5 research outputs found
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Phospholipid fatty acid analysis as part of the Yucca Mountain Project. Final report
In support of the Yucca Mountain subsurface microbial characterization project phospholipid fatty acid (PLFA) analyses for viable microbial biomass, community composition and nutritional status were performed. Results showed a positive correlation between a decrease in viable biomass and increase in depth with the lowest biomass values being obtained from the Topopah Spring geologic horizon. A plot of the ratio of non-viable (diglyceride fatty acids) to viable (PLFA) cells also showed the lowest values to derive from the Topopah Spring horizon. Estimations of microbial community composition, made from the patterns of PLFA recovered from the sediment samples, revealed similarities between samples collected within the same geologic horizons: Tiva Canyon, Pre-Pah Canyon and Topopah Spring. Results indicated the presence of mixed communities composed of gram positive, gram negative, actinomycete and obligate anaerobic bacteria. Culturable organisms, recovered from similar sediments, were representative of the same bacterial classifications although gram positive bacterial isolates typically outnumbered gram negative isolates. Within the gram negative bacterial community, corroborative indicators of physiological stress were apparent in the Topopah Spring horizon
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Quantitative comparison of the in situ microbial communities in different biomes
A system to define microbial communities in different biomes requires the application of non-traditional methodology. Classical microbiological methods have severe limitations for the analysis of environmental samples. Pure-culture isolation, biochemical testing, and/or enumeration by direct microscopic counting are not well suited for the estimation of total biomass or the assessment of community composition within environmental samples. Such methods provide little insight into the in situ phenotypic activity of the extant microbiota since these techniques are dependent on microbial growth and thus select against many environmental microorganisms which are non- culturable under a wide range of conditions. It has been repeatedly documented in the literature that viable counts or direct counts of bacteria attached to sediment grains are difficult to quantitative and may grossly underestimate the extent of the existing community. The traditional tests provide little indication of the in situ nutritional status or for evidence of toxicity within the microbial community. A more recent development (MIDI Microbial Identification System), measure free and ester-linked fatty acids from isolated microorganisms. Bacterial isolates are identified by comparing their fatty acid profiles to the MIKI database which contains over 8000 entries. The application of the MIKI system to the analysis of environmental samples however, has significant drawbacks. The MIDI system was developed to identify clinical microorganisms and requires their isolation and culture on trypticase soy agar at 27{degrees}C. Since many isolates are unable to grow at these restrictive growth conditions, the system does not lend itself to identification of some environmental organisms. A more applicable methodology for environmental microbial analysis is based on the liquid extrication and separation of microbial lipids from environmental samples, followed by quantitative analysis using gas chromatography
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Phospholipid anaysis of extant microbiota for monitoring in situ bioremediation effectiveness
Two sites undergoing bioremediation were studied using the signature lipid biomarker (SLB) technique. This technique isolates microbial lipid moieties specifically related to viable biomass and to both prokaryotic and eukaryotic biosynthetic pathways. The first site was a South Pacific atoll heavily contaminated with petroleum hydrocarbons. The second site was a mine waste reclamation area. The SLB technique was applied to quantitate directly the viable biomass, community structure, and nutritional/physiological status of the microbiota in the soils and subsurface sediments of these sites. All depths sampled at the Kwajalein Atoll site showed an increase in biomass that correlated with the co-addition of air, water, and nutrients. Monoenoic fatty acids increased in abundance with the nutrient amendment, which suggested an increase in gram-negative bacterial population. Ratios of specific phospholipid fatty acids indicative of nutritional stress decreased with the nutrient amendment. Samples taken from the mine reclamation site showed increases in total microbial biomass and in Thiobacillus biomass in the plots treated with lime and bactericide, especially when a cover soil was added. The plot treated with bactericide and buffered lime without the cover soil showed some decrease in Thiobacillus numbers, but was still slightly higher than that observed in the control plots