The measurement of lipid phosphate is proposed as an indicator of microbial biomass in marine and estuarine sediments. This relatively simple assay can be performed on fresh, frozen or frozen-lyophilized sediment samples with chloroform methanol extraction and subsequent phosphate determination. The sedimentary lipid phosphate recovery correlates with the extractible ATP and the rate of DNA synthesis. Pulse-chase experiments show active metabolism of the sedimentary phospholipids. The recovery of added 14C-labeled bacterial lipids from sediments is quantitative. Replicate analyses from a single sediment sample gave a standard deviation of 11%. The lipid extract can be fractionated by relatively simple procedures and the plasmalogen, diacyl phospholipid, phosphonolipid and non-hydrolyzable phospholipid content determined. The relative fatty acid composition can be readily determined by gas-liquid chromatography. The lipid composition can be used to define the microbial community structure. For example, the absence of polyenoic fatty acids indicates minimal contamination with benthic micro-eukaryotes. Therefore the high content of plasmalogen phospholipids in these sediments suggests that the anaerobic prokaryotic Clostridia are found in the aerobic sedimentary horizon. This would require anaerobic microhabitats in the aerated zones.

Determination of the sedimentary microbial biomass by extractible lipid phosphate.

The potential for removing uranium from contaminated groundwater by stimulating the in situ activity of dissimilatory metal-reducing microorganisms was evaluated in a uranium-contaminated aquifer located in Rifle, Colo. Acetate (1 to 3 mM) was injected into the subsurface over a 3-month period via an injection gallery composed of 20 injection wells, which was installed upgradient from a series of 15 monitoring wells. U(VI) concentrations decreased in as little as 9 days after acetate injection was initiated, and within 50 days uranium had declined below the prescribed treatment level of 0.18 μM in some of the monitoring wells. Analysis of 16S ribosomal DNA (rDNA) sequences and phospholipid fatty acid profiles demonstrated that the initial loss of uranium from the groundwater was associated with an enrichment of Geobacter species in the treatment zone. Fe(II) in the groundwater also increased during this period, suggesting that U(VI) reduction was coincident with Fe(III) reduction. As the acetate injection continued over 50 days there was a loss of sulfate from the groundwater and an accumulation of sulfide and the composition of the microbial community changed. Organisms with 16S rDNA sequences most closely related to those of sulfate reducers became predominant, and Geobacter species became a minor component of the community. This apparent switch from Fe(III) reduction to sulfate reduction as the terminal electron accepting process for the oxidation of the injected acetate was associated with an increase in uranium concentration in the groundwater. These results demonstrate that in situ bioremediation of uranium-contaminated groundwater is feasible but suggest that the strategy should be optimized to better maintain long-term activity of Geobacter species.

http://envismadrasuniv.org/Microbes%20and%20Radionuclide%20Pollution/pdf/Stimulating%20the%20In%20Situ%20Activity%20of%20Geobacter.pdf

Stimulating the in situ activity of Geobacter species to remove uranium from the groundwater of a uranium-contaminated aquifer.

Microbial biomass measured as total lipid phosphate in soils of different organic content

The fungi-to-bacteria ratio in soil ecological concepts and its application to explain the effects of land use changes have gained increasing attention over the past decade. Four different main approaches for quantifying the fungal and bacterial contribution to microbial tissue can be distinguished: (1) microscopic methods, (2) selective inhibition, (3) specific cell membrane components and (4) specific cell wall components. In this review, the different methods were compared and we hypothesized that all these approaches result in similar values for the fungal and bacterial contribution to total microbial biomass, activity, and residues (dead microbial tissue) if these methods are evenly reliable for the estimation of fungal biomass. The fungal contribution to the microbial biomass or respiration varied widely between 2 and 95% in different data sets published over the past three decades. However, the majority of the literature data indicated that fungi dominated microbial biomass, respiration or non-biomass microbial residues, with mean percentages obtained by the different methodological approaches varying between 35 and 76% in different soil groups, i.e. arable, grassland, and forest soils and litter layers. Microscopic methods generally gave the lowest average values, especially in arable and grasslands soils. Very low ratios in fungal biomass C-to-ergosterol obtained by microscopic methods suggest a severe underestimation of fungal biomass by certain stains. Relatively consistent ratios of ergosterol to linoleic acid (18:2ω6,9) indicate that both cell membrane components are useful indicators for saprotrophic and ectomycorrhizal fungi. More quantitative information on the PLFA content of soil bacteria and the 16:1ω5 content of arbuscular mycorrhizal fungi is urgently required to fully exploit the great potential of PLFA measurements. The most consistent results have been obtained from the analysis of fungal glucosamine and bacterial muramic acid in microbial residues. Component-specific δ 13 C analyses of PLFA and amino sugars are a promising prospect for the near future.

Quantitative assessment of the fungal contribution to microbial tissue in soil

Improvements in the analysis of lipid-bound phosphates resulted in a simplified and sensitive method for determining microbial biomass in sediments. Sensitivity was enhanced over previous methods by use of a dye, malachite green, which when complexed with phosphomolybdate at low pH has a high extinction coefficient (at 610 nm). The use of a persulfate oxidation technique to liberate phosphate from lipids increased the simplicity and safety of the method relative to the traditional perchloric acid digestions. The modified method was both accurate (yielding quantitative recoveries of cells added to sediments) and precise (coefficient of variation of less than 5% for cells and sediments). A comparison with an epifluorescence technique indicated that the analysis of lipid-bound phosphate was more rapid and less tedious and could be successfully applied to a wider variety of sediment types. An estimate of the lipid-bound phosphate-to-carbon conversion factor based on a diverse enrichment culture from sediments suggested that previous factors for pure cultures may have been too low.

Efficacy of phospholipid analysis in determining microbial biomass in sediments.

Accurate measurement of the mass and activities of environmental microbial assemblies requires methods that differ from those of classical microbiology. Using the leaf-litter detrital microflora recovered after incubationin a semitropical estuary, biochemical assays of multiple components can be made which give reasonably similar estimates of microbial mass, using normalization values from bacterial monocultures. The assays involve recovery of adenosine triphosphate, the unique bacterial cell wall mucopeptide component muramic acid, the uniquely prokaryotic endogenous storage material poly-β-hydroxybutyrate, a series of exoesterase activities, and the extractable lipids, primarily the phospholipids and the glycolipids. If the sampling conditions are such that incorporation of radioisotopes into the microflora is possible, these assays become extraordinarily sensitive. If radioisotope usage can be coupled with the purification of particular lipids, it is possible to gain information on the structure and dynamics of the microbial population.

Biochemical Measurements of Microbial Mass and Activity from Environmental Samples

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Biochemical Measurements of Microbial Mass and Activity from Environmental Samples