Perfusion Method for Assaying Microbial Activities in Sediments: Applicability to Studies of N2 Fixation by C2H2 Reduction

A perfusion method for assaying nitrogenase activity (acetylene reduction) in marine sediments was developed. The method was used to assay sediment cores from Spartina alterniflora (salt marsh), Zostera marina (sea grass), and Thalassia testudinum (sea grass) communities, and the results were compared with those of conventional sealed-flask assays. Rates of ethylene production increased progressively with time in the perfusion assays, reaching plateau values of 2 to 3 nmol · g of dry sediment⁻¹ · h⁻¹ by 10 to 20 h. Depletion of interstitial NH₄⁺ was implicated in this stimulation of nitrogenase activity. Initial acetylene reduction rates determined by the perfusion assay of cores from the Spartina community ranged from 0.15 to 0.60 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹. These rates were similar to those for sediments assayed in sealed flasks without seawater when determined over linear periods of C₂H₄ production. Initial values obtained by using the perfusion method were 0.66 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹ for sediments from Zostera communities and 0.70 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹ for sediments from Thalassia communities. In all cases, rates determined by simultaneous slurry assays were lower than those determined by the perfusion method.     

Populations of Methane-Producing Bacteria and In Vitro Methanogenesis in Salt Marsh and Estuarine Sediments

Most probable numbers (MPNs) of methanogens in various salt marsh and estuarine sediments were determined with an anaerobic, habitat-simulating culture medium with 80% H₂ plus 20% CO₂ as substrate. Average MPNs for the short Spartina (SS) marsh sediments of Sapelo Island, Ga., were maximal at the 5- to 7-cm depth (1.2 × 10⁷/g of dry sediment). Populations decreased to approximately 880/g of dry sediment at the 34- to 36-cm depth. There was no significant difference between summer and winter populations. In tall Spartina (TS) marsh sediments, average populations were maximal (1.2 × 10⁶/g of dry sediment) in the upper 0- to 2-cm zone; populations from the 5- to 36-cm zones were similar (average of 9 × 10⁴/g of dry sediment). Methanogenic populations for TS sediments of James Island Creek marsh, Charleston, S.C., were similar (average of 3 × 10⁶/g of dry sediment) for all depths tested (0 to 22 cm), which was comparable to the trend observed for TS sediments at Sapelo Island, Ga. Sediment grab samples collected along a transect of James Island Creek and its adjacent Spartina marsh had MPNs that were approximately 20 times greater for the region of Spartina growth (average of 10⁶/g of dry sediment) compared with the channel (approximately 5 × 10⁴ methanogens per g of dry sediment). A similar trend was found at Pawley's Island marsh, S.C., but populations were approximately one order of magnitude lower. In vitro rates of methanogenesis with SS sediments incubated under 80% H₂-20% CO₂ showed that the 5- to 7-cm region exhibited maximal activity (51 nmol of CH₄ g⁻¹ h⁻¹), which was greater than rates for sediments above and below this depth. SS sediment samples (5 to 7 cm) incubated under 100% N₂ and supplemented with formate exhibited rates of methanogenesis similar to those generated by samples under 80% H₂-20% CO₂. Replacing the N₂ atmosphere with H₂ resulted in an eightfold decrease in the rate of methanogenesis. In vitro methanogenic activity by TS salt marsh sediments, incubated under 80% H₂-20% CO₂, was similar for all depths tested (0 to 22 cm). TS sediment samples (0 to 7 cm) supplemented with formate and incubated under 100% N₂ had greater rates of methanogenesis compared with unsupplemented samples.     

Coexistence of Microaerophilic,Nitrate-Reducing,and Phototrophic Fe(II) Oxidizers and Fe(III) Reducers in Coastal Marine Sediment

Iron is abundant in sediments, where it can be biogeochemically cycled between its divalent and trivalent redox states. The neutrophilic microbiological Fe cycle involves Fe(III)-reducing and three different physiological groups of Fe(II)-oxidizing microorganisms, i.e., microaerophilic, anoxygenic phototrophic, and nitrate-reducing Fe(II) oxidizers. However, it is unknown whether all three groups coexist in one habitat and how they are spatially distributed in relation to gradients of O₂, light, nitrate, and Fe(II). We examined two coastal marine sediments in Aarhus Bay, Denmark, by cultivation and most probable number (MPN) studies for Fe(II) oxidizers and Fe(III) reducers and by quantitative-PCR (qPCR) assays for microaerophilic Fe(II) oxidizers. Our results demonstrate the coexistence of all three metabolic types of Fe(II) oxidizers and Fe(III) reducers. In qPCR, microaerophilic Fe(II) oxidizers (Zetaproteobacteria) were present with up to 3.2 × 10⁶ cells g dry sediment⁻¹. In MPNs, nitrate-reducing Fe(II) oxidizers, anoxygenic phototrophic Fe(II) oxidizers, and Fe(III) reducers reached cell numbers of up to 3.5 × 10⁴, 3.1 × 10², and 4.4 × 10⁴ g dry sediment⁻¹, respectively. O₂ and light penetrated only a few millimeters, but the depth distribution of the different iron metabolizers did not correlate with the profile of O₂, Fe(II), or light. Instead, abundances were homogeneous within the upper 3 cm of the sediment, probably due to wave-induced sediment reworking and bioturbation. In microaerophilic Fe(II)-oxidizing enrichment cultures, strains belonging to the Zetaproteobacteria were identified. Photoferrotrophic enrichments contained strains related to Chlorobium and Rhodobacter; the nitrate-reducing Fe(II) enrichments contained strains related to Hoeflea and Denitromonas. This study shows the coexistence of all three types of Fe(II) oxidizers in two near-shore marine environments and the potential for competition and interrelationships between them.     

Quantification of Ammonia-Oxidizing Bacteria and Factors Controlling Nitrification in Salt Marsh Sediments

To elucidate the geomicrobiological factors controlling nitrification in salt marsh sediments, a comprehensive approach involving sediment geochemistry, process rate measurements, and quantification of the genetic potential for nitrification was applied to three contrasting salt marsh habitats: areas colonized by the tall (TS) or short (SS) form of Spartina alterniflora and unvegetated creek banks (CBs). Nitrification and denitrification potential rates were strongly correlated with one another and with macrofaunal burrow abundance, indicating that coupled nitrification-denitrification was enhanced by macrofaunal burrowing activity. Ammonia monooxygenase (amoA) gene copy numbers were used to estimate the ammonia-oxidizing bacterial population size (5.6 × 10⁴ to 1.3 × 10⁶ g of wet sediment⁻¹), which correlated with nitrification potentials and was 1 order of magnitude higher for TS and CB than for SS. TS and CB sediments also had higher Fe(III) content, higher Fe(III)-to-total reduced sulfur ratios, higher Fe(III) reduction rates, and lower dissolved sulfides than SS sediments. Iron(III) content and reduction rates were positively correlated with nitrification and denitrification potential and amoA gene copy number. Laboratory slurry incubations supported field data, confirming that increased amounts of Fe(III) relieved sulfide inhibition of nitrification. We propose that macrofaunal burrowing and high concentrations of Fe(III) stimulate nitrifying bacterial populations, and thus may increase nitrogen removal through coupled nitrification-denitrification in salt marsh sediments.     

Estimation of Bacterial Cell Numbers in Humic Acid-Rich Salt Marsh Sediments with Probes Directed to 16S Ribosomal DNA

The feasibility of using probes directed towards ribosomal DNAs (rDNAs) as a quantitative approach to estimating cell numbers was examined and applied to study the structure of a bacterial community in humic acid-rich salt marsh sediments. Hybridizations were performed with membrane-bound nucleic acids by using seven group-specific DNA oligonucleotide probes complementary to 16S rRNA coding regions. These included a general eubacterial probe and probes encompassing most members of the gram-negative, mesophilic sulfate-reducing bacteria (SRB). DNA was extracted from sediment samples, and contaminating materials were removed by a series of steps. Efficiency of DNA extraction was 48% based on the recovery of tritiated plasmid DNA added to samples prior to extraction. Reproducibility of the extraction procedure was demonstrated by hybridizations to replicate samples. Numbers of target cells in samples were estimated by comparing the amount of hybridization to extracted DNA obtained with each probe to that obtained with a standard curve of genomic DNA for reference strains included on the same membrane. In June, numbers of SRB detected with an SRB-specific probe ranged from 6.0 × 10⁷ to 2.5 × 10⁹ (average, 1.1 × 10⁹ ± 5.2 × 10⁸) cells g of sediment⁻¹. In September, numbers of SRB detected ranged from 5.4 × 10⁸ to 7.3 × 10⁹ (average, 2.5 × 10⁹ ± 1.5 × 10⁹) cells g of sediment⁻¹. The capability of using rDNA probes to estimate cell numbers by hybridization to DNA extracted from complex matrices permits initiation of detailed studies on community composition and changes in communities based on cell numbers in formerly intractable environments.     

Genotypic and Phenotypic Responses of a Riverine Microbial Community to Polycyclic Aromatic Hydrocarbon Contamination

The phenotypic and genotypic adaptation of a freshwater sedimentary microbial community to elevated (22 to 217 μg g [dry weight] of sediment⁻¹) levels of polycyclic aromatic hydrocarbons (PAHs) was determined by using an integrated biomolecular approach. Central to the approach was the use of phospholipid fatty acid (PLFA) profiles to characterize the microbial community structure and nucleic acid analysis to quantify the frequency of degradative genes. The study site was the Little Scioto River, a highly impacted, channelized riverine system located in central Ohio. This study site is a unique lotic system, with all sampling stations having similar flow and sediment characteristics both upstream and downstream from the source of contamination. These characteristics allowed for the specific analysis of PAH impact on the microbial community. PAH concentrations in impacted sediments ranged from 22 to 217 μg g (dry weight) of sediment⁻¹, while PAH concentrations in ambient sediments ranged from below detection levels to 1.5 μg g (dry weight) of sediment⁻¹. Total microbial biomass measured by phospholipid phosphate (PLP) analysis ranged from 95 to 345 nmol of PLP g (dry weight) of sediment⁻¹. Nucleic acid analysis showed the presence of PAH-degradative genes at all sites, although observed frequencies were typically higher at contaminated sites. Principal component analysis of PLFA profiles indicated that moderate to high PAH concentrations altered microbial community structure and that seasonal changes were comparable in magnitude to the effects of PAH pollution. These data indicate that this community responded to PAH contamination at both the phenotypic and the genotypic level.     

Evidence for the Cooccurrence of Nitrite-Dependent Anaerobic Ammonium and Methane Oxidation Processes in a Flooded Paddy Field

Anaerobic ammonium oxidation (anammox) and nitrite-dependent anaerobic methane oxidation (n-damo) are two of the most recent discoveries in the microbial nitrogen cycle. In the present study, we provide direct evidence for the cooccurrence of the anammox and n-damo processes in a flooded paddy field in southeastern China. Stable isotope experiments showed that the potential anammox rates ranged from 5.6 to 22.7 nmol N₂ g⁻¹ (dry weight) day⁻¹ and the potential n-damo rates varied from 0.2 to 2.1 nmol CO₂ g⁻¹ (dry weight) day⁻¹ in different layers of soil cores. Quantitative PCR showed that the abundance of anammox bacteria ranged from 1.0 × 10⁵ to 2.0 × 10⁶ copies g⁻¹ (dry weight) in different layers of soil cores and the abundance of n-damo bacteria varied from 3.8 × 10⁵ to 6.1 × 10⁶ copies g⁻¹ (dry weight). Phylogenetic analyses of the recovered 16S rRNA gene sequences showed that anammox bacteria affiliated with “Candidatus Brocadia” and “Candidatus Kuenenia” and n-damo bacteria related to “Candidatus Methylomirabilis oxyfera” were present in the soil cores. It is estimated that a total loss of 50.7 g N m⁻² per year could be linked to the anammox process, which is at intermediate levels for the nitrogen flux ranges of aerobic ammonium oxidation and denitrification reported in wetland soils. In addition, it is estimated that a total of 0.14 g CH₄ m⁻² per year could be oxidized via the n-damo process, while this rate is at the lower end of the aerobic methane oxidation rates reported in wetland soils.     

Role of Sulfate Reduction Versus Methanogenesis in Terminal Carbon Flow in Polluted Intertidal Sediment of Waimea Inlet, Nelson, New Zealand

An investigation of the terminal anaerobic processes occurring in polluted intertidal sediments indicated that terminal carbon flow was mainly mediated by sulfate-reducing organisms in sediments with high sulfate concentrations (>10 mM in the interstitial water) exposed to low loadings of nutrient (equivalent to <10² kg of N · day⁻¹) and biochemical oxygen demand (<0.7 × 10³ kg · day⁻¹) in effluents from different pollution sources. However, in sediments exposed to high loadings of nutrient (>10² kg of N · day⁻¹) and biochemical oxygen demand (>0.7 × 10³ kg · day⁻¹), methanogenesis was the major process in the mediation of terminal carbon flow, and sulfate concentrations were low (≤2 mM). The respiratory index [¹⁴CO₂/(¹⁴CO₂ + ¹⁴CH₄)] for [2-¹⁴C]acetate catabolism, a measure of terminal carbon flow, was ≥0.96 for sediment with high sulfate, but in sediments with sulfate as little as 10 μM in the interstitial water, respiratory index values of ≤0.22 were obtained. In the latter sediment, methane production rates as high as 3 μmol · g⁻¹ (dry weight) · h⁻¹ were obtained, and there was a potential for active sulfate reduction.     

Does Virus-Induced Lysis Contribute Significantly to Bacterial Mortality in the Oxygenated Sediment Layer of Shallow Oxbow Lakes?

Despite the recognition that viruses are ubiquitous components of aquatic ecosystems, the number of studies on viral abundance and the ecological role of viruses in sediments is scarce. In this investigation, the interactions between viruses and bacteria were studied in the oxygenated silty sediment layer of a mesotrophic oxbow lake. A long-term study (13 months) and a diel study revealed that viruses are a numerically important and dynamic component of the microbial community. The abundance and decay rates ranged from 4.3 × 10⁹ to 7.2 × 10⁹ particles ml of wet sediment⁻¹ and from undetectable to 22.2 × 10⁷ particles ml⁻¹ h⁻¹, respectively, and on average the values were 2 orders of magnitude higher than the values for the overlying water. In contrast to our expectations, viruses did not contribute significantly to the bacterial mortality in the sediment, since on average only 6% (range, 0 to 25%) of the bacterial secondary production was controlled by viruses. The low impact of viruses on the bacterial community may be associated with the quantitatively low viral burden that benthic bacteria have to cope with compared to the viral burden with which bacterial assemblages in the water column are confronted. The virus-to-bacterium ratio of the sediment varied between 0.9 and 3.2, compared to a range of 5.0 to 12.4 obtained for the water column. We speculate that despite high numbers of potential hosts, the possibility of encountering a host cell is limited by the physical conditions in the sediment, which is therefore not a favorable environment for viral proliferation. Our data suggest that viruses do not play an important role in the processing and transfer of bacterial carbon in the oxygenated sediment layer of the environment investigated.     

Spatial Heterogeneity of Bacterial Populations along an Environmental Gradient at a Shallow Submarine Hydrothermal Vent near Milos Island (Greece)

The spatial heterogeneity of bacterial populations at a shallow-water hydrothermal vent in the Aegean Sea close to the island of Milos (Greece) was examined at two different times by using acridine orange staining for total cell counts, cultivation-based techniques, and denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA gene fragments. Concurrent with measurements of geochemical parameters, samples were taken along a transect from the center of the vent to the surrounding area. Most-probable-number (MPN) counts of metabolically defined subpopulations generally constituted a minor fraction of the total cell counts; both counting procedures revealed the highest cell numbers in a transition zone from the strongly hydrothermally influenced sediments to normal sedimentary conditions. Total cell counts ranged from 3.2 × 10⁵ cells ml⁻¹ in the water overlying the sediments to 6.4 × 10⁸ cells g (wet weight) of sediment⁻¹. MPN counts of chemolithoautotrophic sulfur-oxidizing bacteria varied between undetectable and 1.4 × 10⁶ cells g⁻¹. MPN counts for sulfate-reducing bacteria and dissimilatory iron-reducing bacteria ranged from 8 to 1.4 × 10⁵ cells g⁻¹ and from undetectable to 1.4 × 10⁶ cells g⁻¹, respectively. DGGE revealed a trend from a diverse range of bacterial populations which were present in approximately equal abundance in the transition zone to a community dominated by few populations close to the center of the vent. Temperature was found to be an important parameter in determining this trend. However, at one sampling time this trend was not discernible, possibly due to storm-induced disturbance of the upper sediment layers.     

Anaerobic Degradation of Flavonoids by Clostridium orbiscindens

An anaerobic, quercetin-degrading bacterium was isolated from human feces and identified as Clostridium orbiscindens by comparative 16S rRNA gene sequence analysis. The organism was tested for its ability to transform several flavonoids. The isolated C. orbiscindens strain converted quercetin and taxifolin to 3,4-dihydroxyphenylacetic acid; luteolin and eriodictyol to 3-(3,4-dihydroxyphenyl)propionic acid; and apigenin, naringenin, and phloretin to 3-(4-hydroxyphenyl)propionic acid, respectively. Genistein and daidzein were not utilized. The glycosidic bonds of luteolin-3-glucoside, luteolin-5-glucoside, naringenin-7-neohesperidoside (naringin), quercetin-3-glucoside, quercetin-3-rutinoside (rutin), and phloretin-2′-glucoside were not cleaved. Based on the intermediates and products detected, pathways for the degradation of the flavonol quercetin and the flavones apigenin and luteolin are proposed. To investigate the numerical importance of C. orbiscindens in the human intestinal tract, a species-specific oligonucleotide probe was designed and tested for its specificity. Application of the probe to fecal samples from 10 human subjects proved the presence of C. orbiscindens in 8 out of the 10 samples tested. The numbers ranged from 1.87 × 10⁸ to 2.50 × 10⁹ cells g of fecal dry mass⁻¹, corresponding to a mean count of 4.40 × 10⁸ cells g of dry feces⁻¹.     

Formation of carbon monoxide and bile pigment in red and blue-green algae

Chromium was not required for normal growth of romaine lettuce (Lactuca sativa L. subsp. longifolia), tomato (Lycopersicon esculentum Mill.), wheat (Triticum aestivum L.), or bean (Phaseolus vulgaris L.) in solution culture containing 3.8 × 10⁻⁴ μM Cr. Plants grown on this purified nutrient solution contained an average of 22 ng Cr/g dry weight. Duckweed (Lemna sp.) grew and reproduced normally on a dilute nutrient solution containing 3.8 × 10⁻⁵ μM Cr.     

High-Frequency Phage-Mediated Gene Transfer among Escherichia coli Cells, Determined at the Single-Cell Level

Recent whole-genome analysis suggests that lateral gene transfer by bacteriophages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency of phage-mediated gene transfer, we employed cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) and investigated the movement of the ampicillin resistance gene among Escherichia coli cells mediated by phage at the single-cell level. Phages P1 and T4 and the newly isolated E. coli phage EC10 were used as vectors. The transduction frequencies determined by conventional plating were 3 × 10⁻⁸ to 2 × 10⁻⁶, 1 × 10⁻⁸ to 4 × 10⁻⁸, and <4 × 10⁻⁹ to 4 × 10⁻⁸ per PFU for phages P1, T4, and EC10, respectively. The frequencies of DNA transfer determined by CPRINS-FISH were 7 × 10⁻⁴ to 1 × 10⁻³, 9 × 10⁻⁴ to 3 × 10⁻³, and 5 × 10⁻⁴ to 4 × 10⁻³ for phages P1, T4, and EC10, respectively. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viabilities. These results revealed that the difference in the number of viable cells carrying the transferred gene and the number of cells capable of growth on the selective medium was 3 to 4 orders of magnitude, indicating that phage-mediated exchange of DNA sequences among bacteria occurs with unexpectedly high frequency.     

myo-Inositol homeostasis in foetal rabbit lung

In several species, lung maturation is accompanied by a decline in the phosphatidylinositol content of lung surfactant and a concomitant increase in its phosphatidylglycerol content. To examine the possibility that this developmental change is influenced by the availability of myo-inositol, potential sources of myo-inositol for the developing rabbit lung were investigated. On day 28 of gestation the myo-inositol content of foetal rabbit lung tissue (2.3±0.5μmol/g of tissue) was not significantly different from that of adult lung tissue but the activity of d-glucose 6-phosphate:1l-myo-inositol 1-phosphate cyclase (cyclase) in foetal lung tissue (81.0±9.0nmol·h⁻¹·g of tissue⁻¹) was higher than that found in adult lung tissue (23.2±1.0nmol·h⁻¹·g of tissue⁻¹). Day 28 foetal rabbit lung tissue was found also to take up myo-inositol by a specific, energy-dependent, Na⁺-requiring mechanism. Half-maximal uptake of myo-inositol by foetal rabbit lung slices was observed when the concentration of myo-inositol in the incubation medium was 85μm. When the myo-inositol concentration was 1mm (but not 100μm) the addition of glucose (5.5mm) stimulated myo-inositol uptake. myo-Inositol uptake was observed also in adult rabbit lung and was found to be sub-maximal at the concentration of myo-inositol found in adult rabbit serum. The concentration of myo-inositol in the serum of pregnant adult rabbits (47.5±5.5μm) was significantly lower than that of non-pregnant adult female rabbits (77.9±9.2μm). On day 28 of gestation the concentration of myo-inositol in foetal serum (175.1±12.0μm) was much less than on day 25, but more than that found on day 30. A transient post-partum increase in the concentration of myo-inositol in serum was followed by a rapid decline. Much of the myo-inositol in foetal rabbit serum probably originates from the placenta, where on day 28 of gestation a high cyclase activity (527±64nmol·h⁻¹·g of tissue⁻¹) was measured. The gestational decline in serum myo-inositol concentration, together with the decreasing cyclase activity of the lungs, is consistent with the view that maturation of the lungs is accompanied by decreased availability of myo-inositol to this tissue.     

Inhibition of glucose phosphate isomerase by metabolic intermediates of fructose

1. Purified rabbit-muscle and -liver glucose phosphate isomerase, free of contaminating enzyme activities that could interfere with the assay procedures, were tested for inhibition by fructose, fructose 1-phosphate and fructose 1,6-diphosphate. 2. Fructose 1-phosphate and fructose 1,6-diphosphate are both competitive with fructose 6-phosphate in the enzymic reaction, the apparent K_i values being 1·37×10⁻³−1·67×10⁻³m for fructose 1-phosphate and 7·2×10⁻³−7·9×10⁻³m for fructose 1,6-diphosphate; fructose and inorganic phosphate were without effect. 3. The apparent K_m values for both liver and muscle enzymes at pH7·4 and 30° were 1·11×10⁻⁴−1·29×10⁻⁴m for fructose 6-phosphate, determined under the conditions in this paper. 4. In the reverse reaction, fructose, fructose 1-phosphate and fructose 1,6-diphosphate did not significantly inhibit the conversion of glucose 6-phosphate into fructose 6-phosphate. 5. The apparent K_m values for glucose 6-phosphate were in the range 5·6×10⁻⁴−8·5×10⁻⁴m. 6. The competitive inhibition of hepatic glucose phosphate isomerase by fructose 1-phosphate is discussed in relation to the mechanism of fructose-induced hypoglycaemia in hereditary fructose intolerance.     

Flowthrough Reactor Flasks for Study of Microbial Metabolism in Sediments

Flowthrough reactor flasks are described that allow continuous low-level nutrient input to mixed anoxic sediments without dilution of the sediment. The flasks were tested by simulating sulfate inputs into sediments collected from a freshwater eutrophic lake. After an initial 2-day adaptation within the reactor system, rates of methane production and sulfate consumption were constant for the duration of a 12-day incubation. A sulfate input rate of 0.15 mmol liter of sediment⁻¹ day⁻¹ resulted in an equivalent rate of sulfate removal, which was unaffected by inputs of acetate (1.0 mmol liter of sediment⁻¹ day⁻¹). The rate of methane production in control reactors, 0.18 mmol liter of sediment⁻¹ day⁻¹, was doubled by the addition of acetate, whereas sulfate consumption was only stimulated by additions of high concentrations of sulfate plus acetate (1.5 and 1.0 mmol liter of sediment⁻¹ day⁻¹, respectively). The reactor system appears to be effective in maintaining the balance between sulfate reduction and methane production in freshwater sediments and is potentially useful for study of the response of sediment populations to varying inputs of naturally occurring substrates, selected inhibitors, or xenobiotic compounds.     

Modeling Reduction of Uranium U(VI) under Variable Sulfate Concentrations by Sulfate-Reducing Bacteria

The kinetics for the reduction of sulfate alone and for concurrent uranium [U(VI)] and sulfate reduction, by mixed and pure cultures of sulfate-reducing bacteria (SRB) at 21 ± 3°C were studied. The mixed culture contained the SRB Desulfovibrio vulgaris along with a Clostridium sp. determined via 16S ribosomal DNA analysis. The pure culture was Desulfovibrio desulfuricans (ATCC 7757). A zero-order model best fit the data for the reduction of sulfate from 0.1 to 10 mM. A lag time occurred below cell concentrations of 0.1 mg (dry weight) of cells/ml. For the mixed culture, average values for the maximum specific reaction rate, V_max, ranged from 2.4 ± 0.2 μmol of sulfate/mg (dry weight) of SRB · h⁻¹) at 0.25 mM sulfate to 5.0 ± 1.1 μmol of sulfate/mg (dry weight) of SRB · h⁻¹ at 10 mM sulfate (average cell concentration, 0.52 mg [dry weight]/ml). For the pure culture, V_max was 1.6 ± 0.2 μmol of sulfate/mg (dry weight) of SRB · h⁻¹ at 1 mM sulfate (0.29 mg [dry weight] of cells/ml). When both electron acceptors were present, sulfate reduction remained zero order for both cultures, while uranium reduction was first order, with rate constants of 0.071 ± 0.003 mg (dry weight) of cells/ml · min⁻¹ for the mixed culture and 0.137 ± 0.016 mg (dry weight) of cells/ml · min⁻¹ (U₀ = 1 mM) for the D. desulfuricans culture. Both cultures exhibited a faster rate of uranium reduction in the presence of sulfate and no lag time until the onset of U reduction in contrast to U alone. This kinetics information can be used to design an SRB-dominated biotreatment scheme for the removal of U(VI) from an aqueous source.     

Methane Oxidation by Nitrosococcus oceanus and Nitrosomonas europaea

Chemolithotrophic ammonium-oxidizing and nitrite-oxidizing bacteria including Nitrosomonas europaea, Nitrosococcus oceanus, Nitrobacter sp., Nitiospina gracilis, and Nitrococcus mobilis were examined as to their ability to oxidize methane in the absence of ammonium or nitrite. All ammonium oxidizers tested had the ability to oxidize significant amounts of methane to CO₂ and incorporate various amounts into cellular components. None of the nitrite-oxidizing bacteria were capable of methane oxidation. The methane-oxidizing capabilities of Nitrosococcus oceanus and Nitrosomonas europaea were examined with respect to ammonium and methane concentrations, nitrogen source, and pH. The addition of ammonium stimulated both CO₂ production and cellular incorporation of methane-carbon by both organisms. Less than 0.1 mM CH₄ in solution inhibited the oxidation of ammonium by Nitrosococcus oceanus by 87%. Methane concentrations up to 1.0 mM had no inhibitory effects on ammonium oxidation by Nitrosomonas europaea. In the absence of NH₄-N, Nitrosococcus oceanus achieved a maximum methane oxidation rate of 2.20 × 10⁻² μmol of CH₄ h⁻¹ mg (dry weight) of cells⁻¹, which remained constant as the methane concentration was increased. In the presence of NH₄-N (10 ppm [10 μg/ml]), its maximum rate was 26.4 × 10⁻² μmol of CH₄ h⁻¹ mg (dry weight) of cells⁻¹ at a methane concentration of 1.19 × 10⁻² mM. Increasing the methane concentration above this level decreased CO₂ production, whereas cellular incorporation of methane-carbon continued to increase. Nitrosomonas europaea showed a linear response throughout the test range, with an activity of 196.0 × 10⁻² μmol of CH₄ h⁻¹ mg (dry weight) of cells ⁻¹ at a methane concentration of 1.38 × 10⁻¹ mM. Both nitrite and nitrate stimulated the oxidation of methane. The pH range was similar to that for ammonium oxidation, but the points of maximum activity were at lower values for the oxidation of methane.     

Genome-wide association identifies a common variant in the reelin gene that increases the risk of schizophrenia only in women

Sex differences in schizophrenia are well known, but their genetic basis has not been identified. We performed a genome-wide association scan for schizophrenia in an Ashkenazi Jewish population using DNA pooling. We found a female-specific association with rs7341475, a SNP in the fourth intron of the reelin (RELN) gene (p = 2.9 × 10⁻⁵ in women), with a significant gene-sex effect (p = 1.8 × 10⁻⁴). We studied rs7341475 in four additional populations, totaling 2,274 cases and 4,401 controls. A significant effect was observed only in women, replicating the initial result (p = 2.1 × 10⁻³ in women; p = 4.2 × 10⁻³ for gene-sex interaction). Based on all populations the estimated relative risk of women carrying the common genotype is 1.58 (p = 8.8 × 10⁻⁷; p = 1.6 × 10⁻⁵ for gene-sex interaction). The female-specific association between RELN and schizophrenia is one of the few examples of a replicated sex-specific genetic association in any disease.     

Characterization of Subsurface Bacteria Associated with Two Shallow Aquifers in Oklahoma

The bacterial microflora of two shallow aquifers (saturated subsurface zones) in Oklahoma was characterized by direct observation with light and electron microscopy, by plating, and by examination of colony morphology and distribution. Isolated bacterial strains were also examined. Total cell counts varied only slightly (2.9 × 10⁶ to 9.8 × 10⁶ g [dry wt]⁻¹) from sample to sample, whereas colony counts varied widely (6.3 × 10² to 6.5 × 10⁶ CFU g [dry wt]⁻¹). Colony counts on nutritionally rich media were lower than on low-nutrient media, especially in samples from the saturated zone. The variety of colony types growing on nutritionally rich media decreased with increasing depth and saturation. Colony counts of anaerobic bacteria also decreased with depth but were at least 100-fold lower than aerobic counts on most media. Cell morphologies of bacteria grown aerobically on plates included short rods, cocci, and actinomycete-like forms. Direct light microscopic observation of sediments revealed short, rod-shaped, and coccoid bacterial cells; endospores, actinomycete spores, and eucaryotic forms were not observed by light microscopy. Electron microscopic observation of bacteria released from the samples revealed that 85 to 90% of them were coccoid, gram-positive, Arthrobacter-like organisms, some of which were dividing or contained completed division septa; other types of gram-positive and gram-negative bacteria were present in lower numbers. Isolated bacterial strains were able to grow on both nutritionally rich and low-nutrient media. A higher proportion of gram-negative organisms was isolated than gram-positive organisms. Most of the isolates were capable of storing polyphosphate, poly-β-hydroxybutyrate, or polysaccharide. The results of this study suggest that the microbial population of these two shallow aquifers is dominated by aerobic, nutritionally versatile bacteria that can subsist on low concentrations of organic compounds without forming specialized resting cells. Other types of microorganisms, such as facultatively anaerobic bacteria and microeucaryotes, may also be present, but they represent only a small fraction of the microflora.