MODELING BACTERIAL GROWTH, ATTACHMENT, EXTRACELLULAR POLYMER PRODUCTION AND INTERACTIONS WITH TOXIC TRANSITION METALS UNDER CONDITIONS OF VARIABLE pH

Abstract

Bacteria are capable of adaptation to a variety of environmental extremes that abound in freshwater aquatic systems. One adaptation is biofilm formation and another is alteration of optimum enzyme activity. For example, enzyme activity is influenced by extremes in hydrogen ion concentration and the presence or absence of toxic trace metals. Most bacteria have a maximum activity over a narrow pH range. Computer modelling is able to quantify the interactive effects of the hydrogen ion and trace metals on cellular activity. A cyclic-flow reactor system was constructed and tested as an experimental means of studying interactions between trace metal adsorption and bacterial attachment to inanimate surfaces under defined chemical conditions. Development of a chemically defined growth medium was required for use in the reactor system. Bacterial strains were screened for their ability to grow on the defined medium, to grow in the presence of toxic trace metals, and to attach and grow on inorganic surfaces. The bacterium Pseudomonas cepacia 17616 was determined to be a well suited for experimental analysis of effects of pH and trace metal interactions with bacterial biofilms. A mechanistic, structured model was developed to simulate growth, biopolymer production and association with solid surfaces of freshwater bacteria under conditions of variable pH. A noncompetitive form of inhibition kinetics was applied to modify the maximum growth rate of suspended and attached cells to predict the effects of pH. The bacterial model was also interactively linked to a chemical equilibrium model to quantify trace metal distribution and metal effects on bacterial components as a function of pH. The output from the integrated model revealed that simulations for cell growth and polymer production were not sensitive to variation in the selected molecular dissociation constants. Model simulations were generated for reactors with a low surface to volume ratio (“Multigen†) and a high surface to volume ratio (bioreactor). In simulations of batch operation of either a Multigen or bioreactor, the highest cell concentrations were predicted at neutral pH values. Model simulations of the same duration at the neutral pH values but using alkaline pH values did not retard cell growth to as great an extent as the simulations using acidic pH values. In either the alkaline or acidic pH simulations, peak concentrations of cells and polymer required much longer simulation run times to achieve than the neutral pH simulations. Polymer production increased as pH values moved above or below neutral values in both reactor types. Simulations of continuous operation to steady state in either reactor type revealed little overall variation in the final cumulative concentration of cells or polymer at acidic or alkaline levels compared to production at neutral pH. However, the simulation time to achieve steady state at acidic or alkaline pH values was much longer than at pH 7. A decrease in the growth rates of cells and polymers was predicted under batch conditions at pH 7 or less as the total metal concentration increased from 1x10-9 M to 1x10-5 M. Under these conditions the predominant form of the metal was in the free metal ionic form which was more toxic to cellular and polymer growth. At pH 9, the free metal had little or no effect on the shape of the growth curve due to very low ion concentrations as a result of the formations of complexes and precipitation. Predictions using continuous growth conditions gave results which followed similar patterns, with the washout dilution rate decreasing with increasing total metal concentration at pH 7 or less and with no effect of the total metal on the dilution rate at pH 9. To test the transient responses to fluctuations in total metal concentrations, step increases in metal levels were simulated in the model. At alkaline pH values, there was little effect on the cells and polymer production after addition of the metal. As the pH decreased to circumneutral or acidic values, the concentrations of cells and polymers became strongly affected by the more prevalent toxic free metal ion form and washout of the cells and polymers would occur at lower dilution rates. Models of the type described in this thesis have potential applications in assessing toxic transition metal behavior in both natural aquatic systems and engineered reactor systems with sufficient laboratory work to evaluate the necessary kinetic constants specific to the particular bacterial strains to be modeled.