Biotransformation of toluene, benzene and naphthalene under anaerobic conditions

Aromatic hydrocarbons are widespread in nature, due to increasing industrial activity, and often contribute to polluted soils, sediments, and groundwater. Most of these compounds are toxic at relatively high concentrations, but some are already carcinogenic at very low concentrations, e.g. benzene. A growing awareness of the health risks associated with contamination has directed research to the removal or degradation of such compounds. The use of microorganisms to degrade toxic compounds (bioremediation) is a relatively slow process compared to traditional, chemical methods, but it is a natural process, mostly very specific and low in costs. A review of the available information on the microbial degradation of aromatic compounds is given in chapter 1. The anaerobic degradation is emphasized, since in many polluted environments oxygen is limiting and anaerobic processes will prevail. In the absence of oxygen, compounds like nitrate, metalions (Fe 3+and Mn 4+), sulfate, and carbondioxide, have taken over the function of oxygen as a terminal electron acceptor. In addition, the first transformation reactions differ from those in aerobic processes. Oxygenases are no longer ftinctioning and the degradation of oxygenated aromatic compounds, like benzoate and phenol, is known to occur via e.g. reduction, dehydroxylation and dehydrogenation of the aromatic ring. Information on the anaerobic degradation of mono- and polycyclic aromatic hydrocarbons without functional groups, like toluene, benzene, and naphthalene, is scarse. To gain more insight in the possibilities and limitations of the anaerobic degradation of these aromatic compounds, their behaviour in anaerobic sediment columns was followed. Toluene, benzene, and naphthalene were chosen as model compounds under methanogenic, sulfate-, iron-, manganese-, and nitrate-reducing conditions (Chapter 2). Toluene was transformed readily (within 1 to 2 months), while benzene was recalcitrant over the test period of 375-525 days under all redox conditions tested. Naphthalene was partly transformed in the column with nitrate or manganese as electron acceptor present; the addition of benzoate had a positive effect on the degradation of naphthalene in the column with nitrate. In the column with sulfate, the majority of the added naphthalene disappeared. No effect on the degradation of naphthalene was observed after adding and omitting an easier degradable substrate. [ 14C]naphthalene was used to confirm the disappearance to be the result of degradation; two third of the naphthalene was converted to CO 2 .Numerous attempts have been made for further enrichment of sulfatereducing, naphthalene degrading bacteria (Chapter 3). Unfortunately, the observed degradation of naphthalene in a sediment column could not be obtained in batch cultures, despite the large variety of tested enrichment conditions (different naphthalene concentrations, inoculum. size, medium composition, extra additions etc.). A toxic effect of naphthalene on sulfate- reducing bacteria could not be found.Toluene degradation in the columns was demonstrated under all redox conditions tested. Chapter 4 describes the degradation of toluene in freshly started sediment columns, to which either amorphous or highly crystalline manganese oxide had been added. In batch experiments with material from these columns as inoculum, the degradation of toluene to C0 2 and the formation of biomass under manganese-reducing conditions was demonstrated. The oxidation of toluene was found to be coupled to the reduction of Mn(IV), and the rate of oxidation was found to be lower with the crystalline than with the amorphous manganese oxide. Upon successive transfers of the enrichment cultures, the toluene degrading activity would decrease in time. The activity could only be maintained in the presence of sterilized Rhine river sediment or its supernatant. Without the sediment, but in the presence of solids like teflon beads, glass beads, bentonite, vermiculite and sterilized granular sludge, the toluene degrading activity completely disappeared after 4 to 5 transfers. Furthermore, a direct contact between the bacteria and the manganese oxide was found to be advantageous for a rapid toluene degradation. The degradation rate could further be increased by adding organic ligands such as oxalic acid or nitrilotriacetic acid (NTA).The highly purified enrichment culture LET-13, which degrades toluene with manganese oxide as electron acceptor, was obtained via repeated dilution series, and is described and characterized in chapter 5. LET-13 was able to degrade a variety of substituted monoaromatic compounds like (p-hydroxy) benzylalcohol, (p-hydroxy) benzaldehyde, (p-hydroxy) benzoate, cresol, and phenol. Benzene, ethylbenzene, xylene and naphthalene were not degraded under the experimental conditions used. The degradation of toluene occurred via hydroxylation of the methyl group to benzoate, and a possible side reaction can lead to the formation of cresol.All organisms in the culture look similar; motile rods which are gram negative, oxidase negative and catalase negative. The culture was partly identified with phylogenetic analysis of cloned rDNA sequences. The phylogenetic analysis showed that at least two major groups of bacteria are present. One group of bacteria belongs to the Bacteroides- Cytophaga group, and one group consists of members of the β-subclass of the Proteobacteria.Finally, the results from this research are discussed in relation to their relevance for soil bioremediation technologies.