The enzymology of enhanced hydrolysis within the biosulphidogenic recycling sludge bed reactor (RSBR)
- Authors: Enongene, Godlove Nkwelle
- Date: 2004
- Subjects: Hydrolysis , Sewage sludge , Sewage -- Purification -- Anaerobic treatment , Water -- Purification -- Biological treatment
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4132 , http://hdl.handle.net/10962/d1015744
- Description: The hydrolysis of complex organic heteropolymers contained in municipal wastewater to simpler monomers by extracellular hydrolytic enzymes is generally considered the rate-limiting step of the biodegradation process. Previous studies of the Recycling Sludge Bed Reactor (RSBR) revealed that the hydrolysis of complex particulate organics, such as those contained in primary sludge (PS), was enhanced under anaerobic biosulphidogenic conditions. Although the mechanism was not fully understood, it appeared to involve the interaction of sulfide and sludge flocs. The current study was conducted using a 3500 ml laboratory-scale RSBR fed sieved PS at a loading rate of 0.5 kg COD/m³.day and an initial chemical oxygen demand (COD) to sulfate ratio (COD:SO₄) of 1:1. There was no significant accumulation of undigested sludge in the reactor over the 60-day experimental period and the quantity of SO₄ reduced indicated that the yield of soluble products from PS was at least as high as those reported previously for this system (> 50%). In the current study, the specific activities of a range of extracellular hydrolytic enzymes (L-alanine aminopeptidase, L-leucine aminopeptidase, arylsulphatase, α-glucosidase, β- glucosidase, protease and lipase) were monitored in a sulfide gradient within a biosulphidogenic RSBR. Data obtained indicated that the specific enzymatic activities increased with the depth of the RSBR and also correlated with a number of the physicochemical parameters including sulfide, alkalinity and sulfate. The activities of α- glucosidase and β-glucosidase were higher than that of the other enzymes studied. Lipase activity was relatively low and studies conducted on the enzyme-enzyme interaction using specific enzyme inhibitors indicated that lipases were probably being digested by the proteases. Further studies to determine the impact of sulfide on the enzymes, showed an increase in the enzyme activity with increasing sulfide concentration. Possible direct affects were investigated by looking for changes in the Michaelis constant (Km) and the maximal velocity (Vmax) of the crude enzymes with varying sulfide concentrations (250, 400 and 500 mg/l) using natural and synthetic substrates. The results showed no significant difference in both the Km and the Vmax for any of the hydrolytic enzymes except for the protease. The latter showed a statistically significant increase in the Km with increasing sulfide concentration. Although this indicated a direct interaction, this difference was not large enough to be of biochemical significance and was consequently not solely responsible for the enhanced hydrolysis observed in the RSBR. Investigation into the floc characteristics indicated that the biosulphidogenic RSBR flocs were generally small in size and became more dendritic with the depth of the RSBR. Based on the above data, the previously proposed descriptive models of enhanced hydrolysis of particulate organic matter in a biosulphidogenic RSBR has been revised. It is thought that the effect of sulfide on the hydrolysis step is primarily indirect and that the reduction in floc size and alteration of the floc shape to a more dendritic form is central to the success of the process.
- Full Text:
- Date Issued: 2004
- Authors: Enongene, Godlove Nkwelle
- Date: 2004
- Subjects: Hydrolysis , Sewage sludge , Sewage -- Purification -- Anaerobic treatment , Water -- Purification -- Biological treatment
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4132 , http://hdl.handle.net/10962/d1015744
- Description: The hydrolysis of complex organic heteropolymers contained in municipal wastewater to simpler monomers by extracellular hydrolytic enzymes is generally considered the rate-limiting step of the biodegradation process. Previous studies of the Recycling Sludge Bed Reactor (RSBR) revealed that the hydrolysis of complex particulate organics, such as those contained in primary sludge (PS), was enhanced under anaerobic biosulphidogenic conditions. Although the mechanism was not fully understood, it appeared to involve the interaction of sulfide and sludge flocs. The current study was conducted using a 3500 ml laboratory-scale RSBR fed sieved PS at a loading rate of 0.5 kg COD/m³.day and an initial chemical oxygen demand (COD) to sulfate ratio (COD:SO₄) of 1:1. There was no significant accumulation of undigested sludge in the reactor over the 60-day experimental period and the quantity of SO₄ reduced indicated that the yield of soluble products from PS was at least as high as those reported previously for this system (> 50%). In the current study, the specific activities of a range of extracellular hydrolytic enzymes (L-alanine aminopeptidase, L-leucine aminopeptidase, arylsulphatase, α-glucosidase, β- glucosidase, protease and lipase) were monitored in a sulfide gradient within a biosulphidogenic RSBR. Data obtained indicated that the specific enzymatic activities increased with the depth of the RSBR and also correlated with a number of the physicochemical parameters including sulfide, alkalinity and sulfate. The activities of α- glucosidase and β-glucosidase were higher than that of the other enzymes studied. Lipase activity was relatively low and studies conducted on the enzyme-enzyme interaction using specific enzyme inhibitors indicated that lipases were probably being digested by the proteases. Further studies to determine the impact of sulfide on the enzymes, showed an increase in the enzyme activity with increasing sulfide concentration. Possible direct affects were investigated by looking for changes in the Michaelis constant (Km) and the maximal velocity (Vmax) of the crude enzymes with varying sulfide concentrations (250, 400 and 500 mg/l) using natural and synthetic substrates. The results showed no significant difference in both the Km and the Vmax for any of the hydrolytic enzymes except for the protease. The latter showed a statistically significant increase in the Km with increasing sulfide concentration. Although this indicated a direct interaction, this difference was not large enough to be of biochemical significance and was consequently not solely responsible for the enhanced hydrolysis observed in the RSBR. Investigation into the floc characteristics indicated that the biosulphidogenic RSBR flocs were generally small in size and became more dendritic with the depth of the RSBR. Based on the above data, the previously proposed descriptive models of enhanced hydrolysis of particulate organic matter in a biosulphidogenic RSBR has been revised. It is thought that the effect of sulfide on the hydrolysis step is primarily indirect and that the reduction in floc size and alteration of the floc shape to a more dendritic form is central to the success of the process.
- Full Text:
- Date Issued: 2004
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:
- Date Issued: 2004
- 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:
- Date Issued: 2004
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