD at 1 increased to 1.77 after 2 d while pH of the neutral control increased to 8.1 after 4 d. A direct comparison between lactate and algae biomass revealed 94 % sulphate removal after 23 d in the presence of algae biomass while 82 % sulphate removal was measured in the presence of lactate. Thus the EBRU 00AB/06 SRB consortium successfully utilized algae biomass for sulphate reduction and sulphide generation. In another experiment to establish if the consortium could remediate simulated AMD (pH 5) containing 0.5 g.L-1 iron and 3 g.L-1 sulphate while utilizing an algae biomass as the carbon source no residual iron was detected after 14 d and by day 23, an 89.07 % reduction in sulphate was measured. The results of this investigation are discussed in terms of utilizing a readily available and renewable biomass in the form of microalgae produced in HRAOPs as an effective carbon source in the SRB catalysed remediation of AMD.
- Full Text:
- Authors: Mambo, Mutsa Prudence
- Date: 2011
- Subjects: Acid mine drainage , Algae culture , Reduction (Chemistry) , Hydrolysis , ASPAM model (Acid mine drainage) , Water -- Purification
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5955 , http://hdl.handle.net/10962/d1006167 , Acid mine drainage , Algae culture , Reduction (Chemistry) , Hydrolysis , ASPAM model (Acid mine drainage) , Water -- Purification
- Description: Acid mine drainage is of growing concern for both developing and developed economies. Thus there is increasing pressure to develop alternative remediation strategies. Biological sulphidogenic mechanisms have long since been studied but, very few have been implemented on a large scale. Limitations are due to the inability to acquire a suitable, low cost, environmentally friendly, renewable carbon source. The present study investigated the use of an algae biomass generated by the HRAOP of an IAPS as a carbon source for the EBRU 00AB/06 SRB consortium. The algae biomass and consortium were utilized together to remediate simulated AMD. Remediation involved decreasing the sulphate and metal concentrations in solution and decreasing the acidity of a simulated AMD. Experiments were carried out to investigate the capability of the EBRU 00AB/06 SRB consortium for sulphate reduction and sulphide generation. The consortium produced colonies when grown under anaerobic conditions in Petri dishes containing modified lactate SRB medium. The SRB consortium reduced the sulphate concentration of modified Postgates medium B and generated sulphide. Further analysis of the EBRU 00AB/06 SRB consortium revealed that the consortium was minimally impacted at pH 5 and by sulphate and iron at 3 g.L-1 and 0.5 g.L-1 respectively. The EBRU 00AB/06 SRB consortium was exposed to Actinomycin D and Ethidium Bromide to determine whether transcription and translation of proteins was required for sulphate reduction. Results indicated that sulphide generation and sulphate reduction were inducible. Analysis of the algae biomass used in this study revealed the empirical formula C1.0H1.91N0.084S0.003O0.36 indicating a carbon source rich in the nutrients required to sustain microbial development. Light microscopy revealed that algae cell walls and in particular those of Pediastrum were susceptible to acid hydrolysis. Dinitrosalicylic acid, Nile red, Bradford and Ninhydrin assays were used to determine the reducing sugar, lipid, protein and amino acid content respectively, of the mixed algae biomass. Results showed that upon exposure of the biomass to simulated AMD at pH 1 and pH 3, the concentration of reducing sugars and amino acids in solution increased. Whereas levels of lipids remained unchanged while the protein concentration decreased, indicating that, upon exposure of algae biomass to AMD, simulated or otherwise, cells ruptured, proteins were hydrolyzed and polysaccharides were broken down to sugars which are immediately available for SRB utilization. Exposure of biomass to simulated AMD revealed further that the presence of algae biomass increased the pH of simulated AMD (pH 3) to pH 7.67 after 4 d. Likewise, the pH of simulated AMD at 1 increased to 1.77 after 2 d while pH of the neutral control increased to 8.1 after 4 d. A direct comparison between lactate and algae biomass revealed 94 % sulphate removal after 23 d in the presence of algae biomass while 82 % sulphate removal was measured in the presence of lactate. Thus the EBRU 00AB/06 SRB consortium successfully utilized algae biomass for sulphate reduction and sulphide generation. In another experiment to establish if the consortium could remediate simulated AMD (pH 5) containing 0.5 g.L-1 iron and 3 g.L-1 sulphate while utilizing an algae biomass as the carbon source no residual iron was detected after 14 d and by day 23, an 89.07 % reduction in sulphate was measured. The results of this investigation are discussed in terms of utilizing a readily available and renewable biomass in the form of microalgae produced in HRAOPs as an effective carbon source in the SRB catalysed remediation of AMD.
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Understanding the complexity of metabolic regulatory systems an investigation into the regulation of hydantoin-hydrolysis in Pseudomonas putida RU-KM3s
- Authors: De la Mare, Jo-Anne
- Date: 2009
- Subjects: Pseudomonas , Hydantoin , Hydrolysis , Enzymes -- Regulation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3993 , http://hdl.handle.net/10962/d1004053 , Pseudomonas , Hydantoin , Hydrolysis , Enzymes -- Regulation
- Description: It has been well-established that Pseudomonas species possess extremely versatile metabolic systems allowing them to utilise a wide range of nutrient sources and, furthermore, that the regulation of these enzyme systems involves highly evolved and sophisticated regulatory machinery. This study examined the complexity of metabolic regulation in Pseudomonas using the hydantoin-hydrolysing system of the environmental isolate, Pseudomonas putida RU-KM3s. In this system, the genes encoding dihydropyrimidinase and β-ureidopropionase (dhp and bup) are arranged divergently on the chromosome, separated by a 616 bp intergenic region involved in the transcriptional regulation of these genes. The focus was on the transcriptional regulation of dhp expression. DHP activity was found to be sensitive to several environmental signals including growth phase, carbon catabolite repression (CCR), substrate induction and quorum sensing (QS). Bioinformatic analysis of the intergenic region upstream of dhp revealed a number of putative binding sites for transcriptional regulators, including recognition sequences for the alternate sigma factors σ54 and σ38, as well as for the global regulators Anr (for anaerobic regulator) and Vfr (for virulence factor regulator). The targeted disruption of the genes encoding the transcriptional regulators, Vfr and the major CCR protein, Crc, resulted in a partial relief from repression for the vfr- mutant under quorum sensing conditions and a general decrease in activity in the crc- mutant. This data suggested that both Vfr and Crc were involved in regulating DHP activity. Mutational analysis of the dhp promoter revealed that at least two sites were involved in regulating transcriptional activity, one which mediated activation and the other repression. These sites were designated as a putative Anr box, situated 232 bp from the start codon of dhp, and a CRP-like binding site, at a position 213 bp upstream of dhp. Taken together, this data shows the involvement of several global regulatory factors in controlling the expression of dhp. A complex synergistic model was proposed for the transcriptional regulation of dhp, involving alternate sigma factors in addition to both global and specific regulators and responding to a number of environmental signals associated with growth phase, including nutrient availability, cell density and oxygen status.
- Full Text:
- Authors: De la Mare, Jo-Anne
- Date: 2009
- Subjects: Pseudomonas , Hydantoin , Hydrolysis , Enzymes -- Regulation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3993 , http://hdl.handle.net/10962/d1004053 , Pseudomonas , Hydantoin , Hydrolysis , Enzymes -- Regulation
- Description: It has been well-established that Pseudomonas species possess extremely versatile metabolic systems allowing them to utilise a wide range of nutrient sources and, furthermore, that the regulation of these enzyme systems involves highly evolved and sophisticated regulatory machinery. This study examined the complexity of metabolic regulation in Pseudomonas using the hydantoin-hydrolysing system of the environmental isolate, Pseudomonas putida RU-KM3s. In this system, the genes encoding dihydropyrimidinase and β-ureidopropionase (dhp and bup) are arranged divergently on the chromosome, separated by a 616 bp intergenic region involved in the transcriptional regulation of these genes. The focus was on the transcriptional regulation of dhp expression. DHP activity was found to be sensitive to several environmental signals including growth phase, carbon catabolite repression (CCR), substrate induction and quorum sensing (QS). Bioinformatic analysis of the intergenic region upstream of dhp revealed a number of putative binding sites for transcriptional regulators, including recognition sequences for the alternate sigma factors σ54 and σ38, as well as for the global regulators Anr (for anaerobic regulator) and Vfr (for virulence factor regulator). The targeted disruption of the genes encoding the transcriptional regulators, Vfr and the major CCR protein, Crc, resulted in a partial relief from repression for the vfr- mutant under quorum sensing conditions and a general decrease in activity in the crc- mutant. This data suggested that both Vfr and Crc were involved in regulating DHP activity. Mutational analysis of the dhp promoter revealed that at least two sites were involved in regulating transcriptional activity, one which mediated activation and the other repression. These sites were designated as a putative Anr box, situated 232 bp from the start codon of dhp, and a CRP-like binding site, at a position 213 bp upstream of dhp. Taken together, this data shows the involvement of several global regulatory factors in controlling the expression of dhp. A complex synergistic model was proposed for the transcriptional regulation of dhp, involving alternate sigma factors in addition to both global and specific regulators and responding to a number of environmental signals associated with growth phase, including nutrient availability, cell density and oxygen status.
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The enzymology of sludge solubilisation under biosulphidogenic conditions : isolation, characterisation and partial purification of endoglucanases
- Authors: Oyekola, Oluwaseun Oyekanmi
- Date: 2004
- Subjects: Sewage -- Purification -- Anaerobic treatment , Anaerobic bacteria , Sewage sludge , Hydrolysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3921 , http://hdl.handle.net/10962/d1003980 , Sewage -- Purification -- Anaerobic treatment , Anaerobic bacteria , Sewage sludge , Hydrolysis
- Description: Endoglucanases play an important function in cellulose hydrolysis and catalyse the initial attack on the polymer by randomly hydrolysing the β-1,4 glucosidic bonds within the amorphous regions of cellulose chains. Cellulolytic bacteria have been isolated and characterised from the sewage sludge and the activation of several hydrolytic enzymes under biosulphidogenic conditions of sewage hydrolysis has been reported. The aims of this study were to: identify, induce production, locate and isolate, characterise (physicochemical and kinetic) and purify endoglucanases from anaerobic biosulphidogenic sludge. The endoglucanase activities were shown to be associated with the pellet particulate matter and exhibited a pH optimum of 6 and temperature optimum of 50 °C. The enzymes were thermally more stable when immobilised to the floc matrix of the sludge than when they were released into the aqueous solution via sonication. For both immobilised and released enzymes, sulphate was slightly inhibitory; activity was reduced to 84 % and 77.5 % of the initial activity at sulphate concentrations between 200 and 1000 mg/l, respectively. Sulphite was stimulatory to the immobilised enzymes between 200 and 1000 mg/l. Sulphide stimulated the activities of the immobilised endoglucanases, but inhibited activities of the soluble enzymes above 200 mg/l. The enzyme fraction did not hydrolyse avicel (a crystalline substrate), indicating the absence of any exocellulase activity. For CMC (carboxymethylcellulose) and HEC (hydroxylethylcellulose) the enzyme had K_m,app_ values of 4 and 5.1 mg/ml respectively and V_max,app_ values of 0.297 and 0.185 μmol/min/ml respectively. Divalent ions (Cu²⁺, Ni²⁺ and Zn²⁺) proved to be inhibitory while Fe²⁺, Mg²⁺ and Ca²⁺ stimulated the enzyme at concentrations between 200 and 1000 mg/l. All the volatile fatty acids studied (acetic acid, butyric acid, propionic acid and valeric acid) inhibited the enzymes, with acetic acid eliciting the highest degree of inhibition. Sonication released ~74.9 % of the total enzyme activities into solution and this was partially purified by PEG 20 000 concentration followed by DEAE-Cellulose ion exchange chromatography, which resulted in an appreciable purity as measured by the purification factor, 25.4 fold.
- Full Text:
- Authors: Oyekola, Oluwaseun Oyekanmi
- Date: 2004
- Subjects: Sewage -- Purification -- Anaerobic treatment , Anaerobic bacteria , Sewage sludge , Hydrolysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3921 , http://hdl.handle.net/10962/d1003980 , Sewage -- Purification -- Anaerobic treatment , Anaerobic bacteria , Sewage sludge , Hydrolysis
- Description: Endoglucanases play an important function in cellulose hydrolysis and catalyse the initial attack on the polymer by randomly hydrolysing the β-1,4 glucosidic bonds within the amorphous regions of cellulose chains. Cellulolytic bacteria have been isolated and characterised from the sewage sludge and the activation of several hydrolytic enzymes under biosulphidogenic conditions of sewage hydrolysis has been reported. The aims of this study were to: identify, induce production, locate and isolate, characterise (physicochemical and kinetic) and purify endoglucanases from anaerobic biosulphidogenic sludge. The endoglucanase activities were shown to be associated with the pellet particulate matter and exhibited a pH optimum of 6 and temperature optimum of 50 °C. The enzymes were thermally more stable when immobilised to the floc matrix of the sludge than when they were released into the aqueous solution via sonication. For both immobilised and released enzymes, sulphate was slightly inhibitory; activity was reduced to 84 % and 77.5 % of the initial activity at sulphate concentrations between 200 and 1000 mg/l, respectively. Sulphite was stimulatory to the immobilised enzymes between 200 and 1000 mg/l. Sulphide stimulated the activities of the immobilised endoglucanases, but inhibited activities of the soluble enzymes above 200 mg/l. The enzyme fraction did not hydrolyse avicel (a crystalline substrate), indicating the absence of any exocellulase activity. For CMC (carboxymethylcellulose) and HEC (hydroxylethylcellulose) the enzyme had K_m,app_ values of 4 and 5.1 mg/ml respectively and V_max,app_ values of 0.297 and 0.185 μmol/min/ml respectively. Divalent ions (Cu²⁺, Ni²⁺ and Zn²⁺) proved to be inhibitory while Fe²⁺, Mg²⁺ and Ca²⁺ stimulated the enzyme at concentrations between 200 and 1000 mg/l. All the volatile fatty acids studied (acetic acid, butyric acid, propionic acid and valeric acid) inhibited the enzymes, with acetic acid eliciting the highest degree of inhibition. Sonication released ~74.9 % of the total enzyme activities into solution and this was partially purified by PEG 20 000 concentration followed by DEAE-Cellulose ion exchange chromatography, which resulted in an appreciable purity as measured by the purification factor, 25.4 fold.
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The hydrolysis of primary sewage sludge under biosulphidogenic conditions
- Molwantwa, Jennifer Balatedi
- Authors: Molwantwa, Jennifer Balatedi
- Date: 2003
- Subjects: Sewage sludge , Hydrolysis , Sewage -- Purification -- Activated sludge process
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3961 , http://hdl.handle.net/10962/d1004020 , Sewage sludge , Hydrolysis , Sewage -- Purification -- Activated sludge process
- Description: The potential for using readily available and cost-effective complex carbon sources such as primary sewage sludge for a range of environmental remediation processes, including biological sulphate reduction, biological nutrient removal and the bioremediation of acid mine drainage, has been constrained by the slow rate of solubilization and low yield of soluble products, which drive the above mentioned processes. Previous work conducted by the Environmental Biotechnology Group at Rhodes University indicated that the degradation of primary sewage sludge was enhanced under sulphate reducing conditions. This was proven in both laboratory and pilot-scale (Reciprocating Sludge Bed Reactor) systems, where the particulate matter accumulated in the sludge bed and the molecules in smaller flocs were rapidly solubilized. The current study was aimed at investigating in more detail the factors that govern the enhanced hydrolysis under sulphate reducing conditions, and to develop a descriptive model to explain the underlying mechanism involved. The solubilization of primary sewage sludge under sulphate reducing conditions was conducted in controlled flask studies and previously reported findings of enhanced hydrolysis were confirmed. The maximum percentage solubilization obtained in this study was 31% and 63% for the methanogenic and sulphidogenic systems respectively, and this was achieved over a period of 10 days. A rate of reducing sugar production and complex molecule breakdown of 51 mg. L⁻¹.hr⁻¹ and 167 mg.L⁻¹.hr⁻¹ was observed for the methanogenic and sulphidogenic systems respectively. The flask studies revealed that during hydrolysis of primary sewage sludge under sulphidogenic conditions there was enhanced production of soluble products, specifically carbohydrates (reducing sugars) and volatile fatty acids, compared to methanogenic conditions. The rate at which these products were utilized was also found to be more rapid under sulphidogenic as compared to methanogenic conditions. A study of the distribution of volatile fatty acids indicated that acetate was utilized preferentially in the methanogenic system, and that propionate, butyrate and valerate accumulated with time. The converse was found to occur in the sulphidogenic system. The descriptive model developed from the results of this study was based on the fact that a consortium of bacteria, composed of hydrolytic, acidogenic and acetogenic species, carries out the solubilization of complex carbon sources. Furthermore, it is essential that equilibrium between product formation and utilization is maintained, and that accumulation of soluble end products impacts negatively on the rate of the hydrolysis step. It is therefore proposed that the relatively poor utilization of VFA and reducing sugars in the methanogenic system activates a negative feedback inhibition on the hydrolytic and/ or acidogenic step. This inhibition is reduced in the sulphidogenic system where the utilization of end products is higher.
- Full Text:
- Authors: Molwantwa, Jennifer Balatedi
- Date: 2003
- Subjects: Sewage sludge , Hydrolysis , Sewage -- Purification -- Activated sludge process
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3961 , http://hdl.handle.net/10962/d1004020 , Sewage sludge , Hydrolysis , Sewage -- Purification -- Activated sludge process
- Description: The potential for using readily available and cost-effective complex carbon sources such as primary sewage sludge for a range of environmental remediation processes, including biological sulphate reduction, biological nutrient removal and the bioremediation of acid mine drainage, has been constrained by the slow rate of solubilization and low yield of soluble products, which drive the above mentioned processes. Previous work conducted by the Environmental Biotechnology Group at Rhodes University indicated that the degradation of primary sewage sludge was enhanced under sulphate reducing conditions. This was proven in both laboratory and pilot-scale (Reciprocating Sludge Bed Reactor) systems, where the particulate matter accumulated in the sludge bed and the molecules in smaller flocs were rapidly solubilized. The current study was aimed at investigating in more detail the factors that govern the enhanced hydrolysis under sulphate reducing conditions, and to develop a descriptive model to explain the underlying mechanism involved. The solubilization of primary sewage sludge under sulphate reducing conditions was conducted in controlled flask studies and previously reported findings of enhanced hydrolysis were confirmed. The maximum percentage solubilization obtained in this study was 31% and 63% for the methanogenic and sulphidogenic systems respectively, and this was achieved over a period of 10 days. A rate of reducing sugar production and complex molecule breakdown of 51 mg. L⁻¹.hr⁻¹ and 167 mg.L⁻¹.hr⁻¹ was observed for the methanogenic and sulphidogenic systems respectively. The flask studies revealed that during hydrolysis of primary sewage sludge under sulphidogenic conditions there was enhanced production of soluble products, specifically carbohydrates (reducing sugars) and volatile fatty acids, compared to methanogenic conditions. The rate at which these products were utilized was also found to be more rapid under sulphidogenic as compared to methanogenic conditions. A study of the distribution of volatile fatty acids indicated that acetate was utilized preferentially in the methanogenic system, and that propionate, butyrate and valerate accumulated with time. The converse was found to occur in the sulphidogenic system. The descriptive model developed from the results of this study was based on the fact that a consortium of bacteria, composed of hydrolytic, acidogenic and acetogenic species, carries out the solubilization of complex carbon sources. Furthermore, it is essential that equilibrium between product formation and utilization is maintained, and that accumulation of soluble end products impacts negatively on the rate of the hydrolysis step. It is therefore proposed that the relatively poor utilization of VFA and reducing sugars in the methanogenic system activates a negative feedback inhibition on the hydrolytic and/ or acidogenic step. This inhibition is reduced in the sulphidogenic system where the utilization of end products is higher.
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Characterization of amide bond hydrolysis in novel hydantoinase-producing bacteria
- Authors: Skepu, Zoleka G
- Date: 2000
- Subjects: Amides , Hydrolysis , Hydantoin , Imides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3911 , http://hdl.handle.net/10962/d1003970 , Amides , Hydrolysis , Hydantoin , Imides
- Description: This thesis describes a series of investigations into the amide bond-hydrolyzing activity of bacterial strains RU-KM1, RU-KM3L, RU-KM3S, and RU-OR, which were previously isolated for their ability to hydrolyze hydantoins to amino acids. The main aim of the study was to develop biotransformations with potential application in the production of enantiomerically pure amino acids and related compounds. Several compounds may be used as substrates by biocatalysts for the production of amino acids, such as hydantoins, amino nitriles, and amides. These compounds are not only important for amino acid production, but they may be used for production of other industrially important compounds, such as 2- arylpropionic acids, which are non-steroidal anti-inflammatory drugs. Thus, the ability of the above-mentioned strains to hydrolyze these substrates was investigated, with the view to utilizing the maximum potential of these biocatalysts. The compounds used as substrates in the investigation are all essentially amides. Thus, the ability of the strains to hydrolyze imides, hydantoins, and amides, was investigated. In particular, imides have a structure which is very similar to that of hydantoins, and thus it was an objective of the study to determine whether these strains could hydrolyze imides. Imidehydrolyzing activity has only recently been discovered in microorganisms. Hydantoin conversion involves a two-step hydrolysis reaction which yields, initially, an Ncarbamylamino acid intermediate, and subsequently, an "-amino acid. The hydantoinhydrolyzing enzymes of a Pseudomonas putida strain, RU-KM3S, were characterized in a crude extract preparation and reaction conditions for its biocatalytic application were optimized. The optimum conditions for conversion of 5-methylhydantoin were found to be 3 hours at 40°C, with conversion yields greater than 50% achieved. The enzymes of RU-KM3S demonstrated considerable stability, retaining 80% of their activity after incubation at 40°C for 3 hours. The activities of the enzymes were increased by the addition of a detergent to the extraction medium, suggesting that the enzymes might be membrane-bound. The results of the determination of the metal-dependence of the hydantoinase and N-carbamylase of RUKM3S suggested that these enzymes required metal ions for activity, with metal ions such as Mg²⁺, Mn²⁺, Zn²⁺, and Co²⁺ resulting in activation of the enzymes. However, Cu²⁺ and Fe²⁺ caused inactivation of these enzymes. The stereoselectivity of the enzymes was investigated, and the results suggested that the hydantoinase was non-selective, whereas the N-carbamylase was L-selective. The hydantoin substrate selectivity of RU-KM3S was compared to that of three other hydantoinase-producing bacteria, RU-KM1, RU-KM3L, and RU-OR. The four strains were able to hydrolyze all of the seven substrates tested. However, there was a difference in activity levels between crude extract preparations and whole cells, with crude extracts generally showing higher activity than whole cells, except in the case of RU-KM1. Some difference was also observed in the order of preference of substrates between whole cells and crude extracts. The preferred substrate for RU-KM1 whole cells was isopropylhydantoin, whereas the crude extract preparation preferentially hydrolyzed p-hydroxyphenylhydantoin. RU-KM3L whole cells achieved a higher conversion yield with isobutylhydantoin, whereas the crude extract achieved a higher yield with 5-t-butylhydantoin. RU-KM3S whole cells and crude extract preferentially hydrolyzed 5-n-butylhydantoin, although the yield was greater with the crude extract. The highest conversion yields were observed with RU-KM3S crude extract, with conversion yields of 71.6% and 100% for n-butylhydantoin and phydroxyphenylhydantoin, respectively.The ability of RU-KM1, RU-KM3L, and RU-KM3S to hydrolyze nitriles, initially to amides and subsequently to carboxylic acids, was investigated. These strains were demonstrated to be unable to utilize acrylonitrile, propionitrile and benzonitrile as nitrogen sources, but were able to hydrolyze acrylonitrile, propionitrile and acetonitrile, in resting cell reactions. Nitrile hydrolysis was demonstrated to be inducible in all three strains, and the enzyme system responsible for nitrile hydrolysis was proposed to be a nitrile hydratase-amidase system. Amidase activity in the four bacterial strains was investigated. The ability of RU-KM1, RUKM3L, RU-KM3S, and RU-OR to utilize amides as a nitrogen source was investigated, and the results showed that propionamide was a good nitrogen source for all four of the strains. Amide-hydrolyzing activity, by resting cells, was shown to be inducible by propionamide in all four strains. RU-KM3S demonstrated superior amide-hydrolyzing ability in that it hydrolyzed propionamide, acetamide, and acrylamide to a greater extent than the other strains. Resting cells of RU-KM1 and RU-OR were demonstrated to have the ability to hydrolyze the imide substrate, succinimide, and this imidase activity was found to be inducible. These strains were also able to utilize this imide as the sole source of nitrogen for growth, which is a novel finding, as to date, bacteria have only be reported to utilize imides as a carbon source. The identity of the enzyme system responsible for succinimide hydrolysis is not yet clear. In conclusion, the hydantoin-hydrolyzing enzymes of RU-KM3S have been shown to be possibly membrane associated, which is a novel finding that has also been proposed in three other hydantoinase-producing strains in our laboratory. This study has shown that the Ncarbamylase of RU-KM3S is L-stereoselective, which, to our knowledge, is the first report of an L-stereospecific N-carbamylase in a Pseudomonas putida. Publication of these findings is already in progress. This is the first report on the study of imide hydrolysis in either an Agrobacterium tumefaciens or a Pseudomonas sp., and publications reporting these results are in preparation.
- Full Text:
- Authors: Skepu, Zoleka G
- Date: 2000
- Subjects: Amides , Hydrolysis , Hydantoin , Imides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3911 , http://hdl.handle.net/10962/d1003970 , Amides , Hydrolysis , Hydantoin , Imides
- Description: This thesis describes a series of investigations into the amide bond-hydrolyzing activity of bacterial strains RU-KM1, RU-KM3L, RU-KM3S, and RU-OR, which were previously isolated for their ability to hydrolyze hydantoins to amino acids. The main aim of the study was to develop biotransformations with potential application in the production of enantiomerically pure amino acids and related compounds. Several compounds may be used as substrates by biocatalysts for the production of amino acids, such as hydantoins, amino nitriles, and amides. These compounds are not only important for amino acid production, but they may be used for production of other industrially important compounds, such as 2- arylpropionic acids, which are non-steroidal anti-inflammatory drugs. Thus, the ability of the above-mentioned strains to hydrolyze these substrates was investigated, with the view to utilizing the maximum potential of these biocatalysts. The compounds used as substrates in the investigation are all essentially amides. Thus, the ability of the strains to hydrolyze imides, hydantoins, and amides, was investigated. In particular, imides have a structure which is very similar to that of hydantoins, and thus it was an objective of the study to determine whether these strains could hydrolyze imides. Imidehydrolyzing activity has only recently been discovered in microorganisms. Hydantoin conversion involves a two-step hydrolysis reaction which yields, initially, an Ncarbamylamino acid intermediate, and subsequently, an "-amino acid. The hydantoinhydrolyzing enzymes of a Pseudomonas putida strain, RU-KM3S, were characterized in a crude extract preparation and reaction conditions for its biocatalytic application were optimized. The optimum conditions for conversion of 5-methylhydantoin were found to be 3 hours at 40°C, with conversion yields greater than 50% achieved. The enzymes of RU-KM3S demonstrated considerable stability, retaining 80% of their activity after incubation at 40°C for 3 hours. The activities of the enzymes were increased by the addition of a detergent to the extraction medium, suggesting that the enzymes might be membrane-bound. The results of the determination of the metal-dependence of the hydantoinase and N-carbamylase of RUKM3S suggested that these enzymes required metal ions for activity, with metal ions such as Mg²⁺, Mn²⁺, Zn²⁺, and Co²⁺ resulting in activation of the enzymes. However, Cu²⁺ and Fe²⁺ caused inactivation of these enzymes. The stereoselectivity of the enzymes was investigated, and the results suggested that the hydantoinase was non-selective, whereas the N-carbamylase was L-selective. The hydantoin substrate selectivity of RU-KM3S was compared to that of three other hydantoinase-producing bacteria, RU-KM1, RU-KM3L, and RU-OR. The four strains were able to hydrolyze all of the seven substrates tested. However, there was a difference in activity levels between crude extract preparations and whole cells, with crude extracts generally showing higher activity than whole cells, except in the case of RU-KM1. Some difference was also observed in the order of preference of substrates between whole cells and crude extracts. The preferred substrate for RU-KM1 whole cells was isopropylhydantoin, whereas the crude extract preparation preferentially hydrolyzed p-hydroxyphenylhydantoin. RU-KM3L whole cells achieved a higher conversion yield with isobutylhydantoin, whereas the crude extract achieved a higher yield with 5-t-butylhydantoin. RU-KM3S whole cells and crude extract preferentially hydrolyzed 5-n-butylhydantoin, although the yield was greater with the crude extract. The highest conversion yields were observed with RU-KM3S crude extract, with conversion yields of 71.6% and 100% for n-butylhydantoin and phydroxyphenylhydantoin, respectively.The ability of RU-KM1, RU-KM3L, and RU-KM3S to hydrolyze nitriles, initially to amides and subsequently to carboxylic acids, was investigated. These strains were demonstrated to be unable to utilize acrylonitrile, propionitrile and benzonitrile as nitrogen sources, but were able to hydrolyze acrylonitrile, propionitrile and acetonitrile, in resting cell reactions. Nitrile hydrolysis was demonstrated to be inducible in all three strains, and the enzyme system responsible for nitrile hydrolysis was proposed to be a nitrile hydratase-amidase system. Amidase activity in the four bacterial strains was investigated. The ability of RU-KM1, RUKM3L, RU-KM3S, and RU-OR to utilize amides as a nitrogen source was investigated, and the results showed that propionamide was a good nitrogen source for all four of the strains. Amide-hydrolyzing activity, by resting cells, was shown to be inducible by propionamide in all four strains. RU-KM3S demonstrated superior amide-hydrolyzing ability in that it hydrolyzed propionamide, acetamide, and acrylamide to a greater extent than the other strains. Resting cells of RU-KM1 and RU-OR were demonstrated to have the ability to hydrolyze the imide substrate, succinimide, and this imidase activity was found to be inducible. These strains were also able to utilize this imide as the sole source of nitrogen for growth, which is a novel finding, as to date, bacteria have only be reported to utilize imides as a carbon source. The identity of the enzyme system responsible for succinimide hydrolysis is not yet clear. In conclusion, the hydantoin-hydrolyzing enzymes of RU-KM3S have been shown to be possibly membrane associated, which is a novel finding that has also been proposed in three other hydantoinase-producing strains in our laboratory. This study has shown that the Ncarbamylase of RU-KM3S is L-stereoselective, which, to our knowledge, is the first report of an L-stereospecific N-carbamylase in a Pseudomonas putida. Publication of these findings is already in progress. This is the first report on the study of imide hydrolysis in either an Agrobacterium tumefaciens or a Pseudomonas sp., and publications reporting these results are in preparation.
- Full Text:
Sulphate reduction utilizing hydrolysis of complex carbon sources
- Authors: Molipane, Ntaoleng Patricia
- Date: 1999
- Subjects: Sewage sludge , Acid mine drainage , Hydrolysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4000 , http://hdl.handle.net/10962/d1004060 , Sewage sludge , Acid mine drainage , Hydrolysis
- Description: Due to environmental pollution caused by acid mine drainage (AMD), the Department of Water Affairs has developed a National Water Bill for managing and controlling the water environment to prevent AMD pollution. The application of sulphate reducing bacteria have been demonstrated for the treatment of AMD. However, the scale-up application of this technology ultimately depends on the cost and availability of a carbon source. This study evaluated the use of sewage sludge to provide a carbon source for sulphate reduction in synthetic drainage wastewaters. The demonstration of this process in a laboratory-scale reactor proved that sewage sludge could provide a useful model and viable carbon source for evaluation of sulphate reduction as a process for treating AMD. Since sewage sludge is a complex carbon source, hydrolysis reactions controlling the anaerobic digestion of particulate substrate from this medium were optimized by evaluating the effect of pH on hydrolysis. Controlled and uncontrolled pH studies were conducted using a three stage mixed anaerobic reactor. Analysis of the degradation behaviour of the three important organic classes (carbohydrate, proteins and lipids) revealed that each class followed an indvidual trend with respect to pH changes. In addition, the solubilization of organic particulate carbon was also shown to be a function of pH. The hydrolysis pattern of organic substrate and COD solublization was induced at pH 6.5 rather than at high pH values (7.5 and 8.5). The biodegradation activity of sewage sludge was characterized by the API-ZYM1N test system to provide rapid semiquantitative information on the activity of hydrolytic enzymes associated with the degradation of carbohydrates, lipids, proteins and nucleic acids. A wide range of enzyme activities with phosphatases, aminopeptidases, and glucosyl hydralases dominating were displayed. The pattern of substrate hydrolysis correlated to the degradation efficiency of each organic class as a function of pH. The evaluation of scale-up application for sulphate reduction utilizing sewage sludge as a carbon source demonstrated that large water volume flows could possibly be treated with this cost-effective technology. Generation of alkalinity and sulphide in this medium was shown to be successful in the removal of heavy metals by precipitation. The use of this technology coupled to reduced cost involved showed that biological sulphate reduction utilizing hydrolysates of complex organic particulate from sewage sludge ss a carbon source has a potential scale-up application for the treatment of AMD.
- Full Text:
- Authors: Molipane, Ntaoleng Patricia
- Date: 1999
- Subjects: Sewage sludge , Acid mine drainage , Hydrolysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4000 , http://hdl.handle.net/10962/d1004060 , Sewage sludge , Acid mine drainage , Hydrolysis
- Description: Due to environmental pollution caused by acid mine drainage (AMD), the Department of Water Affairs has developed a National Water Bill for managing and controlling the water environment to prevent AMD pollution. The application of sulphate reducing bacteria have been demonstrated for the treatment of AMD. However, the scale-up application of this technology ultimately depends on the cost and availability of a carbon source. This study evaluated the use of sewage sludge to provide a carbon source for sulphate reduction in synthetic drainage wastewaters. The demonstration of this process in a laboratory-scale reactor proved that sewage sludge could provide a useful model and viable carbon source for evaluation of sulphate reduction as a process for treating AMD. Since sewage sludge is a complex carbon source, hydrolysis reactions controlling the anaerobic digestion of particulate substrate from this medium were optimized by evaluating the effect of pH on hydrolysis. Controlled and uncontrolled pH studies were conducted using a three stage mixed anaerobic reactor. Analysis of the degradation behaviour of the three important organic classes (carbohydrate, proteins and lipids) revealed that each class followed an indvidual trend with respect to pH changes. In addition, the solubilization of organic particulate carbon was also shown to be a function of pH. The hydrolysis pattern of organic substrate and COD solublization was induced at pH 6.5 rather than at high pH values (7.5 and 8.5). The biodegradation activity of sewage sludge was characterized by the API-ZYM1N test system to provide rapid semiquantitative information on the activity of hydrolytic enzymes associated with the degradation of carbohydrates, lipids, proteins and nucleic acids. A wide range of enzyme activities with phosphatases, aminopeptidases, and glucosyl hydralases dominating were displayed. The pattern of substrate hydrolysis correlated to the degradation efficiency of each organic class as a function of pH. The evaluation of scale-up application for sulphate reduction utilizing sewage sludge as a carbon source demonstrated that large water volume flows could possibly be treated with this cost-effective technology. Generation of alkalinity and sulphide in this medium was shown to be successful in the removal of heavy metals by precipitation. The use of this technology coupled to reduced cost involved showed that biological sulphate reduction utilizing hydrolysates of complex organic particulate from sewage sludge ss a carbon source has a potential scale-up application for the treatment of AMD.
- Full Text:
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