Anaerobic degradation of limonene andp-xylene in freshwater enrichmentcultures

Abstract

The anaerobic degradation of hydrocarbons has been intensively explored in the last decade yielding insights into new physiological capabilities and biochemical pathways. However, for a few hydrocarbons, e.g. p-xylene, it proved to be more difficult to enrich microorganisms and to isolate pure strains. Thermodynamically, the mineralization of hydrocarbons is least favorable under anoxic conditions and especially under methanogenic conditions. In my thesis, two enrichment cultures were characterized, a methanogenic freshwater enrichment culture grown on limonene, the most abundant monoterpene in nature, and a denitrifying freshwater enrichment culture grown on p-xylene. The methanogenic enrichment culture consumed limonene with proportional formation of methane. The full cycle 16S rRNA gene approach revealed the presence of Archaea related to Methanosaeta and Methanoculleus and Bacteria related to Syntrophobacteraceae, Bacteroidetes, and the Candidate Division OP3. This candidate phylum lies within the Planctomycetes, Chlamydiae, Verrucomicrobia, Lentisphaerae superphylum and has no member in culture. Hence nucleic acid probes were developed to target the OP3-phylotype. The probe detected very small spherical cells, which lived alone or attached to larger cells and represented 18% of the total DAPI-stained population. Thus they may play an important role in limonene degradation. The other Bacteria were mainly Deltaproteobacteria (13%) and only 1% were Bacteroidetes. Together with a new EUB-338 probe specific for the OP3 cells, the bacterial 338 probe mixture detected 40% of the total cells whereas the Archaea probe detected 33%. The presence of several phylotypes suggests that more then one syntrophic bacterium and one methanogenic archaeon are involved in limonene degradation to methane gas and carbon dioxide. Syntrophic bacteria were isolated using fumarate and lactate as organic substrates for fermentation. All seven strains belonged phylogenetically to Deltaproteobacteria, with 11% 16S rRNA gene dissimilarity to Desulfoarculus baarsii. Initial co-culture experiments with Methanosarcina mazei showed growth on lactate or fumarate in the presence of limonene. However, their role in limonene degradation could not demonstrated. Denitrifying enrichment cultures were established with a freshwater sediment mix and p-xylene as sole electron donor and carbon source. Several batch transfers and liquid dilution-to-extinction series enriched a curved rod morphotype, 0.5 by 2 μm in size. This dominant morphotype (96% of all cells) was as well identified as the dominant phylotype (91-95%) using the 16S rRNA full cycle approach. The organism affiliated phylogenetically with Betaproteobacteria and not with other anaerobic hydrocarbon degrading microorganisms from the Azoarcus-Thauera clade. Their closest relative was a group of steroid-degraders: Denitratisoma oestradiolicum, Sterolibacterium denitrificans and strain 72Chol.This enrichment cultures coupled complete mineralization of p-xylene to denitrification of nitrate to dinitrogen gas. By gas chromatography mass spectrometric analysis of metabolites found in cell extracts, (4-methylbenzyl)succinate and (4-methylbenzyl)itaconate were identified, supporting an activation mechanism by addition to fumarate. A gene fragment for a benzylsuccinyl synthase could be sequenced, and revealed amino acid similarities with TutD from the well known toluene degrading denitrifier, Thauera aromatica. This study has revealed that anaerobic hydrocarbon degradation involves a broad diversity of microorganism, even outside the phylum Proteobacteria. My thesis established the participation of novel organisms to anaerobic hydrocarbon degradation and revealed for the first time the morphology of Candidate phylum OP3 cells.