Capsule immobilisation of sulphate-reducing bacteria and application in disarticulated systems
- Authors: Sanyahumbi, Douglas
- Date: 2004
- Subjects: Sulfur bacteria , Water -- Purification -- Biological treatment
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3935 , http://hdl.handle.net/10962/d1003994
- Description: Biotechnology of sulphate reducing bacteria has developed rapidly in recent years with the recognition of their extensive and diverse biocatalytic potential. However, their application in a number of areas has been constrained due to problems including poor cell retention within the continuous bioprocess reactor environment, and contamination of the treated stream with residual organic feed components and cell biomass. These problems have so far excluded the application of biological sulphate reduction in the treatment of ‘clean’ inorganic waste streams where components such as sulphate, acidity and heavy metal contamination require treatment. This study investigated the effective immobilisation of sulphate reducing bacterial cultures and proposed that the disarticulation of the electron donor and carbon source supply using such systems would create the basis for their application in the treatment of ‘clean’ inorganic waste streams. A functional and stable sulphate reducing culture was selected and following evaluation using a number of techniques, was immobilised by encapsulation within a calcium-alginate-xanthum gum membrane to give robust capsules with good sulphate reduction activity. The concept of disarticulation was investigated in a swing-back cycle where the carbon source was excluded and the electron donor supplied in the form of hydrogen gas in a continuous up-flow capsule-packed column reactor. Following a period of operation in this mode (4-12 days), the system was swung back to a carbon feed to supply requirements of cell maintenance (2-3 days). Three types of synthetic ‘clean’ inorganic waste stream treatments were investigated, including sulphate removal, neutralisation of acidity and heavy metal (copper and lead) removal. The results showed: • Sulphate removal at a rate of 50 mg SO₄²⁻L/day/g initial wet mass of capsules during three 4-day cycles of electron donor phase. This was comparable to the performance of free cell systems; • Neutralisation of acidity where influent pH values of 2.4 and 4.0 were elevated to above pH 7.5; • Copper removal of 99 and 85 % was achieved with initial copper concentrations of 2 and 60 mg/L respectively; • Percentage lead removal values of 49 and 78 % were achieved; This first report on the application of the concept of capsular immobilisation and disarticulation in the treatment of ‘clean’ inorganic waste streams will require future studies in order to extend the development of the full potential of the concept.
- Full Text:
- Date Issued: 2004
- Authors: Sanyahumbi, Douglas
- Date: 2004
- Subjects: Sulfur bacteria , Water -- Purification -- Biological treatment
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3935 , http://hdl.handle.net/10962/d1003994
- Description: Biotechnology of sulphate reducing bacteria has developed rapidly in recent years with the recognition of their extensive and diverse biocatalytic potential. However, their application in a number of areas has been constrained due to problems including poor cell retention within the continuous bioprocess reactor environment, and contamination of the treated stream with residual organic feed components and cell biomass. These problems have so far excluded the application of biological sulphate reduction in the treatment of ‘clean’ inorganic waste streams where components such as sulphate, acidity and heavy metal contamination require treatment. This study investigated the effective immobilisation of sulphate reducing bacterial cultures and proposed that the disarticulation of the electron donor and carbon source supply using such systems would create the basis for their application in the treatment of ‘clean’ inorganic waste streams. A functional and stable sulphate reducing culture was selected and following evaluation using a number of techniques, was immobilised by encapsulation within a calcium-alginate-xanthum gum membrane to give robust capsules with good sulphate reduction activity. The concept of disarticulation was investigated in a swing-back cycle where the carbon source was excluded and the electron donor supplied in the form of hydrogen gas in a continuous up-flow capsule-packed column reactor. Following a period of operation in this mode (4-12 days), the system was swung back to a carbon feed to supply requirements of cell maintenance (2-3 days). Three types of synthetic ‘clean’ inorganic waste stream treatments were investigated, including sulphate removal, neutralisation of acidity and heavy metal (copper and lead) removal. The results showed: • Sulphate removal at a rate of 50 mg SO₄²⁻L/day/g initial wet mass of capsules during three 4-day cycles of electron donor phase. This was comparable to the performance of free cell systems; • Neutralisation of acidity where influent pH values of 2.4 and 4.0 were elevated to above pH 7.5; • Copper removal of 99 and 85 % was achieved with initial copper concentrations of 2 and 60 mg/L respectively; • Percentage lead removal values of 49 and 78 % were achieved; This first report on the application of the concept of capsular immobilisation and disarticulation in the treatment of ‘clean’ inorganic waste streams will require future studies in order to extend the development of the full potential of the concept.
- Full Text:
- Date Issued: 2004
Characterisation of the J domain aminoacid residues important for the interaction of DNAJ-like proteins with HSP70 chaperones
- Authors: Hennessy, Fritha
- Date: 2004
- Subjects: Heat shock proteins Protein folding Proteins -- Analysis Proteins -- Structure Molecular chaperones
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3937 , http://hdl.handle.net/10962/d1003996
- Description: The 70 kDa heat shock proteins (Hsp70s) are vital for normal protein folding, as they stabilise the unfolded state of nascent polypeptides, allowing these sufficient time to attain a correct tertiary structure. Hsp70s are aided by the DnaJ-like family of proteins, which interact with Hsp70s in order to enhance the chaperone activity of these proteins. DnaJ-like proteins contain a J domain, a seventy amino acid domain consisting of four α-helices, which is defined by the presence of an invariant tripeptide of histidine, proline and aspartic acid (HPD motif). This motif is key to the interaction between DnaJ-like proteins and Hsp70s. This thesis has focused on determining the presence of other conserved residues in the J domain and their role in mediating the interaction of DnaJ-like proteins with partner Hsp70s. DnaJ-like proteins from Agrobacterium tumefaciens RUOR were isolated and used as a model system. A. tumefaciens DnaJ (Agt DnaJ) was able to replace the lack of E. coli DnaJ in an E. coli null mutant strain, however, additional A. tumefaciens DnaJ-like proteins Agt DjC1/DjlA, Agt DjC2 and Agt DjC5 were unable to complement for the lack of E. coli DnaJ. Replacement of the Agt DnaJ J domain with J domains from these proteins resulted in non-functional chimeric proteins, despite some sequence conservation. The kinetics of the basal specific ATPase activity of Agt DnaK, and its ability to have this activity stimulated by Agt DnaJ and Agt DnaJ-H33Q were also investigated. Stimulation of the ATPase activity by Agt DnaJ ranged between 1.5 to 2 fold, but Agt DnaJ-H33Q was unable to stimulate the basal ATPase activity. Conserved amino acids in the J domain were identified in silico, and these residues were substituted in the J domain of Agt DnaJ. The ability of these derivative proteins to replace E. coli DnaJ was investigated. Alterations in the HPD motif gave rise to proteins unable to complement for lack of E. coli DnaJ, consistent with literature. Agt DnaJ-R26A was unable to replace E. coli DnaJ suggesting that Arg26 could be key to the interaction with partner Hsp70s. Agt DnaJ-D59A was unable to replace E. coli DnaJ; substitutions in Asp59 have not previously been shown to impact on the function of DnaJ. Substituting Arg63 in Agt DnaJ abrogated the levels of complementation. Substitution of several structural residues was also found to disrupt the in vivo function of Agt DnaJ suggesting that the maintenance of the structural integrity of the J domain was important for function. This study has identified a number of residues critical to the structure and function of the J domain of Agt DnaJ, and potentially of general importance as molecular determinants for DnaJ-Hsp70 interaction.
- Full Text:
- Date Issued: 2004
- Authors: Hennessy, Fritha
- Date: 2004
- Subjects: Heat shock proteins Protein folding Proteins -- Analysis Proteins -- Structure Molecular chaperones
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3937 , http://hdl.handle.net/10962/d1003996
- Description: The 70 kDa heat shock proteins (Hsp70s) are vital for normal protein folding, as they stabilise the unfolded state of nascent polypeptides, allowing these sufficient time to attain a correct tertiary structure. Hsp70s are aided by the DnaJ-like family of proteins, which interact with Hsp70s in order to enhance the chaperone activity of these proteins. DnaJ-like proteins contain a J domain, a seventy amino acid domain consisting of four α-helices, which is defined by the presence of an invariant tripeptide of histidine, proline and aspartic acid (HPD motif). This motif is key to the interaction between DnaJ-like proteins and Hsp70s. This thesis has focused on determining the presence of other conserved residues in the J domain and their role in mediating the interaction of DnaJ-like proteins with partner Hsp70s. DnaJ-like proteins from Agrobacterium tumefaciens RUOR were isolated and used as a model system. A. tumefaciens DnaJ (Agt DnaJ) was able to replace the lack of E. coli DnaJ in an E. coli null mutant strain, however, additional A. tumefaciens DnaJ-like proteins Agt DjC1/DjlA, Agt DjC2 and Agt DjC5 were unable to complement for the lack of E. coli DnaJ. Replacement of the Agt DnaJ J domain with J domains from these proteins resulted in non-functional chimeric proteins, despite some sequence conservation. The kinetics of the basal specific ATPase activity of Agt DnaK, and its ability to have this activity stimulated by Agt DnaJ and Agt DnaJ-H33Q were also investigated. Stimulation of the ATPase activity by Agt DnaJ ranged between 1.5 to 2 fold, but Agt DnaJ-H33Q was unable to stimulate the basal ATPase activity. Conserved amino acids in the J domain were identified in silico, and these residues were substituted in the J domain of Agt DnaJ. The ability of these derivative proteins to replace E. coli DnaJ was investigated. Alterations in the HPD motif gave rise to proteins unable to complement for lack of E. coli DnaJ, consistent with literature. Agt DnaJ-R26A was unable to replace E. coli DnaJ suggesting that Arg26 could be key to the interaction with partner Hsp70s. Agt DnaJ-D59A was unable to replace E. coli DnaJ; substitutions in Asp59 have not previously been shown to impact on the function of DnaJ. Substituting Arg63 in Agt DnaJ abrogated the levels of complementation. Substitution of several structural residues was also found to disrupt the in vivo function of Agt DnaJ suggesting that the maintenance of the structural integrity of the J domain was important for function. This study has identified a number of residues critical to the structure and function of the J domain of Agt DnaJ, and potentially of general importance as molecular determinants for DnaJ-Hsp70 interaction.
- Full Text:
- Date Issued: 2004
Development of a hydantoin-hydrolysing biocatalyst for the production of optically pure amino acids using Agrobacterium tumefaciens strain RU-ORPN1
- Authors: Foster, Ingrid Margaret
- Date: 2004
- Subjects: Agrobacterium tumefaciens Amino acids Hydantoin Hydrolysis Enzymes
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3943 , http://hdl.handle.net/10962/d1004002
- Description: A calcium alginate bead-immobilised biocatalyst was developed utilising the D-hydantoinase and D-N-carbamoylase from a novel, mutant Agrobacterium tumefaciens strain RU-ORPN1. The growth conditions for the inducer-independent strain were optimised for production of hydantoinase and N-carbamoylase activities. Methods for the preparation of crude enzyme extracts were evaluated in terms of hydantoinase and N-carbamoylase activities produced. After comparison of the enzyme activities and stabilities in various extracts from fresh and frozen cells, sonication of frozen cells for 5 minutes was found to be the best method for the production of the enzyme extract. The optimal pH and temperature for the hydantoinase activity were pH 10 and 30°C, respectively, while pH 9 and 40°C were optimal for Ncarbamoylase activity. The hydantoinase activity was enhanced by the addition of Mg^(2+) ions to the enzyme extract and the N-carbamoylase was enhanced by the addition of Mg^(2+), Mn^(2+) or Zn^(2+) ions to the enzyme extract. The enzyme activities increased in the presence of ATP suggesting that the enzymes may be ATP-dependent. The addition of DTT and PMSF to the enzyme extract enhanced the hydantoinase activity but had no effect on the N-carbamoylase activity. The N-carbamoylase was unstable at 40°C and was almost completely inactivated after 24 hours incubation at this temperature. The hydantoinase and N-carbamoylase appeared to be insoluble. Various techniques were investigated for the solubilisation of the enzymes including various cell lysis methods, cell lysis at extremes of pH and ionic strength, addition of a reducing agent and protease inhibitors, and treatment with hydrolysing enzymes and detergents. Treatment with Triton X-100 was most effective for the solubilisation of the enzymes indicating that the enzymes were membrane-bound. Hydropathy and transmembrane prediction plots of the predicted amino acid sequences for two identified N-carbamoylase genes from A. tumefaciens RU-ORPN1 revealed possible transmembrane regions in the amino acid sequences, and thus supported the hypothesis that the enzymes were membrane-bound. Various methods were evaluated for the immobilisation of the enzymes in whole cells and enzyme extracts. Immobilisation of the enzyme extract in calcium alginate beads was found to be the best method in terms of enzyme activity retention and stability. The hydantoinase retained 55% activity while the N-carbamoylase exhibited a remarkable sevenfold increase in activity after immobilisation by this method. Furthermore, the hydantoinase activity increased after storage at 4°C for 21 days, while the N-carbamoylase retained 30% activity after this storage period. The calcium alginate bead-immobilised enzymes were further biochemically characterised and then applied in a bioreactor system for the production of D-hydroxyphenylglycine (D-HPG) from D,L-5-hydroxyphenylhydantoin (D,L-5-HPH). The pH and temperature optima for the immobilised hydantoinase were pH 7 and 50°C, respectively, while pH 8 and 40°C were optimal for the immobilised N-carbamoylase enzyme. The immobilised enzymes showed improved thermostability at 40°C in comparison to the free enzymes and retained high levels of activity after five repeated batch reactions. Low levels of conversion were obtained in a packed-bed bioreactor containing the A. tumefaciens RU-ORPN1 biocatalyst due to the low hydantoinase activity present in the strain, relative to N-carbamoylase. A novel, packed-bed bioreactor system was therefore developed for the production of D-HPG from D,L-5-HPH using the A. tumefaciens biocatalyst in combination with a Pseudomonas sp. biocatalyst having high hydantoinase activity. A conversion yield of 22 to 30% was achieved for the production of D-HPG from D,L-5-HPH over 5 days operation demonstrating that the hydantoin-hydrolysing enzymes from A. tumefaciens RU-ORPN1 could be stabilised by immobilisation and, in combination with a biocatalyst with high hydantoinase activity, could be applied to the fully enzymatic conversion of D,L-5-HPH to D-HPG.
- Full Text:
- Date Issued: 2004
- Authors: Foster, Ingrid Margaret
- Date: 2004
- Subjects: Agrobacterium tumefaciens Amino acids Hydantoin Hydrolysis Enzymes
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3943 , http://hdl.handle.net/10962/d1004002
- Description: A calcium alginate bead-immobilised biocatalyst was developed utilising the D-hydantoinase and D-N-carbamoylase from a novel, mutant Agrobacterium tumefaciens strain RU-ORPN1. The growth conditions for the inducer-independent strain were optimised for production of hydantoinase and N-carbamoylase activities. Methods for the preparation of crude enzyme extracts were evaluated in terms of hydantoinase and N-carbamoylase activities produced. After comparison of the enzyme activities and stabilities in various extracts from fresh and frozen cells, sonication of frozen cells for 5 minutes was found to be the best method for the production of the enzyme extract. The optimal pH and temperature for the hydantoinase activity were pH 10 and 30°C, respectively, while pH 9 and 40°C were optimal for Ncarbamoylase activity. The hydantoinase activity was enhanced by the addition of Mg^(2+) ions to the enzyme extract and the N-carbamoylase was enhanced by the addition of Mg^(2+), Mn^(2+) or Zn^(2+) ions to the enzyme extract. The enzyme activities increased in the presence of ATP suggesting that the enzymes may be ATP-dependent. The addition of DTT and PMSF to the enzyme extract enhanced the hydantoinase activity but had no effect on the N-carbamoylase activity. The N-carbamoylase was unstable at 40°C and was almost completely inactivated after 24 hours incubation at this temperature. The hydantoinase and N-carbamoylase appeared to be insoluble. Various techniques were investigated for the solubilisation of the enzymes including various cell lysis methods, cell lysis at extremes of pH and ionic strength, addition of a reducing agent and protease inhibitors, and treatment with hydrolysing enzymes and detergents. Treatment with Triton X-100 was most effective for the solubilisation of the enzymes indicating that the enzymes were membrane-bound. Hydropathy and transmembrane prediction plots of the predicted amino acid sequences for two identified N-carbamoylase genes from A. tumefaciens RU-ORPN1 revealed possible transmembrane regions in the amino acid sequences, and thus supported the hypothesis that the enzymes were membrane-bound. Various methods were evaluated for the immobilisation of the enzymes in whole cells and enzyme extracts. Immobilisation of the enzyme extract in calcium alginate beads was found to be the best method in terms of enzyme activity retention and stability. The hydantoinase retained 55% activity while the N-carbamoylase exhibited a remarkable sevenfold increase in activity after immobilisation by this method. Furthermore, the hydantoinase activity increased after storage at 4°C for 21 days, while the N-carbamoylase retained 30% activity after this storage period. The calcium alginate bead-immobilised enzymes were further biochemically characterised and then applied in a bioreactor system for the production of D-hydroxyphenylglycine (D-HPG) from D,L-5-hydroxyphenylhydantoin (D,L-5-HPH). The pH and temperature optima for the immobilised hydantoinase were pH 7 and 50°C, respectively, while pH 8 and 40°C were optimal for the immobilised N-carbamoylase enzyme. The immobilised enzymes showed improved thermostability at 40°C in comparison to the free enzymes and retained high levels of activity after five repeated batch reactions. Low levels of conversion were obtained in a packed-bed bioreactor containing the A. tumefaciens RU-ORPN1 biocatalyst due to the low hydantoinase activity present in the strain, relative to N-carbamoylase. A novel, packed-bed bioreactor system was therefore developed for the production of D-HPG from D,L-5-HPH using the A. tumefaciens biocatalyst in combination with a Pseudomonas sp. biocatalyst having high hydantoinase activity. A conversion yield of 22 to 30% was achieved for the production of D-HPG from D,L-5-HPH over 5 days operation demonstrating that the hydantoin-hydrolysing enzymes from A. tumefaciens RU-ORPN1 could be stabilised by immobilisation and, in combination with a biocatalyst with high hydantoinase activity, could be applied to the fully enzymatic conversion of D,L-5-HPH to D-HPG.
- Full Text:
- Date Issued: 2004
Integrated anaerobic/aerobic bioprocess environments and the biodegradation of complex hydrocarbon wastes
- Authors: Ehlers, George A C
- Date: 2004
- Subjects: Hydrocarbons -- Biodegradation Sewage -- Purification -- Anaerobic treatment Water -- Purification -- Biological treatment Anaerobic bacteria Aerobic bacteria
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4011 , http://hdl.handle.net/10962/d1004071
- Description: An investigation of the biodegradation of complex hydrocarbon wastes, with emphasis on chlorinated aromatic compounds, in an anaerobic/aerobic bioprocess environment was made. A reactor configuration was developed consisting of linked anaerobic and aerobic reactors which served as the model for a proposed bioremediation strategy targeting subterranean soil/sediment/aquifer chlorinated phenol-contaminated environments. Here oxygen is frequently limited and sulphate is readily available, as occurs especially in marine sediment and intertidal habitats. In the anaerobic system the successful transformation and mobilization of the model contaminant, 2,4,6-trichlorophenol, was shown to rely on reductive dechlorination by a sulphate-reducing dependent dechlororespiring co-culture. This was followed in the aerobic system by degradation of the pollutant and its metabolites, 2,4-dichlorophenol, 4-chlorophenol and phenol, by immobilized white-rot fungi.The strategy was initially investigated separately in laboratory bench- and intermediate scale reactors whereafter reactors were linked to simulate the integrated biodegradation strategy. The application of the fungal reactor to treat an actual waste stream by degrading complex mixtures of hydrocarbons in a waste oil recycling effluent was also investigated. The mineralization of phenol and 2,4,6-TCP by immobilized fungal cultures was studied in pinewood chip and foam glass bead-packed trickling reactors. The reactors were operated in sequencing batch format. Removal efficiency increased over time and elevated influent phenol and TCP (800 and 85 mg.L⁻¹) concentrations were degraded by > 98 % in 24 – 30 h batch cycles. Comparable performance between the packing materials was shown. Uptake by the packing was negligible and stripping of compounds induced by aeration had a minimal effect on biodegradation efficiency. Reactor performances are discussed in relation to sequencing batch operation and nutrient requirements necessary to sustain fungal activity in inert vs. organic material packed systems. It was shown that a co-culture consisting of sulphate-reducing and dechlororespiring bacteria established in fed-batch and soil flasks, as well as pine chip-packed fluidized bed reactors. Results showed reductive dechlorination of 2,4,6-TCP to be in strict dependence on the activity of the sulphate-reducing population, sulphate and lactate concentrations. Transformation to 2,4-DCP, 4-CP and phenol was enhanced in sulphate deficient conditions. Dechlororespiring activity was found to be dependent on the fermentative activity of sulphate-reducing bacteria, and the culture was also shown to mobilize and dechlorinate TCP in soils contaminated with the pollutant. Linking the systems achieved degradation of the compound by > 99 % through fungal mineralization of metabolites produced in the dechlororespiring stage of the system. pH correction to the anaerobic reactor was found to be necessary since acidic effluent from the fungal reactor inhibited sulphate reduction and dechlorination. The fungal reactor system was evaluated at intermediate-scale using a complex waste oil recycling effluent. Substantial COD reduction (> 96 % in 48 h batch cycles) and removal of specific effluent hydrocarbon components was shown in diluted, undiluted (COD > 37 g.L⁻¹) and 2,4,6-TCP-spiked effluents. Industrial application of the fungal reactor was evaluated in a 14 m³ pilot plant operated on-site at a waste oil processing plant.
- Full Text:
- Date Issued: 2004
- Authors: Ehlers, George A C
- Date: 2004
- Subjects: Hydrocarbons -- Biodegradation Sewage -- Purification -- Anaerobic treatment Water -- Purification -- Biological treatment Anaerobic bacteria Aerobic bacteria
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4011 , http://hdl.handle.net/10962/d1004071
- Description: An investigation of the biodegradation of complex hydrocarbon wastes, with emphasis on chlorinated aromatic compounds, in an anaerobic/aerobic bioprocess environment was made. A reactor configuration was developed consisting of linked anaerobic and aerobic reactors which served as the model for a proposed bioremediation strategy targeting subterranean soil/sediment/aquifer chlorinated phenol-contaminated environments. Here oxygen is frequently limited and sulphate is readily available, as occurs especially in marine sediment and intertidal habitats. In the anaerobic system the successful transformation and mobilization of the model contaminant, 2,4,6-trichlorophenol, was shown to rely on reductive dechlorination by a sulphate-reducing dependent dechlororespiring co-culture. This was followed in the aerobic system by degradation of the pollutant and its metabolites, 2,4-dichlorophenol, 4-chlorophenol and phenol, by immobilized white-rot fungi.The strategy was initially investigated separately in laboratory bench- and intermediate scale reactors whereafter reactors were linked to simulate the integrated biodegradation strategy. The application of the fungal reactor to treat an actual waste stream by degrading complex mixtures of hydrocarbons in a waste oil recycling effluent was also investigated. The mineralization of phenol and 2,4,6-TCP by immobilized fungal cultures was studied in pinewood chip and foam glass bead-packed trickling reactors. The reactors were operated in sequencing batch format. Removal efficiency increased over time and elevated influent phenol and TCP (800 and 85 mg.L⁻¹) concentrations were degraded by > 98 % in 24 – 30 h batch cycles. Comparable performance between the packing materials was shown. Uptake by the packing was negligible and stripping of compounds induced by aeration had a minimal effect on biodegradation efficiency. Reactor performances are discussed in relation to sequencing batch operation and nutrient requirements necessary to sustain fungal activity in inert vs. organic material packed systems. It was shown that a co-culture consisting of sulphate-reducing and dechlororespiring bacteria established in fed-batch and soil flasks, as well as pine chip-packed fluidized bed reactors. Results showed reductive dechlorination of 2,4,6-TCP to be in strict dependence on the activity of the sulphate-reducing population, sulphate and lactate concentrations. Transformation to 2,4-DCP, 4-CP and phenol was enhanced in sulphate deficient conditions. Dechlororespiring activity was found to be dependent on the fermentative activity of sulphate-reducing bacteria, and the culture was also shown to mobilize and dechlorinate TCP in soils contaminated with the pollutant. Linking the systems achieved degradation of the compound by > 99 % through fungal mineralization of metabolites produced in the dechlororespiring stage of the system. pH correction to the anaerobic reactor was found to be necessary since acidic effluent from the fungal reactor inhibited sulphate reduction and dechlorination. The fungal reactor system was evaluated at intermediate-scale using a complex waste oil recycling effluent. Substantial COD reduction (> 96 % in 48 h batch cycles) and removal of specific effluent hydrocarbon components was shown in diluted, undiluted (COD > 37 g.L⁻¹) and 2,4,6-TCP-spiked effluents. Industrial application of the fungal reactor was evaluated in a 14 m³ pilot plant operated on-site at a waste oil processing plant.
- Full Text:
- Date Issued: 2004
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
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