Comparative transcriptome analysis of Methylibium petroleiphilum PM1 exposed to the fuel oxygenates methyl tert-butyl ether and ethanol

High-density whole-genome cDNA microarrays were used to investigate substrate-dependent gene expression of Methylibium petroleiphilum PM1, one of the best-characterized aerobic methyl tert-butyl ether (MTBE)-degrading bacteria. Differential gene expression profiling was conducted with PM1 grown on MTBE and ethanol as sole carbon sources. Based on microarray high scores and protein similarity analysis, an MTBE regulon located on the megaplasmid was identified for further investigation. Putative functions for enzymes encoded in this regulon are described with relevance to the predicted MTBE degradation pathway. A new unique dioxygenase enzyme system that carries out the hydroxylation of tert-butyl alcohol to 2-methyl-2-hydroxy-1-propanol in M. petroleiphilum PM1 was discovered. Hypotheses regarding the acquisition and evolution of MTBE genes as well as the involvement of IS elements in these complex processes were formulated. The pathways for toluene, phenol, and alkane oxidation via toluene monooxygenase, phenol hydroxylase, and propane monooxygenase, respectively, were upregulated in MTBE-grown cells compared to ethanol-grown cells. Four out of nine putative cyclohexanone monooxygenases were also upregulated in MTBE-grown cells. The expression data allowed prediction of several hitherto-unknown enzymes of the upper MTBE degradation pathway in M. petroleiphilum PM1 and aided our understanding of the regulation of metabolic processes that may occur in response to pollutant mixtures and perturbations in the environment.     

Reduction of perchlorate and nitrate by microbial communities in vadose soil

Perchlorate contamination is a concern because of the increasing frequency of its detection in soils and groundwater and its presumed inhibitory effect on human thyroid hormone production. Although significant perchlorate contamination occurs in the vadose (unsaturated) zone, little is known about perchlorate biodegradation potential by indigenous microorganisms in these soils. We measured the effects of electron donor (acetate and hydrogen) and nitrate addition on perchlorate reduction rates and microbial community composition in microcosm incubations of vadose soil. Acetate and hydrogen addition enhanced perchlorate reduction, and a longer lag period was observed for hydrogen (41 days) than for acetate (14 days). Initially, nitrate suppressed perchlorate reduction, but once perchlorate started to be degraded, the process was stimulated by nitrate. Changes in the bacterial community composition were observed in microcosms enriched with perchlorate and either acetate or hydrogen. Denaturing gradient gel electrophoresis analysis and partial sequencing of 16S rRNA genes recovered from these microcosms indicated that formerly reported perchlorate-reducing bacteria were present in the soil and that microbial community compositions were different between acetate- and hydrogen-amended microcosms. These results indicate that there is potential for perchlorate bioremediation by native microbial communities in vadose soil.     

Impact of trichloroethylene and toluene on nitrogen cycling in soil.

The effects of trichloroethylene (TCE) and toluene on soil nitrogen-cycling activities were examined. Ammonium oxidation potential (AOP) was reduced after incubation with as little as 1 microgram of TCE ml-1, and the effects were generally greater when toluene was present and increased with longer exposure. Arginine ammonification potential and denitrification enzyme activity were constant regardless of TCE concentration or the presence of toluene, while nitrite oxidation potential (NOP) exhibited variable sensitivity. KCl-extractable ammonium levels increased dramatically after exposure to 30 and 60 micrograms of TCE ml-1 in the presence of toluene, whereas gamma-irradiated or sodium azide-treated soil incubated with the same concentrations of TCE and toluene showed no increase. Alfalfa-amended soils showed similar decreases in AOP and increases in extractable ammonium during incubation with 60 micrograms of TCE ml-1 and 20 micrograms of toluene ml-1, although most probable number estimates of the ammonium oxidizer population showed no difference between exposed and unexposed soil. AOP and extractable ammonium returned slowly to control levels after 28 days of incubation in the presence of TCE and toluene. Activity assays to which various TCE and toluene concentrations were added indicated that AOP and NOP were relatively more sensitive to these compounds than was arginine ammonification potential. These results indicate that the soil microbial populations responsible for nitrogen cycling exhibit different sensitivities to TCE and toluene and that they may be more susceptible to adverse effects than previously thought.     

Land use and climatic factors structure regional patterns in soil microbial communities

Aim  Although patterns are emerging for macroorganisms, we have limited understanding of the factors determining soil microbial community composition and productivity at large spatial extents. The overall objective of this study was to discern the drivers of microbial community composition at the extent of biogeographical provinces and regions. We hypothesized that factors associated with land use and climate would drive soil microbial community composition and biomass.
Location  Great Basin Province, Desert Province and California Floristic Province, California, USA.
Methods  Using phospholipid fatty acid analysis, we compared microbial communities across eight land-use types sampled throughout the State of California, USA ( n = 1117).
Results  The main factor driving composition and microbial biomass was land-use type, especially as related to water availability and disturbance. Dry soils were more enriched in Gram-negative bacteria and fungi, and wetter soils were more enriched in Gram-positive, anaerobic and sulphate-reducing bacteria. Microbial biomass was lowest in ecosystems with the wettest and driest soils. Disturbed soils had less fungal and more Gram-positive bacterial biomass than wildland soils. However, some factors known to influence microbial communities, such as soil pH and specific plant taxa, were not important here.
Main conclusions  Distinct microbial communities were associated with land-use types and disturbance at the regional extent. Overall, soil water availability was an important determinant of soil microbial community composition. However, because of the inclusion of managed and irrigated agricultural ecosystems, the effect of precipitation was not significant. Effects of environmental and management factors, such as flooding, tillage and irrigation, suggest that agricultural management can have larger effects on soil microbial communities than elevation and precipitation gradients.     

Effect of nitrogen and phosphorus addition on phenanthrene biodegradation in four soils

Phenanthrene mineralization rates were found to vary widely among four soils; differences in soil nutrient levels was one hypothesis to explain this variation. To test this hypothesis, phenanthrene mineralization rates were measured in these soils with, and without, added nitrogen and phosphorus. Mineralization rates either remained unchanged or were depressed by the addition of nitrogen and phosphorus. Phenanthrene degradation rates remained unchanged in the soil which had the highest indigenous levels of nitrogen and phosphorus and which showed the largest increase in phosphorus levels after nutrients were added. The soils in which degradation rates were depressed had lower initial phosphorus concentrations and showed much smaller or no measurable increase in phosphorus levels after nutrients were added to the soils. To understand the response of phenanthrene degradation rates to added nitrogen and phosphorus, it may be necessary to consider the bioavailability of added nutrients and nutrient induced changes in microbial metabolism and ecology.     

Biodegradation of trichloroethylene and toluene by indigenous microbial populations in vadose sediments

The unsaturated subsurface (vadose zone) receives significant amounts of hazardous chemicals, yet little is known about its microbial communities and their capacity to biodegrade pollutants. Trichloroethylene (TCE) biodegradation occurs readily in surface soils; however, the process usually requires enzyme induction by aromatic compounds, methane, or other cosubstrates. The aerobic biodegradation of toluene and TCE by indigenous microbial populations was measured in samples collected from the vadose zone at unpolluted and gasoline-contaminated sites. Incubation at field moisture levels showed little activity on either TCE or toluene, so samples were tested in soil suspensions. No degradation occurred in samples suspended in water or phosphate buffer solution; however, both toluene and TCE were degraded in samples suspended in mineral salts medium. TCE degradation depended on toluene degradation, and little loss occurred under sterile conditions. Studies with specific nutrients showed that addition of ammonium sulfate was essential for degradation, and addition of other mineral nutrients further enhanced the rate. Additional studies with vadose sediments amended with nutrients showed similar trends to those observed in sediment suspensions. Initial rates of biodegradation in suspensions were faster in uncontaminated samples than in gasolinecontaminated samples, but the same percentages of chemicals were degraded. Biodegradation was slower and less extensive in shallower samples than deeper samples from the uncontaminated site. Two toluene-degrading organisms isolated from a gasoline-contaminated sample were identified as Corynebacterium variabilis SVB74 and Acinetobacter radioresistens SVB65. Inoculation with 10⁶ cells of C. variabilis ml^–1 of soil solution did not enhance the rate of degradation above that of the indigenous population. These results indicate that mineral nutrients limited the rate of TCE and toluene degradation by indigenous populations and that no additional benefit was derived from inoculation with a toluene-degrading bacterial strain. Correspondence to: K.M. Scow     

Effect of carbon:nitrogen ratio on kinetics of phenol biodegradation byAcinetobacter johnsonii in saturated sand

In polluted soil or ground water, inorganic nutrients such as nitrogen may be limiting, so that Monod kinetics for carbon limitation may not describe microbial growth and contaminant biodegradation rates. To test this hypothesis we measured¹⁴CO₂ evolved by a pure culture ofAcinetobacter johnsonii degrading 120 µg¹⁴C-phenol per ml in saturated sand with molar carbon:nitrogen (CN) ratios ranging from 1.5 to 560. We fit kinetics models to the data using non-linear least squares regression. Phenol disappearance and population growth were also measured at CN1.5 and CN560.After a 5- to 10-hour lag period, most of the¹⁴CO₂ evolution curves at all CN ratios displayed a sigmoidal shape, suggesting that the microbial populations grew. As CN ratio increased, the initial rate of¹⁴CO₂ evolution decreased. Cell growth and phenol consumption occurred at both CN1.5 and CN560, and showed the same trends as the¹⁴CO₂ data. A kinetics model assuming population growth limited by a single substrate best fit the¹⁴CO₂ evolution data for CN1.5. At intermediate to high CN ratios, the data were best fit by a model originally formulated to describe no-growth metabolism of one substrate coupled with microbial growth on a second substrate. We suggest that this dual-substrate model describes linear growth on phenol while nitrogen is available and first-order metabolism of phenol without growth after nitrogen is depleted.