C. White

Microbial treatment of metal pollution — a working biotechnology?

Some of the main processes that remove, immobilize or detoxify heavy metals and radionuclides in the natural environment result from microbial activities. These activities can be harnessed to clean up toxic metal wastes before they enter the wider environment. To date, the most successful biotechnological processes utilize biosorption and bioprecipitation, but other processes such as binding by specific macromolecules may have future potential. Technologies using these processes are currently used to control pollution from diverse sources, including smelters and mine workings.

The role of microorganisms in biosorption of toxic metals and radionuclides

Abstract A multiplicity of physico-chemical and biological mechanisms determine the removal of toxic metals, metalloids and radionuclides from contaminated wastes. Physico-chemical mechanisms of removal, which may be encompassed by the general term “biosorption”, include adsorption, ion exchange and entrapment which are features of living and dead biomass as well as derived products. In living cells, biosorption can be directly and indirectly influenced by metabolism. Metabolism-dependent mechanisms of metal removal which occur in living microorganisms include metal precipitation as sulphides, complexation by siderophores and other metabolites, sequestration by metal-binding proteins and peptides, transport and intracellular compartmentation. In addition, transformations of metal species can occur resulting in oxidation, reduction or methylation. For metalloids such as selenium, two main transformation mechanisms are the reduction of oxyanions to elemental forms, and methylation to methylated derivatives which are volatilized. Such mechanisms are important components of natural biogeochemical cycles for metals and metalloids as well as being of potential application for bioremediation.

Metal accumulation by fungi: applications in environmental biotechnology

SummaryFungi can accumulate metal and radionuclide species by physico-chemical and biological mechanisms including extracellular binding by metabolites and biopolymers, binding to specific polypeptides and metabolism-dependent accumulation. Biosorptive processes appear to have the most potential for environmental biotechnology. ‘Biosorption’ consists of accumulation by predominatly metabolism-independent interactions, such as adsorptive or ion-exchange processes: the biosorptive capacity of the biomass can be manipulated by a range of physical and chemical treatments. Immobilized biomass retains biosorptive properties and possesses a number of advantages for process applications. Native or immobilized biomass can be used in fixed-bed, air-lift or fluidized bed bioreactors; biosorbed metal/radionuclide species can be removed for reclamation and the biomass regenerated by simple chemical treatments.

The Uptake and Cellular Distribution of Zinc in Saccharomyces cerevisiae

SUMMARY: Zn2+ uptake by Saccharomyces cerevisiae was biphasic. The first phase was independent of metabolic energy, consisting of adsorption to the cell surface, and followed a Freundlich isotherm. The second phase was dependent on metabolic energy, ATPase activity and the transmembrane proton gradient, and consisted of uptake into the cell. Energy-dependent uptake showed Michaelis-Menten kinetics with a K m of 3·7 μm-Zn2+ and a V max of 1·6 nmol min-1 per 107 cells at Zn2+ concentrations below 80 μm but deviated at higher concentrations. K+ and Mg2+ inhibited energy-dependent Zn2+ uptake while Na+ and Ca2+ did not. The effect of heavy metals was complex and included both inhibition and stimulation of Zn2+ uptake. K+ efflux accompanied Zn2+ uptake at all Zn2+ concentrations but there was no simple stoichiometric relationship between the two. Toxic effects of Zn2+ such as inhibition of H+ efflux and K+ uptake and reduction of viability were observed at all Zn2+ concentrations and toxicity appeared to be a major factor in K+ efflux. Toxicity also affected the kinetics of Zn2+ uptake, being a major cause of deviation from Michaelis-Menten kinetics. Zn2+ was compartmented within the cell: 56% of the total intracellular pool was in the soluble vacuolar fraction, 39% was bound to insoluble components and only 5% was found in the cytosol. Isolated yeast vacuoles possessed an ATP-dependent Zn2+ uptake system whose properties were consistent with a Zn2+/H+ antiport.

A comparison of carbon/energy and complex nitrogen sources for bacterial sulphate-reduction: potential applications to bioprecipitation of toxic metals as sulphides.

Detailed nutrient requirements were determined to maximise efficacy of a sulphate-reducing bacterial mixed culture for biotechnological removal of sulphate, acidity and toxic metals from waste waters. In batch culture, lactate produced the greatest biomass, while ethanol was more effective in stimulating sulphide production and acetate was less effective. The presence of additional bicarbonate and H2 only marginally stimulated sulphide production. The sulphide output per unit of biomass was greatest using ethanol as substrate. In continuous culture, ethanol and lactate were used directly as efficient substrates for sulphate reduction while acetate yielded only slow growth. Glucose was utilised following fermentation to organic acids and therefore had a deleterious effect on pH. Ethanol was selected as the most efficient substrate due to cost and efficient yield of sulphide. On ethanol, the presence of additional carbon sources had no effect on growth or sulphate reduction in batch culture but the presence of complex nitrogen sources (yeast extract or cornsteep) stimulated both. Cornsteep showed the strongest effect and was also preferred on cost grounds. In continuous culture, cornsteep significantly improved the yield of sulphate reduced per unit of ethanol consumed. These results suggest that the most efficient nutrient regime for bioremediation using sulphate-reducing bacteria required both ethanol as carbon source and cornsteep as a complex nitrogen source.

Mixed sulphate-reducing bacterial cultures for bioprecipitation of toxic metals : factorial and response-surface analysis of the effects of dilution rate, sulphate and substrate concentration

The effect of process variables on alkalization and removal of typical contaminating toxic metals from a simulated acid leachate by continuous mixed cultures of sulphate-reducing bacteria was studied. It was shown that the amount of metal removed and rise in pH both varied with the amount of sulphate reduction occurring, the residual sulphate concentration being the main determinant of final pH. Factorial experiments showed that sulphate reduction was enhanced by increasing the substrate concentration and inhibited by the initial sulphate concentration. The dilution rate did not exert a primary effect, but the existence of a significant interactive effect between the substrate and sulphate concentrations and the flow rate was indicative of a quantitative modification of the effect of the former two variables by the latter. The biomass concentration in the cultures was only affected by the substrate concentration indicating that the other variables acted by selection for or against sulphate-reducing components of the mixed culture. A response-surface analysis of the yield of sulphate reduction and alkalization against substrate concentration and dilution rate indicated that sulphate reduction (and alkalization) was sensitive to both of these variables where the substrate: sulphate stoichiometry was in the range 1: 1-3: 1. At lower sulphate concentrations complete reduction occurred at all levels while at higher sulphate concentrations washout occurred in all runs, which indicated that the key variable was the substrate:sulphate stoichiometry and its interaction with the dilution rate. Attention is drawn to the efficiency of the experimental designs employed for elucidating these factors.

Accumulation and effects of cadmium on sulphate-reducing bacterial biofilms

Biofilms comprising a pure and a mixed culture of sulphate-reducing bacteria (SRB) were grown in continuous culture. When exposed to 20 or 200 μM Cd, both cultures accumulated Cd but the mixed culture accumulated more and continued to accumulate Cd during the experiment, whereas accumulation by the pure cultures ceased after 4-6 d. Unlike the pure culture, the mixed culture also accumulated both protein and carbohydrate throughout the experiment proportionally to Cd which showed that accumulation required the production of biofilm material. Electron microscopy showed the presence of polysaccharide and particulates in both pure and mixed cultures, irrespective of the presence of Cd. However, energy-dispersive X-ray analysis (EDXA) showed that accumulation of Cd in the form of CdS occurred in biofilms exposed to Cd while back-scattered electron imaging of sections indicated that the accumulation of Cd was localized in a superficial layer of the biofilm. The mechanism of uptake, therefore, appeared to be entrapment and/or precipitation of CdS at the biofilm surface. The relatively low Cd uptake by the pure culture biofilm was attributed to its less efficient growth and polysaccharide production. These results indicate that mixed SRB cultures are more effective than pure cultures for metal removal and underlines significant differences between the biology of pure and mixed cultures.

Copper Uptake by Penicillium ochro-chloron: Influence of pH on Toxicity and Demonstration of Energy-dependent Copper Influx Using Protoplasts

Summary: The existence of energy-dependent copper influx was demonstrated in protoplasts of Penicillium ochro-chloron. Protoplasts and mycelium were tolerant of copper over the pH range 3·0 to 5·5 but sensitive at pH 6·0. Uptake of copper was approximately ten times greater at pH 6·0 than in the lower range. At pH 3·0, influx showed saturation kinetics with a half-maximal influx at an external Cu2+ concentration of 390 μ;M and a maximum influx rate of 22 nmol h-1 (107 cells)-1. Saturation kinetics were not observed at pH 6·0.

Extracellular metal-binding activity of the sulphate-reducing bacterium Desulfococcus multivorans

Polarography was used to measure the copper-binding ability of culture filtrates from a range of sulphate-reducing bacteria (SRB), including pure cultures and environmental isolates. Of those tested, Desulfococcus multivorans was shown to have the greatest copper-binding capacity and this organism was used for further experiments. Extracellular copper- and zinc-binding activities of Dc. multivorans culture filtrates from batch cultures increased over time and reached a maximum after 10 d growth. The culture filtrate was shown to bind copper reversibly and zinc irreversibly. Twelve-day-old Dc. multivorans culture filtrates were shown to have a copper-binding capacity of 3.64 +/- 0.33 micromol ml(-1) with a stability constant, log10K, of 5.68 +/- 0.64 (n=4). The metal-binding compound was partially purified from culture growth media by dichloromethane extraction followed by HPLC using an acetonitrile gradient.

Uptake and intracellular compartmentation of thorium in Saccharomyces cerevisiae.

When Saccharomyces cerevisiae was cultured in the presence of thorium, the element was accumulated by the cells and was visible in electron micrographs as electron dense granules. When thorium was present during exponential growth, these granules were located mainly in the vacuole, with some present in the cytosol. Where thorium was present only during the stationary phase, there appeared to be greater thorium deposition in the cell wall than during exponential growth and some vacuolar deposits were also evident. Thorium uptake by exponential-phase cells was not stimulated by glucose and was thus independent of metabolic energy.