An integrative approach was used to obtain pure cultures of previously uncultivated members of the divisions Acidobacteria and Verrucomicrobia from agricultural soil and from the guts of wood-feeding termites. Some elements of the cultivation procedure included the following: the use of agar media with little or no added nutrients; relatively long periods of incubation (more than 30 days); protection of cells from exogenous peroxides; and inclusion of humic acids or a humic acid analogue (anthraquinone disulfonate) and quorumsignaling compounds (acyl homoserine lactones) in growth media. The bacteria were incubated in the presence of air and in hypoxic (1 to 2% O 2 [vol/vol]) and anoxic atmospheres. Some bacteria were incubated with elevated concentrations of CO 2 (5% [vol/vol]). Significantly more Acidobacteria were found on isolation plates that had been incubated with 5% CO 2 . A simple, high-throughput, PCR-based surveillance method (plate wash PCR) was developed. This method greatly facilitated detection and ultimate isolation of target bacteria from as many as 1,000 colonies of nontarget microbes growing on the same agar plates. Results illustrate the power of integrating culture methods with molecular techniques to isolate bacteria from phylogenetic groups underrepresented in culture.

To examine phylogenetic identity and metabolic activity of individual cells in complex microbial communities, we developed a method which combines rRNA-based in situ hybridization with stable isotope imaging based on nanometer-scale secondary-ion mass spectrometry (NanoSIMS). Fluorine or bromine atoms were introduced into cells via 16S rRNA-targeted probes, which enabled phylogenetic identification of individual cells by NanoSIMS imaging. To overcome the natural fluorine and bromine backgrounds, we modified the current catalyzed reporter deposition fluorescence in situ hybridization (FISH) technique by using halogencontaining fluorescently labeled tyramides as substrates for the enzymatic tyramide deposition. Thereby, we obtained an enhanced element labeling of microbial cells by FISH (EL-FISH). The relative cellular abundance of fluorine or bromine after EL-FISH exceeded natural background concentrations by up to 180-fold and allowed us to distinguish target from non-target cells in NanoSIMS fluorine or bromine images. The method was optimized on single cells of axenic Escherichia coli and Vibrio cholerae cultures. EL-FISH/NanoSIMS was then applied to study interrelationships in a dual-species consortium consisting of a filamentous cyanobacterium and a heterotrophic alphaproteobacterium. We also evaluated the method on complex microbial aggregates obtained from human oral biofilms. In both samples, we found evidence for metabolic interactions by visualizing the fate of substrates labeled with 13 C-carbon and 15 N-nitrogen, while individual cells were identified simultaneously by halogen labeling via EL-FISH. Our novel approach will facilitate further studies of the ecophysiology of known and uncultured microorganisms in complex environments and communities.

The role of the rRNA gene copy number as a central component of bacterial life histories was studied by using strains of Escherichia coli in which one or two of the seven rRNA operons (rrnA and/or rrnB) were deleted. The relative fitness of these strains was determined in competition experiments in both batch and chemostat cultures. In batch cultures, the decrease in relative fitness corresponded to the number of rRNA operons deleted, which could be accounted for completely by increased lag times and decreased growth rates. The magnitude of the deleterious effect varied with the environment in which fitness was measured: the negative consequences of rRNA operon deletions increased under culture conditions permitting more-rapid growth. The rRNA operon deletion strains were not more effective competitors under the regimen of constant, limited resources provided in chemostat cultures. Enhanced fitness in chemostat cultures would have suggested a simple tradeoff in which deletion strains grew faster (due to more efficient resource utilization) under resource limitation. The contributions of growth rate, lag time, K s , and death rate to the fitness of each strain were verified through mathematical simulation of competition experiments. These data support the hypothesis that multiple rRNA operons are a component of bacterial life history and that they confer a selective advantage permitting microbes to respond quickly and grow rapidly in environments characterized by fluctuations in resource availability.An organism's life history is its lifetime pattern of growth, differentiation, storage of resources, and reproduction (6). Tradeoffs for the optimization of one life history trait at the expense of another have led to the evolution of a magnificent array of ecological strategies in plants and animals. That is why the identification and investigation of life histories remain a cornerstone of contemporary ecology. Since natural selection favors individuals that are better able to survive and leave viable progeny, regardless of whether they are multicellular or single-celled, it should be valuable to consider the life histories of microbes.One readily quantifiable feature of bacteria that has been proposed as a component of bacterial life histories is the number of rRNA genes (25,26,39). This proposal is based on the understanding that multiple copies of rRNA genes allow for increased rates of rRNA synthesis, leading to more rapid synthesis of ribosomes and ultimately conferring the potential for a quicker response to an influx of resources and rapid growth (13). This characteristic fits the classical definition of a component of life history and may also exhibit a tradeoff, namely, a metabolic cost for retention of multiple copies of rRNA genes associated with basal levels of transcription of these genes. This proposed expense would be particularly important in environments characterized by slow and constant flux of resources, where there is little selection for shorter lag times or rapid growth.We have investigated th...

Microbial activity associated with produced water from hydraulic fracturing operations can lead to gas souring and corrosion of carbon-steel equipment. We examined the microbial ecology of produced water and the prospective role of the prevalent microorganisms in corrosion in a gas production field in the Barnett Shale. The microbial community was mainly composed of halophilic, sulfidogenic bacteria within the order Halanaerobiales, which reflected the geochemical conditions of highly saline water containing sulfur species (S2O32-, SO42-, and HS-). A predominant, halophilic bacterium (strain DL-01) was subsequently isolated and identified as belonging to the genus Halanaerobium. The isolate could degrade guar gum, a polysaccharide polymer used in fracture fluids, to produce acetate and sulfide in a 10% NaCl medium at 37°C when thiosulfate was available. To mitigate potential deleterious effects of sulfide and acetate, a quaternary ammonium compound was found to be an efficient biocide in inhibiting the growth and metabolic activity of strain DL-01 relative to glutaraldehyde and tetrakis (hydroxymethyl) phosphonium sulfate. Collectively, our findings suggest that predominant halophiles associated with unconventional shale gas extraction could proliferate and produce sulfide and acetate from the metabolism of polysaccharides used in hydraulic fracturing fluids. These metabolic products might be returned to the surface and transported in pipelines to cause pitting corrosion in downstream infrastructure.

Landfills are the final repository for most of the discarded material from human society and its “built environments.” Microorganisms subsequently degrade this discarded material in the landfill, releasing gases (largely CH4 and CO2) and a complex mixture of soluble chemical compounds in leachate. Characterization of “landfill microbiomes” and their comparison across several landfills should allow the identification of environmental or operational properties that influence the composition of these microbiomes and potentially their biodegradation capabilities. To this end, the composition of landfill microbiomes was characterized as part of an ongoing USGS national survey studying the chemical composition of leachates from 19 non-hazardous landfills across 16 states in the continental U.S. The landfills varied in parameters such as size, waste composition, management strategy, geography, and climate zone. The diversity and composition of bacterial and archaeal populations in leachate samples were characterized by 16S rRNA gene sequence analysis, and compared against a variety of physical and chemical parameters in an attempt to identify their impact on selection. Members of the Epsilonproteobacteria, Gammaproteobacteria, Clostridia, and candidate division OP3 were the most abundant. The distribution of the observed phylogenetic diversity could best be explained by a combination of variables and was correlated most strongly with the concentrations of chloride and barium, rate of evapotranspiration, age of waste, and the number of detected household chemicals. This study illustrates how leachate microbiomes are distinct from those of other natural or built environments, and sheds light on the major selective forces responsible for this microbial diversity.