Spatial Variability in Streambed Microbial Community Structure across Two Watersheds.

Both spatial and temporal variability are key attributes of sedimentary microbial communities, and while spatial effects on beta-diversity appear to dominate at larger distances, the character of spatial variability at finer scales remains poorly understood, especially for headwater stream communities. We investigated patterns of microbial community structure (MCS) in biofilms attached to streambed sediments from two watersheds across spatial scales spanning <1 m within a single stream to several hundred kilometers between watersheds. Analyses of phospholipid fatty acid (PLFA) profiles indicated that the variations in MCS were driven by increases in the relative abundance of microeukaryotic photoautotrophs and their contribution to total microbial biomass. Furthermore, streams within watersheds had similar MCS, underscoring watershed-level controls of microbial communities. Moreover, bacterial community structure assayed as either PCR-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprints or PLFA profiles edited to remove microeukaryotes indicated a distinct watershed-level biogeography. No distinct stream order-level distributions were identified, although DGGE analyses clearly indicated that there was greater variability in community structure among 1st-order streams than among 2nd- and 3rd-order streams. Longitudinal gradients in microbial biomass and structure showed that the greatest variations were associated with 1st-order streams within a watershed, and 68% of the variation in total microbial biomass was explained by sediment atomic carbon-to-nitrogen ratio (C:N ratio), percent carbon, sediment surface area, and percent water content. This study confirms a distinct microbial biogeography for headwater stream communities driven by environmental heterogeneity across distant watersheds and suggests that eukaryotic photoautotrophs play a key role in structuring bacterial communities on streambed sediments. IMPORTANCE Microorganisms in streams drive many biogeochemical reactions of global significance, including nutrient cycling and energy flow; yet, the mechanisms responsible for the distribution and composition of streambed microbial communities are not well known. We sampled sediments from multiple streams in two watersheds (Neversink River [New York] and White Clay Creek [WCC; Pennsylvania] watersheds) and measured microbial biomass and total microbial and bacterial community structures using phospholipid and molecular methods. Microbial and bacterial community structures displayed a distinct watershed-level biogeography. The smallest headwater streams within a watershed showed the greatest variation in microbial biomass, and 68% of that variation was explained by the atomic carbon-to-nitrogen ratio (C:N ratio), percent carbon, sediment surface area, and percent water content. Our study revealed a nonrandom distribution of microbial communities in streambeds, and showed that microeukaryotic photoautotrophs, environmental heterogeneity, and geographical distance influence microbial composition and spatial distribution.

Periphytic algae decouple fungal activity from leaf litter decomposition via negative priming.

1. Well-documented in terrestrial settings, priming effects describe stimulated heterotrophic microbial activity and decomposition of recalcitrant carbon by additions of labile carbon. In aquatic settings, algae produce labile exudates which may elicit priming during organic matter decomposition, yet the directions and mechanisms of aquatic priming effects remain poorly tested. 2. We tested algal-induced priming during decomposition of two leaf species of contrasting recalcitrance, Liriodendron tulipifera and Quercus nigra, in experimental streams under light or dark conditions. We measured litter-associated algal, bacterial, and fungal biomass and activity, stoichiometry, and litter decomposition rates over 43 days. 3. Light increased algal biomass and production rates and increased bacterial abundance 141-733% and fungal production rates 20-157%. Incubations with a photosynthesis inhibitor established that algal activity directly stimulated fungal production rates in the short-term. 4. Algal-stimulated fungal production rates on both leaf species were not coupled to long-term increases in fungal biomass accrual or litter decomposition rates, which were 154-157% and 164-455% greater in the dark, respectively. The similar patterns on fast- vs. slow-decomposing L. tulipifera and Q. nigra, respectively, indicated that substrate recalcitrance may not mediate priming strength or direction. 5. In this example of negative priming, periphytic algae decoupled fungal activity from decomposition, likely by providing labile carbon invested toward greater fungal growth and reproduction instead of recalcitrant carbon degradation. If common, algal-induced negative priming could stimulate heterotrophy reliant on labile carbon yet suppress decomposition of recalcitrant carbon, modifying energy and nutrients available to upper trophic levels and enhancing organic carbon storage or export in well-lit aquatic habitats.

Linking permafrost thaw to shifting biogeochemistry and food web resources in an arctic river.

Rapidly, increasing air temperatures across the Arctic are thawing permafrost and exposing vast quantities of organic carbon, nitrogen, and phosphorus to microbial processing. Shifts in the absolute and relative supplies of these elements will likely alter patterns of ecosystem productivity and change the way carbon and nutrients are delivered from upland areas to surface waters such as rivers and lakes. The ultra-oligotrophic nature of surface waters across the Arctic renders these ecosystems particularly susceptible to changes in productivity and food web dynamics as permafrost thaw alters terrestrial-aquatic linkages. The objectives of this study were to evaluate decadal-scale patterns in surface water chemistry and assess potential implications of changing water chemistry to benthic organic matter and aquatic food webs. Data were collected from the upper Kuparuk River on the North Slope of Alaska by the U.S. National Science Foundation's Long-Term Ecological Research program during 1978-2014. Analyses of these data show increases in stream water alkalinity and cation concentrations consistent with signatures of permafrost thaw. Changes are also documented for discharge-corrected nitrate concentrations (+), discharge-corrected dissolved organic carbon concentrations (-), total phosphorus concentrations (-), and δ13 C isotope values of aquatic invertebrate consumers (-). These changes show that warming temperatures and thawing permafrost in the upland environment are leading to shifts in the supply of carbon and nutrients available to surface waters and consequently changing resources that support aquatic food webs. This demonstrates that physical, geochemical, and biological changes associated with warming permafrost are fundamentally altering linkages between upland and aquatic ecosystems in rapidly changing arctic environments.

Climatic and watershed controls of dissolved organic matter variation in streams across a gradient of agricultural land use.

Human land use has led to significant changes in the character of dissolved organic matter (DOM) in lotic ecosystems. These changes are expected to have important environmental and ecological consequences. However, high spatiotemporal variability has been reported in previous studies, and the underlying mechanisms remain inadequately understood. This study assessed variation in the properties of stream water DOM within watersheds across a gradient of agricultural land use with grazing pasture lands as the dominant agricultural type in the southeastern United States. We collected water samples under baseflow conditions five times over eight months from a regional group of first- to fourth-order streams. Samples were analyzed for dissolved organic carbon (DOC) concentration, DOM quality based on absorbance and fluorescence properties, as well as DOM biodegradability. We found that air temperature and antecedent hydrological conditions (indicated by antecedent precipitation index and stream water sodium concentrations) positively influenced stream water DOC concentration, DOM fluorescence index, and the proportion of soil-derived, microbial humic fluorescence. This observation suggests that elevated production and release of microbial DOM in soils facilitated by high temperature, in conjunction with strong soil-stream hydrological connectivity, were important drivers for changes in the concentration and composition of stream water DOM. By comparison, watersheds with a high percentage of agricultural land use showed higher DOC concentration, larger proportion of soil-derived, humic-like DOM compounds, and higher DOC biodegradability. These observations reflect preferential mobilization of humic DOM compounds from shallow organic matter-rich soils in agricultural watersheds, likely due to enhanced soil erosion, organic matter oxidation and relatively shallow soil-to-stream flow paths.

Mercury levels in largemouth bass (Micropterus salmoides) from regulated and unregulated rivers.

Within areas of comparable atmospheric mercury deposition rates methylmercury burden in largemouth bass populations vary significantly between regulated and unregulated rivers. To investigate if trophic dynamics strongly influenced pollutant body load, we sampled largemouth bass from two adjacent rivers, one regulated and one unregulated, and applied a suite of biochemical and stable isotope assays to compare their trophic dynamics. Total mercury burden in the bass from the unregulated Sipsey River (Elrod, AL, USA) and the regulated Black Warrior River (Demopolis, AL, USA) averaged 0.87 mg kg-1 and 0.19 mg kg-1 wet weight, respectively. For both populations, age, weight, and length were positively correlated with muscle mercury concentration. Compound specific stable isotope analysis of amino acids showed the trophic position of both populations was just under four. Quantitative and isotopic analysis of neutral lipid fatty acid of Sipsey River bass indicated a greater reliance upon the detrital component of the food web compared to Demopolis Reservoir bass which fed within the autochthonous, pelagic component of the food web. Since the close proximity of the rivers makes differences in atmospheric deposition unlikely and both populations had similar trophic position, our findings indicate that food web dynamics should be included among the factors that can strongly influence mercury concentration in fish.

Transcriptomic evaluation of the American oyster, Crassostrea virginica, deployed during the Deepwater Horizon oil spill: Evidence of an active hydrocarbon response pathway.

Estuarine organisms were impacted by the Deepwater Horizon oil spill which released ∼5 million barrels of crude oil into the Gulf of Mexico in the spring and summer of 2010. Crassostrea virginica, the American oyster, is a keystone species in these coastal estuaries and is routinely used for environmental monitoring purposes. However, very little is known about their cellular and molecular responses to hydrocarbon exposure. In response to the spill, a monitoring program was initiated by deploying hatchery-reared oysters at three sites along the Alabama and Mississippi coast (Grand Bay, MS, Fort Morgan, AL, and Orange Beach, AL). Oysters were deployed for 2-month periods at five different time points from May 2010 to May 2011. Gill and digestive gland tissues were harvested for gene expression analysis and determination of aliphatic and polycyclic aromatic hydrocarbon (PAH) concentrations. To facilitate identification of stress response genes that may be involved in the hydrocarbon response, a nearly complete transcriptome was assembled using Roche 454 and Illumina high-throughput sequencing from RNA samples obtained from the gill and digestive gland tissues of deployed oysters. This effort resulted in the assembly and annotation of 27,227 transcripts comprised of a large assortment of stress response genes, including members of the aryl hydrocarbon receptor (AHR) pathway, Phase I and II biotransformation enzymes, antioxidant enzymes and xenobiotic transporters. From this assembly several potential biomarkers of hydrocarbon exposure were chosen for expression profiling, including the AHR, two cytochrome P450 1A genes (CYP1A-like 1 and CYP1A-like 2), Cu/Zn superoxide dismutase (CuZnSOD), glutathione S-transferase theta (GST theta) and multidrug resistance protein 3 (MRP3). Higher expression levels of GST theta and MRP3 were observed in gill tissues from all three sites during the summer to early fall 2010 deployments. Linear regression analysis indicated a statistically significant relationship between total PAH levels in digestive gland tissue samples with CYP1A-like 2, CuZnSOD, GST theta and MRP3 induction. These observations provide evidence of a potentially conserved AHR pathway in invertebrates and yield new insight into the development of novel biomarkers for use in environmental monitoring activities.

Pollutant emissions and environmental assessment of ethyl 3-ethoxybutyrate, a potential renewable fuel.

Renewable and bio-based transportation fuel sources can lower the life-cycle greenhouse gas emissions from vehicles. We present an initial assessment of ethyl 3-ethoxybutyrate (EEB) as a biofuel in terms of its performance as a fuel oxygenate and its persistence in the environment. EEB can be produced from ethanol and poly-3-hydroxybutyrate, a bacterial storage polymer that can be produced from non-food biomass and other organic feedstocks. Physicochemical properties of EEB and fuel-relevant properties of EEB-gasoline blends were measured, emissions of criteria pollutants from EEB as a gasoline additive in a production vehicle were evaluated, and fate and persistence of EEB in the environment were estimated. EEB solubility in water was 25.8 g/L, its Kow was 1.8, and its Henry's Law constant was 1.04 × 10(-5) atm-m(3)/mole. The anti-knock index values for 5 and 20 % v/v EEB-gasoline blends were 91.6 and 91.9, respectively. Reductions in fuel economy were consistent with the level of oxygenation, and criteria emissions were met by the vehicle operated over the urban dynamometer driving cycle (FTP 75). Predicted environmental persistence ranged from 15 to 30 days which indicates that EEB is not likely to be a persistent organic pollutant. In combination, these results suggest a high potential for the use of EEB as a renewable fuel source.

Unique odd-chain polyenoic phospholipid fatty acids present in chytrid fungi.

Chytrid fungi are ubiquitous components of aquatic and terrestrial ecosystems yet they remain understudied. To investigate the use of phospholipid fatty acids as phenotypic characteristics in taxonomic studies and biomarkers for ecological studies, 18 chytrid fungi isolated from soil to freshwater samples were grown in defined media and their phospholipid fatty acid profile determined. Gas chromatographic/mass spectral analysis indicated the presence of fatty acids typically associated with fungi, such as 16:1(n-7), 16:0, 18:2(n-6), 18:3(n-3) 18:1(n-9), and 18:0, as well as, a number of odd-chain length fatty acids, including two polyunsaturated C-17 fatty acids. Conversion to their 3-pyridylcarbinol ester facilitated GC-MS determination of double-bond positions and these fatty acid were identified as 6,9-17:2 [17:2(n-8)] and 6,9,12-17:3 [17:3(n-5)]. To the best of our knowledge, this is the first report of polyunsaturated C-17 fatty acids isolated from the phospholipids of chytrid fungi. Cluster analysis of PLFA profiles showed sufficient correlation with chytrid phylogeny to warrant inclusion of lipid analysis in species descriptions and the presence of several phospholipid fatty acids of restricted phylogenetic distributions suggests their usefulness as biomarkers for ecological studies.

Priming in the microbial landscape: periphytic algal stimulation of litter-associated microbial decomposers.

Microbial communities associated with submerged detritus in aquatic ecosystems often comprise a diverse mixture of autotrophic and heterotrophic microbes, including algae, bacteria, protozoa, and fungi. Recent studies have documented increased rates of plant litter mass loss when periphytic algae are present. We conducted laboratory and field experiments to assess potential metabolic interactions between natural autotrophic and heterotrophic microbial communities inhabiting submerged decaying plant litter of Typha angustifolia and Schoenoplectus acutus. In the field, submerged plant litter was either exposed to natural sunlight or placed under experimental canopies that manipulated light availability and growth of periphytic algae. Litter was collected and returned to the laboratory, where algal photosynthesis was manipulated (light/dark incubation), while rates of bacterial and fungal growth and productivity were simultaneously quantified. Bacteria and fungi were rapidly stimulated by exposure to light, thus establishing the potential for algal priming of microbial heterotrophic decay activities. Experimental incubations of decaying litter with 14C- and 13C-bicarbonate established that inorganic C fixed by algal photosynthesis was rapidly transferred to and assimilated by heterotrophic microbial decomposers. Periphytic algal stimulation of microbial heterotrophs, especially fungal decomposers, is an important and largely unrecognized interaction within the detrital microbial landscape, which may transform our current conceptual understanding of microbial secondary production and organic matter decomposition in aquatic ecosystems.

U(VI) reduction in sulfate-reducing subsurface sediments amended with ethanol or acetate.

An experiment was conducted with subsurface sediments from Oak Ridge National Laboratory to determine the potential for reduction of U(VI) under sulfate-reducing conditions with either ethanol or acetate as the electron donor. The results showed extensive U(VI) reduction in sediments supplied with either electron donor, where geochemical and microbiological analyses demonstrated active sulfate reduction.

Direct and indirect influence of parental bedrock on streambed microbial community structure in forested streams.

A correlative study was performed to determine if variation in streambed microbial community structure in low-order forested streams can be directly or indirectly linked to the chemical nature of the parental bedrock of the environments through which the streams flow. Total microbial and photosynthetic biomass (phospholipid phosphate [PLP] and chlorophyll a), community structure (phospholipid fatty acid analysis), and physical and chemical parameters were measured in six streams, three located in sandstone and three in limestone regions of the Bankhead National Forest in northern Alabama. Although stream water flowing through the two different bedrock types differed significantly in chemical composition, there were no significant differences in total microbial and photosynthetic biomass in the sediments. In contrast, sedimentary microbial community structure differed between the bedrock types and was significantly correlated with stream water ion concentrations. A pattern of seasonal variation in microbial community structure was also observed. Further statistical analysis indicated dissolved organic matter (DOM) quality, which was previously shown to be influenced by geological variation, correlated with variation in bacterial community structure. These results indicate that the geology of underlying bedrock influences benthic microbial communities directly via changes in water chemistry and also indirectly via stream water DOM quality.

Streptomyces scopuliridis sp. nov., a bacteriocin-producing soil streptomycete.

Actinomycete strain RB72(T) was isolated from woodland bluff soil in northern Alabama, USA, and shown to produce a broad spectrum bacteriocin. Based on morphological and chemotaxonomic characteristics, the strain was determined to belong to the genus Streptomyces. Phylogenetic analysis of the near-complete 16S rRNA gene sequence indicated that it differed from those of the described streptomycetes available in public databases. The distinctive white aerial hyphae and lack of sporulation suggest a deficiency in the whi pathway of the organism. A combination of substrate utilization patterns, morphological and chemotaxonomic characteristics and DNA-DNA hybridization results supported the affiliation of strain RB72(T) to the genus Streptomyces and enabled the genotypic and phenotypic differentiation of strain RB72(T) from closely related reference strains. Strain RB72(T) therefore represents a novel species of the genus Streptomyces, for which the name Streptomyces scopuliridis sp. nov. is proposed. The type strain is RB72(T) ( = DSM 41917(T)  = NRRL B-24574(T)).

Investigating bacterial sources of toxicity as an environmental contributor to dopaminergic neurodegeneration.

Parkinson disease (PD) involves progressive neurodegeneration, including loss of dopamine (DA) neurons from the substantia nigra. Select genes associated with rare familial forms of PD function in cellular pathways, such as the ubiquitin-proteasome system (UPS), involved in protein degradation. The misfolding and accumulation of proteins, such as alpha-synuclein, into inclusions termed Lewy Bodies represents a clinical hallmark of PD. Given the predominance of sporadic PD among patient populations, environmental toxins may induce the disease, although their nature is largely unknown. Thus, an unmet challenge surrounds the discovery of causal or contributory neurotoxic factors that could account for the prevalence of sporadic PD. Bacteria within the order Actinomycetales are renowned for their robust production of secondary metabolites and might represent unidentified sources of environmental exposures. Among these, the aerobic genera, Streptomyces, produce natural proteasome inhibitors that block protein degradation and may potentially damage DA neurons. Here we demonstrate that a metabolite produced by a common soil bacterium, S. venezuelae, caused DA neurodegeneration in the nematode, Caenorhabditis elegans, which increased as animals aged. This metabolite, which disrupts UPS function, caused gradual degeneration of all neuronal classes examined, however DA neurons were particularly vulnerable to exposure. The presence of DA exacerbated toxicity because neurodegeneration was attenuated in mutant nematodes depleted for tyrosine hydroxylase (TH), the rate-limiting enzyme in DA production. Strikingly, this factor caused dose-dependent death of human SH-SY5Y neuroblastoma cells, a dopaminergic line. Efforts to purify the toxic activity revealed that it is a highly stable, lipophilic, and chemically unique small molecule. Evidence of a robust neurotoxic factor that selectively impacts neuronal survival in a progressive yet moderate manner is consistent with the etiology of age-associated neurodegenerative diseases. Collectively, these data suggest the potential for exposures to the metabolites of specific common soil bacteria to possibly represent a contributory environmental component to PD.

Mercury in southeastern U.S. riverine fish populations linked to water body type.

We compared Hg concentrations in fishes from the regulated Black Warrior River and the unregulated Sipsey River in west Alabama whose neighboring watersheds receive equivalent atmospheric Hg deposition. Average fish fillet Hg concentrations were 3-fold higher in the unregulated river compared to the regulated river. Between river differences in Hg fish concentrations likely originate from structural (e.g., species composition) and functional (e.g., energy flow pathways) differences between the two ecosystems. We tested the generality of these findings by comparing largemouth bass Hg concentrations among unregulated rivers (n=6) and reservoirs (n=11) throughout the southern Coastal Plain geologic region. ANCOVA revealed that at a given bass length, Hg concentrations were approximately 1.75 times higher in unregulated rivers compared to regulated rivers. Aerial deposition of Hg was not correlated to largemouth bass Hg concentrations. We suggest that the link between atmospheric Hg deposition and fish Hg concentrations is significantly modulated by the structure and function of aquatic ecosystems and this accounts for much of the variation in fish Hg concentrations among systems. Unregulated floodplain-rivers in the south have some of the highest fish Hg concentrations on record and should be intensely monitored to establish human consumption risks.

Geobacter uraniireducens sp. nov., isolated from subsurface sediment undergoing uranium bioremediation.

A Gram-negative, rod-shaped, motile bacterium, strain Rf4T, which conserves energy from dissimilatory Fe(III) reduction concomitant with acetate oxidation, was isolated from subsurface sediment undergoing uranium bioremediation. The 16S rRNA gene sequence of strain Rf4T matched sequences recovered in 16S rRNA gene clone libraries constructed from DNA extracted from groundwater sampled at the same time as the source sediment. Cells of strain Rf4T were regular, motile rods, 1.2-2.0 microm long and 0.5-0.6 microm in diameter, with rounded ends. Cells had one lateral flagellum. Growth was optimal at pH 6.5-7.0 and 32 degrees C. With acetate as the electron donor, strain Rf4T used Fe(III), Mn(IV), anthraquinone-2,6-disulfonate, malate and fumarate as electron acceptors and reduced U(VI) in cell suspensions. With poorly crystalline Fe(III) oxide as the electron acceptor, strain Rf4T oxidized the following electron donors: acetate, lactate, pyruvate and ethanol. Phylogenetic analysis of the 16S rRNA gene sequence of strain Rf4T placed it in the genus Geobacter. Strain Rf4T was most closely related to 'Geobacter humireducens' JW3 (95.9 % sequence similarity), Geobacter bremensis Dfr1T (95.4 %) and Geobacter bemidjiensis BemT (95.1 %). Based on phylogenetic analysis and phenotypic differences between strain Rf4T and closely related Geobacter species, this strain is described as a representative of a novel species, Geobacter uraniireducens sp. nov. The type strain is Rf4T (=ATCC BAA-1134T =JCM 13001T).

Biome-level biogeography of streambed microbiota.

A field study was conducted to determine the microbial community structures of streambed sediments across diverse geographic and climatic areas. Sediment samples were collected from three adjacent headwater forest streams within three biomes, eastern deciduous (Pennsylvania), southeastern coniferous (New Jersey), and tropical evergreen (Guanacaste, Costa Rica), to assess whether there is biome control of stream microbial community structure. Bacterial abundance, microbial biomass, and bacterial and microbial community structures were determined using classical, biochemical, and molecular methods. Microbial biomass, determined using phospholipid phosphate, was significantly greater in the southeastern coniferous biome, likely due to the smaller grain size, higher organic content, and lower levels of physical disturbance of these sediments. Microbial community structure was determined using phospholipid fatty acid (PLFA) profiles and bacterial community structure from terminal restriction fragment length polymorphism and edited (microeukaryotic PLFAs removed) PLFA profiles. Principal component analysis (PCA) was used to investigate patterns in total microbial community structure. The first principal component separated streams based on the importance of phototrophic microeukaryotes within the community, while the second separated southeastern coniferous streams from all others based on increased abundance of fungal PLFAs. PCA also indicated that within- and among-stream variations were small for tropical evergreen streams and large for southeastern coniferous streams. A similar analysis of bacterial community structure indicated that streams within biomes had similar community structures, while each biome possessed a unique streambed community, indicating strong within-biome control of stream bacterial community structure.

Variations in PCB concentrations between genders of six warmwater fish species in Lake Logan Martin, Alabama, USA.

We collected and analyzed 955 individual fish (six species) for sexual differences in PCB bioaccumulations from a southeastern, USA reservoir. Using 2-way ANCOVAs, we found significant differences in fillet PCB concentrations between sexes for channel catfish (Ictalurus punctatus), largemouth bass (Micropterus salmoides) and spotted bass (Micropterus punctulatus). Striped bass (Morone saxatilus), black crappie (Pomoxis nigromaculatus) and freshwater drum (Aplodinotus grunniens) did not display differences between sexes in PCB concentrations. We suspect that sexual differences may be due to biological differences in reproduction, relative motility and lipid deposition. For one species (striped bass), sexual differences in PCB concentrations were inconsistent with a study in the Hudson River suggesting that sexual differences in bioaccumulations can change across ecosystems. Two species which did show sexual differences, largemouth bass and channel catfish, are often chosen as representative species (e.g., "piscivore" and "benthivore") in contaminant monitoring in many USA states indicating human consumption and risk management decisions would be improved if an equal number of male and female fish were included in composite PCBs analysis. This could reduce variability in fish PCBs data from which consumption advisories are based.

Physical and chemical factors affecting microbial biomass and activity in contaminated subsurface riverine sediments.

Over 80 years of direct discharge of industrial effluents into the Mahoning River, located in northeastern Ohio, USA, has led to the accumulation of a wide variety of pollutants within its sediments. This study examined the physical and chemical parameters, including lipophilic pollutants, affecting microbial activity and biomass in subsurface (10-40 cm horizon) sediments. Microbial biomass was higher in anthropogenically contaminated sediments, and step-wise linear regression showed that approximately 82% of the variation in microbial biomass could be explained by total hexane extractable hydrocarbons, sediment particle size, and water content. There was no correlation between microbial activity and biomass. Independent variables influencing anaerobic activity were temperature and water holding capacity. The results of this study indicate that freshwater, sedimentary anaerobic microbial communities respond to a range of environmental parameters, many of which influence subsurface river sediments, and that lipophilic pollutants, when present, can cause increases in total microbial biomass.

Low-temperature growth of Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1 is a mesophilic bacterium with a maximum growth temperature of approximately 35 degrees C but the ability to grow over a wide range of temperatures, including temperatures near zero. At room temperature ( approximately 22 degrees C) MR-1 grows with a doubling time of about 40 min, but when moved from 22 degrees C to 3 degrees C, MR-1 cells display a very long lag phase of more than 100 h followed by very slow growth, with a doubling time of approximately 67 h. In comparison to cells grown at 22 degrees C, the cold-grown cells formed long, motile filaments, showed many spheroplast-like structures, produced an array of proteins not seen at higher temperature, and synthesized a different pattern of cellular lipids. Frequent pilus-like structures were observed during the transition from 3 to 22 degrees C.

Convenient determination of DNA extraction efficiency using an external DNA recovery standard and quantitative-competitive PCR.

Molecular biology techniques have advanced the field of microbial ecology through the analysis of nucleic acids. Most techniques that use DNA or RNA require their extraction from environmental matrices, which can be tedious and inefficient. While a number of extraction methods, both laboratory-based and commercially available, have been developed, none of these include a convenient method to determine extraction efficiency. We have developed an external DNA recovery standard, Lambda DNA (target DNA) contained within pBR322, allowing routine determinations of DNA recovery efficiency. Target DNA was added to sediments as whole cells, total DNA extracted using commercial DNA extraction/purification kits and the amount of target DNA recovered quantified by quantitative-competitive PCR (QC-PCR). Three commercially available kits (UltraClean Soil DNA, FastDNA SPIN and Soil Master DNA Extraction) were evaluated for recovery efficiency. Recoveries for the three kits ranged from undetectable to 43.3% with average recoveries of 14.9+/-16.0%, 28.3+/-10.5% and 2.4+/-0.1% (UltraClean, FastDNA and Soil Master, respectively). Quantification of target DNA proved robust in sediments heavily polluted with polycyclic aromatic hydrocarbons and the external recovery standard could be detected following extraction and amplification from as few as 1 x 10(3) cells added to 0.5 g sediment (wet weight). The external DNA recovery standard was also added directly to the sediment as purified plasmid DNA prior to extraction. It was recovered with similar efficiency as when added as whole cells, suggesting its usefulness in estimating DNA recovery in ribosomal DNA studies. These results show that, while the commercial kits offer expedited sample processing, the extraction efficiencies vary on a sample-by-sample basis and were <100%. Therefore, quantitative DNA studies require an estimation of DNA recovery.