Isolation of a Clostridium Beijerinckii sLM01 cellulosome and the effect of sulphide on anaerobic digestion
- Authors: Mayende, Lungisa
- Date: 2007
- Subjects: Cellulose , Clostridium , Cellulase , Sulfides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3973 , http://hdl.handle.net/10962/d1004032 , Cellulose , Clostridium , Cellulase , Sulfides
- Description: Cellulose is the most abundant and the most resistant and stable natural organic compound on earth. Enzyme hydrolysis is difficult because of its insolubility and heterogeneity. Some (anaerobic) microorganisms have overcome this by having a multienzyme system called the cellulosome. The aims of the study were to isolate a mesophilic Clostridium sp. from a biosulphidogenic bioreactor, to purify the cellulosome from this culture, to determine the cellulase and endoglucanase activities using Avicel and carboxymethylcellulose (CMC) as substrates and the dinitrosalicyclic (DNS) method. The organism was identified using 16S rDNA sequence analysis. The sequence obtained indicated that a strain of Clostridium beijerinckii was isolated. The cellulosome was purified from the putative C. beijerinckii sLM01 host culture using affinity chromatography purification and affinity digestion purification procedures. The cellulosomal and non-cellulosomal fractions of C. beijerinckii sLM01 were separated successfully, but the majority of the endoglucanase activity was lost during the Sepharose 4B chromatography step. These cellulosomal and non-cellulosomal fractions were characterised with regards to their pH and temperature optima and effector sensitivity. Increased additions of sulphide activated the cellulase activity of the cellulosomal and non-cellulosomal fractions up to 700 %, while increased additions of sulphate either increased the activity slightly or inhibited it dramatically, depending on the cellulosomal and non-cellulosomal fractions. Increased additions of cellobiose, glucose and acetate inhibited the cellulase and endoglucanase activities. pH optima of 5.0 and 7.5 were observed for cellulases and 5.0 for endoglucanases of the cellulosomal fraction. The noncellulosomal fraction exhibited a pH optimum of 7.5 for both cellulase and endoglucanase activities. Both fractions and enzymes exhibited a temperature optimum of 30 °C. The fundamental knowledge gained from the characterisation was applied to anaerobic digestion, where the effect of sulphide on the rate-limiting step was determined. Sulphide activated cellulase and endoglucanase activities and increased the % chemical oxygen demand (COD) removal rate. Levels of volatile fatty acids (VFAs) were higher in the bioreactor containing sulphide, substrate and C. beijerinckii. Sulphide therefore accelerated the rate-limiting step of anaerobic digestion.
- Full Text:
- Date Issued: 2007
- Authors: Mayende, Lungisa
- Date: 2007
- Subjects: Cellulose , Clostridium , Cellulase , Sulfides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3973 , http://hdl.handle.net/10962/d1004032 , Cellulose , Clostridium , Cellulase , Sulfides
- Description: Cellulose is the most abundant and the most resistant and stable natural organic compound on earth. Enzyme hydrolysis is difficult because of its insolubility and heterogeneity. Some (anaerobic) microorganisms have overcome this by having a multienzyme system called the cellulosome. The aims of the study were to isolate a mesophilic Clostridium sp. from a biosulphidogenic bioreactor, to purify the cellulosome from this culture, to determine the cellulase and endoglucanase activities using Avicel and carboxymethylcellulose (CMC) as substrates and the dinitrosalicyclic (DNS) method. The organism was identified using 16S rDNA sequence analysis. The sequence obtained indicated that a strain of Clostridium beijerinckii was isolated. The cellulosome was purified from the putative C. beijerinckii sLM01 host culture using affinity chromatography purification and affinity digestion purification procedures. The cellulosomal and non-cellulosomal fractions of C. beijerinckii sLM01 were separated successfully, but the majority of the endoglucanase activity was lost during the Sepharose 4B chromatography step. These cellulosomal and non-cellulosomal fractions were characterised with regards to their pH and temperature optima and effector sensitivity. Increased additions of sulphide activated the cellulase activity of the cellulosomal and non-cellulosomal fractions up to 700 %, while increased additions of sulphate either increased the activity slightly or inhibited it dramatically, depending on the cellulosomal and non-cellulosomal fractions. Increased additions of cellobiose, glucose and acetate inhibited the cellulase and endoglucanase activities. pH optima of 5.0 and 7.5 were observed for cellulases and 5.0 for endoglucanases of the cellulosomal fraction. The noncellulosomal fraction exhibited a pH optimum of 7.5 for both cellulase and endoglucanase activities. Both fractions and enzymes exhibited a temperature optimum of 30 °C. The fundamental knowledge gained from the characterisation was applied to anaerobic digestion, where the effect of sulphide on the rate-limiting step was determined. Sulphide activated cellulase and endoglucanase activities and increased the % chemical oxygen demand (COD) removal rate. Levels of volatile fatty acids (VFAs) were higher in the bioreactor containing sulphide, substrate and C. beijerinckii. Sulphide therefore accelerated the rate-limiting step of anaerobic digestion.
- Full Text:
- Date Issued: 2007
Biological sulphide oxidation in heterotrophic environments
- Authors: Rein, Neil Berthold
- Date: 2002
- Subjects: Acid mine drainage , Oxidation , Sulfides , Oxidation, Physiological
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3919 , http://hdl.handle.net/10962/d1003978 , Acid mine drainage , Oxidation , Sulfides , Oxidation, Physiological
- Description: Acid mine drainage is a major environmental pollution concern associated with the mining of sulphide-containing ore bodies. Both physicochemical and biological options have been investigated for the treatment of acid mine drainage with recent interest in biological processes targeting low-cost and passive treatment applications. All acid mine drainage biological treatment processes are based to some extent on the activity of sulphate reducing bacteria, and their ability to reduce sulphate to sulphide in the presence of a range of carbon and electron donor sources. A portion of the sulphide produced may be consumed in the precipitation of heavy metals present in the mine drainage. Residual sulphide must be removed, not only due to its toxicity, but especially to prevent its reoxidation to sulphate where salinity reduction is a target of the treatment process. The partial oxidation of sulphide to elemental sulphur is an option that has received considerable attention and both physicochemical and biological options have been investigated. Biological processes have substantial potential cost advantages and run at ambient temperatures and pressures. However, the oxidation of sulphide to elemental sulphur is poised over a narrow redox range and process control to maintain optimum conditions remains a serious problem. In addition little has been reported in the literature on process control of sulphide oxidation to elemental sulphur, in the heterotrophic conditions prevailing in the reaction environment following sulphate reduction. This study undertook an investigation of biological sulphide oxidation under heterotrophic conditions in order to establish the effect of organic compounds on biological sulphide oxidation, and to determine whether the presence of organics, and associated heterotrophic oxygen consumption, may be manipulated to maintain the defined redox conditions required for the production of elemental sulphur. Biological sulphide oxidation under heterotrophic conditions was investigated in a series of flask experiments. Based on these results three different reactor configurations, a Fixed-Film Trickle Filter Reactor, Submerged Fixed-Film Reactor and a Silicone Tubular Reactor were used to investigate sulphur production. The flask studies indicated that organics, and associated heterotrophic metabolism in the presence of excess oxygen in the sulphide oxidation reaction environment, did contribute to the poising of redox conditions and thereby enabling the production of elemental sulphur. While the Fixed-Film Trickle Filter Reactor was found to be redox unstable, probably due to excess oxygen ingress to the system, a reduced oxygen challenge in the Submerged Fixed-Film Reactor configuration was found to be more successful for production of elemental sulphur. However, due to the production of a predominantly filamentous sulphur producing microbial population, recovery of sulphur from the column was intermittent and unpredictable. Extended residence times for produced sulphur on the column increased the likelihood for its eventual oxidation to sulphate. The Silicone Tubular Reactor was found to support a vigorous sulphide oxidising biofilm and produced elemental sulphur effectively. Electron microscopic studies showed that this occurred as both biologically produced sulphur and, probably mainly, as crystalline sulphur in the ortho-rhomic form. Given the linear extension of the sulphur production reaction environment it is was possible to investigate the sequence of the reaction mechanism in grater detail than is possible in mixed systems. Based on these findings a model explaining sulphur production under heterotrophic conditions has been proposed and is presented. The commercial implications of the development have also been noted.
- Full Text:
- Date Issued: 2002
- Authors: Rein, Neil Berthold
- Date: 2002
- Subjects: Acid mine drainage , Oxidation , Sulfides , Oxidation, Physiological
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3919 , http://hdl.handle.net/10962/d1003978 , Acid mine drainage , Oxidation , Sulfides , Oxidation, Physiological
- Description: Acid mine drainage is a major environmental pollution concern associated with the mining of sulphide-containing ore bodies. Both physicochemical and biological options have been investigated for the treatment of acid mine drainage with recent interest in biological processes targeting low-cost and passive treatment applications. All acid mine drainage biological treatment processes are based to some extent on the activity of sulphate reducing bacteria, and their ability to reduce sulphate to sulphide in the presence of a range of carbon and electron donor sources. A portion of the sulphide produced may be consumed in the precipitation of heavy metals present in the mine drainage. Residual sulphide must be removed, not only due to its toxicity, but especially to prevent its reoxidation to sulphate where salinity reduction is a target of the treatment process. The partial oxidation of sulphide to elemental sulphur is an option that has received considerable attention and both physicochemical and biological options have been investigated. Biological processes have substantial potential cost advantages and run at ambient temperatures and pressures. However, the oxidation of sulphide to elemental sulphur is poised over a narrow redox range and process control to maintain optimum conditions remains a serious problem. In addition little has been reported in the literature on process control of sulphide oxidation to elemental sulphur, in the heterotrophic conditions prevailing in the reaction environment following sulphate reduction. This study undertook an investigation of biological sulphide oxidation under heterotrophic conditions in order to establish the effect of organic compounds on biological sulphide oxidation, and to determine whether the presence of organics, and associated heterotrophic oxygen consumption, may be manipulated to maintain the defined redox conditions required for the production of elemental sulphur. Biological sulphide oxidation under heterotrophic conditions was investigated in a series of flask experiments. Based on these results three different reactor configurations, a Fixed-Film Trickle Filter Reactor, Submerged Fixed-Film Reactor and a Silicone Tubular Reactor were used to investigate sulphur production. The flask studies indicated that organics, and associated heterotrophic metabolism in the presence of excess oxygen in the sulphide oxidation reaction environment, did contribute to the poising of redox conditions and thereby enabling the production of elemental sulphur. While the Fixed-Film Trickle Filter Reactor was found to be redox unstable, probably due to excess oxygen ingress to the system, a reduced oxygen challenge in the Submerged Fixed-Film Reactor configuration was found to be more successful for production of elemental sulphur. However, due to the production of a predominantly filamentous sulphur producing microbial population, recovery of sulphur from the column was intermittent and unpredictable. Extended residence times for produced sulphur on the column increased the likelihood for its eventual oxidation to sulphate. The Silicone Tubular Reactor was found to support a vigorous sulphide oxidising biofilm and produced elemental sulphur effectively. Electron microscopic studies showed that this occurred as both biologically produced sulphur and, probably mainly, as crystalline sulphur in the ortho-rhomic form. Given the linear extension of the sulphur production reaction environment it is was possible to investigate the sequence of the reaction mechanism in grater detail than is possible in mixed systems. Based on these findings a model explaining sulphur production under heterotrophic conditions has been proposed and is presented. The commercial implications of the development have also been noted.
- Full Text:
- Date Issued: 2002
The structure and microbiology of floating sulphide oxidising biofilms
- Authors: Gilfillan, Joanne Criseyde
- Date: 2000
- Subjects: Biofilms , Sulfides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3903 , http://hdl.handle.net/10962/d1003962 , Biofilms , Sulfides
- Description: Floating sulphur biofilms are observed as surface layers in numerous aquatic sulphide-rich environments, and apparently play an important role in the cycling of sulphur in its various oxidation states. In addition to the conversion of sulphide to sulphur and/or sulphate species, it has been suspected that subsequent reduction back to sulphide may occur within the floating sulphur biofi1m in organic-rich environments. The use of sulphur biofilms for the harvesting of elemental sulphur from wastewater treatment systems has also been suggested. There is, however, little documented information in the literature on the structure of floating sulphur biofilms, or the microbial species responsible for their occurrence. In this study, floating sulphur biofilms were generated in a continuous flow baflle reactor and their structure was examined using scanning electron microscopy. It was found that they occur as layered structures with morphologically distinct bacterial forms present in different layers of the biofilm. The biofilpl structure was also found to be dynamic, with structural changes observed as feed conditions were altered. An enriched culture derived from the biofi1m demonstrated rates of sulphide oxidation comparable to values reported in the literature for liquid culture systems. The microbiology of the biofi1m was studied using traditional plate culture techniques and analysis ofrRNA genes. Identification of plate culture isolates as representatives of the biofi1m community proved to be limited, leading to a PeR-based cloning approach. The majority of the organisms present in the sulphur biofi1m were classified as species in the genus ~eudomonas, and a number of other bacterial species whose sulphide oxidising capacity has been noted previously. Surprisingly, only 2% of the clone library consisted of Thiobacillus spp., and no sulphate reducing bacteria were identified in the biofilm at all. These results indicate that in organic sulphide-rich environments facultative chemolithoheterotrophic bacterial forms predominate in floating sulphur biofilms, and that the complete biological cycling of sulphur may not occur in these systems.
- Full Text:
- Date Issued: 2000
- Authors: Gilfillan, Joanne Criseyde
- Date: 2000
- Subjects: Biofilms , Sulfides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3903 , http://hdl.handle.net/10962/d1003962 , Biofilms , Sulfides
- Description: Floating sulphur biofilms are observed as surface layers in numerous aquatic sulphide-rich environments, and apparently play an important role in the cycling of sulphur in its various oxidation states. In addition to the conversion of sulphide to sulphur and/or sulphate species, it has been suspected that subsequent reduction back to sulphide may occur within the floating sulphur biofi1m in organic-rich environments. The use of sulphur biofilms for the harvesting of elemental sulphur from wastewater treatment systems has also been suggested. There is, however, little documented information in the literature on the structure of floating sulphur biofilms, or the microbial species responsible for their occurrence. In this study, floating sulphur biofilms were generated in a continuous flow baflle reactor and their structure was examined using scanning electron microscopy. It was found that they occur as layered structures with morphologically distinct bacterial forms present in different layers of the biofilm. The biofilpl structure was also found to be dynamic, with structural changes observed as feed conditions were altered. An enriched culture derived from the biofi1m demonstrated rates of sulphide oxidation comparable to values reported in the literature for liquid culture systems. The microbiology of the biofi1m was studied using traditional plate culture techniques and analysis ofrRNA genes. Identification of plate culture isolates as representatives of the biofi1m community proved to be limited, leading to a PeR-based cloning approach. The majority of the organisms present in the sulphur biofi1m were classified as species in the genus ~eudomonas, and a number of other bacterial species whose sulphide oxidising capacity has been noted previously. Surprisingly, only 2% of the clone library consisted of Thiobacillus spp., and no sulphate reducing bacteria were identified in the biofilm at all. These results indicate that in organic sulphide-rich environments facultative chemolithoheterotrophic bacterial forms predominate in floating sulphur biofilms, and that the complete biological cycling of sulphur may not occur in these systems.
- Full Text:
- Date Issued: 2000
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