The pollution of our environment with a large number of synthetic organic chemicals has raised serious concern about their toxicity to existing life forms. Microorganisms play an essential role in the breakdown of xenobiotic compounds by using them as a carbon and energy source. However, pollutants that are intrinsically biodegradable are still widespread in soil, groundwater and natural waters at typical concentrations at the nanomolar to micromolar level. This has raised questions about microbial degradation kinetics at low concentration levels and about factors which prevent complete biotransformation.
The project of this thesis was initiated to study the biodegradation of xenobiotic compounds in the low concentration range. The work presented here focussed on three main topics: (i) biotransformation kinetics at low concentration levels, (ii) the lowest attainable or residual concentration, and (iii) the effects of experimental conditions on the kinetics and residual concentrations. We examined the influence of the growth state of the organisms, the presence of additional readily degradable substrate, the temperature, the liquid flow regime and the process of mass transfer. The studies were done using a pure culture of Pseudomonas sp. strain B13 in a defined medium. 3-Chlorobenzoate (3CB) was used as a model pollutant and acetate as a model compound for easily degradable substrate. We used experimental systems with different levels of complexity, this to be able to distinguish the influence of intrinsic microbial properties and system-linked factors on the biotransformation kinetics and residual concentrations.
Batch experiments with resting cell suspensions of strain B13 are presented in Chapter 2. Transformation of 3CB; is described by Michaelis-Menten kinetics in the wide concentration range of 1.5 μM to 16 mM ( K m , 0.13 mM; V max , 24 nmol mg protein -1min -1). Experiments at the nanomolar and low micromolar concentration level indicated that probably a second system for uptake or transformation at low concentrations is present in strain B13. The system operating above 1 μM 3CB possessed an apparent threshold concentration of 0.50 ± 0. 11 μM. The system that was active below 1 μM showed first order kinetics and no saturation, as is typical for Michaelis-Menten kinetics. This system had a rate constant of 0.076 liter g protein -1min -1and no detectable threshold concentration. No residual concentrations could be detected for 3CB and acetate when they were spiked as single substrates (detection limits, 1.0 and 0.5 nM, respectively). The addition of various concentrations of acetate as a second, readily degradable substrate neither affected the 3CB transformation kinetics, nor did it generate a detectable residual concentration of either substrate.
Residual concentrations of 3CB and acetate were measured in well-mixed suspended cell fermentors with continuous substrate supply (Chapter 3). Growth kinetic parameters of stain B13 determined the steady-state residual substrate concentrations in chemostats. In a fermentor with 100% biomass retention (recycling fermentor), the lowest residual concentration measured was S min , the minimum concentration required for growth. S min was measured in a stationary phase of net zero growth, and was determined by the maintenance requirement and the transformation kinetic parameters of strain B13 in the recycling fermentor. Both maintenance coefficient and S min were lower in recycling fermentors at 20°C than at 30°C. When provided as a mixture, the individual S min values of 3CB and acetate were lower than the measured S min values during single substrate use. The S min values with the mixture reflected the relative energy contribution of the two substrates in the fermentor feed. The kinetic parameters of 3CB transformation, and thus predicted S min values, varied remarkably with the different growth states of strain B13 in batch (resting state), recycling fermentor (very slow growth) and chemostat (exponential growth).
The kinetics of substrate transfer to the microorganisms, and not the intrinsic microbial kinetics, may under certain conditions determine the biodegradation rate. The possible effect of mass transfer on residual concentrations of 3CB was studied in percolated soil columns with attached resting cells of strain B13 (Chapter 4). The rate of mass transfer was varied by applying different flow rates and biomass levels. The observed residual concentrations in steady state were compared with predictions from spreadsheet models describing the combined action of microbial and mass transfer kinetics. The effluent concentrations were successfully predicted above a critical ratio of flow rate and biomass. Below this critical point, the experimental concentrations were higher than predicted and this deviation increased with decreasing flow rate:biomass ratios. These results corresponded remarkably with literature data on 3-chlorodibenzofuran degradation in percolation columns with Sphingomonas sp. strain HH19k. It was calculated that 3CB transformation was probably limited by convective- diffusive transport of both 3CB and oxygen to the cells.
The relevance of the results presented in this thesis is discussed in Chapter 5. The results are included in a short review of literature data reporting on the kinetics of uptake and transformation, on residual concentrations and on the factors which are of influence, such as concentration level, bacterial growth state, thresholds, maintenance energy requirement, other carbon substrates and mass transfer kinetics.
The gathered knowledge on these topics supports the following concluding remarks for pollutant degradation in natural and engineered environments. S min can be considered as the lower concentration limit of a compound in relatively stable or steady-state environments. In non-stable systems like batch incubations, even lower residual concentrations are expected, providing that bioavailability of the pollutant or thresholds for utilization are not limiting the degradation. The significance for bioremediation techniques may lie in the application of batch- like or pulsing systems (Chapter 2) and/or the provision of low concentrations of additional substrates (Chapter 3) to reduce residual pollutant concentrations.
Microbially determined S min values are probably much lower in natural carbon-limited environments than in studies with one organism/substrate combination. Such S min values may even be far below a detection limit, if we consider the 1000 times lower in situ maintenance coefficients compared to pure culture suspensions (Chapter 3). Intrinsically biodegradable pollutants are still detected in many environments, however. It is therefore suggested that mass transfer kinetics (Chapter 4), and not microbial kinetics determine the S min in these environments. Residual pollutant concentrations can then only be reduced by improving the bioavailability of the compounds.
|Doctor of Philosophy
|4 Dec 1996
|Place of Publication
|Published - 4 Dec 1996
- microbial degradation