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The integrated biomass beneath the surface horizon in unsaturated soils is large and potentially important in nutrient and carbon cycling. Compared to surface soils, the ecology of these subsurface soils is weakly understood, particularly in terms of the composition of bacterial communities. We compared soil bacterial communities along two vertical transects by terminal restriction fragment length polymorphisms (TRFLPs) of PCR-amplified 16S rRNA genes to determine how surface and deep bacterial communities differ. DNA yield from soils collected from two Mediterranean grassland transects decreased exponentially from the surface to 4 m deep. Richness, as assessed by the number of peaks obtained after restriction with HhaI, MspI, RsaI, or HaeIII, and diversity, as assessed by the Shannon diversity indices, were lowest in the deepest sample. Lower diversity at depth is consistent with species-energy theory, which would predict relatively low diversity in the low organic matter horizons. Principal components analysis suggested that, in terms of HhaI and HaeIII generated TRFLPs, bacterial communities differed between depths. The most abundant amplicons cloned from the deepest sample contained sequences with restriction sites consistent with the largest peaks observed in TRFLPs generated from deep samples. These more abundant operational taxonomic units (OTUs) appeared related to Pseudomonas and Variovorax. Several OTUs were more related to each other than any previously described ribotypes. These OTUs showed similarity to bacteria from the divisions Actinobacteria and Firmicutes.

Culture-independent fecal source tracking methods have many potential advantages over library-dependent, isolate-culture methods, but they have been subjected to limited testing. The purpose of this study was to compare culture-independent, library-independent methods of fecal source tracking. Five laboratories analysed identical sets of aqueous samples that contained one or more of the following sources: sewage, human feces, dog feces, cattle feces and gull feces. Two investigators used methods based on PCR amplification of Bacteroidetes marker genes and both successfully discriminated between samples that did or did not contain human fecal material. One of these investigators was also able to identify the remaining sources, except for gull, with a low rate of false positives. A method based on E. coli toxin genes successfully identified samples containing sewage and cattle feces, but missed some samples with human feces because of low marker prevalence in individual human fecal samples. Researchers who used community terminal restriction fragment length polymorphism (T-RFLP) were limited by the amount of DNA recovered from samples, but they correctly identified human and cattle fecal contamination when sufficient DNA was obtained. Culture independent methods show considerable promise; further research is needed to develop markers for additional fecal sources and to understand the correlation of these source-tracking indicators to measures of human and environmental health.

The Pacific Estuarine Ecosystem Indicators Research Consortium seeks to develop bioindicators of toxicantinduced stress and bioavailability for wetland biota. Within this framework, the effects of environmental and pollutant variables on microbial communities were studied at different spatial scales over a 2-year period. Six salt marshes along the California coastline were characterized using phospholipid fatty acid (PLFA) analysis and terminal restriction fragment length polymorphism (TRFLP) analysis. Additionally, 27 metals, six currently used pesticides, total polychlorinated biphenyls and polycyclic aromatic hydrocarbons, chlordanes, nonachlors, dichlorodiphenyldichloroethane, and dichlorodiphenyldichloroethylene were analyzed. Sampling was performed over large (between salt marshes), medium (stations within a marsh), and small (different channel depths) spatial scales. Regression and ordination analysis suggested that the spatial variation in microbial communities exceeded the variation attributable to pollutants. PLFA analysis and TRFLP canonical correspondence analysis (CCA) explained 74 and 43% of the variation, respectively, and both methods attributed 34% of the variation to tidal cycles, marsh, year, and latitude. After accounting for spatial variation using partial CCA, we found that metals had a greater effect on microbial community composition than organic pollutants had. Organic carbon and nitrogen contents were positively correlated with PLFA biomass, whereas total metal concentrations were positively correlated with biomass and diversity. Higher concentrations of heavy metals were negatively correlated with branched PLFAs and positively correlated with methyl-and cyclo-substituted PLFAs. The strong relationships observed between pollutant concentrations and some of the microbial indicators indicated the potential for using microbial community analyses in assessments of the ecosystem health of salt marshes.

We analyzed, by terminal restriction fragment length polymorphisms (T-RFLPs) of PCR-amplified 16S rDNA, microbial diversity in water collected during the dry and wet seasons in a human-impacted coastal lagoon. Water samples were fractionated by prefiltration to differentiate particle-associated and free-living microbes. From a sample collected during the dry season, prefiltration removed 23 to 44% of bacteria, as assessed by direct counts and MPN, and 99% of phytoplankton, as assessed by chlorophyll a. Restriction with RsaI yielded fewer peaks than restriction with HhaI. Diversity indices calculated from T-RFLPs were higher in the lagoon than adjoining coastal waters and higher in the particle-associated than the free-living fraction. In the dry season, peaks found only in bulk and particle-associated T-RFLPs were consistent with plastid and cyanobacterial ribotypes. These peaks matched those observed in the sequence of a clone generated from the bulk fraction with plastid and cyanobacterial specific primers. This clone appeared related to plastids found in the diatom genus Skeletonema. Principal component analysis of T-RFLPs suggested that the difference between the free-living and particle-associated fractions in the dry season was less than temporal variability in this lagoon and that these fractions varied significantly only in the wet season. This fractionation of microbial populations into particle-associated and free-living guilds during the wet season, when water residence time in the lagoon is relatively low, suggests an external source of particle-associated bacteria such as erosion of upland soils by runoff.

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Hurricanes have the potential to alter the structures of coastal ecosystems and generate pathogen-laden floodwaters that threaten public health. To examine the impact of hurricanes on urban systems, we compared microbial community structures in samples collected after Hurricane Katrina and before and after Hurricane Rita. We extracted environmental DNA and sequenced small-subunit ribosomal RNA (SSU rRNA) gene clone libraries to survey microbial communities in floodwater, water and sediment samples collected from Lake Charles, Lake Pontchartrain, the 17th Street and Industrial Canals in New Orleans and raw sewage. Correspondence Analysis showed that microbial communities associated with sediments formed one cluster while communities associated with lake and Industrial Canal water formed a second. Communities associated with water from the 17th Street Canal and floodwaters collected in New Orleans showed similarity to communities in raw sewage and contained a number of sequences associated with possible pathogenic microbes. This suggests that a distinct microbial community developed in floodwaters following Hurricane Katrina and that microbial community structures as a whole might be sensitive indicators of ecosystem health and serve as “sentinels” of water quality in the environment.