Search

Polychlorinated dibenzo-p-dioxins (PCDDs) are released into the environment from a variety of both anthropogenic and natural sources. While highly chlorinated dibenzo-p-dioxins are persistent under oxic conditions, in anoxic environments, these organohalogens can be reductively dechlorinated to less chlorinated compounds that are then more amenable to subsequent aerobic degradation. Identifying the microorganisms responsible for dechlorination is an important step in developing bioremediation approaches. In this study, we demonstrated the use of a DNA-stable isotope probing (SIP) approach to identify the bacteria active in dechlorination of PCDDs in river sediments, with 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TeCDD) as a model. In addition, pyrosequencing of reverse transcribed 16S rRNA of TeCDD dechlorinating enrichment cultures was used to reveal active members of the bacterial community. A set of operational taxonomic units (OTUs) responded positively to the addition of 1,2,3,4-TeCDD in SIP microcosms assimilating 13C-acetate as the carbon source. Analysis of bacterial community profiles of the 13C labeled heavy DNA fraction revealed that an OTU corresponding to Dehalococcoides mccartyi accounted for a significantly greater abundance in cultures amended with 1,2,3,4-TeCDD than in cultures without 1,2,3,4-TeCDD. This implies the involvement of this Dehalococcoides mccartyi strain in the reductive dechlorination of 1,2,3,4-TeCDD and suggests the applicability of SIP for a robust assessment of the bioremediation potential of organohalogen contaminated sites.

There are many choices for methods of extracting bacterial DNA for Next Generation Sequencing (NGS) from fecal samples. Here, we compare our modifications of a phenol/chloroform extraction method plus an inhibitor removal solution (C3) (ph/Chl+C3) to the PowerFecal® DNA Isolation Kit (MoBio-K). DNA quality and quantity coupled to NGS results were used to assess differences in relative abundance, Shannon diversity index, unique species, and principle coordinate analysis (PCoA) between biological replicates. Six replicate samples, taken from a single ball of horse feces manually collected from the rectum, were subjected to each extraction method. The Ph/Chl+C3 method produced 100× higher DNA yields with less shearing than the MoBio-K method. To assess the methods, the two method samples were sent for sequencing of the bacterial V3-V4 region of 16S rRNA gene using the Illumina MiSeq platform. The relative abundance of Bacteroidetes was greater and there were more unique species assigned to this group in MoBio-K than in Ph/Chl+C3 (P

Anaerobic aniline biodegradation was investigated under different electron-accepting conditions using contaminated canal and groundwater aquifer sediments from an industrial site. Aniline loss was observed in nitrate- and sulfate-amended microcosms and in microcosms established to promote methanogenic conditions. Lag times of 37 days (sulfate amended) to more than 100 days (methanogenic) were observed prior to activity. Time-series DNA-stable isotope probing (SIP) was used to identify bacteria that incorporated (13)C-labeled aniline in the microcosms established to promote methanogenic conditions. In microcosms from heavily contaminated aquifer sediments, a phylotype with 92.7% sequence similarity to Ignavibacterium album was identified as a dominant aniline degrader as indicated by incorporation of (13)C-aniline into its DNA. In microcosms from contaminated canal sediments, a bacterial phylotype within the family Anaerolineaceae, but without a match to any known genus, demonstrated the assimilation of (13)C-aniline. Acidovorax spp. were also identified as putative aniline degraders in both of these two treatments, indicating that these species were present and active in both the canal and aquifer sediments. There were multiple bacterial phylotypes associated with anaerobic degradation of aniline at this complex industrial site, which suggests that anaerobic transformation of aniline is an important process at the site. Furthermore, the aniline degrading phylotypes identified in the current study are not related to any known aniline-degrading bacteria. The identification of novel putative aniline degraders expands current knowledge regarding the potential fate of aniline under anaerobic conditions.

Polychlorinated dibenzo-p-dioxins and -furans (PCDD/Fs) are persistent organic pollutants whose main removal process in the environment is due to biodegradation, and particularly anaerobic reductive dechlorination. Since PCDD/F congeners that are substituted in the lateral 2, 3, 7, and 8 positions are the most toxic, removal of these chlorines is advantageous, but previous studies have only demonstrated their removal under laboratory conditions. We evaluated a concentration data set of PCDD/F congeners with four or more chlorines along with all 209 polychlorinated biphenyl (PCB) congeners in surface water, treated and untreated wastewater, landfill leachate, and biosolids (NY CARP data set) to determine whether peri and peri/lateral dechlorination of PCDD/Fs occurs in these environments. Positive Matrix Factorization (PMF) applied to the data set revealed a factor indicative of the microbial dechlorination of PCBs, and this factor also contained a variety of non-2,3,7,8 substituted PCDD/F congeners. These results suggest that dechlorination of PCDD/Fs at the lateral positions is facile if not preferred in these environments. The relative lack of tetra- and penta-chlorinated PCDD/Fs suggested that dechlorination proceeds to PCDD/F congeners with less than four chlorines. The PMF results were confirmed by examining three samples that contained >90% PCB dechlorination products from the Fresh Kills Landfill and the Hudson River. Even without factor analysis, these samples demonstrated almost identical PCDD/F congener patterns. This study suggests that PCDD/Fs are reductively dechlorinated to nontoxic non-2,3,7,8 PCDD/F congeners in sewers and landfills as well as in the sediment of the Upper Hudson River.

The sensitivity of bacteria to the marine neurotoxins, brevetoxins, produced by the dinoflagellate Karenia brevis and raphidophytes Chattonella spp. remains an open question. We investigated the bacteriocidal effects of brevetoxin (PbTx-2) on the abundance and community composition of natural microbial communities by adding it to microbes from three coastal marine locations that have varying degrees of historical brevetoxin exposure: (1) Great Bay, New Jersey, (2) Rehoboth Bay, Delaware and (3) Sarasota Bay, Florida. The populations with limited or no documented exposure were more susceptible to the effects of PbTx-2 than the Gulf of Mexico populations which are frequently exposed to brevetoxins. The community with no prior documented exposure to brevetoxins showed significant (p = 0.03) changes in bacterial abundance occurring with additions greater than 2.5 μg PbTx-2 L−1. Brevetoxin concentrations during K. brevis blooms range from ∼2.5 to nearly 100 μg L−1 with typical concentrations of ∼10–30 μg L−1. In contrast to the unexposed populations, there was no significant decrease in bacterial cell number for the microbial community that was frequently exposed to brevetoxins, which implies variable sensitivity in natural communities. The diversity in the bacterial communities that were sensitive to PbTx-2 declined upon exposure. This suggests that the PbTx-2 was selecting for or against specific species. Mortality was much higher in the 200 μg PbTx-2 L−1 treatment after 48 h and >37% of the species disappeared in the bacterial communities with no documented exposure. These results suggest that toxic red tides may play a role in structuring bacterial communities.

Field laboratories interested in using the MinION often need the internet to perform sample analysis. Thus, the lack of internet connectivity in resource-limited or remote locations renders downstream analysis problematic, resulting in a lack of sample identification in the field. Due to this dependency, field samples are generally transported back to the lab for analysis where internet availability for downstream analysis is available. These logistics problems and the time lost in sample characterization and identification, pose a significant problem for field scientists. To address this limitation, we have developed a stand-alone data analysis packet using open source tools developed by the Nanopore community that does not depend on internet availability. Like Oxford Nanopore Technologies’ (ONT) cloud-based What’s In My Pot (WIMP) software, we developed the offline MinION Detection Software (MINDS) based on the Centrifuge classification engine for rapid species identification. Several online bioinformatics applications have been developed surrounding ONT’s framework for analysis of long reads. We have developed and evaluated an offline real time classification application pipeline using open source tools developed by the Nanopore community that does not depend on internet availability. Our application has been tested on ATCC’s 20 strain even mix whole cell (ATCC MSA-2002) sample. Using the Rapid Sequencing Kit (SQK-RAD004), we were able to identify all 20 organisms at species level. The analysis was performed in 15 min using a Dell Precision 7720 laptop. Our offline downstream bioinformatics application provides a cost-effective option as well as quick turn-around time when analyzing samples in the field, thus enabling researchers to fully utilize ONT’s MinION portability, ease-of-use, and identification capability in remote locations.

Mesophilic Crenarchaeota (also known as Thaumarchaeota) are ubiquitous and abundant in marine habitats. However, very little is known about their metabolic function in situ. In this study, salt marsh sediments from New Jersey were screened via stable isotope probing (SIP) for heterotrophy by amending with a single (13)C-labeled compound (acetate, glycine or urea) or a complex (13)C-biopolymer (lipids, proteins or growth medium (ISOGRO)). SIP incubations were done at two substrate concentrations (30-150 μM; 2-10 mg ml(-1)), and (13)C-labeled DNA was analyzed by terminal restriction fragment length polymorphism (TRFLP) analysis of 16S rRNA genes. To test for autotrophy, an amendment with (13)C-bicarbonate was also performed. Our SIP analyses indicate salt marsh crenarchaea are heterotrophic, double within 2-3 days and often compete with heterotrophic bacteria for the same organic substrates. A clone library of (13)C-amplicons was screened to find matches to the (13)C-TRFLP peaks, with seven members of the Miscellaneous Crenarchaeal Group and seven members from the Marine Group 1.a Crenarchaeota being discerned. Some of these crenarchaea displayed a preference for particular carbon sources, whereas others incorporated nearly every (13)C-substrate provided. The data suggest salt marshes may be an excellent model system for studying crenarchaeal metabolic capabilities and can provide information on the competition between crenarchaea and other microbial groups to improve our understanding of microbial ecology.

Field biostimulation experiments at the U.S. Department of Energy's Integrated Field Research Challenge (IFRC) site in Rifle, Colorado, have demonstrated that uranium concentrations in groundwater can be decreased to levels below the U.S. Environmental Protection Agency's (EPA) drinking water standard (0.126 μM). During successive summer experiments – referred to as “Winchester” (2007) and “Big Rusty” (2008) - acetate was added to the aquifer to stimulate the activity of indigenous dissimilatory metal-reducing bacteria capable of reductively immobilizing uranium. The two experiments differed in the length of injection (31 vs. 110 days), the maximum concentration of acetate (5 vs. 30 mM), and the extent to which iron reduction (“Winchester”) or sulfate reduction (“Big Rusty”) was the predominant metabolic process. In both cases, rapid removal of U(VI) from groundwater occurred at calcium concentrations (6 mM) and carbonate alkalinities (8 meq/L) where Ca-UO2-CO3 ternary complexes constitute >90% of uranyl ...

Little is known about the relative importance of inorganic and organic nitrogen (N) sources in fueling production of phytoplankton versus heterotrophic bacteria on the continental shelf. This issue was addressed during two diel experiments conducted in the Mid-Atlantic Bight at the Long-term Ecosystem Observatory, LEO-15, off southern New Jersey. Uptake of 15 N-labeled ammonium (NH 4 + ), nitrate (NO 3 − ), and nitrite (NO 2 − ), and dual-labeled ( 15 N and 13 C) urea and dissolved free amino acids was measured in water taken from the surface and bottom mixed layers approximately every 4 h over two 24 h periods in July 2002. Two methods were used to quantify 15 N uptake rates: (1) traditional filtration into various phytoplankton and bacterial size classes, and (2) flow cytometric (FCM) sorting of autotrophic cells based on the presence of chlorophyll autofluorescence. Due to a strong pycnocline, the nutrient composition was quite distinct between the surface and bottom mixed layers. Dissolved organic N (DON) comprised >99% of the total dissolved N (TDN) pool in surface waters, whereas the bottom-water TDN pool was roughly divided between NH 4 + , NO 3 − , and DON. Urea was the dominant N form used by all fractions at the surface, and although phytoplankton >3 μm was responsible for most of the urea uptake, bacterial use was detected using stable isotopes and also suggested by ure C sequence analysis. The majority of ure C sequences recovered from the 0.2–0.8 μm fraction belonged to members of the Alphaproteobacteria (46%), whereas those of the 0.8–3.0 μm size class consisted primarily of Cyanobacteria (70%). In contrast to the surface, N uptake in the bottom layer was dominated by NH 4 + . The bacterial fraction was responsible for 20–49% of the size-fractionated NH 4 + and NO 3 − uptake in surface samples and 36–93% at the bottom. These results suggest that organic N, such as urea, is a viable source of N nutrition to phytoplankton forced to compete with heterotrophic bacteria for limited inorganic N.

There is a growing need for a better understanding of the biogeochemical dynamics involved in microbial U(VI) reduction due to an increasing interest in using biostimulation via electron donor addition as a means to remediate uranium contaminated sites. U(VI) reduction has been observed to be maximized during iron-reducing conditions and to decrease upon commencement of sulfate-reducing conditions. There are many unknowns regarding the impact of iron/sulfate biogeochemistry on U(VI) reduction. This includes Fe(III) availability as well as the microbial community changes, including the activity of iron-reducers during the uranium biostimulation period even after sulfate reduction becomes dominant. Column experiments were conducted with Old Rifle site sediments containing Fe-oxides, Fe-clays, and sulfate rich groundwater. Half of the columns had sediment that was augmented with small amounts of Fe(III) in the form of (57)Fe-goethite, allowing for a detailed tracking of minute changes of this added phase to study the effects of increased Fe(III) levels on the overall biostimulation dynamics. Mössbauer spectroscopy showed that the added (57)Fe-goethite was bioreduced only during the first thirty days of biostimultuion, after which it remained constant. Augmentation with Fe(III) had a significant effect on the total flux of electrons towards different electron acceptors; it suppressed the degree of sulfate reduction, had no significant impact on Geobacter-type bacterial numbers but decreased the bacterial numbers of sulfate reducers and affected the overall microbial community composition. The addition of Fe(III) had no noticeable effect on the total uranium reduction.

In situ treatment of PCB contaminated sediments via microbial dechlorination is a promising alternative to dredging, which may be reserved for only the most contaminated areas. Reductive dechlorination of low levels of weathered PCB mixtures typical of urban environments may occur at slow rates. Here, we report that biostimulation and bioaugmentation enhanced dechlorination of low concentration (2.1 mg PCBs/kg dry weight) historical PCBs in microcosms prepared with Anacostia River, Washington, DC, sediment. Treatments included electron donors butyrate, lactate, propionate and acetate (1 mM each); alternate halogenated electron acceptors (haloprimers) tetrachlorobenzene (TeCB, 25 microM), pentachloronitrobenzene (PCNB, 25 microM), or 2,3,4,5,6-PCB (PCB116, 2.0 microM); and/or bioaugmentation with a culture containing Dehalococcoides ethenogenes strain 195 (3 x 10(6)cells/mL). Dechlorination rates were enhanced in microcosms receiving bioaugmentation, PCNB and PCNB plus bioaugmentation, compared to other treatments. Microcosm subcultures generated after 415 days and spiked with PCB116 showed sustained capacity for dechlorination of PCB116 in PCNB, PCNB plus bioaugmentation, and TeCB treatments, relative to other treatments. Analysis of Chloroflexi 16S rRNA genes showed that TeCB and PCNB increased native Dehalococcoides spp. from the Pinellas subgroup; however this increase was correlated to enhanced dechlorination of low concentration weathered PCBs only in PCNB-amended microcosms. D. ethenogenes strain 195 was detected only in bioaugmented microcosms and decreased over 281 days. Bioaugmentation with D. ethenogenes strain 195 increased PCB dechlorination rates initially, but enhanced capacity for dechlorination of a model congener, PCB116, after 415 days occurred only in microcosms with enhanced native Dehalococcoides spp.

Technology for accessing the genomic DNA of microorganisms, directly from environmental samples without prior cultivation, has opened new vistas to understanding microbial diversity and functions. Especially as applied to soils and the oceans, environments on Earth where microbial diversity is vast, metagenomics and its emergent approaches have the power to transform rapidly our understanding of environmental microbiology. Here we explore select recent applications of the metagenomic suite to ocean microbiology.

Although open ocean time-series sites have been areas of microbial research for years, relatively little is known about the population dynamics of bacterioplankton communities in the coastal ocean on kilometer spatial and seasonal temporal scales. To gain a better understanding of microbial community variability, monthly samples of bacterial biomass were collected in 1995–1996 along a 34-km transect near the Long-Term Ecosystem Observatory (LEO-15) off the New Jersey coast. Surface and bottom sampling was performed at seven stations along a transect line with depths ranging from 1 to 35 m (n=178). Microbial populations were fingerprinted using ribosomal 16S rRNA genes and terminal restriction fragment length polymorphism analysis. Results from cluster analysis revealed distinct temporal patterns among the bacterioplankton communities in the Mid-Atlantic Bight rather than grouping by sample location or depth. Principal components analysis models supported the temporal patterns. In addition, partial least squares regression modeling could not discern a significant correlation from traditional oceanographic physical and phytoplankton nutrient parameters on overall bacterial community variability patterns at LEO-15. These results suggest factors not traditionally measured during oceanographic studies are structuring coastal microbial communities.

Sandy or permeable sediment deposits cover the majority of the shallow ocean seafloor, and yet the associated bacterial communities remain poorly described. The objective of this study was to expand the characterization of bacterial community diversity in permeable sediment impacted by advective pore water exchange and to assess effects of spatial, temporal, hydrodynamic, and geochemical gradients. Terminal restriction fragment length polymorphism (TRFLP) was used to analyze nearly 100 sediment samples collected from two northeastern Gulf of Mexico subtidal sites that primarily differed in their hydrodynamic conditions. Communities were described across multiple taxonomic levels using universal bacterial small subunit (SSU) rRNA targets (RNA- and DNA-based) and functional markers for nitrification ( amoA ) and denitrification ( nosZ ). Clonal analysis of SSU rRNA targets identified several taxa not previously detected in sandy sediments (i.e., Acidobacteria , Actinobacteria , Chloroflexi , Cyanobacteria , and Firmicutes ). Sequence diversity was high among the overall bacterial and denitrifying communities, with members of the Alphaproteobacteria predominant in both. Diversity of bacterial nitrifiers ( amoA ) remained comparatively low and did not covary with the other gene targets. TRFLP fingerprinting revealed changes in sequence diversity from the family to species level across sediment depth and study site. The high diversity of facultative denitrifiers was consistent with the high permeability, deeper oxygen penetration, and high rates of aerobic respiration determined in these sediments. The high relative abundance of Gammaproteobacteria in RNA clone libraries suggests that this group may be poised to respond to short-term periodic pulses of growth substrates, and this observation warrants further investigation.

It is widely believed that the vast majority of microbes in the environment have-yet-to-be cultured using standard techniques. Bulk DNA from microbial communities is therefore often cloned into large insert vectors (e.g. bacterial artificial chromosomes [BAC] or cosmids) in order to study the genetic properties of these as yet (un)-cultured bacteria. In a typical BAC experiment, tens of thousands of clones are generated with only a small fraction of colonies containing the target(s) of interest. Efficient screening methodologies are therefore needed to allow targeted clone isolation. In this paper, we describe a rapid, inexpensive protocol that allows for the identification of specific 16S ribosomal RNA genes in a metagenomic library arrayed into 384-well microtiter plates. The rapid screening protocol employs Terminal Restriction Fragment Length Polymorphism (TRFLP) analysis to identify wells containing specific T-RF peaks. A nested approach using multiplexed samples of 384, 48, 8, and single colony analysis is described and applied in order to survey a BAC library generated from a marine microbial community off the coast of New Jersey. Screening revealed a total of 50 different 16 rRNA genes within the BAC library. Overall, the multiplexing format provided a simple, cost effective methodology for detecting clones bearing a target gene of interest in a large clone library. However, the limitations of screening BAC libraries using PCR methodologies and recommendations for improved screening efficiency using this approach are also discussed.

A suite of experiments were conducted to ascertain whether dehalogenation of a model dioxin compound could be stimulated in marine sediments by supplementation with halogenated analogues to enrich for dehalogenating bacteria and if growth by members of the Chloroflexi-like group was associated with dioxin removal. Five halogenated compounds (tetrachlorobenzene, tetrachloroanisole, tetrachlorophenol, tetrachlorobenzoic acid and trichloroacetophenone) were added with 1,2,3,4-tetrachlorodibenzo-p-dioxin (TeCDD) to estuarine sediments from four sites in San Diego Bay and the coast of southern New Jersey to test for dioxin dehalogenation. Most of the halogenated additives were found to stimulate dechlorination of the model dioxin. Molecular analysis of the bacterial population using 16S rRNA and reductive dehalogenase genes indicated that distinct microbial populations were enriched with each halogenated co-amendment. Additionally, Chloroflexi-like ribosomal genes associated with dehalogenation were detected. For example, quantitative real-time PCR analysis of 16S rRNA and reductive dehalogenase gene copy number in the microcosms showed a positive correlation with 1,2,3,4-TeCDD reductive dechlorination in coastal sediments amended with different halogenated additives. These results suggest that specific Chloroflexi-like microorganisms related to Dehalococcoides are involved in 1,2,3,4-TeCDD reductive dechlorination.

Soil is a highly heterogeneous matrix, which can contain thousands of different bacterial species per gram. Only a small component of this diversity (maybe

Sediment samples were collected worldwide from 16 locations on four continents (in New York, California, New Jersey, Virginia, Puerto Rico, Venezuela, Italy, Latvia, and South Korea) to assess the extent of the diversity and the distribution patterns of sulfate-reducing bacteria (SRB) in contaminated sediments. The SRB communities were examined by terminal restriction fragment (TRF) length polymorphism (TRFLP) analysis of the dissimilatory sulfite reductase genes ( dsrAB ) with NdeII digests. The fingerprints of dsrAB genes contained a total of 369 fluorescent TRFs, of which dsrAB gene sequences corresponding to hydrocarbon-degrading isolates or consortium members were detected in geographically widely separated polluted sites. Finally, a cluster analysis of the TRFLP fingerprints indicated that many SRB microbial communities were most similar on the basis of close geographic proximity (tens of kilometers). Yet, on larger scales (hundreds to thousands of kilometers) SRB communities could cluster with geographically widely separated sites and not necessarily with the site with the closest proximity. These data demonstrate that SRB populations do not adhere to a biogeographic distribution pattern similar to that of larger eukaryotic organisms, with the greatest species diversity radiating from the Indo-Pacific region. Rather, a patchy SRB distribution is encountered, implying an initially uniform SRB community that has differentiated over time.

Terminal restriction fragment length polymorphism analysis of reverse-transcribed 16S rRNA during periods of community flux was used as a tool to delineate the roles of the members of a 2-bromophenol-degrading, sulfate-reducing consortium. Starved, washed cultures were amended with 2-bromophenol plus sulfate, 2-bromophenol plus hydrogen, phenol plus sulfate, or phenol with no electron acceptor and were monitored for substrate use. In the presence of sulfate, 2-bromophenol and phenol were completely degraded. In the absence of sulfate, 2-bromophenol was dehalogenated and phenol accumulated. Direct terminal restriction fragment length polymorphism fingerprinting of the 16S rRNA in the various subcultures indicated that phylotype 2BP-48 (aDesulfovibrio-like sequence) was responsible for the dehalogenation of 2-bromophenol. A stable coculture was established which contained predominantly 2BP-48 and a secondDesulfovibrio-like bacterium (designated BP212 based on terminal restriction fragment length polymorphism fingerprinting) that was capable of dehalogenating 2-bromophenol to phenol. Strain 2BP-48 in the coculture could couple reductive dehalogenation to growth with 2-bromophenol, 2,6-dibromophenol, or 2-iodophenol and lactate or formate as the electron donor. In addition to halophenols, strain 2BP-48 appears to use sulfate, sulfite, and thiosulfate as electron acceptors and is capable of simultaneous sulfidogenesis and reductive dehalogenation in the presence of sulfate.

Biomass samples from the Black Sea collected in 1988 were analyzed for SSU genes from Bacteria and Archaea after 10 years of storage at −80°C. Both clonal libraries and direct fingerprinting by terminal restriction fragment length polymorphism (T-RFLP) analyses were used to assess the microbial community. Uniform and discrete depth distributions of different SSU phylotypes were observed. However, most recombinant clones were not restricted to a specific depth in the water column, and many of the major T-RFLP peaks remain uncharacterized. Of the clones obtained, an ε- Proteobacteria and a Pseudoalteromonas -like clone accounted for major peaks in the fingerprint, while deeply branching lineages of α- and γ- Proteobacteria were associated with smaller peaks. Additionally, members were found among both the δ- Proteobacteria related to sulfate reducers and the Archaea related to phylotypes from the ANME groups that anaerobically oxidize methane.

Polymerase chain reaction (PCR) primers were developed based on three known reductive dehalogenase (RDH) genes (pceA from Dehalospirillum multivorans, cprA from Desulfitobacterium dehalogenans, tceA from Dehalococcoides ethenogenes) and used to amplify bands of the appropriate size from a microbial consortium inoculated with contaminated estuarine sediment and enriched under sulfidogenic conditions using 2-bromophenol (2-BP) as the sole carbon source. These PCR fragments were found to contain many of the conserved amino acids and motifs present in the known RDH genes. The three cloned PCR products (2bprdh61, 2bprdh63, 2bprdh81 - designated 2-bromophenol RDH) shared 21 of the 31 conserved amino acids present in the C-terminus of the RDHs in GenBank. (The N-terminus of the RDH protein shares very little homology for the known RDH genes.) All 2-BP PCR products and the known RDH genes were found to contain two iron-sulfur cluster binding domains as well as conserved PCR priming sites. Southern hybridization of genomic DNA revealed multiple bands, implying additional RDH-like motifs in the sulfidogenic 2-BP-degrading consortium. In order to gain information on upstream regions of the RDH-like motifs, DNA fragments containing 2bprdh61, 2bprdh63, and 2bprdh81 were cloned and sequenced via an inverse PCR approach. The results indicated the presence of transposase gene homologues upstream of 2-bprdh61A and 2-bprdh81A. Therefore, some RDH-like PCR products in our consortium are possibly pseudogenes and some RDH genetic diversity may be generated by transposition.

Chlorobenzoate and 4-bromobenzoate were readily degraded in denitrifying enrichment cultures established with river sediment, estuarine sediment or agricultural soil as inoculum. Stable denitrifying consortia were obtained and maintained by serial dilution and repeated feeding of substrates. Microbial community analyses were performed to characterize the 4-chlorobenzoate and 4-bromobenzoate degrading consortia with terminal restriction fragment length polymorphism (T-RFLP) and cloning of 16S rRNA genes from the cultures. Interestingly, two major terminal restriction fragments (T-RFs) in the 4-chlorobenzoate degrading consortia and one T-RF in the 4-bromobenzoate utilizing consortium were observed from T-RFLP analysis regardless of their geographical and ecological origins. The two T-RFs (clones 4CB1 and 4CB2) in 4-chlorobenzoate degrading consortia were identified as members of the L-subunit of the Proteobacteria on the basis of 16S rRNA sequencing analysis. Phylogenetic analysis of 16S rRNA genes showed that clone 4CB1 was closely related to Thauera aromatica while clone 4CB2 was distantly related to the genera Limnobacter and Ralstonia. The 4-bromobenzoate utilizing consortium mainly consisted of one T-RF, which was identical to clone 4CB2 in spite of different enrichment substrate. This suggests that degradation of 4-chlorobenzoate and 4-bromobenzoate under denitrifying conditions was mediated by bacteria belonging to the L-subunit of the Proteobacteria. 7 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

The degradation of a range of aromatic compounds under selenate-reducing conditions was examined. Enrichment cultures were initiated with a 10% sediment slurry from the Arthur Kill, an intertidal strait in the New York/New Jersey harbor, and from the Kesterson Reservoir, California, with selenate as the sole electron acceptor. After repeated feeding, activity was maintained with either benzoate, 3-hydroxybenzoate, or 4-hydroxybenzoateas thesole carbonsubstrate.Degradationofthebenzoicacidderivatives did not occur without the addition of selenate, or insterile controls, indicating that substrate utilization was dependent on selenate reduction. Degradation of each of these aromatic compounds was coupled stoichiometrically to the reduction of selenate to selenite. Two selenate-reducing bacterial strains that utilized 4-hydroxybenzoate as a carbon source were isolated from the Arthur Kill and the Kesterson Reservoir, respectively. Based on analysis of their 16S rRNA gene sequences these isolates were 98.3% similar to one another, but distinct from other known Se(VI)-reducing bacteria. Phylogenetic analysis indicated that they were afeliated with the gamma subgroup of the Proteobacteria, with uncultured sulfur-oxidizing symbionts as their closest neighbors.

The characterization of sulfate-reducing bacteria (SRBs) is presented using the dissimilatory sulfite reductase (dsrAB) gene from various samples capable of mineralizing petroleum components. These samples include several novel, sulfidogenic pure cultures which degrade alkanes, toluene, and tribromophenol. Additionally, we have sulfidogenic consortia which re-mineralize benzene, naphthalene, 2-methylnaphthalene, and phenanthrene as a sole carbon source. In this study, 22 new dsrAB genes were cloned and sequenced. The dsrAB genes from our pollutant-degrading cultures or consortia were distributed among known SRBs and previously described dsrAB environmental clones, suggesting that many biodegradative SRBs are phylogenetically distinct and geographically wide spread. Specifically, the same dsrAB gene was discovered in independently established consortia capable of benzene, phenanthrene, and methylnaphthalene degradation, indicating that this particular SRB may be a key player in anaerobic degradation of hydrocarbons in the environment.

Nine strains of marine Proteobacteria were assayed for nucleic acid content during non-steady-state growth to assess whether a species-specific growth rate based on rRNA content is feasible for environmental samples. The large and small ribosomal subunits and genomic DNA were quantified using image analysis. It was found that the maximal intracellular concentration of 16S rRNA during batch growth for the bacteria averaged 155 fg+/-60 (S.D.) per cell for eight of the nine marine bacteria in the exponential phase (with the exception of one strain, Pac 218). The dilution/decay of 16S rRNA/cell was rapid with a return to pre-shift up values within 6-12 h for all strains except Vibrio fisherii. An overall relationship between the RNA:DNA ratio and the specific growth rate for non-steady-state growth for all bacterial strains was not observed as previously described for other Proteobacteria during steady-state growth. However, a predictable relationship between rRNA content and growth rate for many isolates during batch growth was observed. Furthermore, the rapid kinetics of intracellular rRNA levels indicates it will be feasible to assess whether specific bacteria are in steady state or non-steady state in the marine environment. If the condition of steady state is met for a specific Proteobacterial group in an environmental sample, it will be possible to estimate species-specific growth rates by measuring rRNA content.

Microbial consortia were enriched under sulfidogenic conditions using a common estuarine sediment inoculum with 2-bromophenol or phenol as the sole the carbon source. Stable consortia were maintained over a 3-year period with repeated feeding and serial dilution into fresh medium. 2-Bromophenol was initially dehalogenated to phenol. Degradation of phenol was dependent on sulfate reduction and inhibited by molybdate, a specific inhibitor of sulfate reduction. Reductive dehalogenation of 2-bromophenol, however, was not dependent on, or inhibited by sulfate. The 2-bromophenol- and phenol-degrading sulfidogenic consortia were characterized using 16S rRNA restriction fragment length polymorphism analysis and unique clones were sequenced. Terminal restriction fragment length polymorphism of all individual clones and both microbial consortia indicated that all 16S rRNA types present in the consortia were cloned and characterized. Four phylotypes were identified from the 2-bromophenol-utilizing consortium which based upon their 16S rRNA sequences clustered into three major groups: one sequence was related to the ϵ-subgroup of the Proteobacteria, two clones clustered within the sulfate-reducers (δ-subgroup of Proteobacteria), the fourth phylotype was divergent from previously described bacteria and was most closely related to the genus Planctomycetes. None of the clones from the 2-bromophenol-degrading consortium are close to previously described aryl-dehalogenating bacteria which predominantly comprise the genera Desulfitobacterium and Desulfomonile. In contrast, the phenol-degrading consortium yielded only two clonal types. One was placed within the ϵ-sub-division of the Proteobacteria with Thiomicrospira denitrificans as its closest neighbor. The other clone was closest to the genus Cytophaga with Anaeroflexus maritimus as its closest neighbor.

Diversity of the nitrous oxide reductase ( nosZ ) gene was examined in sediments obtained from the Atlantic Ocean and Pacific Ocean continental shelves. Approximately 1,100 bp of the nosZ gene were amplified via PCR, using nosZ gene-specific primers. Thirty-seven unique copies of the nosZ gene from these marine environments were characterized, increasing the nosZ sequence database fourfold. The average DNA similarity for comparisons between all 49 variants of the nosZ gene was 64% ± 10%. Alignment of the derived amino acid sequences confirmed the conservation of important structural motifs. A highly conserved region is proposed as the copper binding, catalytic site (Cu Z ) of the mature protein. Phylogenetic analysis demonstrated three major clusters of nosZ genes, with little overlap between environmental and culture-based groups. Finally, the two non-culture-based gene clusters generally corresponded to sampling location, implying that denitrifier communities may be restricted geographically.

A stable and sediment-free, benzene mineralizing, sulfate-reducing culture that resisted repeated attempts at isolation was examined using molecular approaches such as traditional cloning and sequencing and a direct PCR fingerprinting method for 16S rRNA genes. Despite the culture's long exposure to benzene as the only carbon and energy source (over 3 years) and repeated dilutions of the original enrichment, this consortium has remained relatively complex. Cloning and sequence analysis identified 12 unique small subunit rRNA genes. The 16S rRNA genes belong to different eubacterial phyla, including Proteobacteria, Cytophagales and Gram-positives. There is one deeply branching clone which is not closely related to any known, sequenced, bacterium. A different clone, however, is closely related to a known sulfidogenic, aromatic hydrocarbon degrader. To assess 16S rRNA gene cloning efficiency, a fingerprinting method based on fluorescent, end-labeling of PCR product (16S rRNA genes) and screening by restriction length polymorphism analysis (RFLP) was employed. The data obtained indicated that we had cloned and characterized nearly all of the eubacterial 16S rRNA genes amplified from the consortia.

Previous experiments at the Rifle, Colorado Integrated Field Research Challenge (IFRC) site demonstrated that field-scale addition of acetate to groundwater reduced the ambient soluble uranium concentration. In this report, sediment samples collected before and after acetate field addition were used to assess the active microbes via (13)C acetate stable isotope probing on 3 phases [coarse sand, fines (8-approximately 150 μm), groundwater (0.2-8 μm)] over a 24-day time frame. TRFLP results generally indicated a stronger signal in (13)C-DNA in the "fines" fraction compared to the sand and groundwater. Before the field-scale acetate addition, a Geobacter-like group primarily synthesized (13)C-DNA in the groundwater phase, an alpha Proteobacterium primarily grew on the fines/sands, and an Acinetobacter sp. and Decholoromonas-like OTU utilized much of the (13)C acetate in both groundwater and particle-associated phases. At the termination of the field-scale acetate addition, the Geobacter-like species was active on the solid phases rather than the groundwater, while the other bacterial groups had very reduced newly synthesized DNA signal. These findings will help to delineate the acetate utilization patterns of bacteria in the field and can lead to improved methods for stimulating distinct microbial populations in situ.

Continuous culture of Pseudomonas stutzeri Zobell, a marine denitrifying bacterium, was used to determine the relationship between growth rate and nucleic acid content. The trend of decreasing RNA content with decreasing growth rate, well known for enteric organisms, was found to occur in P. stutzeri Zobell as well, even at very long generation times such as those thought to occur in the oligotrophic ocean. When assayed by ethidium bromide fluorescence, the total RNA/DNA ratio was linear for generation times between 6 and 60 h. We also developed a 200-bp nucleic acid probe (with species-specific potential) for a portion of the 23S rRNA gene of P. stutzeri Zobell, which was used to quantify rRNA and rDNA by hybridization in the same continuous cultures. The rRNA/rDNA ratio also exhibited a decrease with decreasing growth rate, but the relationship, although significant, was not simply linear. The sensitivity and accuracy of the two methods are compared, and the potential for species specificity in future hybridizations is discussed.

Methane concentrations and oxidation rates were measured in the water column and sediments of the Black Sea at a central station during leg 5 of the 1988 U.S.—Turkey Black Sea Expedition. Methane concentrations were 10 nM in the upper 100 m, increased to 11 μM at 550 m, and were uniform to the bottom. Water column methane oxidation rates were measured using two independent radiotracer techniques: tracer level additions of 3H−CH4, and non-tracer level additions of 14C−CH4. The methods agree within a factor of two. Methane oxidation rates were low in the surface 100 m and increased to relatively uniform values of 0.6μM y−1 below 500 m. Sediment methane concentration and oxidation rate distributions showed that shelf and slope sediments were methane sources, while deep basin sediments were methane sinks. These measurements were used to construct a methane budget for Black Sea waters. Microbially mediated anaerobic methane oxidation is the dominant water column methane sink, followed by evasion to the atmosphere, abyssal plain sediment consumption and outflow at the Bosporus. The source of methane appears to be anoxic, high deposition rate shelf and slope sediments. The water column oxidation rate measurements suggest a short (5–20 year) residence time for methane in the Black Sea, indicating a higher geochemical activity than previously believed. The quantity of carbon participating in the Black Sea methane cycle is equivalent to about 0.5% of the primary production.

A suite of experiments were conducted to ascertain whether dehalogenation of a model dioxin compound could be stimulated in marine sediments by supplementation with halogenated analogues to enrich for dehalogenating bacteria and if growth by members of the Chloroflexi-like group was associated with dioxin removal. Five halogenated compounds (tetrachlorobenzene, tetrachloroanisole, tetrachlorophenol, tetrachlorobenzoic acid and trichloroacetophenone) were added with 1,2,3,4-tetrachlorodibenzo-p-dioxin (TeCDD) to estuarine sediments from four sites in San Diego Bay and the coast of southern New Jersey to test for dioxin dehalogenation. Most of the halogenated additives were found to stimulate dechlorination of the model dioxin. Molecular analysis of the bacterial population using 16S rRNA and reductive dehalogenase genes indicated that distinct microbial populations were enriched with each halogenated co-amendment. Additionally, Chloroflexi-like ribosomal genes associated with dehalogenation were detected. For example, quantitative real-time PCR analysis of 16S rRNA and reductive dehalogenase gene copy number in the microcosms showed a positive correlation with 1,2,3,4-TeCDD reductive dechlorination in coastal sediments amended with different halogenated additives. These results suggest that specific Chloroflexi-like microorganisms related to Dehalococcoides are involved in 1,2,3,4-TeCDD reductive dechlorination.

Microbially mediated dehalogenation processes contribute to the global cycling of both biogenic and anthropogenic halogenated organic compounds. Detailed information on biodegradation mechanisms for a variety of organohalides and on the microorganisms mediating these processes has greatly increased our understanding of the cycling and fate of these unique and widespread compounds in our environment. The marine environment appears to be a particularly rich source of dehalogenating microorganisms. It is well established by laboratory and field studies that anaerobic dehalogenation of sediment contaminants, such as PCBs, pesticides, and dioxins, occurs intrinsically and can be enhanced via various methods. Specific dehalogenating bacterial populations can be enriched on various organohalides. Biodehalogenation processes are likely to be significantly affected by the prevailing terminal electron-accepting condition, and thus, biotransformation of organohalide contaminants in marine and estuarine environments will vary as a function of the redox conditions within the sediment profile. Fundamental knowledge of the activities and interactions of dehalogenating microorganisms is providing a strong basis for development of new bioremediation technologies for removal of harmful halogenated compounds from our environment.

A “snap shot” survey of the Mississippi estuary was made during a period of low river discharge, when the estuarine mixing zone was within the deltaic channels. Concentrations of H+, Ca2+, inorganic phosphorus and inorganic carbon suggest that the waters of the river and the low salinity ( 5‰). The phytoplankton abundance (measured as chlorophylla) increased as the depth of the mixed layer decreased in a manner consistent with that expected for a light-limited ecosystem. Fluxes of NO3−+NO2− and soluble inorganic phosphorus to the Gulf of Mexico were estimated to be 3.4±0.2×103 g N s−1 and 1.9±0.2 g P s−1 respectively, at the time of this study.