An investigation into the use of batch experiments in the determination of the kinetics of ferrous-iron oxidation by Leptospirillum ferrooxidans

Master Thesis


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University of Cape Town

The objectives of this research were to investigate the effect of temperature and pH on the bacterial ferrous-iron oxidation by Leptospirillum ferrooxidans in batch culture and compare these results with those previously obtained in continuous culture. An additional objective was to assess the validity of a variable maintenance equation by a re-evaluation of previously published literature and to evaluate this equation for use during bacterial ferrous-iron oxidation with more than one limiting factor. The ferrous-iron oxidation kinetics of a predominantly Leptospirillum ferrooxidans culture were studied in batch bioreactors. The inoculum for the batches was obtained from steady state continuous cultures of predominantly Leptospirillum ferrooxidans grown on ferrous-iron medium at a dilution rate of 0.04 h-¹. The continuous cultures were grown at the conditions (temperature and pH) at which the batches were operated. The batches were started with a inoculum of 500 ml and 500 ml of a salts medium containing 12g.l-¹ ferrous-iron. The batches were run at temperatures ranging from 30 to 40°C and pH values ranging from pH 1.10 to pH 1.70. The growth rate and the oxygen and ferrous-iron utilisation rates of the bacteria were monitored by means of off-gas analysis and redox potential measurement. The re-evaluation of previously published data proved that the bio-oxidation of ferrous-iron can be represented using a variable maintenance equation proposed by Pirt (1982). An estimation of the maximum bacterial specific ferrous-iron utilisation rate and the maximum bacterial specific growth rate can be obtained from batch or continuous experiments that have been limited by more than one factor e.g. ferrous-iron, arsenic, ammonia, phosphate, sulfate, growth rate. The maximum bacterial specific ferrous-iron utilisation rates and the maximum bacterial specific growth rates calculated from the variable maintenance model are very similar to those values previously published and affirms the validity of the equation. The batch growth curves follow expected trends and have a very long exponential phase and a very short deceleration phase. The bacterial growth during the exponential phase follows first order kinetics with respect to biomass concentration and is independent of the substrate concentration. It was not possible to determine the maximum bacterial yields and the maintenance coefficients during the batch bacterial ferrous-iron oxidation using the constant maintenance energy equation of Pirt (1965). The reason for this is that at the beginning of a batch the ferrous-iron concentration is very high and the culture is limited by the growth rate, while at the end of a batch it is limited by ferrous-iron and inhibited by the ferric-iron product. A variable maintenance energy equation, proposed by Pirt (1982), had to be used to calculate the maximum bacterial yields and the constant-and variable maintenance coefficients. The maximum bacterial yields on ferrous-iron and oxygen and the respective constant, and variable maintenance coefficients did not vary significantly with temperature or pH. The average maximum yield and constant maintenance coefficient calculated were similar to those reported previously for Leptospirillum ferrooxidans in continuous culture and for Acidithiobacillus ferrooxidans in continuous and batch culture. The maximum bacterial specific ferrous-iron utilisation rate and the maximum bacterial specific growth rate calculated from the variable maintenance equation are very similar to those previously published. The constant maintenance equation is only valid for bacterial ferrous-iron oxidation if the culture is only limited by the substrate during the whole experiment. If at any stage the culture is limited by another factor then the bioenergetics must be represented using the variable maintenance equation. The variable maintenance equation should therefore replace the constant maintenance equation when quantifying the bioenergetics of bacterial ferrous-iron oxidation and the bioleaching of sulfide minerals. The variable maintenance equation is especially relevant to the bioleaching of sulfide minerals at low redox potentials (high ferrousiron concentrations) where the bacterial culture may not be limited by the energy source (ferrous-iron). The growth rate during the deceleration phase in a batch culture is limited by the substrate, ferrous-iron, and follows Michaelis-Menten based kinetics. The kinetic parameters calculated from batch experiments were very similar to those calculated by previous researchers in continuous experiments. The variation of temperature and pH, however produced different results in batch and continuous experiments. The continuous experiments are more accurate for representing bacterial ferrous-iron oxidation kinetics limited by ferrous-iron because batch experiments have transient conditions and the limitation by ferrous-iron occurs over a very short time, hence a limited number of data points can be obtained. Batch experiments, however are useful for initial short-term analysis of a system and to provide an initial indication of the kinetic modelling parameters.

Bibliography: leaves 60-64.