We performed two experiments to assess the methodological variability of microbial community-level physiological profiles (CLPP, using the BiOLOG assay) in soil from a California annual grassland ecosystem. In a study to assess the impact of sample preparation, we found that bacteria adhered to soil surfaces are numerically dominant and have CLPPs indistinguishable from that of intact soil. Studies that allow soil particles to settle prior to dilution or plating may not accurately reflect the substrate utilization pattern of whole soil. In a hierarchical ANOVA, we found that nearly all of the methodological variability in the CLPP assay comes from soil replicates rather than plate replicates. Many laboratories replicate at the level of the CLPP plate. Our results indicate that to best represent a given soil sample, it is important to replicate soil subsamples, rather than CLPP plates.

Methodological variability in microbial community level physiological profiles

Application of multivariate analysis of variance and related techniques in soil studies with substrate utilization tests

The kinetics of response curves from Biolog community profiles for heterotrophic bacteria from a river in Nova Scotia, Canada have been analyzed to generate lag, slope, and asymptote parameters. The river water samples were treated with one of three supplements of Escherichia coli (in situ levels, 10(3) CFU/mL, or 10(6) CFU/mL) and one of five concentrations of chlorine (0, 1, 3, 5, or 7 ppm) to satisfy a full factorial design. The chlorine treatments decreased the inoculum density by up to 2 log values and decreased the species evenness. The E. coli supplements increased the inoculum density and decreased the species richness. Examination of the asymptotes did not reveal any significant effects owing to E. coli, but differences owing to the chlorine were detected. Analyses of the slopes showed a similar insignificance of the effects of E. coli and a lack of treatment effect owing to chlorine. The lag analyses also showed no significant E. coli effects, but showed a significant effect owing to chlorine. The discrepancy produced with the slope analysis (i.e., no chlorine effect) may represent an anomaly of the Biolog community approach. The use of lag phase was impaired because of the problem of infinite lags from wells that had no response, but a principle component analysis with a reduced set of substrates did suggest some influence of E. coli on the community profile. An examination of the substrates metabolized by the river water compared with pure E. coli revealed that the Biolog profiles of the river communities were not a simple summation of the component parts. In light of the lack of uniformity between these analyses, where the outcome depended on which parameter was used, caution is advised in interpreting Biolog community profiles on the basis of only one parameter.

Kinetic analyses of Biolog community profiles to detect changes in inoculum density and species diversity of river bacterial communities.

Determining replication for discrimination among microbial communities in environmental samples using community‐level physiological profiles

Bioregenerative components for advanced life support (ALS) systems will need to be reliable and stable for longduration space travel. To examine the stability and resilience of microbial communities that recover nutrients from inedible wheat residues, we maintained 4 bacterial strains in mixed communities for 7 weeks. After 3 weeks of incubation, aeration was stopped for several days. Although the abundance of each isolate declined during the perturbation, all strains persisted throughout the experiments. However, only 80% of functions lost during perturbation were recovered afterward. Thus, persistence of strains in a community did not guarantee the persistence of metabolic functions which those strains could perform. Niche partitioning of the heterogeneous molecules in the wheat residue apparently contributed to stable coexistence of the 4 strains. A different community, composed of 4 isolates from a hydroponic study grown under either simulated microgravity or 1×g control conditions in a comparatively homogeneous 1/2-TSB medium, was less stable. Increased diversity of microbial strains and complexity of the substrates upon which they were maintained seemed to increase the stability of microbial communities in ALS bioreactors.

Reliability and Resilience of Populations and Metabolic Functions within Defined Microbial Communities in Biological Reactors

The ability of the community level physiological profiling approach to analyzing microbial community structure was examined in a constructed community. Four bacterial strains, characterized by BIOLOG®-GN profiles, were combined into 2 communities which were maintained for 7 weeks on 2% wheat residue in distilled water with 10% daily removal and replenishment of medium, after 3 weeks incubation, aeration was stopped for 5 days. Although the abundance of all the isolates decreased during the perturbation, all the strains persisted throughout the experiments. Only 80% of functions lost during perturbation were recovered afterward. Persistence of strains in a community did not guarantee persistence of metabolic functions which those strains could perform.

Strain and Function Stability in Gnotobiotic Reactors

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Strain and Function Stability in Gnotobiotic Reactors