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Hundreds of active and dormant geothermal vents have been located on the floor of Yellowstone Lake, although characterization of the associated biology (macro or micro) has been extremely limited. Herein, we describe an aquatic moss ( Fontinalis) colony closely associated with vent emissions that considerably exceeded known temperature maxima for this plant. Vent waters were supersaturated with CO2, likely accommodating a CO2 compensation point that would be expected to be quite elevated under these conditions. The moss was colonized by metazoa, including the crustaceans Hyalella and Gammarus, a segmented worm in the Lumbriculidae family, and a flatworm specimen tentatively identified as Polycelis. The presence of these invertebrates suggest a highly localized food chain that derives from the presence of geothermal inputs and thus is analogous to the deep marine vents that support significant biodiversity. [ABSTRACT FROM AUTHOR]

The hot springs in Yellowstone National Park, USA, provide concentrated microbial biomass and associated mineral crusts from which surface functional group (FG) concentrations and pKa distributions can be determined. To evaluate the importance of substratum surface reactivity for solute adsorption in a natural setting, samples of iron-rich sediment were collected from three different springs; two of the springs were acid-sulfate-chloride (ASC) in composition, while the third was neutral-chloride (NC). At one of the ASC springs, mats of Sº-rich Hydrogenobaculum-like streamers and green Cyanidia algae were also collected for comparison to the sediment. All samples were then titrated over a pH range of 3–11, and comparisons were made between the overall FG availability and the concentration of solutes bound to the samples under natural conditions. Sediments from ASC springs were composed of hydrous ferric oxides (HFO) that displayed surface FGs typical of synthetic HFO, while sediments from the NC spring were characterized by a lower functional group density, reflected by decreased excess charge over the titration range (i.e., lower surface reactivity). The latter also showed a lower apparent point of zero charge (PZC), likely due the presence of silica (up to 78 wt. %) in association with HFO. Variations in the overall HFO surface charge are manifest in the quantities and types of solutes complexed; the NC sediments bound more cations, while the ASC sediments retained significantly more arsenic, presumably in the form of arsenate (H2AsO4−). When the microbial biomass samples were analyzed, FG concentrations summed over the titratable range were found to be an order of magnitude lower for the Sº-rich mats, relative to the algal and HFO samples that displayed similar FG concentrations on a dry weight basis. A diffuse-layer surface complexation model was employed to further illustrate the importance of surface chemical parameters on adsorption reactions in complex natural systems. [ABSTRACT FROM AUTHOR]

Chemolithotrophic micro-organisms are important primary producers in high-temperature geothermal environments and may catalyse a number of different energetically favourable redox reactions as a primary energy source. Analysis of geochemical constituents followed by chemical speciation and subsequent calculation of reaction free energies (Δ Grxn) is a useful tool for evaluating the thermodynamic favourability and potential energy available for microbial metabolism. The primary goal of this study was to examine relationships among geochemical gradients and microbial population distribution, and to evaluate the utility of energetic approaches for predicting microbial metabolism from free-energy calculations, utilizing as examples, several geothermal habitats in Yellowstone National Park where thorough geochemical and phylogenetic analyses have been performed. Acidic (pH ∼ 3) and near-neutral (pH ∼ 6–7) geothermal springs were chosen for their range in geochemical properties. Aqueous and solid phase samples obtained from the source pools and the outflow channels of each spring were characterized for all major chemical constituents using laboratory and field methods to accurately measure the concentrations of predominant oxidized and reduced species. Reaction free energies (Δ Grxn) for 33 oxidation–reduction reactions potentially important to chemolithotrophic micro-organisms were calculated at relevant spring temperatures after calculating ion activities using an aqueous equilibrium model. Free-energy values exhibit significant variation among sites for reactions with pH dependence. For example, free-energy values for reactions involving Fe3+ are especially variable across sites due in large part to the pH dependence of Fe3+ activity, and exhibit changes of up to 40 kJ mol−1 electron from acidic to near neutral geothermal springs. Many of the detected 16S rRNA gene sequences represent organisms whose metabolisms are consistent with exergonic processes. However, sensitivity analyses demonstrated that reaction free energies do not generally represent the steep gradients in local geochemical conditions resulting from air–water gas exchange and solid phase deposition that are important in defining microbial habitats and 16S rRNA gene sequence distribution within geothermal outflow channels. [ABSTRACT FROM AUTHOR]

Accurate assessment of the fate of contaminants in soil relies on precise estimation of solute transport parameters under field conditions. Traditionally, transport parameters are estimated from measured solute transport data using the least-squares optimization (LSO) technique or the method of moments (MOM). Considerable mismatch between the parameters estimated by these two methods has been reported in the solute transport literature. In this study, the MOM and LSO approaches were examined for estimating pore water velocity (v) and dispersion coefficient (D) from 85 laboratory- and field-measured breakthrough curves (BTCs). The two methods yielded similar estimates of v and D for BTCs with 100% mass recovery. They yielded similar estimates of v for BTCs with incomplete mass recovery. However, estimates of D obtained by the MOM departed significantly from those estimated using LSO for STCs with incomplete mass recovery. Analyses of truncated BTCs showed that 91% mass recovery resulted in errors of 138 and 57% in D values estimated by the MOM for repacked and undisturbed soil columns, respectively. Corresponding errors in estimated D values were below 5% for the LSO approach. Although it may be possible to normalize or extrapolate the BTC using its zeroth experimental moment to ensure 100% mass recovery, the use of either LSO or MOM would yield a different set of parameters representing a new set of experimental conditions and, therefore, would lead to further complications in obtaining a unique set of transport parameters. This suggests that where the MOM is indispensable, 100% experimental mass balance should be ensured. [ABSTRACT FROM AUTHOR]

The source waters of acid-sulphate-chloride (ASC) geothermal springs located in Norris Geyser Basin, Yellowstone National Park contain several reduced chemical species, including H2, H2S, As(III), and Fe(II), which may serve as electron donors driving chemolithotrophic metabolism. Microorganisms thriving in these environments must also cope with high temperatures, low pH (∼3), and high concentrations of sulphide, As(III), and boron. The goal of the current study was to correlate the temporal and spatial distribution of bacterial and archaeal populations with changes in temperature and geochemical energy gradients occurring throughout a newly formed (redirected) outflow channel of an ASC spring. A suite of complimentary analyses including aqueous geochemistry, microscopy, solid phase identification, and 16S rDNA sequence distribution were used to correlate the appearance of specific microbial populations with biogeochemical processes mediating S, Fe, and As cycling and subsequent biomineralization of As(V)-rich hydrous ferric oxide (HFO) mats. Rapid As(III) oxidation (maximum first order rate constants ranged from 4 to 5 min−1, t1/2 = 0.17 − 0.14 min) was correlated with the appearance ofHydrogenobaculumandThiomonas–like populations, whereas the biogenesis of As(V)-rich HFO microbial mats (mole ratios of As:Fe∼0.7) was correlated with the appearance ofMetallosphaera,Acidimicrobium, andThiomonas–like populations. Several 16S sequences detected near the source were closely related to sequences of chemolithotrophic hyperthermophilic populations includingStygiolobusandHydrogenobaculumorganisms that are known H2 oxidizers. The use of H2, reduced S(–II,0), Fe(II) and perhaps As(III) by different organisms represented throughout the outflow channel was supported by thermodynamic calculations, confirming highly exergonic redox couples with these electron donors. Results from this work demonstrated that chemical energy gradients play an important role in establishing distinct community structure as a function of distance from geothermal spring discharge. [ABSTRACT FROM AUTHOR]

The influence of a nonionic (alcohol ethoxylate) surfactant (Witconol SN70) on biodegradation of phenanthrene and hexadecane (nonaqueous-phase liquid) in soil was studied in batch and transport systems. Simultaneous enhancement of phenanthrene and hexadecane degradation was noted at surfactant doses resulting in aqueous-phase surfactant concentrations below the critical micelle concentration (CMC). Conversely,degradation rates of both compounds declined to essentially zero at supra-CMC doses, suggesting that distinct mechanisms of inhibition and enhancement were operating depending on the effective surfactant concentration (i.e., accounting for surfactant sorption, log KD = 2.2 L/kg). Surfactant doses resulting in enhanced degradation correlated with enhanced gross microbial activity as determined using total CO2 evolution rates. Supra-CMC doses that resulted in inhibited degradation did not suppress gross microbial activity. Furthermore, measurements of phenanthrene solubilization and surface tension indicated that phenanthrene was solubilized at supra-CMC levelsof surfactant. Mechanisms of inhibition of phenanthrene and hexadecane degradation at supra-CMC surfactant concentrations may include changes in interfacial chemistry and subsequent mass transfer processes due to sorbed surfactant, reduced bioavailability of micelle-bound phenanthrene and hexadecane, or inhibition of specific members of the microbial community responsible for hydrophobic organic compound degradation. [ABSTRACT FROM AUTHOR]

Arsenic concentrations in 850 km of the Madison and Upper Missouri Rivers exceed the Montana State human health standard (0.24 Mu M). In addition, ground water As concentrations in the northern portion of the Lower Madison River Valley are above the federal drinking water standard (0.67 Mu M) and correlate with high soluble As levels in overlying soils. The objectives of this study were to determine processes affecting As solubility in soils of the Madison and Upper Missouri River Basins and assess potential impacts of long-term irrigation with As-rich river water on As mobility. Sixteen irrigated (>20 yr of irrigation) and nonirrigated (never irrigated) soils were sampled in fourmajor regions adjacent to the Madison and Upper Missouri Rivers. There were no significant differences (Alpha = 0.05) in total or solubleAs levels between irrigated and nonirrigated soils within any of thefour regions. In addition, sorption coefficients (Kd values) in six paired soils selected for additional chemical characterization were not significantly different (Alpha = 0.05) between irrigated and nonirrigated soils. Ammonium oxalate extractable Fe and Mn concentrations were found to be positively correlated with Kd and Olsen P concentration was found to be negatively correlated with Kd based on a multiple linear regression (r2 = 0.92). Sequential extractions performed for a subset of soils showed that labile As concentrations were similar between irrigated and nonirrigated soils. Soils that had been irrigated for >100 yr had additional As sorption capacities of at least 3 mg kg -1 in saturated column studies following 30 yr of simulated irrigation. Based onthis suite of data, it was determined that irrigation history has not significantly affected As solubility or attenuation capacities in soils of the study area. [ABSTRACT FROM AUTHOR]

The practice of liming to remediate contaminated soils and mine tailings has the potential to mobilize arsenic (As), due to the pH dependence of As sorption reactions on oxide minerals and layer silicates. The objectives of this study were to determine the effects of liming on As mobility in mine tailings and identify possible mechanisms controlling As mobilization with Increased pH. Six mine tailing samples obtained from an abandoned copper smelter near Anaconda, Montana were analyzed for total As and soluble constituents using saturated paste extractions. Concentrations of soluble As among the six samples did not correlate with total As, but were more closely related to pH. Saturation indices with respect to known metal arsenate solid phases suggested that metal arsenate solid phases were not controlling soluble As in these samples. Two low pH samples (pond tailings and reprocessedtailings) were chosen for more detailed chemical characterization and unsaturated column transport experiments before and after liming. Soluble As concentrations measured in column effluent increased by factors of 10 (reprocessed tailings, RT) to 400 (pond tailings, PT) following liming. Sequential extractions of these tailings samples showedthat the PT contained significantly higher 'labile' As relative to the RT, consistent with amounts of As mobilized after liming. Further characterization of these samples using scanning electron microscopy (SEM) and energy dispersive analysis of x-rays (EDAX) suggested that these samples do not contain discrete metal arsenate solid phases. Based on this suite of experimental data, increased mobility of As withliming appears to be consistent with the pH dependence of sorption reactions of As on Fe oxide minerals rather than dissolution-precipitation reactions involving As. [ABSTRACT FROM AUTHOR]

Thermoproteales (phylum Crenarchaeota) populations are abundant in high-temperature (>70°C) environments of Yellowstone National Park (YNP) and are important in mediating the biogeochemical cycles of sulfur, arsenic, and carbon. The objectives of this study were to determine the specific physiological attributes of the isolate Pyrobaculum yellowstonensis strain WP30, which was obtained from an elemental sulfur sediment (Joseph's Coat Hot Spring [JCHS], 80°C, pH 6.1, 135 μM As) and relate this organism to geochemical processes occurring in situ. Strain WP30 is a chemoorganoheterotroph and requires elemental sulfur and/or arsenate as an electron acceptor. Growth in the presence of elemental sulfur and arsenate resulted in the formation of thioarsenates and polysulfides. The complete genome of this organism was sequenced (1.99 Mb, 58% G+C content), revealing numerous metabolic pathways for the degradation of carbohydrates, amino acids, and lipids. Multiple dimethyl sulfoxide-molybdopterin (DMSO-MPT) oxidoreductase genes, which are implicated in the reduction of sulfur and arsenic, were identified. Pathways for the de novo synthesis of nearly all required cofactors and metabolites were identified. The comparative genomics of P. yellowstonensis and the assembled metagenome sequence from JCHS showed that this organism is highly related (∼95% average nucleotide sequence identity) to in situ populations. The physiological attributes and metabolic capabilities of P. yellowstonensis provide an important foundation for developing an understanding of the distribution and function of these populations in YNP. [ABSTRACT FROM AUTHOR]

High-temperature (> 70°C) ecosystems in Yellowstone National Park (YNP) provide an unparalleled opportunity to study che-motrophic archaea and their role in microbial community structure and function under highly constrained geochemical conditions. Acidilobus spp. (order Desulfurococcales) comprise one of the dominant phylotypes in hypoxic geothermal sulfur sediment and Fe(III)-oxide environments along with members of the Thermoproteales and Sulfolobales. Consequently, the primary goals of the current study were to analyze and compare replicate de novo sequence assemblies of Acidilobus-like populations from four different mildly acidic (pH 3.3 to 6.1) high-temperature (72°C to 82°C) environments and to identify metabolic pathways and/or protein-encoding genes that provide a detailed foundation of the potential functional role of these populations in situ. De novo assemblies of the highly similar Acidilobus-like populations (>99% 16S rRNA gene identity) represent near-complete consensus genomes based on an inventory of single-copy genes, deduced metabolic potential, and assembly statistics generated across sites. Functional analysis of coding sequences and confirmation of gene transcription by Acidilobus-like populations provide evidence that they are primarily chemoorganoheterotrophs, generating acetyl coenzyme A (acetyl-CoA) via the degradation of carbohydrates, lipids, and proteins, and auxotrophic with respect to several external vitamins, cofactors, and metabolites. No obvious pathways or protein-encoding genes responsible for the dissimilatory reduction of sulfur were identified. The presence of a formate dehydrogenase (Fdh) and other protein-encoding genes involved in mixed-acid fermentation supports the hypothesis that Acidilobus spp. function as degraders of complex organic constituents in high-temperature, mildly acidic, hypoxic geothermal systems. [ABSTRACT FROM AUTHOR]

"Metallosphaera yellowsionensis" is a thermoacidophilic archaeon isolated from Yellowstone National Park that is capable of autotrophic growth using Fe(II), elemental S, or pyrite as electron donors. Analysis of the draft genome sequence from M. yellowsionensis strain MK1 revealed seven different copies of heme copper oxidases (subunit I) in a total of five different terminal oxidase complexes, including doxBCEF, foxABCDEF- GHIJ, soxABC, and the soxM supercomplex, as well as a novel hypothetical two-protein doxB-like polyferredoxin complex. Other genes found in M. yellowstonensis with possible roles in S and or Fe cycling include a thiosulfate oxidase (tqoAB), a sulfite oxidase (som), a cbsA cytochrome b5581566, several small blue copper proteins, and a novel gene sequence coding for a putative multicopper oxidase (Mco). Results from gene expression studies, including reverse transcriptase (RT) quantitative PCR (qPCR) of cultures grown autotrophically on either Fe(II), pyrite, or elemental S showed that thefox gene cluster and mco are highly expressed under conditions where Fe(II) is an electron donor. Metagenome sequence and gene expression studies of Fe-oxide mats confirmed the importance offox genes (e.g.,foxA andfoxC) and mco under Fe(II)-oxidizing conditions. Protein modeling of FoxC suggests a novel lysine-lysine or lysine-arginine heme B binding domain, indicating that it is likely the cytochrome component of a heterodimer complex with foxG as a ferredoxin subunit. Analysis of mco shows that it encodes a novel multicopper blue protein with two plastocyanin type I copper domains that may play a role in the transfer of electrons within the Fox protein complex. An understanding of metabolic pathways involved in aerobic iron and sulfur oxidation in Sulfolobales has broad implications for understanding the evolution and niche diversification of these thermophiles as well as practical applications in fields such as bioleaching of trace metals from pyritic ores. [ABSTRACT FROM AUTHOR]

Novel thermophilic crenarchaea have been observed in Fe(III) oxide microbial mats of Yellowstone National Park (YNP); however, no definitive work has identified specific microorganisms responsible for the oxidation of Fe(II). The objectives of the current study were to isolate and characterize an Fe(II)-oxidizing member of the Sulfolobales observed in previous 16S rRNA gene surveys and to determine the abundance and distribution of close relatives of this organism in acidic geothermal springs containing high concentrations of dissolved Fe(II). Here we report the isolation and characterization of the novel, Fe(II)-oxidizing, thermophilic, acidophilic organism Metallosphaera sp. strain MK1 obtained from a well-characterized acid-sulfate-chloride geothermal spring in Norris Geyser Basin, YNP. Full-length 16S rRNA gene sequence analysis revealed that strain MK1 exhibits only 94.9 to 96.1% sequence similarity to other known Metallosphaera spp. and less than 89.1% similarity to known Sulfolobus spp. Strain MK1 is a facultative chemolithoautotroph with an optimum pH range of 2.0 to 3.0 and an optimum temperature range of 65 to 75°C. Strain MK1 grows optimally on pyrite or Fe(II) sorbed onto ferrihydrite, exhibiting doubling times between 10 and 11 h under aerobic conditions (65°C). The distribution and relative abundance of MK1-like 16S rRNA gene sequences in 14 acidic geothermal springs containing Fe(III) oxide microbial mats were evaluated. Highly related MK1-like 16S rRNA gene sequences (>99% sequence similarity) were consistently observed in Fe(III) oxide mats at temperatures ranging from 55 to 80°C. Quantitative PCR using Metallosphaera-specific primers confirmed that organisms highly similar to strain MK1 comprised up to 40% of the total archaeal community at selected sites. The broad distribution of highly related MK1-Iike 16S rRNA gene sequences in acidic Fe(III) oxide microbial mats is consistent with the observed characteristics and growth optima of Metallosphaera-like strain MK1 and emphasizes the importance of this newly described taxon in Fe(II) chemolithotrophy in acidic high-temperature environments of YNP. [ABSTRACT FROM AUTHOR]

Geothermal and hydrothermal waters often contain high concentrations of dissolved sulfide, which reacts with oxygen (abiotically or biotically) to yield elemental sulfur and other sulfur species that may support microbial metabolism. The primary goal of this study was to elucidate predominant biogeochemical processes important in sulfur biogeochemistry by identifying predominant sulfur species and describing microbial community structure within high-temperature, hypoxic, sulfur sediments ranging in pH from 4.2 to 6.1. Detailed analysis of aqueous species and solid phases present in hypoxic sulfur sediments revealed unique habitats containing high concentrations of dissolved sulfide, thiosulfate, and arsenite, as well as rhombohedral and spherical elemental sulfur and/or sulfide phases such as orpiment, stibnite, and pyrite, as well as alunite and quartz. Results from 16S rRNA gene sequencing show that these sediments are dominated by Crenarchaeota of the orders Desulfurococcales and Thermoproteales. Numerous cultivated representatives of these lineages, as well as the Thermoproteales strain (WP30) isolated in this study, require complex sources of carbon and respire elemental sulfur. We describe a new archaeal isolate (strain WP30) belonging to the order Thermoproteales (phylum Crenarchaeota, 98% identity to Pyrobaculum/Thermoproteus spp. 16S rRNA genes), which was obtained from sulfur sediments using in situ geochemical composition to design cultivation medium. This isolate produces sulfide during growth, which further promotes the formation of sulfide phases including orpiment, stibnite, or pyrite, depending on solution conditions. Geochemical, molecular, and physiological data were integrated to suggest primary factors controlling microbial community structure and function in high-temperature sulfur sediments., (© 2012 Blackwell Publishing Ltd.)

Microbial reduction of arsenate [As(V)] to arsenite [As(III)] and the subsequent effects on As mobilization in contaminated mine tailings were studied under transport conditions. Molecular analysis of bacterial populations and traditional isolation techniques were used in conjunction with column experiments designed to observe relationships among pH (limed vs unlimed treatments), redox potential (Pt electrode), and mobilization of As. Liming increased pH values from approximately 4 to 8, resulting in a 5-fold increase in total As eluted from sterile columns. Elution of As from limed columns was further enhanced by microbial activity. As(III) was the predominant As species eluted from oxic, nonsterile columns. Conversely, in sterile treatments, As(V) was the predominant valence state in column effluent. Denaturing gradient gel electrophoresis coupled with sequence and phylogenetic analysis of 16S rRNA gene segments revealed that liming of the mine tailings stimulated specific Caulobacter-, Sphingomonas-, and Rhizobium-like populations. Pure culture isolates of these bacteria demonstrated the ability to rapidly reduce As(V) in aerated serum bottles. An intracellular As detoxification pathway was implicated in the reduction of As(V) by these isolates. These results indicate that microbial reduction of As(V) in As-contaminated soils may occur under aerobic conditions over relatively short time scales resulting in enhanced As mobilization.

Geothermal springs within Yellowstone National Park (YNP) often contain arsenic (As) at concentrations of 10-40 microM, levels that are considered toxic to many organisms. Arsenite (As(III)) is often the predominant valence state at the point of discharge but is rapidly oxidized to arsenate (As(V)) during transport in shallow surface water. The current study was designed to establish rates and possible mechanisms of As(III) oxidation and to characterize the geochemical environment associated with predominant microbial mats in a representative acid-sulfate-chloride (pH 3.1) thermal (58-62 degrees C) spring in Norris Basin, YNP. At the spring origin, total soluble As was predominantly As(III) at concentrations of 33 microM. No oxidation of As(III) was detected over the first 2.7 m downstream from the spring source, corresponding to an area dominated by a yellow filamentous S0-rich microbial mat However, rapid oxidation of As(III) to As(V) was observed between 2.7 and 5.6 m, corresponding to termination of the S0-rich mats, decreases in dissolved sulfide, and commencement of a brown Fe/As-rich mat. Rates of As(II) oxidation were estimated, yielding an apparent first-order rate constant of 1.2 min(-1) (half-life = 0.58 min). The oxidation of As(III) was shown to require live organisms present just prior to and within the Fe/As-rich mat. Complementary analytical tools used to characterize the brown mat revealed an As:Fe molar ratio of 0.7 and suggested that this filamentous microbial mat contains iron(III) oxyhydroxide coprecipitated with As(V). Results from the current work are the first to provide a comprehensive characterization of microbially mediated As(III) oxidation and the geochemical environments associated with microbial mats in acid-sulfate-chloride springs of YNP.

Electron microscopy (EM), denaturing gradient gel electrophoresis (DGGE) and 16S rDNA sequencing were used to examine the structure and diversity of microbial mats present in an acid-sulphate-chloride (pH 3.1) thermal (58-62 degrees C) spring in Norris Basin, Yellowstone National Park, WY, USA, exhibiting rapid rates of arsenite oxidation. Initial visual assessments, scanning EM and geochemical measurements revealed the presence of three distinct mat types. Analysis of 16S rDNA fragments with DGGE confirmed the presence of different bacterial and archaeal communities within these zones. Changes in the microbial community appeared to coincide with arsenite oxidation activity. Phylogenetic analysis of 1400 bp 16S rDNA sequences revealed that clone libraries prepared from both arsenic redox active and inactive bacterial communities were dominated by sequences phylogenetically related to Hydrogenobacter acidophilus and Desulphurella sp. The appearance of archaeal 16S rDNA sequences coincided with the start of arsenite oxidation, and sequences were obtained showing affiliation with both Crenarchaeota and Euryarchaeota. The majority of archaeal sequences were most similar to sequences obtained from marine hydrothermal vents and other acidic hot springs, although the level of similarity was typically just 90%. Arsenite oxidation in this system may result from the activities of these unknown archaeal taxa and/or the previously unreported arsenic redox activity of H. acidophilus- or Desulphurella-like organisms. If the latter, arsenite oxidation must be inhibited in the initial high-sulphide zone of the spring, where no change in the distribution of arsenite versus arsenate was observed.

We analyzed the impact of surfactant addition on hydrocarbon mineralization kinetics and the associated population shifts of hydrocarbon-degrading microorganisms in soil. A mixture of radiolabeled hexadecane and phenanthrene was added to batch soil vessels. Witconol SN70 (a nonionic, alcohol ethoxylate) was added in concentrations that bracketed the critical micelle concentration (CMC) in soil (CMC') (determined to be 13 mg g(-1)). Addition of the surfactant at a concentration below the CMC' (2 mg g(-1)) did not affect the mineralization rates of either hydrocarbon. However, when surfactant was added at a concentration approaching the CMC' (10 mg g(-1)), hexadecane mineralization was delayed and phenanthrene mineralization was completely inhibited. Addition of surfactant at concentrations above the CMC' (40 mg g(-1)) completely inhibited mineralization of both phenanthrene and hexadecane. Denaturing gradient gel electrophoresis of 16S rRNA gene segments showed that hydrocarbon amendment stimulated Rhodococcus and Nocardia populations that were displaced by Pseudomonas and Alcaligenes populations at elevated surfactant levels. Parallel cultivation studies revealed that the Rhodococcus population can utilize hexadecane and that the Pseudomonas and Alcaligenes populations can utilize both Witconol SN70 and hexadecane for growth. The results suggest that surfactant applications necessary to achieve the CMC alter the microbial populations responsible for hydrocarbon mineralization.

Reduced bioavailability of nonpolar contaminants due to sorption to natural organic matter is an important factor controlling biodegradation of pollutants in the environment. We established enrichment cultures in which solid organic phases were used to reduce phenanthrene bioavailability to different degrees (R. J. Grosser, M. Friedrich, D. M. Ward, and W. P. Inskeep, Appl. Environ. Microbiol. 66:2695-2702, 2000). Bacteria enriched and isolated from contaminated soils under these conditions were analyzed by denaturing gradient gel electrophoresis (DGGE) and sequencing of PCR-amplified 16S ribosomal DNA segments. Compared to DGGE patterns obtained with enrichment cultures containing sand or no sorptive solid phase, different DGGE patterns were obtained with enrichment cultures containing phenanthrene sorbed to beads of Amberlite IRC-50 (AMB), a weak cation-exchange resin, and especially Biobead SM7 (SM7), a polyacrylic resin that sorbed phenanthrene more strongly. SM7 enrichments selected for mycobacterial phenanthrene mineralizers, whereas AMB enrichments selected for a Burkholderia sp. that degrades phenanthrene. Identical mycobacterial and Burkholderia 16S rRNA sequence segments were found in SM7 and AMB enrichment cultures inoculated with contaminated soil from two geographically distant sites. Other closely related Burkholderia sp. populations, some of which utilized phenanthrene, were detected in sand and control enrichment cultures. Our results are consistent with the hypothesis that different phenanthrene-utilizing bacteria inhabiting the same soils may be adapted to different phenanthrene bioavailabilities.

The sorption of organic contaminants by natural organic matter (NOM) often limits substrate bioavailability and is an important factor affecting microbial degradation rates in soils and sediments. We hypothesized that reduced substrate bioavailability might influence which microbial assemblages are responsible for contaminant degradation under enrichment culture conditions. Our primary goal was to characterize enrichments in which different model organic solid phases were used to establish a range of phenanthrene bioavailabilities for soil microorganisms. Phenanthrene sorption coefficients (expressed as log K(D) values) ranged from 3.0 liters kg(-1) for Amberlite carboxylic acid cation-exchange resin (AMB) to 3.5 liters kg(-1) for Biobeads polyacrylic resin (SM7) and 4.2 liters kg(-1) for Biobeads divinyl benzene resin (SM2). Enrichment cultures were established for control (no sorptive phase), sand, AMB, SM7, and SM2 treatments by using two contaminated soils (from Dover, Ohio, and Libby, Mont.) as the initial inocula. The effects of sorption by model phases on the degradation of phenanthrene were evaluated for numerous transfers in order to obtain stable microbial assemblages representative of sorptive and nonsorptive enrichment cultures and to eliminate the effects of the NOM present in the initial inoculum. Phenanthrene degradation rates were similar for each soil inoculum and ranged from 4 to 5 micromol day(-1) for control and sand treatments to approximately 0.4 micromol day(-1) in the presence of the SM7 sorptive phase. The rates of phenanthrene degradation in the highly sorptive SM2 enrichment culture were insignificant; consequently, stable microbial populations could not be obtained. Bacterial isolates obtained from serial dilutions of enrichment culture samples exhibited significant differences in rates of phenanthrene degradation performed in the presence of SM7, suggesting that enrichments performed in the presence of a sorptive phase selected for different microbial assemblages than control treatments containing solid phase phenanthrene.

We found inconsistencies in the commonly used data for chlorophyll analysis in 80% acetone. Recently developed extinction coefficients for chlorophyll b in N,N-dimethylformamide (DMF) based on values from 80% acetone are low as a result of these inconsistencies. We determined extinction coefficients of chlorophyll a (Chl a) and chlorophyll b (Chl b) in DMF for wavelengths of 618 to 665 nanometers. The simultaneous equations necessary for quantifying Chl a, Chl b, or total Chl in DMF in the absence of other chlorophyllous pigments are: Chl a = 12.70A(664.5) - 2.79A(647); Chl b = 20.70 A(647) - 4.62A(664.5); total Chl = 17.90A(647) + 8.08A(664.5), where A = absorbance in 1.00 centimeter cuvettes and Chl = milligrams per liter.N,N-Dimethylformamide is a very convenient solvent for Chl extraction since it is effective on intact plant parts and Chl is quite stable in DMF. There was no difference in the amount of Chl extracted when plant tissue was stored for 1 or 3 days at three temperatures, with or without solvent added.