Quantitative information on microbial processes in the field is important. Here we propose a new field method, the "gas push-pull test" (GPPT) for the in-situ quantification of microbial activities in the vadose zone. To evaluate the new method, we studied microbial methane oxidation above an anaerobic, petroleum-contaminated aquifer. A GPPT consists of the injection of a gas mixture of reactants (e.g., methane, oxygen) and nonreactive tracer gases (e.g., neon, argon) into the vadose zone and the subsequent extraction of the injection gas mixture together with soil air from the same location. Rate constants of gas conversion are calculated from breakthrough curves of extracted reactants and tracers. In agreement with expectations from previously measured gas profiles, we determined first-order rate constants of 0.68 h(-1) at 1.1 m below soil surface and 2.19 h(-1) at 2.7 m, close to the groundwater table. Co-injection of a specific inhibitor (acetylene) for methanotrophs showed that the observed methane consumption was microbially mediated. This was confirmed by increases of stable carbon isotope ratios in methane by up to 42.6 %. In the future, GPPTs should provide useful quantitative information on a range of microbial processes in the vadose zone.

Aerobic granules are dense microbial aggregates with the potential to replace floccular sludge for the treatment of wastewaters. In bubble-column sequencing batch reactors, distinct microbial populations dominated propionate-and acetate-cultivated aerobic granules after 50 days of reactor operation when only carbon removal was detected. Propionate granules were dominated by Zoogloea (40%), Acidovorax, and Thiothrix, whereas acetate granules were mainly dominated by Thiothrix (60%). Thereafter, an exponential increase in enhanced biological phosphorus removal (EBPR) activity was observed in the propionate granules, but a linear and erratic increase was detected in the acetate ones. Besides Accumulibacter and Competibacter, other bacterial populations found in both granules were associated with Chloroflexus and Acidovorax. The EBPR activity in the propionate granules was high and stable, whereas EBPR in the acetate granules was erratic throughout the study and suffered from a deterioration period that could be readily reversed by inducing hydrolysis of polyphosphate in presumably saturated Accumulibacter cells. Using a new ppk1 gene-based dual terminalrestriction fragment length polymorphism (T-RFLP) approach revealed that Accumulibacter diversity was highest in the floccular sludge inoculum but that when granules were formed, propionate readily favored the dominance of Accumulibacter type IIA. In contrast, acetate granules exhibited transient shifts between type I and type II before the granules were dominated by Accumulibacter type IIA. However, ppk1 gene sequences from acetate granules clustered separately from those of propionate granules. Our data indicate that the mere presence of Accumulibacter is not enough to have consistently high EBPR but that the type of Accumulibacter determines the robustness of the phosphate removal process.In sequencing batch reactors operated under high shear stress and short setting times, microorganisms involved in wastewater treatment can form structures called aerobic granules (1). Aerobic granules are compact quasispherical aggregates formed by self-immobilized mixed microbial communities embedded in a matrix of extracellular biopolymeric substances (35). These microbial aggregates can accommodate a biomass concentration five times higher than that of activated sludge flocs (13) and settle 10 times faster (42, 60). These characteristics translate into small-footprint treatment plants, and therefore the use of aerobic granular sludge-based systems has recently been proposed as a promising alternative technology to conventional activated sludge treatment systems, with annual maintenance costs and land requirements that would be reduced by 17% and

The metabolic properties and ultrastructure of mesophilic aggregates from a full-scale expanded granular sludge bed reactor treating brewery wastewater are described. The aggregates had a very high methanogenic activity on acetate (17.19 mmol of CH 4 /g of volatile suspended solids [VSS]⅐day or 1.1 g of CH 4 chemical oxygen demand/g of VSS⅐day). Fluorescent in situ hybridization using 16S rRNA probes of crushed granules showed that 70 and 30% of the cells belonged to the archaebacterial and eubacterial domains, respectively. The spherical aggregates were black but contained numerous whitish spots on their surfaces. Cross-sectioning these aggregates revealed that the white spots appeared to be white clusters embedded in a black matrix. The white clusters were found to develop simultaneously with the increase in diameter. Energy-dispersed X-ray analysis and back-scattered electron microscopy showed that the whitish clusters contained mainly organic matter and no inorganic calcium precipitates. The white clusters had a higher density than the black matrix, as evidenced by the denser cell arrangement observed by high-magnification electron microscopy and the significantly higher effective diffusion coefficient determined by nuclear magnetic resonance imaging. Highmagnification electron microscopy indicated a segregation of acetate-utilizing methanogens (Methanosaeta spp.) in the white clusters from syntrophic species and hydrogenotrophic methanogens (Methanobacterium-like and Methanospirillum-like organisms) in the black matrix. A number of physical and microbial ecology reasons for the observed structure are proposed, including the advantage of segregation for high-rate degradation of syntrophic substrates.The view of the structure of biofilms has dramatically changed during the last decade. Until the early 1990s, biofilms were considered more or less homogeneous layers of microorganisms embedded in a matrix of extracellular polymeric substances (23, 32). The application of one-dimensional models to calculate concentration profiles in these biofilms is straightforward. In general, an excellent agreement between observed concentration gradients (16) and predicted ones was obtained using one-dimensional models (34) for the biofilm systems studied.More-detailed investigations using advanced microscopic techniques revealed that biofilm morphology can be much more complex. Confocal scanning laser microscopy and computerized image analysis tools were used to show that glucosegrown P. aeruginosa biofilms were composed of cell clusters separated by interstitial voids and channels (6,7,26,39,46). Based on these observations, biofilms containing these clusters were referred to as having a cluster-and-channel morphology and the clusters were visualized as "mushrooms" (3). Also other aerobic, multispecies biofilms have been found to contain a structured cell cluster-and-channel arrangement (10).Anaerobic aggregates from anaerobic wastewater treatment plants are a special type of biofilms. These spherical biofilms are formed sp...

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