Effects of BT Maize (MON810) crop and its residues on selected soil biological properties and N and P release in a sandy loam soil from Alice, Eastern Cape, South Africa
- Authors: Landzela, Besule
- Date: 2013
- Subjects: Bacillus (Bacteria) , Bacillus thuringiensis , Corn -- Planting , Biomass , Plant proteins , Enzymes , Soil fertility , Crop residue management
- Language: English
- Type: Thesis , Masters , MSc Agric (Crop Science)
- Identifier: vital:11870 , http://hdl.handle.net/10353/d1007542 , Bacillus (Bacteria) , Bacillus thuringiensis , Corn -- Planting , Biomass , Plant proteins , Enzymes , Soil fertility , Crop residue management
- Description: There are apprehensions that genetic modification of maize with Bacillus thuringiensis (Bt) may have negative effects on soil biodiversity, ecosystem processes and functions. This study aimed at determining the effect of Bt maize crop, Bt maize residues and its genetic modification on microbial biomass carbon (MBC), selected enzyme activities, vesicular arbuscular mycorrhizal (VAM) fungi and N and P release patterns. The study was conducted under field, glasshouse and laboratory conditions. In 2010/2011 season, four maize cultivars; DKC 61-25B (Bt), DKC 61-24 (non-Bt), PAN 6Q-321B (Bt) and PAN6777 (non-Bt) were planted. Determination of MBC, enzyme activities and fungal spore count was done at 42, 70, and 105 days after planting (DAP). A loam soil amended with Bt or non-Bt maize leaf residues from a study of 2009/2010 season was incubated to investigate effects of Bt maize residues on MBC and soil enzyme activities. Leaf residues of Bt and non-Bt maize cultivars (DKC 61-25B, DKC 61-24, PAN 6Q-321B and PAN6777) were used and soil without residues was used as a control. Samples were collected at 7, 28 and 56 days of incubation (DOI). An incubation study was also carried out in the laboratory to determine the effect of Bt maize residues (i.e. leaf, stem and root) and its genetic modification on N and P release patterns. Residues of DKC 61-25B, DKC 61-24, PAN 6Q-321B and PAN6777and soil without residues as a control were incubated in the laboratory. After destructive sampling at 0, 7, 14, 28, and 56 DOI, N in the form of NH4-N and NO3-N and P mineralisation were determined. Amendment of soil with residues enhanced MBC (p < 0.05) at all the sampling dates. For example MBC increased from 95 in the control to 146.3 mg/kg in the DKC 61-25B treatment at the end of the glasshouse trial. In the field DKC 61-25B had 9.1 mg/kg greater MBC than DKC 61-24, while PAN 6Q-321B had 23.9 mg/kg more MBC than PAN6777 at the end of the trial. However, no differences (p < 0.05) were observed in enzyme activities under field and glasshouse conditions except for dehydrogenase that had greater activity where DKC 61-25B and PAN 6777 were grown. There were no differences between the type of residues (Bt and non-Bt) on enzyme activities tested. However, differences were observed among the sampling dates. No effects of Bt maize crop on fungal spore count were observed. Similarly no differences were observed in leaf, stem and root tissues composition between Bt and non-Bt maize cultivars. Net N and P mineralisation from Bt maize cultivars did not differ from that of non-Bt maize cultivars. However, differences were observed among the cultivars. The results of this study suggested that Bt maize with Bt MON810 event can be grown in the central region of the Eastern Cape (EC), South Africa without affecting MBC, soil enzyme activities, VAM, and release of N and P nutrients from its residues.
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- Date Issued: 2013
Enzymatic recovery of rhodium(III) from aqueous solution and industrial effluent using sulphate reducing bacteria: role of a hydrogenase enzyme
- Authors: Ngwenya, Nonhlanhla
- Date: 2005
- Subjects: Enzymes , Rhodium , Enzymes -- Industrial applications , Sulfur bacteria , Hydrogenation , Hydragenase , Factory and trade waste -- Purification
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3956 , http://hdl.handle.net/10962/d1004015 , Enzymes , Rhodium , Enzymes -- Industrial applications , Sulfur bacteria , Hydrogenation , Hydragenase , Factory and trade waste -- Purification
- Description: In an attempt to overcome the high maintenance and costs associated with traditional physico-chemical methods, much work is being done on the application of enzymes for the recovery of valuable metals from solutions and industrial effluents. One of the most widely studied enzymatic metal recovery systems uses hydrogenase enzymes, particularly from sulphate reducing bacteria (SRB). While it is known that hydrogenases from SRB mediate the reductive precipitation of metals, the mechanism of enzymatic reduction, however, is not yet fully understood. The main aim of the present study was to investigate the role of a hydrogenase enzyme in the removal of rhodium from both aqueous solution and industrial effluent. A quantitative analysis of the rate of removal of rhodium(III) by a resting SRB consortium under different initial rhodium and biomass concentrations, pH, temperature, presence and absence of SRB cells and electron donor, was studied. Rhodium speciation was found to be the main factor controlling the rate of removal of rhodium from solution. SRB cells were found to have a higher affinity for anionic rhodium species, as compared to both cationic and neutral species, which become abundant when speciation equilibrium was reached. Consequently, a pH-dependant rate of rhodium removal from solution was observed. The maximum SRB uptake capacity for rhodium was found to be 66 mg rhodium per g of resting SRB biomass. Electron microscopy studies revealed a time-dependant localization and distribution of rhodium precipitates, initially intracellularly and then extracellularly, suggesting the involvement of an enzymatic reductive precipitation process. A hydrogenase enzyme capable of reducing rhodium(III) from solution was isolated and purified by PEG, DEAE-Sephacel anion exchanger and Sephadex G200 gel exclusion. A distinct protein band with a molecular weight of 62kDa was obtained when the hydrogenase containing fractions were subjected to a 10% SDS-PAGE. Characterization studies indicated that the purified hydrogenase had an optimum pH and temperature of 8 and 40°C, respectively. A maximum of 88% of the initial rhodium in solution was removed when the purified hydrogenase was incubated under hydrogen. Due to the low pH of the industrial effluent (1.31), the enzymatic reduction of rhodium by the purified hydrogenase was greatly retarded. It was apparent that industrial effluent pretreatment was necessary before the application an enzymatic treatment method. In the present study, however, it has been established that SRB are good candidates for the enzymatic recovery of rhodium from both solution and effluent.
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- Date Issued: 2005
Enzymes with biocatalytic potential from Sorghum bicolor
- Authors: Nganwa, Patience Jennifer Kengyeya
- Date: 2000
- Subjects: Enzymes , Sorghum
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3908 , http://hdl.handle.net/10962/d1003967 , Enzymes , Sorghum
- Description: Sorghum is a staple food in the semi-arid tropics of Asia and Africa, sustaining the lives of the poorest rural people. This project set out to improve the potential economic value of Sorghum bicolor as a crop. The task was undertaken by screening for selected enzymes in the plant that would have a potential market for use in industrial applications and in biotransformations, specifically proteases, polyphenol oxidases and peroxidases. Asurveywas conducted using standard enzyme assays and crude plant extracts, to determine whether the selected enzymes were present. Grain tissue did not appear to have significant protease or polyphenoloxidase activity, but high levels of peroxidases were detected, withthe young grain extracts showing more activity(4.63U/mL)thanripegrain extracts (0.62 U/mL). Leaf tissue extracts contained low levels of protease activity, a considerable amount of polyphenol oxidase (0.127 U/mL), and peroxidase (4.7 U/mL) activities comparable with that found in grain tissue. Root tissue extract was found to contain the highest levels of peroxidase activity (7.8 U/mL) compared to the other extracts. Therefore, sorghum peroxidase from the root was isolated, purified, characterized and applied to biotransformation reactions. Different sorghum strains,withvaryinggraincolour, (Zimbabwe - bronze, Seredo - brown and Epurpur - cream/white) were investigated for the presence of polyphenol oxidase and peroxidase activities. Results of spectrophotometric analysis showed that the enzymes did not appear to be strain specific. However, gel electrophoresis analysis revealed differences in band patterns among the strains. Partial purification of sorghum root peroxidase was achieved after centrifugation, extraction with polyvinylpolypyrrolidone (PVPP), ultrafiltration, and hydrophobic chromatography with phenyl Sepharose, followed by polyacrylamidegelelectrophoresis (PAGE). The specific activity of the 5-fold purified enzyme was found to be 122.3 U/mg. After PAGE analysis, two bands with molecular weights of approximately 30 000 and 40 000 were detected, which compares well with horse radish peroxidase (HRP) which has a molecular weight of approximately 44 000. The colour intensity of the bands in the activity gels indicated that sorghum root peroxidase had apparently higher levels of peroxidase activity than commercial horseradish peroxidase (HRP). Characterizationexperiments revealed that sorghumroot peroxidase is active over a broad temperature range and remains active at temperatures up to 100°C. It also has a broad substrate range. The optimum pH of the enzyme was found to be pH 5 - 6. Under standardized assay conditions, the optimal substrate concentration, using o-dianisidine as substrate, was 50 mM, and the optimal H2O2 concentration under these conditions was found to be 100 mM. Sorghum root peroxidase was applied in a preliminary investigation into the oxidative biotransformationof a number of aromatic compounds. The products obtained were comparable withthose whenthe compounds are reacted with HRP which is the most commonly used commercial peroxidase and has been extensively studied. However, HRP is relatively costly, and the use of peroxidase from sorghum roots as an alternative source, appears to be promising. A patent has been provisionally registered, covering application of sorghum root peroxidase for biotransformations.
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- Date Issued: 2000
Evaluation of a 'defouling on demand' strategy for the ultrafiltration of brown water using activatable enzymes
- Authors: Buchanan, K
- Date: 1999
- Subjects: Water -- Purification , Ultrafiltration , Enzymes , Membranes (Technology)
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3904 , http://hdl.handle.net/10962/d1003963 , Water -- Purification , Ultrafiltration , Enzymes , Membranes (Technology)
- Description: New approaches to the application of membranes for the production of potable water are constantly being sought after in anticipation of future demands for increasingly rigorous water quality standards and reduced environmental impact. A major limitation, however, is membrane fouling, which manifests itself as a continual reduction in flux over time and thus restricts the practical implementation to restore flux. Mechanical and chemical methods have been implemented to restore flux to ultrafiltration systems, but these either result in a break in the process operation or lead to membrane damage or additional pollution problems. This project was aimed to develop a 'defouling on demand' stategy for cleaning membranes used during brown water ultrafiltration. The process involves the use of activatable peroxidase enzymes, which were immobilised onto flat sheet polysulphone membranes. Following flux decline which reaches a critical level with the build-up of the foulant layer, the immobilised enzyme layer was activated by the addition of a chemical activator solution, in this case hydrogen peroxidase and manganous sulphate. Manganese peroxidase was found to be the most effective enzyme at alleviating fouling by degrading the foulant layer formed on the membrane surface and hence restored flux to the ultrafiltration system. A 93% flux improvement was observed when manganese peroxidase was activated when 800uM manganous sulphate, 100mM hydrogen peroxide were added in the presence of a manganese chelator, lactate. The concept and the potential benefits this system holds will be discussed in further detail.
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- Date Issued: 1999
The isolation and characterisation of thermostable hydantoinases from hydantoinase-producing bacteria
- Authors: Phehane, Vuyisile Ntosi
- Date: 1999
- Subjects: Hydantoin , Bacteria -- Physiology , Enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3998 , http://hdl.handle.net/10962/d1004058 , Hydantoin , Bacteria -- Physiology , Enzymes
- Description: In order to characterise thermostable hydantoin-hydrolysing enzymes from bacteria, locally-isolated thermophilic organisms were screened for the ability to convert hydantoin to N-carbamylglycine at 55°C using the hydantoinase enzyme. Cell disruption of a selected strain, RU-20-15, was conducted by French pressing to release enzyme from within the cell. In all of the experiments conducted, the amounts of product were low. In view of the low yields of products formed by the thermophiles, a previously-isolated Gram negative strain, RU-KM3L was selected from a number of mesophiles by screening for hydantoinase and carbamylase activity over a 40-55°C temperature range. Hydantoin conversion at 40°C using crude extract from pressed cells of this organism was similar to conversion at 50°C, and therefore subsequent assays were conducted at the higher temperature. The growth kinetics of RU-KM3L cells were studied and the enzyme activities of the extracts were compared in complete and chemically-defined media. The results suggested that the optimal time to harvest cells was at early stationary phase, when using complete medium for culture of cells; the specific activity of enzyme extracts produced by culture in complete medium was higher than that obtained in chemically-defined medium. 5-methylhydantoin was shown to be the preferred substrate for both the hydantoinase and carbamylase enzymes in the crude extract of RU-KM3L. The substrate specificity of the hydantoinase and carbamylase enzymes of the crude RU-KM3L extract was observed to be altered in the presence of increasing amounts of hydantoin, 5,5-dihydrouracil (DHU) and 5-thiouracil (TU) as inducers, showing selectivity for 5-methylhydantoin over hydantoin at inducer concentrations of 0.1 to 1%. A limiting effect on the hydrolysis of 5-methylhydantoin was observed when DHU and 5,5-dimethylhydantoin (DMH) were used as inducers, while the limiting effect on hydantoin specificity was observed when DHU and TU were used as inducers. The limiting effect was observed to be dependent upon the concentration of inducer, and was not observed when hydantoin was used as an inducer. The optimal time for assay of the hydantoinase enzyme in crude extract preparations at 50°C was observed to be 3h. Alkaline conditions were shown to be optimal for both the hydantoinase and carbamylase enzymes of RU-KM3L. Assay for enzyme activities of RU-KM3L extract in the presence of metal ions showed Mn²⁺ ions (and to a lesser extent, Co²⁺) to activate both the hydantoinase and carbamylase activities. Cu²⁺ ions were observed to inhibit the hydantoinase enzyme. In order to determine the location of the enzymes within the cell, cell debris from disrupted cells of RU-KM3L was removed by centrifugation. A decrease in enzyme activity in the supernatant was observed, and suggested association of the enzymes with the cell membrane. Ammonium sulfate fractionation experiments conducted on the crude extract provided further evidence for this result. Sonication of the crude enzyme extract was the only successful method for the releasing of membrane-associated enzyme. Of a number of strategies investigated, the use of sucrose at 50% (w/v) concentration was shown to preserve the hydantoinase and carbamylase enzyme activities during lyophilisation. Furthermore, assay for these enzyme activities showed the activities to be higher after lyophilisation in the presence of sucrose. However, sucrose did not increase the thermostability of lyophilised crude enzyme extracts. Water-miscible organic solvents at 1% concentration were shown to be inhibitory to the hydantoinase and carbamylase enzymes of RU-KM3L, and the inhibition was also observed to increase with increasing concentrations of these solvents. Hydantoinase activity in the presence of water-immiscible organic solvents was shown to increase with an increase in the hydrophobicity of these solvents, but the activity observed was not significantly higher than activity in the absence of solvent when hydantoin and 5-methylhydantoin were used as substrates. The possibility of reversing the hydantoinase enzyme reaction by water-immiscible organic solvents was investigated, and the results obtained suggested that the reaction could be reversed. It was thought that the partitioning of substrates or products into hydrophobic organic solvents could influence the reaction equilibrium, but the partitioning observed was not sufficient to affect reaction rates. Peptide synthesis was shown to have occurred in small amounts when the hydantoinase reaction was carried out in the presence of water-immiscible organic solvents. In conclusion, the hydantoin-hydrolyzing enzyme activity of a crude extract preparation from the bacterial strain RU-KM3L was characterised at elevated temperatures, and in the presence of watermiscible and -immiscible organic solvents.
- Full Text:
- Date Issued: 1999