Phytochemical analysis of both HEA1 and the crude plant extract showed the presence of phenolics, tannins and saponins. Saponins were the predominant secondary metabolites and were mostly abundant in the plant extract and to a lesser extent in the active compound. Steroidal saponins, tannins and phenolics were also detected in the plant extract, but only the phenolics were detected in the active compound. The results of the phytochemical analysis showed that those compounds that were not present in the active compound could be removed from the crude extract during the TLC purification process. Investigation on the mechanism of action of the crude plant extract on the sterol production by A. flavus showed that the plant extract affected ergosterol biosynthesis by causing an accumulation of oxidosqualene in the ergosterol biosynthetic pathway resulting in a decline in ergosterol production. An oscillatory response in lanosterol production was observed in the presence of the plant extract, which may be an adaptation mechanism of A. flavus to unfavourable conditions and compensation for the loss of enzyme activity which may have occurred as a result of the accumulation of oxidosqualene. The antifungal activity of the plant extract on ergosterol production by A. flavus may also be due to saponins which target the cell membrane and ergosterol production in fungi. The effect of the plant extract on the fungal cell wall of A. flavus also showed that the plant extract caused a decline in β-(1, 3) glucan production by inhibiting β-glucan synthase. The plant extract also affected the chitin synthesis pathway of A. flavus, by causing a decline in chitin production, which was due to the inhibition of chitin synthase. Investigation of chitinase production using 4MU substrates showed that the plant extract caused an accumulation of chitobioses, by activating chitobiosidases and endochitinases. A decline in N-acetylglucosaminidase activity in the presence of the plant extract was observed and this prevented the formation of N-acetylglucosamine. The accumulation of chitobiosidase and endochitinase may be as a result of autolysis that may be triggered by A. flavus as a survival mechanism in the presence of the plant extract and as a compensatory mechanism for the loss of β-glucans and chitin. The antifungal effect of the plant extract on various components of the cell wall of A. flavus, makes T. violacea aqueous plant extract an ideal chemotherapeutic agent against both human and plant pathogens of Aspergillus. The broad spectrum of antifungal activity of T. violacea against A. flavus also eliminates any chances of the fungus developing resistance towards it and would make it a candidate for use as a potential antifungal agent. Further identification and possible chemical synthesis is needed to shed light on the safety and efficacy of the active compound for further development as a chemotherapeutic agent.

Submitted in fulfilment for the requirements for the degree of Doctor of Technology: Biotechnology, Durban University of Technology, 2010. / South African edible oil processing plants produce approximately 3 x 105 tonnes of oil annually with up to 3 tonnes of water for every tonne of oil produced. Wastewater that contains oil extracts varies in organic loading from 30,000 to 60,000 mg.l-1 COD. This wastewater can be used to grow oleophilic fungi to produce valuable industrial products. The global vitamin B market is approximately R25.5 billion with 4500 metric tonnes being produced. A large proportion of this is produced using the fungus Eremothecium gossypii using oil substrates. The aim of this study was to to develop a novel method to produce riboflavin with the aid of fungi, using edible oil effluent (EOE) as substrate, and to optimize the production thereof by statistical experimental design. Four fungi were surveyed for their growth potential on EOE and two, E. gossypii (CBS109.51) and C. famata (ATCC 208.50) were found to produce sufficient riboflavin for further study. Mutation of these organisms using ethylmethane sulphonate (EMS) increased riboflavin production from 3.52 mg.l-1 to 38.98 mg.l-1, an 11-fold increase. An enzyme pathway responsible for this was found to involve isocitrate lyase and comparison of this enzyme’s activity in the mutant against the wild-type using Michaelis-Menten kinetics showed a higher reaction velocity (Vmax) with a reduced substrate affinity (Km) indicating that the mutation was associated with this enzyme. Biomass comparisons were fitted to the sigmoid Gompertz model which was used to compare the wild-type to the mutant and increased specific growth rates and doubling times were observed in mutated cultures of E. gossypi. A strategy of statistical experimental design was pursued to optimize media components and iterative fractional factorial experiments culminating in a central composite optimization experiment were conducted. Statistically verified mathematical models were developed at each stage to identify important media components, predict media interactions, show directions for improvement and finally, predict maximum riboflavin production. An eight-factor resolution IV fractional factorial increased riboflavin production to 112 mg.l-1 followed by a four-factor resolution V experimental design which increased riboflavin production to 123 mg.l-1. A two-factor (yeast extract and NaCl) central composite experimental design predicted a maximum riboflavin production of 136 mg.l-1 which was a 3.5-fold increase from the mutant, and 38.6-fold higher than the E. gossypii wild-type. The optimized value was achieved within predicted confidence intervals in confirmatory experiments. Cost implications for production of riboflavin on EOE were calculated and a 10% technology uptake by the edible oil industry could yield a riboflavin industry with a 63.65 million rand turnover and a potential 24.96 million rand gross profit margin. / National Research Fund.

Submitted in fulfillment of the requirements of the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2011. / Riboflavin (vitamin B2), an essential water-soluble vitamin is commercially produced because it cannot be synthesized by vertebrates. Although this vitamin is produced chemically, bioproduction is a better option since it is more economical, requires less energy, produces less waste and can use renewable sources. In this study we investigated spent oil from the food and motor industries as alternative cheap carbon sources for the bioproduction of this vitamin. Commercial fungal strains namely; Eremothecium gossypii ATCC 10895, Eremothecium gossypii CBS 109.51, Eremothecium ashbyi CBS 206.58 and the yeast, Candida famata ATCC 20850, as well as a laboratory mutated Eremothecium gossypii EMS 30/1 strain were used. Statistical experimental design using a series of fractional factorial experimental designs was used to optimize the effect of yeast extract, peptone, malt extract, K2HPO4 and MgSO4.7H2O to supplement the used oils for optimum riboflavin production. Response surface methodology based on central composite experimental designs was then applied and together with the point predictions made, production media for both substrates were further optimized. The optimized conditions were then tested with laboratory experiments. Results showed that mutant E. gossypii EMS 30/1 produced the most riboflavin in spent motor oil (20.45 mg.l-1) while Candida famata ATCC 20850 produced the highest concentration (16.99 mg.l-1) in spent vegetable oil. With these strains and using the experimental designs from the fractional factorial experiments, supplemented spent motor and spent vegetable oils produced 66.27 mg.l-1 and 72.50 mg.l-1 riboflavin, respectively. The central composite optimization results showed that 0.18 g.l-1 and 0.45 g.l-1 K2HPO4 and 12.5 g.l-1 malt extract increased the production to 91.88 mg.l-1 and 78.68 mg.l-1 in spent vegetable oil and motor oil respectively. A point prediction from the response surface methodology was used to validate these and it was found that 103.59 mg.l-1 riboflavin was produced by mutant E. gossypii EMS 30/1 using 2.5 g.l-1 yeast extract, 0.5 g.l-1 peptone, 12.5 g.l-1 malt extract, 0.18 g.l-1 K2HPO4 and 0.3 g.l-1 MgSO4.7H2O. After optimizing K2HPO4 in a one-factor-at-a-time experiment, 82.75 mg.l-1 riboflavin was produced by C. famataon v SVO using 6.5 g.l-1 peptone, 12.5 g.l-1 malt extract 0.15 g.l-1 K2HPO4 and 1.75 g.l-1 MgSO4.7H2O. This is a 5.08 and 4.87 fold increase respectively when compared to spent oil prior to optimization. This shows that spent motor oil and mutant E. gossypii produces 103.59 mg.l-1 riboflavin while spent vegetable oil and C. famata produces 82.75 mg.l-1 riboflavin. Hence, E. gossypii can be used to generate riboflavin using spent motor oil and C. famata, using spent vegetable oil.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Scotch whiskey is of two main types, namely Scotch malt whiskey, made from malted barley alone, or Scotch grain whiskey, made from cereals, such as wheat or maize, together with malted barley. In both processes, the enzymes from the barley are responsible for starch conversion and should always be derived entirely from the malted barley. No exogenous enzymes are allowed to be added to any mashing. The enzymes involved in the conversion process to fermentable sugars, are the aand p-amylases, limit dextrinase and p-glucosidase. Maize, on the other hand, contains no enzyme activity, therefore enzymes need to be added when producing whiskey from maize alone. In other whiskey-producing countries where maize is freely available and cheaper than barley, the use of exogenous enzymes are allowed in the mashing process and is crucial for the formation of fermentable sugars from complex carbohydrates. The cost of the enzymes, however, can push the production cost of whiskey to higher levels. Saccharomyces cerevisiae does not have any amylolytic activity, but is an excellent fermenter and produces favourable organoleptic notes, which makes it very suitable for producing potable spirit. Efforts have been made to genetically improve industrial strains, relying on classical genetic techniques followed by the selection of broad traits, such as ethanol tolerance, absence of off-flavours and carbohydrate/starch utilisation. No strain has thus far been selected for total starch degradation during the fermentation of whiskey mash. Over the last decade, considerable progress has been made in the development of genetically improved strains for the distilling, wine, brewing and baking industries. The expression of heterologous genes introduced a new dimension in approaches to the genetic improvement of industrial strains. It would therefore be cost-effective to use a yeast strain that can produce active and sufficient enzymes to ferment raw starch efficiently to alcohol without lowering the quality of the end product. No such strain has been developed to date, but the continuous improvement of starch-utilising strains has made this goal more achievable. Two a-amylase genes, namely LKA 1 and LKA2, were previously isolated from Lipomyces kanonenkoae. In this study, we selected 4 strains on the basis of criteria that are important for whiskey-specific strains. The selected strains were transformed with LKA 1, as well as with a combination of LKA 1 and LKA2 genes. The wine yeast VIN13 was included in the transformation of LKA1 and LKA2 because of its rapid fermentation rate. The genes were integrated into the genomes of the yeast strains and were stable after many generations. Assays showed that a significant increase in enzyme activity was induced in the whiskey strains, compared to the untransformed strains. The strains also showed good fermentation ability in whiskey fermentations, although optimum alcohol production was still not achieved. / AFRIKAANSE OPSOMMING: Skotse whiskey bestaan uit 2 tipes, nl. mout whiskey, gemaak slegs van mout d.w.s. gars wat die mout proses ondergaan het, en graan whiskey wat gemaak word van gewasse soos mielies of koring, waarby mout gevoeg word. Die ensieme afkomstig van die mout is verantwoordelik vir die omsetting van stysel na fermenteerbare suikers en geen eksogene ensieme mag by die gars- of graanmengsel gevoeg word nie. Die ensieme wat betrokke is by die omsetting van stysel, is die a- en ~- arnitases, limiet dekstrinase en ~-glukosidase. Mielies bevat geen ensiemaktiwiteit nie, dus moet ensieme by die proses gevoeg word indien slegs mielies vir die vervaardiging van whiskey gebruik word. In whiskey produserende lande waar mielies vryelik beskikbaar is en goedkoper is as gars, word eksogene ensieme by die graanmengsel gevoeg vir die vrystelling van fermenteerbare suikers vanaf komplekse koolhidrate. Die hoë koste van die ensieme kan egter die produksiekoste van whiskey verhoog. Saccharomyces cerevisiae besit geen amilolitiese aktiwiteit nie, maar is 'n uitstekende fermenteerder en produseer gewensde organoleptiese geure. Om hierdie redes is S. cerevisiae baie geskik vir die produksie van drinkbare etanol. Navorsingspogings om industriële rasse geneties m.b.v. klassieke genetiese metodes te verbeter, kom wydverspreid in die literatuur voor. Dit sluit in die seleksie van rasse met 'n verskeidenheid van eienskappe soos etanol toleransie, die afwesigheid van afgeur produksie en koolhidraat/stysel benutting. Geen ras is egter tot op hede geselekteer vir totale stysel afbraak gedurende fermentasie nie. Groot vordering is gedurende die laaste dekade gemaak in die ontwikkeling van genetiese verbeterde rasse vir die wyn- stokery- en brouers industrieë. Die uitdruk van heterogene gene in gisrasse gee 'n nuwe dimensie aan die genetiese verbetering van industriële rasse. Die gebruik van 'n gisras wat aktiewe en genoegsame ensieme produseer om rou stysel te fermenteer, sonder om die kwalitiet van die eindproduk nadelig te beïnvloed, kan die produksiekoste van whiskey aansienlik verminder. Geen gisras met hierdie eienskap is tot op hede ontwikkel nie, maar die voortdurende verbetering van rasse om stysel af te breek maak hierdie doel meer bereikbaar. Twee a-amilase gene, nl. LKA 1 en LKA2 is voorheen uit Lipomyces kononenkoae geïsoleer. In hierdie studie is 4 gisrasse geselekteer op grond van die kriteria wat nodig is vir whiskey giste. Die geselekteerde rasse is getransformeer met LKA 1 sowel as 'n kombinasie van LKA 1 en LKA2 gene. Die wyngis VIN13 is ingesluit by die transformasie met die LKA1 en LKA2 gene, omrede VIN13 bekend is as 'n vinnige fermenteerder. Die gene is geïntegreer in die genoom van die verskillende gisrasse en is stabiel na vele generasies. Die getransformeerde rasse het 'n betekenisvolle verhoging in ensiemaktiwiteit teenoor die nie-getransformeerde rasse getoon. AI die transformante het ook goeie fermentasie vermoë getoon in whiskey fermentasie proewe. Optimum alkoholproduksie is egter nie verkry nie.

No abstract available. / Thesis (M.Sc.Eng.)-University of Durban-Westville, 2000.

Thesis (MTech (Food Technology)--Cape Peninsula University of Technology, 2009 / The primary aim of this study was to establish the effects of both cavitation, (20 KHZ), and heat (55°C or 60•C) on Saccharomyces cerevisiae GC210 (MATa lys2) suspended in physiological saline. Fluorescent flow cytometry was used to determine the effects of moist heat and acoustic cavitation on S. cerevisiae cells. Results from this study could be used as a guide for use by the food industry for the combined use of heat and sonication to disinfect various solutions contaminated with S. cerevisiae.

Yeast are exposed to many physical and chemical stresses when used in large-scale industrial fermentations, particularly the initial stages in which yeast are shifted from anaerobic storage to aerated wort. This work investigated the transcriptional and physiological responses of yeast that had been shifted from anaerobic to aerobic growth conditions. Microarray technology was employed to determine the transcriptional changes that occurred in the first hour of a pilot-plant fermentation compared to the 23rd hour. It was found that over 100 genes were up-regulated initially including genes involved in the synthesis of the essential membrane sterol ergosterol and genes for the protection of cells against oxidative stress. It was also determined that cells which accumulate ergosterol precursors in the absence of ergosterol were more sensitive to exogenous oxidative stresses, indicating a role for ergosterol in oxidative stress tolerance. Aeration of anaerobically grown cells did not affect their growth kinetics or viability. However, anaerobically grown cells were hypersensitive to exogenous oxidative stress compared to their aerobic counterparts. Anaerobic cells that underwent a short period of aeration prior to treatment with hydrogen peroxide generated a tolerance to the oxidant, indicating that the period of aeration produced an adaptive-like response. Microarray analysis of the cells during the period of aeration showed that representative genes from the oxidative stress response family were up-regulated rapidly and it was determined that the response was controlled by the Yap1p and Skn7p transcription factors. Deletion of the transcription factor genes indicated that they were responsible for the creation of tolerance to oxidant. Target gene products of the two transcription factors (Gpx2p, Gsh1p and Trx2p) were shown to be induced during the shift to aeration; however, the glutathione redox balance did not seem to be affected as the cells were shifted from highly reduced to oxidising environments. Unexpectedly, it was discovered that genes involved in the synthesis of amino acids were up-regulated during anaerobic growth and stringently downregulated upon aeration of cells. The transcriptional activator of those genes (Gcn4p) was essential for growth in anaerobic media which included amino acid supplementation.

The yeast expression system Pichia pastoris was investigated as an encapsulation technology capable of serving as a rumen escape vehicle. Cellularly encapsulated protein is protected from the ruminal environment so long as the cell membrane, which surrounds and isolates the intracellular protein is physically intact. Intracellular expression of Green Fluorescent Protein (GFP) allows for the monitoring of cellular integrity as necessary for the protection of encapsulated protein from ruminal proteases. Upon cellular lysis GFP is exposed to extracellular proteases which result in both the proteolytic degradation of the protein-based GFP chromophore and its associated fluorescence. Visualization of rumen fluid under epifluorescent microscopy revealed a high level of background autofluorescence owing to the fluorescent plant particles, microbes, and fluorescent compounds therein. Visualization of GFP in rumen fluid can be optimized through GFP variant selection, filter set design, and light source selection based on bulb emission spectra. Incubation of intracellular GFP expressing P. pastoris in batch culture ruminal in vitro simulations demonstrated that 93%, 97%, and 25% of the P. pastoris inoculum maintained cellular integrity in clarified rumen fluid, bacterial fraction of rumen fluid, and whole rumen fluid, respectively, when incubated over 36 to 48 h. Continuous fermentation in vitro rumen simulations (Rusitec) demonstrated a P. pastoris escape rate of 19% when added daily to fully adapted Rusitec vessels having a dilution rate of 0.75d-1. Abomasal in vitro simulations demonstrated that 84% of the P. pastoris inoculum was lysed within 12 h, as necessary for the release of encapsulated protein. P.pastoris may be an effective post-fuminal delivery vehicle, provided that similar results are obtained in vivo. / xiv, 120 leaves : ill. ; 28 cm.

The possibility of developing an immobilised-enzyme bioprobe, based on mushroom polyphenol oxidase, for the purely biological detection and quantification of phenolic pollutants in water was investigated. Polyphenol oxidase catalyses the bioconversion of many phenolic compounds into quinone-related coloured products. Thus, in an immobilised form, the enzyme serves as a visible indicator of the presence and concentration of phenolic pollutants in water. The objective of this research was to develop a portable, disposable bioprobe incorporating polyphenol oxidase for this purpose. The intensity of the colour changes produced by the enzyme on reaction with p-cresol, p-chlorophenol and phenol was found to increase proportionally with increasing concentrations of these substrates in solution. Immobilisation of the enzyme on various supports did not appear to significantly affect the catalytic activity of the enzyme. The enzyme was immobilised by adsorption and cross-linking on polyethersulphone, nitrocellulose and nylon membranes with the production of various colour ranges on reaction with the phenolic substrates. The most successful immobilisation of the enzyme, in terms of quantity and distribution of enzyme immobilised and colour production, was obtained with the enzyme immobilised by adsorption on nylon membranes in the presence of 3-methyl-2-benzothiazolinone hydrazone (MBTH). The enzyme, immobilised using this method, produced ranges of maroon colours in phenolic solutions and orange colours in cresylic solutions. The colour intensities produced were found to increase proportionally with increasing substrate concentration after 5 minutes exposure to the substrates. The bioprobe had a broad substrate specificity and was sensitive to substrate concentrations down to 0.05 mg/L. The enzyme activity of the bioprobe was not significantly affected in a pH range from 4 to 10 and in a temperature range from 5-25⁰C. The bioprobe activity was not affected by various concentrations of salt and metal ions and the bioprobe was able to detect and semi-quantify phenolic substrates in industrial effluent samples. These features of the bioprobe indicate that the commercialisation of such a bioprobe is feasible and this technology has been patented (Patent No. SA 97/0227). / KMBT_363 / Adobe Acrobat 9.54 Paper Capture Plug-in

This project considered the use of the yeast Saccharomyces cerevisiae as a biosorbent for the removal and recovery of a range of metals from contaminated waters. S. cerevisiae, as a biosorbent, has the potential to provide a cost effective, selective and highly efficient purification system. Initial studies focused on metal accumulation by an immobilized baker's S. cerevisiae biosorbent. The parameters affecting metal uptake were investigated, these included metal concentration, time and solution pH. Metal uptake was rapid. Gold and cobalt reached saturation within 5 min of contact with the biosorbent in batch reactors. Copper, zinc, nickel, cadmium and chromium reached saturation within 30 min of contact. Metal accumulation was pH dependent and was generally unaffected at a solution pH ≥ 4, and was substantially decreased at pH ≤ 2. The exception was gold which was preferentially accumulated at a solution pH of 2. The immobilized baker's yeast accumulated metals with maximum binding capacities in the order of gold > cadmium > cobalt > zinc > copper > chromium > nickel. A rapid method to assess metal recovery was developed. Bioaccumulated metal was efficiently recovered using dilute mineral acids. Copper recovery of ≥ 80 % was achieved by decreasing the solution pH of the reaction mixture to 2 with the addition of nominal quantities of HCl, H₂SO₄ or RNO₃. Adsorption-desorption over 8 cycles had no apparent adverse effect on metal uptake or recovery in batch reactors. Transmission electron microscopy showed no evidence of damage to cells used in copper adsorption-desorption investigations. Biosorption columns were investigated as bioreactors due to their application potential. The metals investigated were effectively removed from solution. At a saturation threshold, metal uptake declined rapidly. Most metals investigated were desorbed from the columns by eluting with 0.1 M HCl. Initially recoveries of copper, cobalt and cadmium were as high as 100%. Desorbed copper, zinc, cadmium, nickel and cobalt were concentrated in 10 to 15 ml of eluent, representing up to a 40 fold decrease in solution volume. Cadmium, nickel and zinc uptake increased with the second application to the columns. Initial accumulation of gold and chromium was 42.2 μmol/g and 28.6 μmol/g, however, due to the low recoveries of these two metals, a second application was not investigated. Copper was applied to a single column for 8 consecutive adsorption-desorption cycles. Uptake increased from an initial 31.3 μmol/g to 47.8 μmol/g at cycle 7. The potential for selective metal recovery was demonstrated using two biosorption columns in series. Copper was accumulated and recovered most efficiently. Zinc, cobalt and cadmium were displaced to the second column. Copper bound preferentially to zinc at a ratio of 6:1. Copper bound preferentially to cobalt at a ratio of 4:1. Cadmium was only displaced at a ratio of 2:1. The successful transfer of the bioremediation technology from the laboratory to an industrial application has yet to be realized. Bioremediation of a Plaatjiesvlei Black Mountain mine effluent, which contained copper, zinc, lead and iron, was investigated in this project. The removal of the metals was most effective at pH 4. A combined strategy of pH adjustment and bioremediation using immobilized S. cerevisiae decreased the copper concentration by 92.5%, lead was decreased by 90% and zinc was decreased by 60%. Iron was mostly precipitated from solution at pH ≥ 4. An ageing pond at the mine with conditions such as; pH, water volume and metal concentration, which were more conducive to biological treatment was subsequently identified. The investigation indicated a possible application of the biomass as a supplement to chemical remediation. The metal removal capability of a waste brewer's yeast was subsequently investigated. A yeast conditioning step increased metal uptake up to 100% and enhanced reproducibility. Metal removal from solution was rapid and pH dependent. The metals were efficiently removed from solution at pH ≥ 4. Uptake was substantially inhibited at pH ≤ 3. The waste brewer's yeast accumulated metals with maximum binding capacities in the order of copper (25.4 μmol/g) > lead (19.4 μmol/g) > iron (15.6 μmol/g) > zinc (12.5 μmol/g). No correlation between cell physiology and metal uptake was observed. Uptake of the four metals was confirmed by energy dispersive X-ray microanalysis. The interference of lead, zinc and iron on copper uptake by the waste brewer's yeast, and the interference of copper on the uptake of lead, zinc and iron was investigated. Maximum copper uptake was not decreased in the presence of lead. The Bmax remained constant at approximately 25 μmol/g. The dissociation constants increased with increasing lead concentrations. Lead bioaccumulation was significantly decreased in the presence of copper. The type of inhibition was dependent on the initial copper concentrations. Zinc had a slight synergistic effect on copper uptake. The copper Bmax increased from 30.8 μmol/g in a single-ion system to 34.5 μmol/g in the presence of 200 μmol/l of zinc. Zinc uptake was severely inhibited in the presence of copper. The maximum uptake and dissociation constant values were decreased in the presence of copper, which suggested an uncompetitive inhibition. The affinity of copper was substantially higher than zinc. The presence of higher levels of copper than zinc in the yeast cells was confirmed by energy dispersive microanalysis. Copper uptake was decreased in the presence of iron, with the copper Bmax being decreased from 25.4 μmol/g in a single-ion system to 20.1 μmol/g in the presence of 200 μmol/l iron. Iron Bmax values remained constant at 16.0 μmol/g. Combined biosorption and EDXA results suggested the iron bound at a higher affinity than copper to the cell wall. Total copper removal was higher as larger quantities of copper were deposited in the cell cytoplasm. Metal removal from the Plaatjiesvlei effluent by free cell suspensions of the waste brewer's yeast was satisfactory. Copper levels were decreased by 96%, iron by 42%, lead 25% and zinc 2%. Waste brewer's yeast is a cheap source of biomass in South Africa, and could potentially provide the basis for the development of an innovative purification system for metal-contaminated waters.