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
Stratigraphy and geochemistry of the Makganyene formation, Transvaal supergroup, Northern Cape, South Africa
- Authors: Polteau, Stéphane
- Date: 2001
- Subjects: Geology, Stratigraphic -- South Africa -- Northern Cape , Geochemistry -- South Africa -- Northern Cape
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5003 , http://hdl.handle.net/10962/d1005616 , Geology, Stratigraphic -- South Africa -- Northern Cape , Geochemistry -- South Africa -- Northern Cape
- Description: The Makganyene Formation forms the base of the Postmasburg Group in the Transvaal Supergroup of the Northern Cape Province. The Makganyene Formation has diamictite as the main rock type, but siltstone, sandstone, shale, and iron-formations are also present. A glacial origin has been proposed in the past due to the presence of dropstones, faceted and striated pebbles. Typically, the Makganyene Formation contains banded iron-formations interbedded with clastic rocks (shale, siltstone, sandstone and diamictites) at the contact with the underlying iron-formations. This transitional zone is generally overlain by massive or layered diamictites which contain poorly sorted clasts (mainly chert) within a shaly matrix. Striated pebbles have been found during field work, and dropstones have been observed in diamictites and banded iron-formations during the study. The top of the Makganyene Formation contains graded cycles interbedded with diamictites and thin layers of andesitic lavas from the Ongeluk Formation. The basal contact of the Makganyene Formation with the underlying Koegas Subgroup was described as unconformable by previous workers. However field work localised in the Rooinekke area shows a broadly conformable and interbedded contact with the underlying Koegas Subgroup. As described above, banded iron-formations are interbedded with the clastic rocks of the Makganyene Formation. Moreover, boreholes from the Sishen area display the same interbedding at the base of the Makganyene Formation. This suggests that no significant time gap is present in the whole succession between the Ghaap and Postmasburg Group. The Transvaal Supergroup in the Northern Cape displays the following succession : carbonates-BIFs-diamictites/ lava-BIFs-carbonates. The Makganyene Formation is thus at the centre of a symmetrical lithologic succession. Bulk rock compositions show that the diamictites have a similar composition to banded iron-formation with regard to their major element contents. Banded iron-formations acted as a source for the diamictites with carbonates and igneous rocks representing minor components. Differences in bulk composition between the Sishen and Matsap areas emphasize that the source of the diamictite was very localised. The Chemical Index of Alteration (CIA) has been calculated, but since the source dominant rock was iron-formation, this index cannot be usefully applied to the diamictites. ACN, A-CN-K, and A-CNK-FM diagrams confer a major importance in sorting processes due to the separation between the fine and coarse diamictites. The interbedded iron-formations display little clastic contamination indicating deposition in clear water conditions. However, dropstones are present in one borehole from the Matsap area, indicating that iron-formation took place under ice cover, or at least under icebergs. Stable isotope studies show that the iron-formations, interbedded towards the base of the Makganyene Formation, have similar values to the iron-formations of the Koegas Subgroup. As a result of the above observations, new correlations are proposed in this study, relating the different Transvaal Supergroup basins located on the Kaapvaal Craton. The Pretoria Group of the Transvaal Basin has no correlative in the Griqualand West Basin, and the Postmasburg Group of the Northern Cape Basin has no lateral equivalent in the Transvaal Basin. These changes have been made to overcome problems present in the current correlations between those two basins. The Makganyene Formation correlates with the Huronian glaciations which occurred between 2.4 and 2.2 Ga ago in North America. Another Precambrian glaciation is the worldwide and well-studied Neoproterozoic glaciation (640 Ma). At each of these glaciations, major banded iron-formation deposition took place with associated deposition of sedimentary manganese in post-glacial positions. The central position of the Makganyene Formation within the Transvaal Supergroup in the Northern Cape emphasizes this glacial climatic dependence of paleoproterozoic banded iron-formation and manganese deposition. However these two Precambrian glaciations are interpreted in paleomagnetic studies as having occurred near to the equator. The controversial theory of the Snowball Earth has been proposed which proposes that the Earth was entirely frozen from pole to pole. Results from field work, sedimentology, petrography and geochemistry were integrated in a proposed depositional model of the Makganyene Formation occurring at the symmetrical centre of the lithologic succession of the Transvaal Supergroup. At the beginning of the Makganyene glaciation, a regression occurred and glacial advance took place. The diamictites are mostly interpreted as being deposited from wet-based glaciers, probably tidewater glaciers, where significant slumping and debris flows occurred. Any transgression would cause a glacial retreat by rapid calving, re-establishing the chemical sedimentation of banded iron-formations. These sea-level variations are responsible for the interbedding of these different types of rocks (clastic and chemical). The end of the Makganyene glacial event is characterised by subaerial eruptions of andesitic lava of the Ongeluk Formation bringing ashes into the basin. Banded iron-formation and associated manganese accumulations are climate-dependant. Glacial events are responsible for the build up of metallic ions such as iron and manganese in solution in deep waters. A warmer climate would induce a transgression and precipitation of these metallic ions when Eh conditions are favourable. In the Transvaal Supergroup, the climatic variations from warm to cold, and cold to warm are expressed by the lithologic succession. The warm climates are represented by carbonates. Cold climates are represented by banded iron-formations and the peak in cold climate represented by the diamictites of the Makganyene Formation. These changes in climate are gradual, which contradict the dramatic Snowball Earth event: a rapid spread of glaciated areas over low-latitudes freezing the Earth from pole-to-pole. Therefore, to explain low-latitude glaciations at sea-level, a high obliquity of the ecliptic is most likely to have occurred. This high obliquity of the ecliptic was acquired at 4.5 Ga when a giant impactor collided into the Earth to form the Moon. Above the critical value of 54° of the obliquity of the ecliptic, normal climatic zonation reverts, and glaciations will take place preferentially at low-latitudes only when favourable conditions are gathered (relative position ofthe continents and PC02 in the atmosphere).
- Full Text:
- Date Issued: 2001
- Authors: Polteau, Stéphane
- Date: 2001
- Subjects: Geology, Stratigraphic -- South Africa -- Northern Cape , Geochemistry -- South Africa -- Northern Cape
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5003 , http://hdl.handle.net/10962/d1005616 , Geology, Stratigraphic -- South Africa -- Northern Cape , Geochemistry -- South Africa -- Northern Cape
- Description: The Makganyene Formation forms the base of the Postmasburg Group in the Transvaal Supergroup of the Northern Cape Province. The Makganyene Formation has diamictite as the main rock type, but siltstone, sandstone, shale, and iron-formations are also present. A glacial origin has been proposed in the past due to the presence of dropstones, faceted and striated pebbles. Typically, the Makganyene Formation contains banded iron-formations interbedded with clastic rocks (shale, siltstone, sandstone and diamictites) at the contact with the underlying iron-formations. This transitional zone is generally overlain by massive or layered diamictites which contain poorly sorted clasts (mainly chert) within a shaly matrix. Striated pebbles have been found during field work, and dropstones have been observed in diamictites and banded iron-formations during the study. The top of the Makganyene Formation contains graded cycles interbedded with diamictites and thin layers of andesitic lavas from the Ongeluk Formation. The basal contact of the Makganyene Formation with the underlying Koegas Subgroup was described as unconformable by previous workers. However field work localised in the Rooinekke area shows a broadly conformable and interbedded contact with the underlying Koegas Subgroup. As described above, banded iron-formations are interbedded with the clastic rocks of the Makganyene Formation. Moreover, boreholes from the Sishen area display the same interbedding at the base of the Makganyene Formation. This suggests that no significant time gap is present in the whole succession between the Ghaap and Postmasburg Group. The Transvaal Supergroup in the Northern Cape displays the following succession : carbonates-BIFs-diamictites/ lava-BIFs-carbonates. The Makganyene Formation is thus at the centre of a symmetrical lithologic succession. Bulk rock compositions show that the diamictites have a similar composition to banded iron-formation with regard to their major element contents. Banded iron-formations acted as a source for the diamictites with carbonates and igneous rocks representing minor components. Differences in bulk composition between the Sishen and Matsap areas emphasize that the source of the diamictite was very localised. The Chemical Index of Alteration (CIA) has been calculated, but since the source dominant rock was iron-formation, this index cannot be usefully applied to the diamictites. ACN, A-CN-K, and A-CNK-FM diagrams confer a major importance in sorting processes due to the separation between the fine and coarse diamictites. The interbedded iron-formations display little clastic contamination indicating deposition in clear water conditions. However, dropstones are present in one borehole from the Matsap area, indicating that iron-formation took place under ice cover, or at least under icebergs. Stable isotope studies show that the iron-formations, interbedded towards the base of the Makganyene Formation, have similar values to the iron-formations of the Koegas Subgroup. As a result of the above observations, new correlations are proposed in this study, relating the different Transvaal Supergroup basins located on the Kaapvaal Craton. The Pretoria Group of the Transvaal Basin has no correlative in the Griqualand West Basin, and the Postmasburg Group of the Northern Cape Basin has no lateral equivalent in the Transvaal Basin. These changes have been made to overcome problems present in the current correlations between those two basins. The Makganyene Formation correlates with the Huronian glaciations which occurred between 2.4 and 2.2 Ga ago in North America. Another Precambrian glaciation is the worldwide and well-studied Neoproterozoic glaciation (640 Ma). At each of these glaciations, major banded iron-formation deposition took place with associated deposition of sedimentary manganese in post-glacial positions. The central position of the Makganyene Formation within the Transvaal Supergroup in the Northern Cape emphasizes this glacial climatic dependence of paleoproterozoic banded iron-formation and manganese deposition. However these two Precambrian glaciations are interpreted in paleomagnetic studies as having occurred near to the equator. The controversial theory of the Snowball Earth has been proposed which proposes that the Earth was entirely frozen from pole to pole. Results from field work, sedimentology, petrography and geochemistry were integrated in a proposed depositional model of the Makganyene Formation occurring at the symmetrical centre of the lithologic succession of the Transvaal Supergroup. At the beginning of the Makganyene glaciation, a regression occurred and glacial advance took place. The diamictites are mostly interpreted as being deposited from wet-based glaciers, probably tidewater glaciers, where significant slumping and debris flows occurred. Any transgression would cause a glacial retreat by rapid calving, re-establishing the chemical sedimentation of banded iron-formations. These sea-level variations are responsible for the interbedding of these different types of rocks (clastic and chemical). The end of the Makganyene glacial event is characterised by subaerial eruptions of andesitic lava of the Ongeluk Formation bringing ashes into the basin. Banded iron-formation and associated manganese accumulations are climate-dependant. Glacial events are responsible for the build up of metallic ions such as iron and manganese in solution in deep waters. A warmer climate would induce a transgression and precipitation of these metallic ions when Eh conditions are favourable. In the Transvaal Supergroup, the climatic variations from warm to cold, and cold to warm are expressed by the lithologic succession. The warm climates are represented by carbonates. Cold climates are represented by banded iron-formations and the peak in cold climate represented by the diamictites of the Makganyene Formation. These changes in climate are gradual, which contradict the dramatic Snowball Earth event: a rapid spread of glaciated areas over low-latitudes freezing the Earth from pole-to-pole. Therefore, to explain low-latitude glaciations at sea-level, a high obliquity of the ecliptic is most likely to have occurred. This high obliquity of the ecliptic was acquired at 4.5 Ga when a giant impactor collided into the Earth to form the Moon. Above the critical value of 54° of the obliquity of the ecliptic, normal climatic zonation reverts, and glaciations will take place preferentially at low-latitudes only when favourable conditions are gathered (relative position ofthe continents and PC02 in the atmosphere).
- Full Text:
- Date Issued: 2001
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