Evaluation of gaseous substrates for microbial immobilization of contaminant mixtures in unsaturated subsurface sediments.

Extensive vadose zone metals and organic contamination remains at many former industrial and defense manufacturing sites, and effective remedial solutions are needed to slow or prevent its migration to groundwater. In this study, the application of gaseous substrates to stimulate microbial respiratory reduction of comingled radioisotopes and nitrate under unsaturated conditions was examined for possible application at the Hanford Site, a former nuclear production facility in southeastern WA, USA. First, screening studies were performed to qualitatively measure the sediment respiratory response to 14 gaseous or volatile organic substrates at two moisture contents, 4% and 8%. Volatile substrates produced the strongest respiratory response, among them were butyrate, pentane, butyl acetate. Ethane and butane were the most effective gaseous substrates but only at 8% water content. Hanford sediment from two waste sites with distinctive chemistries were wetted to 7% moisture content, packed into columns, and treated with ethane or butane. After 4 weeks, columns were then leached to quantify retardation in the mobility of aqueous contaminant concentrations compared to no gas control columns. Treatment with both gases resulted in >80% removal of Cr from the aqueous phase. However, NO3 concentration and a waste sites exposure history to NO3 had a major effect on U and Tc reduction. Incomplete nitrate reduction outcompeted U and Tc in waste site sediments having limited prior exposure to NO3. Conversely, waste site sediments co-contaminated with NO3 were able to achieve highly reduced conditions resulting in complete denitrification of NO3, and delayed leaching of U and Tc. This implied effective reduction of both contaminants to less mobile species. This study demonstrates that unsaturated vadose sediments at Hanford waste sites have the capacity for a sustained respiratory response to gaseous substrate injection, which could potentially be deployed as part of an overall strategy to reduce the flux of long-lived radionuclides to groundwater at Hanford and other legacy waste sites.

Microbial Methylation of Iodide in Unconfined Aquifer Sediments at the Hanford Site, USA.

Incomplete knowledge of environmental transformation reactions limits our ability to accurately inventory and predictably model the fate of radioiodine. The most prevalent chemical species of iodine include iodate (IO3 -), iodide (I-), and organo-iodine. The emission of gaseous species could be a loss or flux term but these processes have not previously been investigated at radioiodine-impacted sites. We examined iodide methylation and volatilization for Hanford Site sediments from three different locations under native and organic substrate amended conditions at three iodide concentrations. Aqueous and gaseous sampling revealed methyl-iodide to be the only iodinated compound produced under biotic conditions. No abiotic transformations of iodide were measured. Methyl-iodide was produced by 52 out of 54 microcosms, regardless of prior exposure to iodine contamination or the experimental concentration. Interestingly, iodide volatilization activity was consistently higher under native (oligotrophic) Hanford sediment conditions. Carbon and nutrients were not only unnecessary for microbial activation, but supplementation resulted in >three-fold reduction in methyl-iodide formation. This investigation not only demonstrates the potential for iodine volatilization in deep, oligotrophic subsurface sediments at a nuclear waste site, but also emphasizes an important role for biotic methylation pathways to the long-term management and monitoring of radioiodine in the environment.

A review of the behavior of radioiodine in the subsurface at two DOE sites.

Multiple processes affect the fate of the radioactive isotope 129I in the environment. Primary categories of these processes include electron transfer reactions mediated by minerals and microbes, adsorption to sediments, interactions with organic matter, co-precipitation, and volatilization. A description of dominant biogeochemical processes is provided to describe the interrelationship of these processes and the associated iodine chemical species. The majority of the subsurface iodine fate and transport studies in the United States have been conducted at U.S. Department of Energy (DOE) sites where radioisotopes of iodine are present in the environment and stored waste. The DOE Hanford Site and Savannah River Site (SRS) are used to illustrate how the iodine species and dominant processes at a site are controlled by the prevailing site biogeochemical conditions. These sites differ in terms of climate (arid vs. sub-tropical), major geochemical parameters (e.g., pH ~7.5 vs. 4), and mineralogy (carbonate vs. Fe/Al oxide dominated). The iodine speciation and dominant processes at a site also have implications for selection and implementation of suitable remedy approaches for 129I.

Amplicon Sequencing Reveals Microbiological Signatures in Spent Nuclear Fuel Storage Basins.

Water quality is an important determinant for the structural integrity of alloy cladded fuels and assemblies during long-term wet storage. Detailed characterization of a water filled storage basin for spent nuclear reactor fuel was performed following the formation and proliferation of an amorphous white flocculent. White precipitant was sampled throughout the storage basin for chemical and spectroscopic characterization, and environmental DNA was extracted for 454 pyrosequencing of bacterial 16S rRNA gene diversity. Accordingly, spectroscopic analyses indicated the precipitant to be primarily amorphous to crystalline aluminum (oxy) hydroxides with minor associated elemental components including Fe, Si, Ti, and U. High levels of organic carbon were co-localized with the precipitant relative to bulk dissolved organic concentrations. Bacterial densities were highly variable between sampling locations and with depth within the water filled storage basin; cell numbers ranged from 4 × 103to 4 × 104 cells/mL. Bacterial diversity that was physically associated with the aluminum (oxy) hydroxide complexes exceeded an estimated 4,000 OTUs/amplicon library (3% cutoff) and the majority of sequences were aligned to the families Burkholderiaceae (23%), Nitrospiraceae (23%), Hyphomicrobiaceae (17%), and Comamonadaceae (6%). We surmise that episodic changes in the physical and chemical properties of the basin contribute to the polymerization of aluminum (oxy) hydroxides, which in turn can chemisorb nutrients, carbon ligands and bacterial cells from the surrounding bulk aqueous phase. As such, these precipitants should establish favorable microhabitats for bacterial colonization and growth. Comparative analyses of 16S rRNA gene amplicon libraries across a selection of natural and engineered aquatic ecosystems were performed and microbial community and taxonomic signatures unique to the spent nuclear fuel (SNF) storage basin environment were revealed. These insights could spur the development of tractable bio-indicators that are specific of and diagnostic for water quality at discrete locations and finer scales of resolution, marking an important contribution for improved water quality and management of SNF storage facilities.

Discovery of Bioactive Metabolites in Biofuel Microalgae That Offer Protection against Predatory Bacteria.

Microalgae could become an important resource for addressing increasing global demand for food, energy, and commodities while helping to reduce atmospheric greenhouse gasses. Even though Chlorophytes are generally regarded safe for human consumption, there is still much we do not understand about the metabolic and biochemical potential of microscopic algae. The aim of this study was to evaluate biofuel candidate strains of Chlorella and Scenedesmus for the potential to produce bioactive metabolites when grown under nutrient depletion regimes intended to stimulate production of triacylglycerides. Strain specific combinations of macro- and micro-nutrient restricted growth media did stimulate neutral lipid accumulation by microalgal cultures. However, cultures that were restricted for iron consistently and reliably tested positive for cytotoxicity by in vivo bioassays. The addition of iron back to these cultures resulted in the disappearance of the bioactive components by LC/MS fingerprinting and loss of cytotoxicity by in vivo bioassay. Incomplete NMR characterization of the most abundant cytotoxic fractions suggested that small molecular weight peptides and glycosides could be responsible for Chlorella cytotoxicity. Experiments were conducted to determine if the bioactive metabolites induced by Fe-limitation in Chlorella sp. cultures would elicit protection against Vampirovibrio chlorellavorus, an obligate predator of Chlorella. Introduction of V. chlorellavorus resulted in a 72% decrease in algal biomass in the experimental controls after 7 days. Conversely, only slight losses of algal biomass were measured for the iron limited Chlorella cultures (0-9%). This study demonstrates a causal linkage between iron bioavailability and bioactive metabolite production in strains of Chlorella and Scenedesmus. Further study of this phenomenon could contribute to the development of new strategies to extend algal production cycles in open, outdoor systems while ensuring the protection of biomass from predatory losses.

A diverse assemblage of indole-3-acetic acid producing bacteria associate with unicellular green algae.

Microalgae have tremendous potential as a renewable feedstock for the production of liquid transportation fuels. In natural waters, the importance of physical associations and biochemical interactions between microalgae and bacteria is generally well appreciated, but the significance of these interactions to algal biofuels production have not been investigated. Here, we provide a preliminary report on the frequency of co-occurrence between indole-3-acetic acid (IAA)-producing bacteria and green algae in natural and engineered ecosystems. Growth experiments with unicellular algae, Chlorella and Scenedesmus, revealed IAA concentration-dependent responses in chlorophyll content and dry weight. Importantly, discrete concentrations of IAA resulted in cell culture synchronization, suggesting that biochemical priming of cellular metabolism could vastly improve the reliability of high density cultivation. Bacterial interactions may have an important influence on algal growth and development; thus, the preservation or engineered construction of the algal-bacterial assembly could serve as a control point for achieving low input, reliable production of algal biofuels.

Kinetics of 1,2-dichloroethane and 1,2-dibromoethane biodegradation in anaerobic enrichment cultures.

1,2-Dichloroethane (1,2-DCA) and 1,2-dibromoethane (ethylene dibromide [EDB]) contaminate groundwater at many hazardous waste sites. The objectives of this study were to measure yields, maximum specific growth rates (µ), and half-saturation coefficients (K(S)) in enrichment cultures that use 1,2-DCA and EDB as terminal electron acceptors and lactate as the electron donor and to evaluate if the presence of EDB has an effect on the kinetics of 1,2-DCA dehalogenation and vice versa. Biodegradation was evaluated at the high concentrations found at some industrial sites (>10 mg/liter) and at lower concentrations found at former leaded-gasoline sites (1.9 to 3.7 mg/liter). At higher concentrations, the Dehalococcoides yield was 1 order of magnitude higher when bacteria were grown with 1,2-DCA than when they were grown with EDB, while µ's were similar for the two compounds, ranging from 0.19 to 0.52 day(-1) with 1,2-DCA to 0.28 to 0.36 day(-1) for EDB. K(S) was larger for 1,2-DCA (15 to 25 mg/liter) than for EDB (1.8 to 3.7 mg/liter). In treatments that received both compounds, EDB was always consumed first and adversely impacted the kinetics of 1,2-DCA utilization. Furthermore, 1,2-DCA dechlorination was interrupted by the addition of EDB at a concentration 100 times lower than that of the remaining 1,2-DCA; use of 1,2-DCA did not resume until the EDB level decreased close to its maximum contaminant level (MCL). In lower-concentration experiments, the preferential consumption of EDB over 1,2-DCA was confirmed; both compounds were eventually dehalogenated to their respective MCLs (5 µg/liter for 1,2-DCA, 0.05 µg/liter for EDB). The enrichment culture grown with 1,2-DCA has the advantage of a more rapid transition to 1,2-DCA after EDB is consumed.

Celerinatantimonas diazotrophica gen. nov., sp. nov., a nitrogen-fixing bacterium representing a new family in the Gammaproteobacteria, Celerinatantimonadaceae fam. nov.

Five strains representing a novel family within the Gammaproteobacteria were isolated from the estuarine grasses Spartina alterniflora and Juncus roemerianus. All strains were facultatively anaerobic, Gram-negative, short, motile, polar monotrichous rods that were mesophilic, oxidase-negative, catalase-positive, had DNA G+C contents of 41.5-44.4 mol% and required seawater salts or NaCl. Growth was observed at pH 3.5-8.0. Polar lipids diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, aminophospholipid, phospholipids and unidentified aminolipids were found in the representative strain S-G2-2(T). The major menaquinone and ubiquinone were MK-8 (100 %) and Q-8 (93 %), respectively. Predominant fatty acids present were C(12 : 0) aldehyde and/or unknown fatty acid 10.9525 (MIDI designation) and/or iso-C(16 : 1) I/C(14 : 0) 3-OH, C(16 : 1)ω7c/C(16 : 1)ω6c, C(16 : 0), C(17 : 0) cyclo and C(18 : 1)ω7c and/or C(18 : 1)ω6c. The nearly full-length 16S rRNA gene sequences of the strains were very similar (99-100 % similarity), and the strains were identified as members of the same species by DNA-DNA relatedness measurements. 16S rRNA gene sequence analysis revealed that the strains formed a monophyletic lineage within the order Alteromonadales. All five strains fixed N(2). Analysis of partial nifH gene sequences also revealed a monophyletic lineage within the Gammaproteobacteria, and the sequences were dissimilar to those of any previously described diazotroph. Differences between the novel strains and other members of the Alteromonadales include the inability to produce cytochrome oxidase. The novel strains were metabolically versatile. On the basis of the information described above, the new genus and species Celerinatantimonas diazotrophica gen. nov., sp. nov. are proposed to accommodate the five strains within a new family, Celerinatantimonadaceae fam. nov. The type strain of Celerinatantimonas diazotrophica is S-G2-2(T) ( = ATCC BAA-1368(T)  = DSM 18577(T)).

Responses of salt marsh plant rhizosphere diazotroph assemblages to changes in marsh elevation, edaphic conditions and plant host species.

An important source of new nitrogen in salt marsh ecosystems is microbial diazotrophy (nitrogen fixation). The diazotroph assemblages associated with the rhizospheres (sediment directly affected by the roots) of salt marsh plants are highly diverse, somewhat stable, and consist mainly of novel organisms. In Crab Haul Creek Basin, North Inlet, SC, the distribution of plant types into discrete zones is dictated by relatively minor differences in marsh elevation and it was hypothesized that the biotic and abiotic properties of the plant zones would also dictate the composition of the rhizosphere diazotroph assemblages. Over a period of 1 year, rhizosphere sediments were collected from monotypic stands of the black needlerush, Juncus roemerianus, the common pickleweed, Salicornia virginica, the short and tall growth forms of the smooth cordgrass Spartina alterniflora, and a mixed zone of co-occurring S. virginica and short form, S. alterniflora. DNA was extracted, purified and nifH sequences PCR amplified for denaturing gradient gel electrophoresis (DGGE) analysis to determine the composition of the diazotroph assemblages. The diazotroph assemblages were strongly influenced by season, abiotic environmental parameters and plant host. Sediment chemistry and nitrogen fixation activity were also significantly influenced by seasonal changes. DGGE bands that significantly affected seasonal and zone specific clustering were identified and most of these sequences were from novel diazotrophs, unaffiliated with any previously described organisms. At least one third of the recovered nifH sequences were from a diverse assemblage of Chlorobia, and γ-, α-, ß- and δ-Proteobacteria. Diazotrophs that occurred throughout the growing season and among all zones (frequently detected) were also mostly novel. These significant sequences indicated that diazotrophs driving the structure of the assemblages were diverse, versatile, and some were ubiquitous while others were seasonally responsive. Several ubiquitous sequences were closely related to sequences of actively N(2) fixing diazotrophs previously recovered from this system. These sequences from ubiquitous and versatile organisms likely indicate the diazotrophs in these rhizosphere assemblages that significantly contribute to ecosystem function.

Proteomic and physiological responses of Kineococcus radiotolerans to copper.

Copper is a highly reactive, toxic metal; consequently, transport of this metal within the cell is tightly regulated. Intriguingly, the actinobacterium Kineococcus radiotolerans has been shown to not only accumulate soluble copper to high levels within the cytoplasm, but the phenotype also correlated with enhanced cell growth during chronic exposure to ionizing radiation. This study offers a first glimpse into the physiological and proteomic responses of K. radiotolerans to copper at increasing concentration and distinct growth phases. Aerobic growth rates and biomass yields were similar over a range of Cu(II) concentrations (0-1.5 mM) in complex medium. Copper uptake coincided with active cell growth and intracellular accumulation was positively correlated with Cu(II) concentration in the growth medium (R(2)=0.7). Approximately 40% of protein coding ORFs on the K. radiotolerans genome were differentially expressed in response to the copper treatments imposed. Copper accumulation coincided with increased abundance of proteins involved in oxidative stress and defense, DNA stabilization and repair, and protein turnover. Interestingly, the specific activity of superoxide dismutase was repressed by low to moderate concentrations of copper during exponential growth, and activity was unresponsive to perturbation with paraquot. The biochemical response pathways invoked by sub-lethal copper concentrations are exceptionally complex; though integral cellular functions are preserved, in part, through the coordination of defense enzymes, chaperones, antioxidants and protective osmolytes that likely help maintain cellular redox. This study extends our understanding of the ecology and physiology of this unique actinobacterium that could potentially inspire new biotechnologies in metal recovery and sequestration, and environmental restoration.

Seasonal variability of diazotroph assemblages associated with the rhizosphere of the salt marsh cordgrass, Spartina alterniflora.

Nitrogen fixation is the primary N source in the highly productive but N-limited North Inlet, SC, USA salt marsh system. The diverse assemblages of nitrogen-fixing (diazotrophic) bacteria associated with the rhizospheres of the short and tall growth forms of Spartina alterniflora were analyzed at two sites, Crab Haul Creek and Goat Island, which are in different tidal creek drainage systems in this marsh. The sites differed in proximity to the main channel for tidal intrusion and in several edaphic parameters. We hypothesized that either the differing abiotic environmental regimes of the two sites or the variation due to seasonal effects result in differences in the diazotroph assemblage. Rhizosphere samples were collected seasonally during 1999 and 2000. DNA was purified and nifH amplified for denaturing gradient gel electrophoresis (DGGE) analysis of diazotroph assemblage composition. Principal components analysis was used to analyze the binary DGGE band position data. Season strongly influenced assemblage composition and biplots were used to identify bands that significantly affected the seasonal and site-specific clustering. The types of organisms that were most responsive to seasonal or site variability were identified on the basis of DGGE band sequences. Seasonally responsive members of the anaerobic diazotrophs were detected during the winter and postsenescence conditions and may have been responsible for elevated pore water sulfide concentrations. Sequences from a diverse assemblage of Gammaproteobacteria were predominant during growth periods of S. alterniflora. Abiotic environmental parameters strongly influenced both the S. alterniflora and the diazotrophic bacterial assemblages associated with this keystone salt marsh plant species.

Direct analysis of sulfate reducing bacterial communities in gas hydrate-impacted marine sediments by PCR-DGGE.

Molecular investigations of the sulfate reducing bacteria that target the dissimilatory sulfite-reductase subunit A gene (dsr A) are plagued by the nonspecific performance of conventional PCR primers. Here we describe the incorporation of the FailSafe PCR System to optimize environmental analysis of dsr A by PCR amplification and denaturing gradient gel electrophoresis. PCR-DGGE analysis of dsr A composition revealed that SRB diversity was greater and more variable throughout the vertical profile of a marine sediment core obtained from a gas hydrate site (GC234) in the Gulf of Mexico than in a sediment core collected from a nearby site devoid of gas hydrates (NBP). Depth profiled dsr B abundance corresponded with sulfate reduction rates at both sites, though measurements were higher at GC234. This study exemplifies the numerical and functional importance of sulfate reducing bacteria in deep-sea sedimentary environments, and incremental methodological advancements, as described herein, will continue to streamline the analysis of sulfate reducer communities in situ.

Survival in nuclear waste, extreme resistance, and potential applications gleaned from the genome sequence of Kineococcus radiotolerans SRS30216.

Kineococcus radiotolerans SRS30216 was isolated from a high-level radioactive environment at the Savannah River Site (SRS) and exhibits gamma-radiation resistance approaching that of Deinococcus radiodurans. The genome was sequenced by the U.S. Department of Energy's Joint Genome Institute which suggested the existence of three replicons, a 4.76 Mb linear chromosome, a 0.18 Mb linear plasmid, and a 12.92 Kb circular plasmid. Southern hybridization confirmed that the chromosome is linear. The K. radiotolerans genome sequence was examined to learn about the physiology of the organism with regard to ionizing radiation resistance, the potential for bioremediation of nuclear waste, and the dimorphic life cycle. K. radiotolerans may have a unique genetic toolbox for radiation protection as it lacks many of the genes known to confer radiation resistance in D. radiodurans. Additionally, genes involved in the detoxification of reactive oxygen species and the excision repair pathway are overrepresented. K. radiotolerans appears to lack degradation pathways for pervasive soil and groundwater pollutants. However, it can respire on two organic acids found in SRS high-level nuclear waste, formate and oxalate, which promote the survival of cells during prolonged periods of starvation. The dimorphic life cycle involves the production of motile zoospores. The flagellar biosynthesis genes are located on a motility island, though its regulation could not be fully discerned. These results highlight the remarkable ability of K radiotolerans to withstand environmental extremes and suggest that in situ bioremediation of organic complexants from high level radioactive waste may be feasible.

Analysis of a diverse assemblage of diazotrophic bacteria from Spartina alterniflora using DGGE and clone library screening.

Methods to assess the diversity of the diazotroph assemblage in the rhizosphere of the salt marsh cordgrass, Spartina alterniflora were examined. The effectiveness of nifH PCR-denaturing gradient gel electrophoresis (DGGE) was compared to that of nifH clone library analysis. Seventeen DGGE gel bands were sequenced and yielded 58 nonidentical nifH sequences from a total of 67 sequences determined. A clone library constructed using the GC-clamp nifH primers that were employed in the PCR-DGGE (designated the GC-Library) yielded 83 nonidentical sequences from a total of 257 nifH sequences. A second library constructed using an alternate set of nifH primers (N-Library) yielded 83 nonidentical sequences from a total of 138 nifH sequences. Rarefaction curves for the libraries did not reach saturation, although the GC-Library curve was substantially dampened and appeared to be closer to saturation than the N-Library curve. Phylogenetic analyses showed that DGGE gel band sequencing recovered nifH sequences that were frequently sampled in the GC-Library, as well as sequences that were infrequently sampled, and provided a species composition assessment that was robust, efficient, and relatively inexpensive to obtain. Further, the DGGE method permits a large number of samples to be examined for differences in banding patterns, after which bands of interest can be sampled for sequence determination.

Microarray-based whole-genome hybridization as a tool for determining procaryotic species relatedness.

The definition and delineation of microbial species are of great importance and challenge due to the extent of evolution and diversity. Whole-genome DNA-DNA hybridization is the cornerstone for defining procaryotic species relatedness, but obtaining pairwise DNA-DNA reassociation values for a comprehensive phylogenetic analysis of procaryotes is tedious and time consuming. A previously described microarray format containing whole-genomic DNA (the community genome array or CGA) was rigorously evaluated as a high-throughput alternative to the traditional DNA-DNA reassociation approach for delineating procaryotic species relationships. DNA similarities for multiple bacterial strains obtained with the CGA-based hybridization were comparable to those obtained with various traditional whole-genome hybridization methods (r=0.87, P<0.01). Significant linear relationships were also observed between the CGA-based genome similarities and those derived from small subunit (SSU) rRNA gene sequences (r=0.79, P<0.0001), gyrB sequences (r=0.95, P<0.0001) or REP- and BOX-PCR fingerprinting profiles (r=0.82, P<0.0001). The CGA hybridization-revealed species relationships in several representative genera, including Pseudomonas, Azoarcus and Shewanella, were largely congruent with previous classifications based on various conventional whole-genome DNA-DNA reassociation, SSU rRNA and/or gyrB analyses. These results suggest that CGA-based DNA-DNA hybridization could serve as a powerful, high-throughput format for determining species relatedness among microorganisms.

Effects of legacy nuclear waste on the compositional diversity and distributions of sulfate-reducing bacteria in a terrestrial subsurface aquifer.

The impact of legacy nuclear waste on the compositional diversity and distribution of sulfate-reducing bacteria in a heavily contaminated subsurface aquifer was examined. dsrAB clone libraries were constructed and restriction fragment length polymorphism (RFLP) analysis used to evaluate genetic variation between sampling wells. Principal component analysis identified nickel, nitrate, technetium, and organic carbon as the primary variables contributing to well-to-well geochemical variability, although comparative sequence analysis showed the sulfate-reducing bacteria community structure to be consistent throughout contaminated and uncontaminated regions of the aquifer. Only 3% of recovered dsrAB gene sequences showed apparent membership to the Deltaproteobacteria. The remainder of recovered sequences may represent novel, deep-branching lineages that, to our knowledge, do not presently contain any cultivated members; although corresponding phylotypes have recently been reported from several different marine ecosystems. These findings imply resiliency and adaptability of sulfate-reducing bacteria to extremes in environmental conditions, although the possibility for horizontal transfer of dsrAB is also discussed.

Influence of host plant-derived and abiotic environmental parameters on the composition of the diazotroph assemblage associated with roots of Juncus roemerianus.

Environmental factors governing the distributions of plant root-associated bacteria are poorly understood. Most plant species occurring in salt marsh estuaries are restricted to very specific habitats within the marsh and plant-derived and abiotic environmental features covary. We examined diazotrophic bacteria inhabiting the rhizoplanes of different populations of the black needlerush, Juncus roemerianus , growing in two different habitats, in order to examine the relative influence of plant-derived and abiotic environmental parameters on diazotroph assemblage composition. Juncus roots were collected from a monotypic Juncus patch in the low intertidal marsh, and from the main monotypic Juncus stand in the high marsh. A total of 235 bacterial pure cultures were isolated from the roots using combined nitrogen-free media. Physiologically similar strains were grouped, producing 58 different groups. Strains representing 49 of these groups tested positive for nifH , and substrate utilization profiles of these strains were compared quantitatively. Three major substrate utilization clusters were identified and all contained both Juncus patch and main stand isolates. Denaturing gradient gel electrophoresis analysis of nifH amplicons recovered from roots and from vegetated sediments taken from the main stand and from two patches was also performed. Juncus root nifH amplicon profiles from all three sampling sites were very similar. Profiles of amplicons from vegetated sediments were also similar across sites, but less similar than the root profiles. Results from two independent methodological approaches indicated a strong impact of the plant host relative to that of the abiotic environment on the composition of the root-associated diazotroph assemblage.

Involvement of linear plasmids in aerobic biodegradation of vinyl chloride.

Pseudomonas putida strain AJ and Ochrobactrum strain TD were isolated from hazardous waste sites based on their ability to use vinyl chloride (VC) as the sole source of carbon and energy under aerobic conditions. Strains AJ and TD also use ethene and ethylene oxide as growth substrates. Strain AJ contained a linear megaplasmid (approximately 260 kb) when grown on VC or ethene, but it contained no circular plasmids. While strain AJ was growing on ethylene oxide, it was observed to contain a 100-kb linear plasmid, and its ability to use VC as a substrate was retained. The linear plasmids in strain AJ were cured, and the ability of strain AJ to consume VC, ethene, and ethylene oxide was lost following growth on a rich substrate (Luria-Bertani broth) through at least three transfers. Strain TD contained three linear plasmids, ranging in size from approximately 90 kb to 320 kb, when growing on VC or ethene. As with strain AJ, the linear plasmids in strain TD were cured following growth on Luria-Bertani broth and its ability to consume VC and ethene was lost. Further analysis of these linear plasmids may help reveal the pathway for VC biodegradation in strains AJ and TD and explain why this process occurs at many but not all sites where groundwater is contaminated with chloroethenes. Metabolism of VC and ethene by strains AJ and TD is initiated by an alkene monooxygenase. Their yields during growth on VC (0.15 to 0.20 mg of total suspended solids per mg of VC) are similar to the yields reported for other isolates (i.e., Mycobacterium sp., Nocardioides sp., and Pseudomonas sp.).

Development and evaluation of microarray-based whole-genome hybridization for detection of microorganisms within the context of environmental applications.

The detection and identification of microorganisms in natural communities is a great challenge to biologists. Microarray-based genomic technology provides a promising high-throughput alternative to traditional microbial characterization. A novel prototype microarray containing whole genomic DNA, termed community genome array (CGA), was constructed and evaluated. Microarray hybridizations at 55 degrees C using 50% formamide permitted the examined bacteria to be distinguished at the species level, while strain-level differentiation was obtained at hybridization temperatures of 65 or 75 degrees C. The detection limit was estimated to be approximately 0.2 ng with genomic DNA from a single pure culture using a reduced hybridization volume (3 microL). Using mixtures of known amounts of DNA or a known number of cells from 14 or 16 different species, respectively, about 5 ng of genomic DNA or 2.5 x 10(5) cells were detected under the hybridization conditions used. In addition, strong linear relationships were observed between hybridization signal intensity and target DNA concentrations for pure cultures, a mixture of DNA templates, and a population of mixed cells (r2 = 0.95-0.98, P < 0.01). Finally, the prototype CGA revealed differences in microbial community composition in soil, river, and marine sediments. The results suggest that CGA hybridization has potential as a specific, sensitive, and quantitative tool for detection and identification of microorganisms in environmental samples.

Molecular diversity of sulfate-reducing bacteria from two different continental margin habitats.

This study examined the natural diversity and distributions of sulfate-reducing bacteria along a natural carbon gradient extending down the shelf-slope transition zone of the eastern Pacific continental margin. Dissimilatory (bi)sulfite reductase gene sequences (dsrAB) were PCR amplified and cloned from five different sampling sites, each at a discrete depth, from two different margin systems, one off the Pacific coast of Mexico and another off the coast of Washington State. A total of 1,762 clones were recovered and evaluated by restriction fragment length polymorphism (RFLP) analysis. The majority of the gene sequences recovered showed site and depth restricted distributions; however, a limited number of gene sequences were widely distributed within and between the margin systems. Cluster analysis identified 175 unique RFLP patterns, and nucleotide sequences were determined for corresponding clones. Several different continental margin DsrA sequences clustered with those from formally characterized taxa belonging to the delta subdivision of the class Proteobacteria (Desulfobulbus propionicus, Desulfosarcina variabilis) and the Bacillus-Clostridium (Desulfotomaculum putei) divisions, although the majority of the recovered sequences were phylogenetically divergent relative to all of the other DsrA sequences available for comparison. This study revealed extensive new genetic diversity among sulfate-reducing bacteria in continental margin sedimentary habitats, which appears to be tightly coupled to slope depth, specifically carbon bioavailability.