Toluene : biological waste-gas treatment, toxicity and microbial adaptation

Due to the increasing stringent legislation concerning the emission of volatile organic compounds, there is nowadays a growing interest to apply biological waste-gas treatment techniques for the removal of higher concentrations of specific contaminants from waste gases. Fluctuations in the contaminant concentrations can strongly affect the performance of bioreactors used for the treatment of waste-gas streams. Temporary high concentrations can be toxic for the microorganisms in the reactor, resulting in inactivation of the system. Furthermore, for a reliable operation the design of the reactor should be based on the peak concentrations in the waste gas, which is not an economically favourable situation. Therefore, it would be desirable to buffer the fluctuations in the contaminant concentration by means of an adsorbent, so that a constant supply of contaminants to the bioreactor can be achieved. The buffer capacity of a number of activated carbons and other adsorbents was tested (Chapter 2). Using one selected type of activated carbon it was demonstrated that fluctuations between 0 and 1000 mg toluene per m 3 could be transformed to an average value of about 300 mg/m 3 , which was subsequently completely degraded in a biofilter. Without an activated-carbon column significant amounts of toluene were not degraded in the biofilter when the inlet concentration was 1000 mg/m3, A disadvantage of trickie-bed reactors for biological waste-gas treatment is the reduction in reactor performance which is sometimed observed due to the formation of an excessive amount of biomass resulting in clogging. By limiting the amount of nutrients available for biomass formation it was attempted to prevent clogging of the reactor (Chapter 3). As a consequence of this nutrient limitation a reduced removal rate of toluene was observed. However, when a fungal culture was used to inoculate the reactor, the toluene removal rate under nutrient limiting conditions was almost twice as high. Over a period of 375 days an average removal rate of 27 g-C/(m 3 h) was obtained with this fungal culture. These results clearly show that, even under non-sterile conditions, inoculation of the reactor with a specific starter culture can influence the reactor performance over a prolonged period of time. From the carbon balance over the reactor and the nitrogen availability it was concluded that under these nutrient-limited conditions large amounts of carbohydrates are formed. Even under nutrientlimited conditions the biomass content (including extracellular polysaccharides and other polymers) of the reactor increased, which eventually can result in clogging of the reactor. In order to prevent clogging eventually biomass has to be removed regularly from the reactor. We therefore studied the application of a NaOH- wash to remove excess biomass. Using regular NaOH-washes an average toluene removal rate of 35 g-C/(m 3 h) was obtained with a mixed culture of bacteria. After about 50 days there was no longer a net increase in the biomass content of the reactor. The amount of biomass which was formed in the reactor equalled the amount removed by the NaOH-wash Under these conditions it should be possible to maintain a high toluene removal rate without clogging of the reactor taking place for a long period of time. From a biofilter used for the removal of toluene from waste gases we have isolated the fungus, Cladosporium sphaerospermum, which is able to grow on toluene as the sole source of carbon and energy (Chapter 4). To our knowledge this is the first report of toluene catabolism by an eukaryotic microorganism. The oxygen-consumption rates as well as the measured enzyme activities of toluenegrown C. sphaerospermum indicate that toluene is degraded by an initial attack on the methyl group. The toxicity of various pollutants in a waste-gas stream for microorganisms could limit the application of biological waste-gas treatment techniques. Especially compounds, with a good solubility in water can be expected to accumulate in the water-phase of the reactor during the start-up period. This accumulation can result in the inactivation of the biomass in the reactor as the contaminant concentrations reach toxic levels. The toxicity of various volatile organic compounds frequently present as contaminants in waste gases has been determined (Chapter 5). For both the Gram-positive Rhodococcus S5 and the Gram-negative Pseudomonas S12 the toxicity was assessed as the concentration which reduced the growth rate of the bacterium with 50%. No significant differences were observed between the IC50% values for these two bacteria. A relationship between the toxicity and hydrophobicity of various substituted benzene compounds was observed. Surprisingly, one of the selected bacteria, Pseudomonas putida S12 was able to adapt to the presence of high concentrations of contaminants. This adaptation resulted in the capacity of this strain to grow in the presence of supersaturating amounts of toluene (Chapter 6). In general, the toxicity of organic solvents is caused by the accumulation of these lipophilic solvents in the membrane lipid bilayer, affecting structural and functional properties of the membrane. The accumulation of solvents in the cell membrane can affect both the membrane fluidity and the bilayer stability. The physico-chemical effects of alkanols, alkanes and other hydrocarbons on biomembranes are summarized in Chapter 8. Although organic solvents can be highly toxic for microorganisms, some microorganisms are able to grow in the presence of concentrations of these solvents which are generally toxic (Chapter 8). P. putida S12 and two other solvent tolerant P. putida strains reacted to toxic concentrations of toluene by accumulating trans unsaturated fatty acids in the membrane instead of the cis isomers. This higher trans / cis ratio of the unsaturated fatty acids in toluene-adapted cells resulted a higher lipid-ordering since the gel to liquid-crystalline transition temperature was about 7-9 °C higher compared to the non- adapted cells. This cis / trans isomerization of fatty acids is probably a mechanism to very quickly compensate for the increase in membrane fluidity, and destabilization of the bilayer structure caused by toluene accumulating in the membrane (Chapter 7 & 8). Apart from changes in the fatty acid composition also changes in the phospholipid composition were observed when the organism was grown in the presence of toluene. The decreased incorporation of phosphaticlylethanolamine and the increased incorporation of diphosphatidylethanolamine (cardiolipin) are expected to prevent the formation of non-bilayer phospholipids configurations which might be caused by toluene (Chapter 8). In Chapter 9 the results presented in this thesis are discussed in relation to biotechnological applications.