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
Enzymology of activated sewage sludge during anaerobic treatment of wastewaters : identification, characterisation, isolation and partial purification of proteases
- Tshivhunge, Azwiedziswi Sylvia
- Authors: Tshivhunge, Azwiedziswi Sylvia
- Date: 2001
- Subjects: Sewage sludge , Sewage sludge -- Environmental aspects , Sewage sludge digestion , Anaerobic bacteria
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4012 , http://hdl.handle.net/10962/d1004072 , Sewage sludge , Sewage sludge -- Environmental aspects , Sewage sludge digestion , Anaerobic bacteria
- Description: During anaerobic digestion bacteria inside the digester require a carbon source for their growth and metabolism, sewage sludge was used as a carbon source in this study. The COD content was used to measure the disappearance of the substrate. COD content was reduced by 48.3% and 49% in the methanogenic and sulphidogenic bioreactors, respectively, while sulphate concentration was reduced by 40%, producing 70mg/L of hydrogen sulphide as the end product over the first 5-7 days. Sulphate (which is used as a terminal electron acceptor of sulphur reducing bacteria) has little or no effect on the sulphidogenic and methanogenic proteases. Sulphite and sulphide (the intermediate and end product of sulphate reduction) increased protease activity by 20% and 40%-80%, respectively. Maximum protease activity occurred on day 21 in the methanogenic reactor and on day 9 in the sulphidogenic reactor. The absorbance, which indicates the level of amino acid increased to 2 and 9 for methanogenic and sulphidogenic bioreactors, respectively. Proteases that were active during anaerobic digestion were associated with the pellet (organic particulate matter) of the sewage. These enzymes have an optimum activity at pH 10 and at temperature of 50°C. The proteases that were active at pH 5 and 7, had optimum temperatures at 30°C and 60°C, respectively. Due to their association with organic particulate matter, these enzymes were stable at their optimum temperatures for at least five hours at their respective pH. Inhibition by PMSF, TPCK and 1.10-phenanthroline suggested that proteases inside the anaerobic digester are a mixture of cysteine, serine and metalloproteases. At pH 5, however, EDTA appeared to enhance protease activity by 368% (three-fold). Acetic acid decreased protease activity by 21%, while both propionic and butyric acid at 200 mg/L cause total inhibition of protease activity while these acids at higher pH (where they exist as their corresponding salts) exerted little effect. Copper, iron and zinc inhibited protease activity by 85% at pH 5 with concentrations ranging between 200 and 600 mg/L. On the other hand, nickel, showed an increase in protease activity of nearly 250%. At pH 7 and 10, copper had no effect on protease activity while iron, nickel and zinc inhibited these enzymes by 20-40%. Proteases at pH 7 were extracted from the pellet by sonication, releasing 50% of the total enzymes into the solution. The enzymes were precipitated by ammonium sulphate precipitation, and further purified by ion exchange chromatography and gel filtration. Ion exchange chromatography revealed that most of the enzymes that hydrolyse proteins are negatively charged while gel filtration showed that their molecular weight is approximately 500 kDa.
- Full Text:
- Date Issued: 2001
- Authors: Tshivhunge, Azwiedziswi Sylvia
- Date: 2001
- Subjects: Sewage sludge , Sewage sludge -- Environmental aspects , Sewage sludge digestion , Anaerobic bacteria
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4012 , http://hdl.handle.net/10962/d1004072 , Sewage sludge , Sewage sludge -- Environmental aspects , Sewage sludge digestion , Anaerobic bacteria
- Description: During anaerobic digestion bacteria inside the digester require a carbon source for their growth and metabolism, sewage sludge was used as a carbon source in this study. The COD content was used to measure the disappearance of the substrate. COD content was reduced by 48.3% and 49% in the methanogenic and sulphidogenic bioreactors, respectively, while sulphate concentration was reduced by 40%, producing 70mg/L of hydrogen sulphide as the end product over the first 5-7 days. Sulphate (which is used as a terminal electron acceptor of sulphur reducing bacteria) has little or no effect on the sulphidogenic and methanogenic proteases. Sulphite and sulphide (the intermediate and end product of sulphate reduction) increased protease activity by 20% and 40%-80%, respectively. Maximum protease activity occurred on day 21 in the methanogenic reactor and on day 9 in the sulphidogenic reactor. The absorbance, which indicates the level of amino acid increased to 2 and 9 for methanogenic and sulphidogenic bioreactors, respectively. Proteases that were active during anaerobic digestion were associated with the pellet (organic particulate matter) of the sewage. These enzymes have an optimum activity at pH 10 and at temperature of 50°C. The proteases that were active at pH 5 and 7, had optimum temperatures at 30°C and 60°C, respectively. Due to their association with organic particulate matter, these enzymes were stable at their optimum temperatures for at least five hours at their respective pH. Inhibition by PMSF, TPCK and 1.10-phenanthroline suggested that proteases inside the anaerobic digester are a mixture of cysteine, serine and metalloproteases. At pH 5, however, EDTA appeared to enhance protease activity by 368% (three-fold). Acetic acid decreased protease activity by 21%, while both propionic and butyric acid at 200 mg/L cause total inhibition of protease activity while these acids at higher pH (where they exist as their corresponding salts) exerted little effect. Copper, iron and zinc inhibited protease activity by 85% at pH 5 with concentrations ranging between 200 and 600 mg/L. On the other hand, nickel, showed an increase in protease activity of nearly 250%. At pH 7 and 10, copper had no effect on protease activity while iron, nickel and zinc inhibited these enzymes by 20-40%. Proteases at pH 7 were extracted from the pellet by sonication, releasing 50% of the total enzymes into the solution. The enzymes were precipitated by ammonium sulphate precipitation, and further purified by ion exchange chromatography and gel filtration. Ion exchange chromatography revealed that most of the enzymes that hydrolyse proteins are negatively charged while gel filtration showed that their molecular weight is approximately 500 kDa.
- Full Text:
- Date Issued: 2001
Characterization and mode of action of a bacteriocin produced by a Bacteroides Fragilis strain
- Mossie, Godwin Mxolisi Kevin
- Authors: Mossie, Godwin Mxolisi Kevin
- Date: 1980
- Subjects: Bacteroides , Anaerobic bacteria , Trypsin , Dinitrophenol , Proteins -- Synthesis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4124 , http://hdl.handle.net/10962/d1013543
- Description: Bacteroides fragilis strain Bf-1 produces an extracellular bacteriocin at the beginning of the stationary growth phase. Production is not inducible by either ultraviolet light or mitomycin C. The low molecular weight bacteriocin (MW estimates of 13 500 and 18 800 obtained from Sephadex G-100 chromatography and SDS-PAGE electrophoresis respecively) is stable between pH 7 - 9 and is inactivated on incubation with trypsin and pronase. An unusual feature of the Bf-1 bacteriocin is its apparent biphasic temperature stability: while the majority of the activity (97%) is destroyed by heating at 60ºC (t [subscript] 1/2 = 2.5 min at 60ºC), a small proportion (3%) is stable even after autoclaving at 121ºC for 15 min. The killing of sensitive cells occurs in 2 stages and the killing action is reversed by incubation with trypsin. The transition from stage I to stage II is dependent on the temperature of incubation and the growth state of sensitive cells. 2,4-Dinitrophenol prevents this transition. The Bf-1 bacteriocin has an unusual mode of action. It specifically inhibits RNA synthesis whilst having no effect on protein or DNA synthesis. No effect on intracellular ATP levels were observed. The heat-stable (3%) fraction had a similar biochemical effect. In vitro studies involving RNA polymerase indicated that the bacteriocin and the antibiotic rifampicin have similar effects on RNA synthesis. The bacteriocinogenic strain (Bf-1) is insensitive to its own bacteriocin both in vivo and in vitro, although this immunity is overcome in vitro by the addition of higher concentrations of the Bf-1 bacteriocin. The bacteriocinogenic strain (Bf-1) harbors a cryptic plasmid (or plasmids) which on a neutral sucrose gradient, sediments faster than the Col E1 marker plasmid DNA. Attempts to cure this strain of its bacteriocinogenic phenotype were unsuccessful.
- Full Text:
- Date Issued: 1980
- Authors: Mossie, Godwin Mxolisi Kevin
- Date: 1980
- Subjects: Bacteroides , Anaerobic bacteria , Trypsin , Dinitrophenol , Proteins -- Synthesis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4124 , http://hdl.handle.net/10962/d1013543
- Description: Bacteroides fragilis strain Bf-1 produces an extracellular bacteriocin at the beginning of the stationary growth phase. Production is not inducible by either ultraviolet light or mitomycin C. The low molecular weight bacteriocin (MW estimates of 13 500 and 18 800 obtained from Sephadex G-100 chromatography and SDS-PAGE electrophoresis respecively) is stable between pH 7 - 9 and is inactivated on incubation with trypsin and pronase. An unusual feature of the Bf-1 bacteriocin is its apparent biphasic temperature stability: while the majority of the activity (97%) is destroyed by heating at 60ºC (t [subscript] 1/2 = 2.5 min at 60ºC), a small proportion (3%) is stable even after autoclaving at 121ºC for 15 min. The killing of sensitive cells occurs in 2 stages and the killing action is reversed by incubation with trypsin. The transition from stage I to stage II is dependent on the temperature of incubation and the growth state of sensitive cells. 2,4-Dinitrophenol prevents this transition. The Bf-1 bacteriocin has an unusual mode of action. It specifically inhibits RNA synthesis whilst having no effect on protein or DNA synthesis. No effect on intracellular ATP levels were observed. The heat-stable (3%) fraction had a similar biochemical effect. In vitro studies involving RNA polymerase indicated that the bacteriocin and the antibiotic rifampicin have similar effects on RNA synthesis. The bacteriocinogenic strain (Bf-1) is insensitive to its own bacteriocin both in vivo and in vitro, although this immunity is overcome in vitro by the addition of higher concentrations of the Bf-1 bacteriocin. The bacteriocinogenic strain (Bf-1) harbors a cryptic plasmid (or plasmids) which on a neutral sucrose gradient, sediments faster than the Col E1 marker plasmid DNA. Attempts to cure this strain of its bacteriocinogenic phenotype were unsuccessful.
- Full Text:
- Date Issued: 1980
Studies on anaerobic R factor transfer in facultative and anaerobic enteric bacteria
- Authors: Moodie, Hildegard Laura
- Date: 1974
- Subjects: Anaerobic bacteria , R factors
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4252 , http://hdl.handle.net/10962/d1007684 , Anaerobic bacteria , R factors
- Description: Introduction: R factor mediated transfer of antibiotic resistance between Enterobacteriaceae has been reported to occur in the mammalian gastrointestinal tract (Farrar et al, 1972; Guinée, 1970; Kasuya, 1964; Reed et al, 1969; Wiedemann et al, 1970). In vivo conjugal transfer of genetic material has also been demonstrated with F¹, F⁺ and Hfr Escherichia coli strains (Jones & Curtiss, 1970). The environment in the lower gastrointestinal tract, where bacteria are abundant, is mainly anaerobic. This is demonstrated by the dominance of obligately anaerobic bacteria such as Bacteroides species (Finegold, 1969; Moore et al, 1969) and direct studies of intestinal gas composition (Askevold, 1956). However, most laboratory investigations of the incidence of R factors and their transfer frequencies have been performed under aerobic conditions using faecal facultative strains. The only investigation of resistance transfer under anaerobic conditions in vitro is that of Mitsuhashi (1965), who reported complete inhibition of transfer of an R factor from a Shigella flexneri donor to an Escherichia coli recipient. In addition, Fisher (1957) reported restriction of chromosomal transfer by an E. coli Hfr strain under anaerobic conditions in various media. On the basis of these results, it could be questioned whether in vivo R factor transfer is in fact possible (Chabbert et al, 1969). The contradictory situation prompted a reexamination of conjugation in facultative strains under anaerobic conditions. Both Fisher (1957) and Mitsuhashi (1965) obtained anaerobic conditions by evacuation. In this investigation, both mating and selection of recombinants were performed under stringent anaerobic conditions using methods developed for the isolation of obligate anaerobes (Hungate, 1969) to obtain a degree of anaerobiosis similar to that found in vivo.
- Full Text:
- Date Issued: 1974
- Authors: Moodie, Hildegard Laura
- Date: 1974
- Subjects: Anaerobic bacteria , R factors
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4252 , http://hdl.handle.net/10962/d1007684 , Anaerobic bacteria , R factors
- Description: Introduction: R factor mediated transfer of antibiotic resistance between Enterobacteriaceae has been reported to occur in the mammalian gastrointestinal tract (Farrar et al, 1972; Guinée, 1970; Kasuya, 1964; Reed et al, 1969; Wiedemann et al, 1970). In vivo conjugal transfer of genetic material has also been demonstrated with F¹, F⁺ and Hfr Escherichia coli strains (Jones & Curtiss, 1970). The environment in the lower gastrointestinal tract, where bacteria are abundant, is mainly anaerobic. This is demonstrated by the dominance of obligately anaerobic bacteria such as Bacteroides species (Finegold, 1969; Moore et al, 1969) and direct studies of intestinal gas composition (Askevold, 1956). However, most laboratory investigations of the incidence of R factors and their transfer frequencies have been performed under aerobic conditions using faecal facultative strains. The only investigation of resistance transfer under anaerobic conditions in vitro is that of Mitsuhashi (1965), who reported complete inhibition of transfer of an R factor from a Shigella flexneri donor to an Escherichia coli recipient. In addition, Fisher (1957) reported restriction of chromosomal transfer by an E. coli Hfr strain under anaerobic conditions in various media. On the basis of these results, it could be questioned whether in vivo R factor transfer is in fact possible (Chabbert et al, 1969). The contradictory situation prompted a reexamination of conjugation in facultative strains under anaerobic conditions. Both Fisher (1957) and Mitsuhashi (1965) obtained anaerobic conditions by evacuation. In this investigation, both mating and selection of recombinants were performed under stringent anaerobic conditions using methods developed for the isolation of obligate anaerobes (Hungate, 1969) to obtain a degree of anaerobiosis similar to that found in vivo.
- Full Text:
- Date Issued: 1974
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