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The minimum substrate concentration required for growth, Smin, was measured for Pseudomonas sp. strain B13 with 3-chlorobenzoate (3CB) and acetate in a recycling fermentor. The substrates were provided alone or in a mixture. Smin values predicted with kinetic parameters from resting-cell batches and chemostat cultures differed clearly from the values measured in the recycling fermentor. When 3CB and acetate were fed as single substrates, the measured Smin values were higher than the individual Smin values in the mixture. The Smin in the mixture reflected the relative energy contributions of the two substrates in the fermentor feed. The energy-based maintenance coefficients during zero growth in the recycling fermentor were comparable for all influent compositions (mean +/- standard deviation, 0.34 +/- 0.07 J mg [dry weight]-1 h-1). Maintenance coefficient values for acetate were significantly higher in chemostat experiments than in recycling-fermentor experiments. 3CB maintenance coefficients were comparable in both experimental systems. The parameters for 3CB consumption kinetics varied remarkably with the experimental growth conditions in batch, chemostat, and recycling-fermentor environments. The results demonstrate that the determination of kinetic parameters in the laboratory for prediction of microbial activity in complex natural systems should be done under conditions which best mimic the system under consideration.

Soil column experiments were performed to obtain insight in the different biological and physico-chemical processes affecting biodegradation of halogenated compounds under natural conditions in a water infiltration site. Lower chlorinated aromatic compounds could be degraded under aerobic conditions, whereas highly chlorinated compounds and chlorinated aliphatic compounds were mainly transformed under anaerobic conditions. Microorganisms which derive energy from reductive dechlorination were enriched and characterized. It was found that microbes could adapt to using chlorinated benzenes by evolution of new enzyme specificities and by exchange of genetic material. For halogenated pollutants, which are generally hydrophobic, sorption processes control the concentration available for biodegradation. The effects of very low concentrations of halogenated compounds on their biodegradability are described. The use of isolated bacterial strains to enhance biodegradation was evaluated with respect to their temperature-related activity and to their adhesion properties.

A soil from a former chemical redistribution company, contaminated with mainly chlorinated aliphatics, was studied for bioremediation purposes. Groundwater analyses revealed that the original pollutants, i.e. tetrachloroethene (PCE) and trichloroethene (TCE), were present at levels ranging from 2.3 to 122 mg/L. Dichloroethene (DCE), vinylchloride (VC), ethene and ethane were also detected at significant concentrations although they had never been introduced to the soil. Relatively high concentrations of cis-DCE as compared to trans-DCE and 1,1-DCE indicated that a slow in situ biodegradation had taken place by reductive dechlorination. Laboratory experiments with flow-through soil columns were performed to determine the optimal conditions for the enhancement of reductive dechlorination by the indigenous dechlorinating population. The addition of single electron donors to artificial groundwater resulted in the dechlorination of PCE to TCE and cis-DCE, whereas complete dechlorination to ethene was solely achieved with compost extract added to native groundwater.

The biotransformation of 3-chlorobenzoate (3CB) by attached cells of Pseudomonas sp. strain B13 was studied in percolation columns operated at various flow rates and biomass contents. Steady-state residual effluent concentrations 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 steady-state residual effluent concentrations were higher than predicted and this deviation increased with decreasing flow rate:biomass ratios. The results are discussed in the light of convection-diffusion processes limiting the transport of both substrate and oxygen to the attached cells.

In this paper a model is presented which can be used to simulate the behaviour of xenobiotic chemicals in soil columns with respect to their physical and chemical properties. Terms describing biological transformation of xenobiotics are also included in the model. It incorporates microbial growth following Monod kinetics and a chemotactic response of the transforming bacteria towards the xenobiotic substrate. The model appeared to yield good simulations of an experiment by Van der Meer et al. (1987) who investigated the degradation of 1,2-dichlorobenzene in soil columns inoculated with Pseudomonas sp. strain P51. The behaviour and the fate of 1,4-dichlorobenzene as found by Kuhn et al. (1985) can also be simulated using this model but their results were also adequately simulated using a simple second order model. The results generated by the model correspond to kinetic parameters obtained in other studies. It is concluded that the model is a useful tool for the investigation of the activity of bacteria degrading xenobiotics in soil columns, provided that the microbial parameters can be determined in independent experiments, and that the active microbial mass in the soil can be measured.

All three isomers of trichlorobenzene were reductively dechlorinated to monochlorobenzene via dichlorobenzenes in anaerobic sediment columns. The dechlorination was specific: 1,2,3- and 1,3,5-trichlorobenzene were solely transformed to 1,3-dichlorobenzene, while 1,4-dichlorobenzene was the only product of 1,2,4-trichlorobenzene transformation. Microorganisms were responsible for the observed transformations. Since monochlorobenzene and dichlorobenzene are mineralized by bacteria in the presence of oxygen, the process of reductive dechlorination may be an important initial step to obtain complete mineralization of otherwise recalcitrant trichlorobenzenes. This is especially true for the 1,3,5-isomer, which seems to resist biodegradation in oxic environments.

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 1.5μM 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 1μM 3CB possessed an apparent threshold concentration of 0.50 ± 0. 11μM. The system that was active below 1μM 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.