Ammonium oxidation by autotrophic ammonia-oxidizing bacteria (AOB) is a key process in agricultural and natural ecosystems and has a large global impact. In the past, the ecology and physiology of AOB were not well understood because these organisms are notoriously difficult to culture. Recent applications of molecular techniques have advanced our knowledge of AOB, but the necessity of using PCR-based techniques has made quantitative measurements difficult. A quantitative real-time PCR assay targeting part of the ammoniamonooxygenase gene (amoA) was developed to estimate AOB population size in soil. This assay has a detection limit of 1.3 ؋ 10 5 cells/g of dry soil. The effect of the ammonium concentration on AOB population density was measured in soil microcosms by applying 0, 1.5, or 7.5 mM ammonium sulfate. AOB population size and ammonium and nitrate concentrations were monitored for 28 days after (NH 4 ) 2 SO 4 application. AOB populations in amended treatments increased from an initial density of approximately 4 ؋ 10 6 cells/g of dry soil to peak values (day 7) of 35 ؋ 10 6 and 66 ؋ 10 6 cells/g of dry soil in the 1.5 and 7.5 mM treatments, respectively. The population size of total bacteria (quantified by real-time PCR with a universal bacterial probe) remained between 0.7 ؋ 10 9 and 2.2 ؋ 10 9 cells/g of soil, regardless of the ammonia concentration. A fertilization experiment was conducted in a tomato field plot to test whether the changes in AOB density observed in microcosms could also be detected in the field. AOB population size increased from 8.9 ؋ 10 6 to 38.0 ؋ 10 6 cells/g of soil by day 39. Generation times were 28 and 52 h in the 1.5 and 7.5 mM treatments, respectively, in the microcosm experiment and 373 h in the ammonium treatment in the field study. Estimated oxidation rates per cell ranged initially from 0.5 to 25.0 fmol of NH 4 ؉ h ؊1 cell ؊1 and decreased with time in both microcosms and the field. Growth yields were 5.6 ؋ 10 6 , 17.5 ؋ 10 6 , and 1.7 ؋ 10 6 cells/mol of NH 4 ؉ in the 1.5 and 7.5 mM microcosm treatments and the field study, respectively. In a second field experiment, AOB population size was significantly greater in annually fertilized versus unfertilized soil, even though the last ammonium application occurred 8 months prior to measurement, suggesting a long-term effect of ammonium fertilization on AOB population size.Ammonium oxidation by autotrophic ammonia-oxidizing bacteria (AOB) is a key process in agricultural and natural ecosystems, with a large global impact. The product of this process, nitrite, is immediately oxidized by nitrite-oxidizing bacteria to nitrate, a nitrogen form susceptible to leaching. Nitrogen leaching can lead to groundwater pollution and surface and groundwater eutrophication. Nitrous oxide and nitric oxide, by-products of ammonia oxidation, contribute to the greenhouse effect and ozone layer depletion. On a local scale, loss of nitrate to groundwater and nitrous oxide and nitric oxide to the atmosphere reduces the amount of nitrogen available ...

Methyl tert-butyl ether (MTBE) is a widespread groundwater contaminant that does not respond well to conventional treatment technologies. Growing evidence indicates that microbial communities indigenous to groundwater can degrade MTBE under aerobic and anaerobic conditions. Although pure cultures of microorganisms able to degrade or cometabolize MTBE have been reported, to date the specific organisms responsible for MTBE degradation in various field studies have not be identified. We report that DNA sequences almost identical (99% homology) to those of strain PM1, originally isolated from a biofilter in southern California, are naturally occurring in an MTBE-polluted aquifer in Vandenberg Air Force Base (VAFB), Lompoc, California. Cell densities of native PM1 (measured by TaqMan quantitative PCR) in VAFB groundwater samples ranged from below the detection limit (in anaerobic sites) to 10 3 to 10 4 cells/ml (in oxygenamended sites). In groundwater from anaerobic or aerobic sites incubated in microcosms spiked with 10 g of MTBE/liter, densities of native PM1 increased to approximately 10 5 cells/ml. Native PM1 densities also increased during incubation of VAFB sediments during MTBE degradation. In controlled field plots amended with oxygen, artificially increasing the MTBE concentration was followed by an increase in the in situ native PM1 cell density. This is the first reported relationship between in situ MTBE biodegradation and densities of MTBE-degrading bacteria by quantitative molecular methods.The fuel oxygenate, methyl tert-butyl ether (MTBE), is a widespread groundwater contaminant in the United States and parts of Europe. MTBE is a water soluble and mobile compound that has the potential to generate long pollution plumes in aquifers impacted by gasoline releases from leaking tanks. In California, at least 10,000 leaking underground storage tank sites are contaminated with MTBE (14).MTBE biodegradation has been reported in a number of environments, including sewage sludge (29), soils (37), river sediments (3, 4), and biofilters (9, 11). Numerous MTBEdegrading isolates have been discovered, including direct metabolizers and cometabolizers (5,7,12,13,15,16,18,20,26,30,31,34). Among these isolates, strain PM 1 is a member of the beta-Proteobacteria and closely related to Aquabacterium, Leptothrix, and Rubrivivax (5). Strain PM1 rapidly degrades MTBE, mineralizes [14 C]MTBE to 14 CO 2 , and uses the compound as a sole carbon and energy source (8, 13).Field evidence indicates that, at many contaminated sites, natural degradation rates are not sufficiently rapid to prevent MTBE plumes from continuing to elongate, except where sufficient concentrations of oxygen are present. In situ technologies for active bioremediation of MTBE-contaminated aquifers have been evaluated at several field sites (3,4,6,23,31,36). Oxygen has been added by sparging (31), by oxygen-releasing compounds (23), and via diffusive release across permeable membranes (24,25,36). At Vandenberg Air Force Base (VAFB) in Lompoc, California, Mack...

Microbial communities in subsurface environments are poorly characterized and the impacts of anthropogenic contamination on their structure and function have not been adequately addressed. The release of contaminant(s) to a previously unexposed environment is often hypothesized to decrease the diversity of the affected community. We characterized the structure of microbial communities along a gradient of benzene, toluene, ethylbenzene, and xylene (BTEX) and methyl-tert-butyl-ether (MTBE) contamination, resulting from a petroleum spill, within a shallow sandy aquifer at Vandenberg Air Force Base (VAFB) in Lompoc, CA. Differences in microbial community composition along the contaminant plume were assessed via a combinatorial approach utilizing denaturing gradient gel electrophoresis (DGGE), cloning and sequencing, intergenic transcribed spacer analysis (ITS), and comparative phylogenetic analysis of partial 16S rDNA sequences. Substantial bacterial sequence diversity, similar levels of species richness, and similar phylo-groups (including the Cytophaga-Flavobacterium-Bacteroidetes group and numerous members of the alpha-, beta-, gamma-, delta-, and epsilon-groups of the proteobacteria) were observed in both uncontaminated and contaminated regions of the aquifer. High-resolution measures (ITS fingerprinting and phylogenetic inference) readily separated communities impacted by the original petroleum spill (in source zone) from those in other parts of the aquifer and indicated that communities exposed to MTBE only were similar to communities in uncontaminated regions. Collectively, these data suggest that petroleum contamination alters microbial community structure at the species and subspecies level. Further study is required to determine whether these changes have an impact on the functioning of this subsurface ecosystem.