Peter Dale Rose

A capillary membrane bioreactor using immobilized polyphenol oxidase for the removal of phenols from industrial effluents

Abstract A capillary membrane bioreactor has been developed and tested for the removal of phenolic compounds from synthetic and industrial effluents. Polyphenol oxidase was immobilized on single capillary membranes in a small-scale bioreactor using two morphologically different polymeric membranes. One has a novel structure with no external supporting skin layer. This membrane allows greater flux and was shown to facilitate high efficiency in removal of reaction products from the reactor. Using this high flux membrane, 949 μmol phenolics were removed from a solution containing 4 m m total phenolics using 45 U polyphenol oxidase in 8 h as compared with 120 μmol removed using non-immobilized enzyme which was inactivated (due to product inhibition) after 7 h. No precipitated melanoid products were observed in the permeate from the capillary membrane bioreactor, and in order to remove the colored quinone-type products of the reaction, a packed column containing chitosan was integrated into the system. Almost complete removal of the colored quinones and associated polymers from the permeate was observed.

Immobilization of polyphenol oxidase on chitosan-coated polysulphone capillary membranes for improved phenolic effluent bioremediation

Abstract Internally skinned polysulphone capillary membranes were coated with a viscous chitosan gel and used as an immobilization matrix for polyphenol oxidase. Bench-scale, single-capillary membrane bioreactors then were used to determine the influence of the chitosan coating on product removal after substrate conversion by immobilized polyphenol oxidase during the treatment of industrial phenolic effluents. The results indicate that greater efficiency was achieved in the removal of polyphenol oxidase-generated products by the chitosan membrane coating, as compared with chitosan flakes. This facilitated an increase in the productivity of the immobilized enzyme.

A continuous process for the biological treatment of heavy metal contaminated acid mine water

Abstract Alkaline precipitation of heavy metals from acidic water streams is a popular and long standing treatment process. While this process is efficient it requires the continuous addition of an alkaline material, such as lime. In the long term or when treating large volumes of effluent this process becomes expensive, with costs in the mining sector routinely exceeding millions of rands annually. The process described below utilises alkalinity generated by the alga Spirulina sp., in a continuous system to precipitate heavy metals. The design of the system separates the algal component from the metal containing stream to overcome metal toxicity. The primary treatment process consistently removed over 99% of the iron (98.9 mg/l) and between 80 and 95% of the zinc (7.16 mg/l) and lead (2.35 mg/l) over a 14-day period (20 l effluent treated). In addition the pH of the raw effluent was increased from 1.8 to over 7 in the post-treatment stream. Secondary treatment and polishing steps depend on the nature of the effluent treated. In the case of the high sulphate effluent the treated stream was passed into an anaerobic digester at a rate of 4 l/day. The combination of the primary and secondary treatments effected a removal of over 95% of all metals tested for as well as a 90% reduction in the sulphate load. The running cost of such a process would be low as the salinity and nutrient requirements for the algal culture could be provided by using tannery effluent or a combination of saline water and sewage. This would have the additional benefit of treating either a tannery or sewage effluent as part of an integrated process.

Biotransformation of phenols using immobilised polyphenol oxidase

Polyphenol oxidase (PPO), obtained from Agaricus bisporus, can be used in hydroxylating a range of phenolic substrates to yield catechols which are then oxidised by the enzyme to give o-quinone products. The objective of this study was to develop systems whereby phenols could be transformed by PPO, and the products of the biotransformation could be isolated and characterised. By comparing the product mixtures obtained using soluble PPO and various forms of immobilised PPO, in aqueous and non-aqueous media, we have found significant differences in reaction rates and in the proportions of catechol and quinone produced. PPO in solution is inactivated by the reaction products, but when it is immobilised, the separation of products from the enzyme reduces this inhibition. Immobilisation also leads to increased stability, and allows continuous use of the enzyme. In bioreactors containing customised novel asymmetric capillary membranes as the enzyme support, high concentrations of phenolic substrates were converted. The addition of a chitosan-containing column downstream from the capillary membrane bioreactor facilitated the removal of the coloured quinone products from the permeate, and recycling of the substrate solution.

Fungal biodegradation of hard coal by a newly reported isolate, Neosartorya fischeri

Cynodon dactylon (Bermuda grass) has been observed to grow sporadically on the surface of coal dumps in the Witbank coal mining area of South Africa. Root zone investigation indicated that a number of fungal species may be actively involved in the biodegradation of hard coal, thus enabling the survival of the plant, through mutualistic interaction, in this extreme environment. In an extensive screening program of over two thousand samples, the Deuteromycete, Neosartorya fischeri, was isolated and identified. The biodegradation of coal by N. fischeri was tested in flask studies and in a perfusion fixed-bed bioreactor used to simulate the coal dump environment. The performance of N. fischeri was compared to Phanaerochaete chrysosporium and Trametes (Polyporus) versicolor, previously described in coal biodegradation studies. Fourier transform infrared spectrometry and pyrolysis gas chromatography mass spectrometry of the biodegradation product indicated oxidation of the coal surface and nitration of the condensed aromatic structures of the coal macromolecule as possible reaction mechanisms in N. fischeri coal biodegradation. This is a first report of N. fischeri-mediated coal biodegradation and, in addition to possible applications in coal biotechnology, the findings may enable development of sustainable technologies in coal mine rehabilitation.

The enzymology of sludge solubilisation utilising sulphate-reducing systems: the properties of lipases

The first stage in the degradation and recycling of particulate organic matter is the solubilisation and enhanced hydrolysis of complex polymeric organic carbon structures associated with the sulphidogenic environment. An investigation into the enzymology of these processes has shown that lipase enzyme activities were found predominantly associated with the organic particulate matter of the sewage sludge. Sonication of the sludge gave an increase in enzyme activity as the enzymes were released into the supernatant. pH and temperature optimisation studies showed optima between 6.5 and 8 and 50-60 degrees C, respectively. All the lipase enzymes from the methanogenic bioreactors indicated extensive stability for at least an hour at their respective optimum temperatures and pH; sulphidogenic lipases reflected limited stability at these temperatures and pH during this time period. Though sulphate showed inhibitory properties towards lipases both sulphide and sulphite appeared to enhance the activity of the enzymes. It is argued that these sulphur species, liberated at different times during the sulphate reduction process, disrupt the integrity of the organic particulate floc by neutralising acidic components on the surface. The release of further entrapped enzymes from the organic particulate matter result in a subsequent enhancement of hydrolysis of polymeric material.

Immobilisation of polyphenol oxidase on nylon and polyethersulphone membranes: Effect on product formation

Preliminary studies were made of the potential use of a membrane-immobilised enzymatic method for the treatment of p-cresol-containing wastewater. The enzyme polyphenol oxidase was immobilised onto hydrophobic polyethersulphone capillary membranes and hydrophilic nylon flat-sheet membranes, by adsorption, and by adsorption with glutaraldehyde cross-linking, respectively. It was found that the intermediate product of the polyphenol oxidase reaction, 4-methylcatechol, was detected when the enzyme was immobilised on the nylon membranes or was not immobilised, but only the o-quinone final product was detected when polyethersulphone was used as the immobilisation matrix.

An integrated anaerobic/aerobic bioprocess for the remediation of chlorinated phenol-contaminated soil and groundwater.

An investigation of biodegradation of chlorinated phenol in an anaerobic/aerobic bioprocess environment was made. The reactor configuration used consisted of linked anaerobic and aerobic reactors, which served as a model for a proposed bioremediation strategy. The proposed strategy was studied in two reactors before linkage. In the anaerobic compartment, the transformation of the model contaminant, 2,4,6-trichlorophenol (2,4,6-TCP), to lesser-chlorinated metabolites was shown to occur during reductive dechlorination under sulfate-reducing conditions. The consortium was also shown to desorb and mobilize 2,4,6-TCP in soils. This was followed, in the aerobic compartment, by biodegradation of the pollutant and metabolites, 2,4-dichlorophenol, 4-chlorophenol, and phenol, by immobilized white-rot fungi. The integrated process achieved elimination of the compound by more than 99% through fungal degradation of metabolites produced in the dechlorination stage. pH correction to the anaerobic reactor was found to be necessary because acidic effluent from the fungal reactor inhibited sulfate reduction and dechlorination.

Specific sulphur metabolites stimulate β-glucosidase activity in an anaerobic sulphidogenic bioreactor

The activities of β-glucosidases are stimulated by specific sulphur metabolites during the hydrolysis of complex polymeric organic carbon in an anaerobic sulphidogenic environment. While sulphate had little or no influence on enzyme activity, sulphite increased the activity of β-glucosidases by 2.5 fold and sulphide increased the activity by six fold. A hypothetical model is proposed in which sulphur species (HSO3− and HS−), liberated at different times during the sulphate reduction process, activate the β-glucosidases which are associated with the organic particulate matter leading to a subsequent enhancement of hydrolysis of polymeric carbohydrate material.

Phyto-bioconversion of hard coal in the Cynodon dactylon/coal rhizosphere.

Fundamental processes involved in the microbial degradation of coal and its derivatives have been well documented. A mutualistic interaction between plant roots and certain microorganisms to aid growth of plants such as Cynodon dactylon (Bermuda grass) on hard coal dumps has recently been suggested. In the present study coal bioconversion activity of nonmycorrhizal fungi was investigated in the C. dactylon/coal rhizosphere. Fungal growth on 2% Duff‐agar, gutation formation on nitric acid treated coal and submerged culture activity in nitrogen‐rich and ‐deficient broth formed part of the screening and selection of the fungi. The selected fungal isolates were confirmed to be found in pristine C. dactylon/coal rhizosphere. To simulate bioconversion, a fungal aliquot of this rhizosphere was used as inoculum for a Perfusate fixed bed bioreactor, packed with coal. The results demonstrate an enhanced coal bioconversion facilitated by low molecular weight organics and the bioconversion of coal may be initiated by an introduction of nitrogen moieties to the coal substrate. These findings suggest a phyto‐bioconversion of hard coal involving plant and microbes occurring in the rhizosphere to promote the growth of C. dactylon. An understanding of this relationship can serve as a benchmark for coal dumps rehabilitation as well as for the industrial scale bioprocessing of hard coal.