The degradation of lignocellulose in a biologically-generated sulphidic environment
- Authors: Roman, Henry James
- Date: 2005
- Subjects: Lignocellulose Sulfides Lignin Lignocellulose -- Biodegradation Mines and mineral resources -- Waste disposal Acid mine drainage
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3997 , http://hdl.handle.net/10962/d1004057
- Description: South Africa is renowned for its mining industry. The period over which the polluted waters from the existing and abandoned mines will require treatment has driven research into the development of passive treatment systems. These waters are characterised by a low pH, high concentrations of heavy metals, high levels of sulphate salts and low concentrations of organic material. The biological treatment of these waters has been a subject of increasing focus as an alternative to physicochemical treatment. The utilisation of lignocellulose as a carbon source has been restricted by the amount of reducing equivalents available within the lignocellulose matrix. After a few months of near 100% sulphate reduction, it was found that although there was a large fraction of lignin and cellulose remaining, sulphate reduction was reduced to less than 20%. The present study demonstrated that lignocellulose can be utilised as a carbon source for sulphate reduction. It was established that lignocellulose degradation was enhanced under biosulphidogenic conditions and that lignin could be degraded by a sulphate reducing microbial consortium. It was established using lignin model compounds synthesized in our laboratory, that the bonds within the lignin polymer can be cleaved within the sulphidic environment. The presence of cellulolytic enzymes, using CMCase as a marker enzyme, was detected within the sulphate reducing microbial consortium. Based on the results obtained a descriptive model was formulated for the degradation of lignocellulose under biosulphidogenic conditions. It was determined that the initial reduction in sulphate observed using lignocellulose as a carbon source was due to the easily extractable components. The degradation of which resulted in the production of sulphide, which aided in the degradation of lignin, allowing greater access to cellulose. Once the easily extractable material is exhausted, the cycle is halted, unless the sulphide production can be maintained. This is the focus of an ongoing project, testing the hypothesis that an easy to assimilate carbon source added after exhaustion of the easily extractable material, can maintain the sulphide production.
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- Date Issued: 2005
Ostrich calpastatin purification and partial characterization of the liver inhibitor
- Authors: Roman, Henry James
- Date: 2000
- Subjects: Calpastatin , Protease inhibitors , Ion exchange chromatography , Ostriches
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:11090 , http://hdl.handle.net/10948/d1015522
- Description: The isolation and purification of calpastatin from ostrich liver is presented, along with its physicochemical and kinetic properties. By using extraction from liver, ion-exchange chromatography on DEAE-Toyopearl, heating to 90 °C for 10 min and rechromatography on Toyopearl Super-Q 650 S, ostrich calpastatin was isolated and purified from ostrich liver. The purified intact calpastatin showed homogeneity on SDS-PAGE (Mr of 105.6 K). Amino acid analysis showed that ostrich calpastatin resembled that of rabbit liver and human erythrocyte calpastatin. An N-terminal sequence could not be obtained because the N-terminus was found to be blocked by an as yet unknown amino acid residue. The Mr values of degradative forms of ostrich liver calpastatin were determined to be 56 K and 90 K. By using PAG-IEF the pI of the intact form was determined to be 5.1. Ostrich liver calpastatin behaved characteristically like other calpastatins during kinetic analysis. Calpastatin inhibited calpain from pH 6 to 9 and was found to be unaffected by temperatures as high as 100 °C. Calpastatin also inhibited calpain activity at Ca2+ concentrations ranging from 1 to 10 mM. The inhibitor was shown to be phosphorylated because after incubation with alkaline phosphatase there was a decrease in inhibitory activity. No inhibitory effects were detected against other proteases such as chymotrypsin and trypsin, with both proteases inactivating calpastatin completely. Ostrich liver calpain was shown to have a pH optimum of 7.5 and a temperature optimum of 30 °C. In terms of its thermodynamic properties it resembled that of other ostrich proteases; DH, DS and DG being 47.07 kJ/mol, -91.1 J/mol/K and 74.237 kJ/mol, respectively. Ostrich liver calpain showed a Km of 0.14 % (w/v). The enzyme was active at both milli- and micro-molar concentrations of Ca2+. Ostrich liver calpastatin showed many physical, chemical and kinetic properties similar to those of other known calpastatins.
- Full Text: false
- Date Issued: 2000