Three novel, anaerobic cultures that degrade MTBE with Fe(III)/AQDS, sulfate, and fumarate as electron acceptors, respectively

A major obstacle in understanding methyl tert-butyl ether (MTBE) biodegradation under anoxic conditions has been the lack of model anaerobic cultures with stable MTBE-degrading activity. The primary limitation to understanding microbial physiology, biochemistry, and molecular ecology of anaerobic MTBE metabolism has been the lack of liquid cultures that maintain consistent activity with repeated transfers, in a reasonable timeframe for experimentation. Although anaerobic MTBE biodegradation has been reported in sediment or soil incubations (microcosms), to date stable liquid cultures have not been characterized in terms of activity or microbial community composition. Aerobic MTBE and TBA biodegradation fundamental research is significantly ahead of anaerobic research due to the number of available pure or mixed cultures; it is this gap between aerobic and anaerobic knowledge we intend to close. In this work, three distinct sediment- free, MTBE-degrading, anaerobic cultures were derived from MTBE-contaminated aquifer material: strains NW1, NW2 and NW3. The electron acceptors used are anthraquinone- 2,6-disulfonate (AQDS; NW1), sulfate (NW2) and fumarate (NW3), respecttively. 1-2mM MTBE is consistently degraded within 20 to 30 days in each culture. MTBE degradation is continuous with each MTBE re-amendment within the same culture or upon transfer to new medium. In addition, molecular analyses using amplified ribosomal DNA restriction analysis (ARDRA) indicate that the dominant organisms are truly novel, in that they are not more than 91% related to any known isolates. MTBE-contaminated aquifer sediment was obtained from a British Petroleum site in California. Enrichments were started in bicarbonate-buffered freshwater medium with several electron acceptors. Three cultures developed with the electron acceptors listed above. Each individual culture was re-spiked with MTBE five times, and was transferred to fresh medium when MTBE reached zero. The cultures have all been transferred more than five times, so there is no residual sediment remaining. Each culture degrades MTBE with tert-butyl alcohol (TBA) production. TBA production is stoichiometric with AQDS and fumarate, but it begins to decline in sulfate-amended cultures if no MTBE is re-added. AQDS-grown strain NW1 also oxidizes benzene, ethanol, lactate, and formate as sole electron donors and carbon. Sulfate grown strain NW2 only utilizes lactate and formate. The only other growth substrate for fumarate-grown strain NW3 identified thus far is toluene. All strains have phylogenetic relatives within the proteobacteria and within the firmicutes that are 97-99% related, but these are all uncultured clones with no genus affiliation. The nearest isolated relatives are 84-92% related, which likely means unique genera: in strain NW1 (∼50% of total community) it is Desulfuromusa kysingii at 84.7% related; in strain NW2 (∼25% of total community) it is Rubrobacter taiwanensis at 91.4% related; in strain NW3 (∼16% of total community) it is Owenweeksia hongkongensis at 86.1% related.