David C. White

The genus Sphingomonas: physiology and ecology

Exploitation of the metabolic capabilities of the genus Sphingomonas could provide important commercial benefits to biotechnology. Recent advances have demonstrated that these organisms have unique abilities to degrade refractory contaminants, to serve as bacterial antagonists to phytopathogenic fungi, and to secrete the highly useful gellan exopolysaccharides. Unfortunately, Sphingomonas are also animal pathogens and can readily degrade the copper pipes in drinking water distribution systems. The closely related Zymomonas could be important for commercial ethanol production. These Gram-negative aerobic bacteria are characterized by an outer membrane that contains glycosphingolipids, but lacks lipopolysaccharide. Their distribution in environmental samples has not been systematically examined as yet.

Microbial activities in deep subsurface environments

Abstract Activities of microorganisms residing in terrestrial deep subsurface sediments were examined in 46 sediment samples from three boreholes. Radiolabeled time course experiments assessing in situ microbial activities were initiated within 30 min of core recovery. [1‐C4] Acetate incorporation into lipids, [ methyl‐3H] thymidine incorporation into DNA, [2‐14C]acetate, and [U‐14C]glucose mineralization in addition to microbial enrichment and enumeration studies were examined in surface and subsurface sediments. Surface soils contained the greatest biomass and activities, followed by the shallow aquifer zones. Water‐saturated subsurface sands exhibited three to four orders of magnitude greater activity and culturable microorganisms than the dense clay zones, which had low permeability. Regardless of depth, sediments that contained more than 20% clays exhibited the lowest activities and culturable microorganisms.

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.

In situ measurement of microbial biomass, community structure and nutritional status

In sediments and soils the extant microbiota that can be counted by direct microscopy have proved exceedingly difficult to isolate and culture. Classical tests are time consuming and provide little indication of the interactions within the community, the community nutritional status or metabolic activity. The in situ method is based on the extraction of ‘signature’ lipid biomarkers (SLB) from the cell membranes and walls of microorganisms. Lipids are cellular components that are recoverable by extraction with organic solvents. Lipids are an essential component of the membrane of all cells and play a role as storage materials. Extraction of the lipid components of the microbiota from soils and sediments provides both purification and concentration together with an in situ quantitative analysis of the microbial biomass, community structure, and nutritional status. The determination of the total phospholipid ester-linked fatty acids (PLFA) provides a quantitative measure of the viable biomass. Viable microbes have an intact membrane which contains phospholipids (and PLFA). With cell death enzymes hydrolyze the phosphate group within minutes to hours. The lipid core remains as diglyceride (DG). The resulting DG has the same signature fatty acids as the phospholipids (until it degrades) so a comparison of the ratio of PLFA to DG provides an indication of the viable and nonviable microbes. Analysis by SLB technique provides a quantitative definition of the microbial community structure as specific groups of microbes contain characteristic PLFA patterns. The analysis of other lipids such as the sterols (for the microeukaryotes -nematodes, algae, protozoa), glycolipids (for the phototrophs, gram-positive bacteria), or the hydroxy fatty acids in the lipopolysaccharide of the lipid A (gramnegative bacteria) can provide more detailed community structure analysis. The formation of poly (3-hydroxyalkanoic acid (PHA) in bacteria or triglyceride (TG) in the microeukaryotes relative to the PLFA provides a measure of the nutritional status. Bacteria grown with adequate carbon and terminal electron acceptors form PHA when they cannot divide, because some essential component is missing. Rates of incorporation of 14C-acetate into PHA relative to PLFA is a sensitive indicator of disturbance artifacts in estimates of metabolic activity in sediments with redox gradients. Exposure to toxic environments can lead to minicell formation and increases in specific PLFAS. Respiratory quinone structure indicates the proportions of aerobic/anaerobic activities in the community. The SLB technology provides quantitative in situ information that define the microbial ecology in sedimentary geochemical processes.

Factors Influencing the Abundance and Metabolic Capacities of Microorganisms in Eastern Coastal Plain Sediments

The abundance and metabolic capacities of microorganisms residing in 49 sediment samples from 4 boreholes in Atlantic Coastal Plain sediments were examined. Radiolabeled time-course experiments assessing in situ mirobial capacities were initiated within 30 min of core recovery. Acetate (1-14C- and3H-) incorporation into lipids, microbial colony forming units, and nutrient limitations were examined in aliquots of subsurface sediments. Water-saturated sands exhibited activity and numbers of viable microorganisms that were orders of magnitude greater than those of the low permeability dense clays. Increased radioisotope utilization rates were observed after 6–24-h incubation times when sediments were amended with additional water and/or nutrients. Supplements of water, phosphate, nitrate, sulfate, glucose, or minerals resulted in the stimulation of microbial activities, as evidenced by the rate of acetate incorporation into microbial lipids. Additions of water or phosphate resulted in the greatest stimulation of microbial activities. Regardless of depth, sediments that contained >20% clay particles exhibited lower activities and biomass densities, and greater stimulation with abundant water supplementation than did sediments containing >66% sands and hydraulic conductivities > 200 μm sec.−1.

Reversible Acceleration of the Corrosion of AISI 304 Stainless Steel Exposed to Seawater Induced by Growth and Secretions of the Marine Bacterium Vibrio natriegens

Abstract Growth of the nonsulfate-reducing marine bacterium Vibrio natriegens increased the corrosion current density (CD) of AISI 304 stainless steel (SS) coupons when grown in a marine medium. The corrosion rate, estimated as the corrosion CD (Icorr), calculated from the Tafel constants and polarization resistance, increased from 230 to 2900 nA/cm2 during a 6-day incubation period with V. natriegens. Just before the rapid increase in the corrosion CD, bacterial cells were seen by epifluorescent microscopy after acridine orange staining to colonize the SS surface. On the third day of exposure to seawater, the rapid increase in the corrosion CD correlated with the appearance of extracellular material from the colonizing bacteria, as observed by (1) epifluorescent microscopy, (2) scanning electron microscopy (SEM), and (3) nondestructive analysis of lyophilized biofilms by Fourier transform-infrared spectroscopy (FT-IR) using diffuse reflectance. The extracellular products from the colonizing bacteria corr...

Microbial biomass and activities associated with subsurface environments contaminated with chlorinated hydrocarbons

Abstract Soil microcosms and enrichment cultures from subsurface sediments and groundwaters contaminated with trichloroethylene (TCE) were examined. Total lipids, [I‐‘4C]acetate incorporation into lipids, and [Me‐3H]thymidine incorporation into DNA were determined in these subsurface environments. In heavily TCE‐contam‐inated zones (greater than 500 mg/L) radioisotopes were not incorporated into lipids or DNA. Radioisotope incorporation occurred in sediments both above and below the TCE plume. Phospholipid fatty acids (PLFA) were not detected, i.e., less than 0.5 pmol/L in heavily contaminated groundwater samples. In less contaminated waters, extracted PLFA concentrations were greater than 100 pmollL and microbial isolates were readily obtained. Degradation of 30–100 mg/L TCE was observed when sediments were amended with a variety of energy sources. Microorganisms in these subsurface sediments have adapted to degrade TCE at concentrations greater than 50 mg/L.

Sterols and phylogeny of the acidophilic hot springs algae Cyanidium caldarium and Galdieria sulphuraria

Abstract The sterols of Cyanidium caldarium and Galdieria sulphuraria were analysed. These unicellular blue-green eukaryotic algae are acido-thermophili

Triacylglycerol Fatty Acid and Sterol Composition of Sediment Microorganisms from McMurdo Sound, Antarctica*

SummaryThe triacylglycerol fatty acid and sterol profiles of microorganisms from three McMurdo Sound sediment sites, collected during the austral summer of 1984–1985, were determined using gas chromatography and gas chromatography-mass spectrometry. Comparison of the three sites indicated that Cape Evans contained the greatest concentration of triacylglycerol (TG) (220 nmoles/gram dry weight (gdw) of sediment), approximately six to seven times that determined for sediment microorganisms from the Cape Armitage and New Harbor sites. The relative proportion of triacylglycerolderived polyunsaturated fatty acids (PUFA) revealed a somewhat different trend. New Harbor sediment contained the greatest relative proportion of PUFA (22% of triacylglycerol fatty acids), followed by Cape Evans (16%) and Cape Armitage (11%). The proportion of unsaturated fatty acids (poly-and monounsaturated) was relatively constant and ranged from 63% to 71% of the triacylglycerol fatty acids for the three sites. Sterol concentrations varied from 610 pmoles/gdw at Cape Evans, to 370 and 240 pmoles/gdw for Cape Armitage and New Harbor respectively, and was approximately 1% of the total determined lipid. Cholesterol was the major sterol component detected, occurring at similar relative levels (29%) for all three sites. Other sterols present in decreasing order of abundance were 22-dehydrocholesterol, brassicasterol, 24-ethylcholesterol and 24-methylcholesterol. 5α-stanols were only minor components of the three sediments, indicating that in situ biohydrogenation of stenols was not a major sterol transformation process in these recent surface oxic sediments.

A total-recycle expanded-bed bioreactor design which allows direct headspace sampling of volatile chlorinated aliphatic compounds

Abstract A two-phase column was developed, allowing direct headspace sampling of volatile chlorinated aliphatic compounds, including trichloroethylene, cis- and trans-1,2-dichloroethylene and vinyl chloride. Propane, methane and CO 2 were sampled in the same fashion. A comparison of two solid support matrices (crushed glass and anthracite coal) was performed. > 97 ± 2% of the trichloroethylene added to control reactors was still present following 12 days of operation. The bioreactor consisted of an expanded-bed column containing 70 g of 60 80 - mesh substratum. The liquid phase was recirculated through the expanded-bed column into a gas-recharge column fitted with a headspace sampling port which allowed direct sampling and subsequent quantification by gas-chromatography techniques.