Sarah J. Macnaughton

Diversity and Characterization of Sulfate-Reducing Bacteria in Groundwater at a Uranium Mill Tailings Site

ABSTRACT Microbially mediated reduction and immobilization of U(VI) to U(IV) plays a role in both natural attenuation and accelerated bioremediation of uranium-contaminated sites. To realize bioremediation potential and accurately predict natural attenuation, it is important to first understand the microbial diversity of such sites. In this paper, the distribution of sulfate-reducing bacteria (SRB) in contaminated groundwater associated with a uranium mill tailings disposal site at Shiprock, N.Mex., was investigated. Two culture-independent analyses were employed: sequencing of clone libraries of PCR-amplified dissimilatory sulfite reductase (DSR) gene fragments and phospholipid fatty acid (PLFA) biomarker analysis. A remarkable diversity among the DSR sequences was revealed, including sequences from δ-Proteobacteria, gram-positive organisms, and theNitrospira division. PLFA analysis detected at least 52 different mid-chain-branched saturate PLFA and included a high proportion of 10me16:0. Desulfotomaculum andDesulfotomaculum-like sequences were the most dominant DSR genes detected. Those belonging to SRB within δ-Proteobacteria were mainly recovered from low-uranium (≤302 ppb) samples. OneDesulfotomaculum-like sequence cluster overwhelmingly dominated high-U (>1,500 ppb) sites. Logistic regression showed a significant influence of uranium concentration over the dominance of this cluster of sequences (P = 0.0001). This strong association indicates that Desulfotomaculum has remarkable tolerance and adaptation to high levels of uranium and suggests the organisms possible involvement in natural attenuation of uranium. The in situ activity level of Desulfotomaculum in uranium-contaminated environments and its comparison to the activities of other SRB and other functional groups should be an important area for future research.

Rapid extraction of lipid biomarkers from pure culture and environmental samples using pressurized accelerated hot solvent extraction

Lipid biomarker analysis is a quantitative and sensitive method for the in situ analysis of microbial communities in environmental samples (e.g. soil, water, air). The one-phase modified Bligh and Dyer solvent extraction is a commonly used method for obtaining phospholipid fatty acid biomarkers used in such community analysis. This method, however, is relatively labor intensive and slow, often taking up to 24 h for the initial extraction. Using a pressurized hot solvent extractor, we have been able to extract lipid biomarkers from selected vegetative and/or sporulated biomass (Escherichia coli, Staphylococcus aureus, Mycobacterium fortuitum, Bacillus subtilis, Saccharomyces cerevisiae and Aspergillus niger) as well as from environmental samples collected from water, soil and air. Depending on sample type, the automated extraction procedure took ∼35–45 min per sample. Compared to the modified Bligh and Dyer extraction, phospholipid fatty acid lipid yields obtained using the pressurized hot solvent extraction were not significantly different for the vegetative biomass or water and soil samples (P>0.05), but were significantly higher for the spores and the airborne biomass (P<0.05 for both sample types). The advantage of using accelerated hot solvent extraction is that by increasing the speed and decreasing the labor involved, pressurized hot solvent extraction should enable the rapid and improved extraction of lipids from large numbers of environmental samples providing data essential for total microbial community analysis.

Competitive PCR-DGGE analysis of bacterial mixtures: an internal standard and an appraisal of template enumeration accuracy.

Analysis of polymerase chain reaction (PCR) amplified 16S rDNA fragments from environmental samples by denaturing gradients of chemicals or heat [denaturing gradient gel electrophoresis (DGGE) and thermal gradient gel electrophoresis (TGGE)] within polyacrylamide gels is a popular tool in microbial ecology. Difficulties in acceptance of the technique and interpretation of the results remain, due to its qualitative nature. In this study we have addressed this problem by the construction and evaluation of a quantitative standard for incorporation into test DNA samples. The standard was based on a naturally occurring 16S rRNA gene carried by the X-endosymbiont of the psyllid Anomoneura mori, a gamma-proteobacterium. This sequence is the most AT-rich 16S rDNA gene recovered from any cultured organism or environmental sample described to date, and a specifically amplified rDNA fragment denatured under exceptionally low stringency denaturing conditions. The native sequence was modified to incorporate perfect matches to the PCR primers used. The efficiency of amplification of this standard in comparison to a range of 16S rDNA sequences and the errors involved in enumerating template molecules under a range of PCR conditions are demonstrated and quantified. Tests indicated that highly accurate counts of released target molecules from a range of bacterial cells could be achieved in both laboratory mixtures and compost.

Measuring soil microbial community diversity using polar lipid fatty acid and denaturing gradient gel electrophoresis data.

The possibility of calculating useful microbial community diversity indices from environmental polar lipid fatty acid and 16S rDNA PCR-DGGE data was investigated. First, the behavior of the species richness, Shannons, and Simpsons diversity indices were determined on polar lipid fatty acid profiles of 115 pure cultures, communities constructed from those profiles with different numbers of species, and constructed communities with different distributions of species. Differences in the species richness of these artificial communities was detected by all three diversity indices, but they were insensitive to the evenness of the distribution of species. Second, data from a field experiment with substrate addition to soil was used to compare the methods developed for lipid- and DNA-based diversity indices. Very good agreement was found between indices calculated from environmental polar lipid fatty acid profiles and denaturing gradient gel electrophoresis profiles from matched samples (Pearsons correlation coefficient r=0.95-0.96). A method for data pre-treatment for diversity calculations is described.

A survey of 16S rRNA and amoA genes related to autotrophic ammonia-oxidizing bacteria of the β-subdivision of the class proteobacteria in contaminated groundwater.

In this study, we investigated the size and structure of autotrophic ammonia oxidizer (AAO) communities in the groundwater of a contamination plume originating from a mill-tailings disposal site. The site has high levels of dissolved N from anthropogenic sources, and exhibited wide variations in the concentrations of NO3- and NH3 + NH4+. Community structures were examined by PCR-DGGE targeting 16S rDNA with band excision and sequence analysis, and by analysis of amoA fragment clone libraries. AAO population sizes were estimated by competitive PCR targeting the gene amoA, and correlated significantly with nitrate concentration. Most samples revealed novel diversity in AAO 16S rDNA and amoA gene sequences. Both 16S rDNA and amoA analyses suggested that all samples were dominated by Nitrosomonas sp., Nitrosospira sp. being detected in only 3 of 15 samples. This study indicated numerical dominance of Nitrosomonas over Nitrosospira in groundwater, and suggests that groundwater ammonia oxidizers are more similar to those dominating freshwater sediments than bulk soil.

Quantitative lipid biomarker detection of unculturable microbes and chlorine exposure in water distribution system biofilms

Abstract Biofilms in the drinking water distribution system can protect pathogens from disinfection and provide the inocula for periodic infestations. Assessing these biofilms can be difficult, as the plate counts of pelagic bacteria may bear little relationship to the biofilm load. Culturing the water at the outlet most often does not reflect the biofilm composition. Herein we show that analysis of polar lipid fatty acids recovered from biofilms on devices possessing a large surface area provides quantitative analysis of the viable biomass, community composition, and nutritional status that is independent of the recovery and culturability. Analysis of the polar lipid fatty acids indicated the biofilm contained a stressed and predominantly Gram-negative bacterial community. The composition was not significantly different whether collected in the summer or winter. Oxirane (epoxide) fatty acids were detected in the polar lipids of the biofilm, indicating exposure to chlorine and loss of viability within the biofilm. Tests with monocultures of Escherichia coli and Sphingomonas paucimobilis exposed to chlorine resulted in oxirane fatty acid generation and rendered them nonculturable.

Detection of Sphingomonas spp in soil by PCR and sphingolipid biomarker analysis.

Sphingomonas spp possess unique abilities to degrade refractory contaminants and are found ubiquitously in the environment. We developed Sphingomonas genus-specific PCR primers (SPf-190 and SPr1-852) which showed specific amplification of a 627-bp 16S rDNA fragment from Sphingomonas spp. A PCR assay using these Sphingomonas specific primers was developed to detect Sphingomonas aromaticivorans B0695R in three texturally distinct soil types, showing detection limits between 1.3–2.2 × 103 CFU g−1 dry soil. A sphingolipid extraction protocol was also developed to monitor Sphingomonas populations in soil quantitatively. The detection limit of the assay was 20 pmol g−1 dry soil, equivalent to about 3 × 105 cells g−1 dry soil. Survival of S. aromaticivorans B0695R was monitored in the three different soils by antibiotic selective plate counting, PCR and sphingolipid analysis. All three approaches showed that the B0695R cells persisted in the low biomass Sequatchie sub-soil at about 3–5 × 107cells g−1 dry soil. In comparison to the plate counting assay, both the PCR and sphingolipid analysis detected a significantly higher level of B0695R cells in the clay soil and Sequatchie top-soil, indicating the possibility of the presence of viable but non-culturable B0695R cells in the soils. The combination of PCR and sphingolipid analysis may provide a more realistic estimation of Sphingomonas population in the environment.

Mapping Changes in Soil Microbial Community Composition Signaling Bioremediation

Abstract Chemical signatures of biological processes reflect their complex interrelationships. The chemical profile is rich in information but poor in content due to the complex processes underlying the chemical composition of natural biological communities. A nonlinear mapping technique, based on artificial neural networks (ANNs), was proposed to highlight information coded in lipid signatures in soil by demonstrating the biological response to hydrocarbon contamination. ANNs do not require mechanistic assumptions, and they can cope with nonlinear associations. Soil sample lipid signatures were mapped using ANNs to recover information on exposure to contamination, to assess the potential for bioremediation as assessed by polymerase chain reaction (PCR)/deoxyribonucleic acid (DNA) gene probes, and to monitor the effects of selected inocula. A two-coordinate system was built from signature lipid biomarkers containing 64 components from which the values of target parameters (6 components) could be recovered...