Molecular characterization of a phenanthrene degradation pathway in Mycobacterium vanbaalenii PYR-1

Mycobacterium vanbaalenii PYR-1 is capable of degrading a number of polycyclic aromatic hydrocarbons (PAHs) to ring cleavage metabolites via multiple pathways. Genes for the large and small subunits of a pyrene dioxygenase, nidA and nidB, respectively, were previously identified in M. vanbaalenii PYR-1 [Appl. Environ. Microbiol. 67 (2001) 3577]. A library of the M. vanbaalenii PYR-1 genome was constructed in a fosmid vector to identify additional genes involved in PAH degradation. Twelve fosmid clones containing nidA were identified by Southern hybridization. Sequence analysis of one nidA-positive clone, pFOS608, revealed a number of additional genes involved in PAH degradation. At this locus, one putative operon contained genes involved in phthalate degradation, and another contained genes encoding a putative ABC transporter(s). A number of the genes found in this region are homologous to those involved in phenanthrene degradation via the phthalic acid pathway. The majority of phenanthrene degradation genes were located between putative transposase genes. In Escherichia coli, pFOS608 converted phenanthrene into phenanthrene cis-3,4-dihydrodiol, and converted 1-hydroxy-2-naphthoic acid into 2'-carboxybenzalpyruvate, 2-carboxybenzaldehyde, and phthalic acid. A subclone containing nidA and nidB converted phenanthrene into phenanthrene cis-3,4-dihydrodiol, suggesting that the NidAB dioxygenase is responsible for an initial attack on phenanthrene. This study is the first to identify genes responsible for the degradation of phenanthrene via the phthalic acid pathway in Mycobacterium species.     

Microbial consortia involved in the anaerobicdegradation of hydrocarbons

In this review, we examine the energetics of well-characterized biodegradation pathways and explore the possibilities for these to support growth of multiple organisms interacting in consortia. The relevant phenotypic and/or phylogenetic characteristics of isolates and consortia mediating hydrocarbon degradation coupled with different terminal electron-acceptingprocesses (TEAP) are also reviewed. While the information on metabolic pathways has been gained from the analysis of individual isolates, the energetic framework presented here demonstrates that microbial consortia could be readily postulated for hydrocarbon degradation coupled to any TEAP. Several specialized reactions occur within these pathways, and the organisms mediating these are likely to play a key role in defining the hydrocarbon degradation characteristics of the community under a given TEAP. Comparing these processes within and between TEAPs reveals biological unity in that divergent phylotypes display similar degradation mechanisms and biological diversity in that hydrocarbon-degraders closely related as phylotypes differ in the type and variety of hydrocarbon degradation pathways they possess. Analysis of microcosms and of field samples suggests that we have only begun to reveal the diversity of organisms mediating anaerobic hydrocarbon degradation. Advancements in the understanding of how hydrocarbon-degrading communities function will be significantly affected by the extent to which organisms mediating specialized reactions can be identified, and tools developed to allow their study in situ.     

Phylogeography of sulfate-reducing bacteria among disturbed sediments, disclosed by analysis of the dissimilatory sulfite reductase genes (dsrAB)

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 <20% were present in the GenBank database. The global sulfidogenic communities appeared to be significantly different among the anthropogenically impacted (petroleum-contaminated) sites, but nearly all were less diverse than pristine habitats, such as mangroves. A global SRB indicator species of petroleum pollution was not identified. However, several 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.     

Analysis of diversity and activity of sulfate-reducing bacterial communities in sulfidogenic bioreactors using 16S rRNA and dsrB genes as molecular markers

Here we describe the diversity and activity of sulfate-reducing bacteria (SRB) in sulfidogenic bioreactors by using the simultaneous analysis of PCR products obtained from DNA and RNA of the 16S rRNA and dissimilatory sulfite reductase (dsrAB) genes. We subsequently analyzed the amplified gene fragments by using denaturing gradient gel electrophoresis (DGGE). We observed fewer bands in the RNA-based DGGE profiles than in the DNA-based profiles, indicating marked differences in the populations present and in those that were metabolically active at the time of sampling. Comparative sequence analyses of the bands obtained from rRNA and dsrB DGGE profiles were congruent, revealing the same SRB populations. Bioreactors that received either ethanol or isopropanol as an energy source showed the presence of SRB affiliated with Desulfobulbus rhabdoformis and/or Desulfovibrio sulfodismutans, as well as SRB related to the acetate-oxidizing Desulfobacca acetoxidans. The reactor that received wastewater containing a diverse mixture of organic compounds showed the presence of nutritionally versatile SRB affiliated with Desulfosarcina variabilis and another acetate-oxidizing SRB, affiliated with Desulfoarculus baarsii. In addition to DGGE analysis, we performed whole-cell hybridization with fluorescently labeled oligonucleotide probes to estimate the relative abundances of the dominant sulfate-reducing bacterial populations. Desulfobacca acetoxidans-like populations were most dominant (50 to 60%) relative to the total SRB communities, followed by Desulfovibrio-like populations (30 to 40%), and Desulfobulbus-like populations (15 to 20%). This study is the first to identify metabolically active SRB in sulfidogenic bioreactors by using the functional gene dsrAB as a molecular marker. The same approach can also be used to infer the ecological role of coexisting SRB in other habitats.     

Cr(VI) Reduction by Sulfidogenic and Nonsulfidogenic Microbial Consortia

In time course experiments, bacterial community compositions were compared between a sulfidogenic and two nonsulfidogenic Cr(VI)-reducing consortia enriched from metal-contaminated sediments. The consortia were subjected to 0 and 0.85 mM or 1.35 mM Cr(VI), and Cr(VI) reduction, growth, and denaturing gradient gel electrophoresis profiles of PCR products of small-subunit (16S) ribosomal genes were compared. Results showed that although Cr(VI) was completely reduced by the three consortia, Cr(VI) inhibited cell growth, with sulfate-reducing bacteria being particularly sensitive to Cr(VI) toxicity relative to other bacteria in the consortia.     

Degradation of organic pollutants by methane grown microbial consortia

Microbial consortia were enriched from various environmental samples with methane as the sole carbon and energy source. Selected consortia that showed a capacity for co-oxidation of naphthalene were screened for their ability to degrade methyl-tert-butyl-ether (MTBE), phthalic acid esters (PAE), benzene, xylene and toluene (BTX). MTBE was not removed within 24 h by any of the consortia examined. One consortium enriched from activated sludge (AAE-A2), degraded PAE, including (butyl-benzyl)phthalate (BBP), and di-(butyl)phthalate (DBP). PAE have not previously been described as substrates for methanotrophic consortia. The apparent K_m and V_max for DBP degradation by AAE-A2 at 20 °C was 3.1 ± 1.2 mg l^–1 and 8.7 ± 1.1 mg DBP (g protein × h)^–1, respectively. AAE-A2 also showed fast degradation of BTX (230 ± 30 nmol benzene (mg protein × h)^–1 at 20 °C). Additionally, AAE-A2 degraded benzene continuously for 2 weeks. In contrast, a pure culture of the methanotroph Methylosinus trichosporium OB3b ceased benzene degradation after only 2 days. Experiments with methane mono-oxygenase inhibitors or competitive substrates suggested that BTX degradation was carried out by methane-oxidizing bacteria in the consortium, whereas the degradation of PAE was carried out by non-methanotrophic bacteria co-existing with methanotrophs. The composition of the consortium (AAE-A2) based on polar lipid fatty acid (PLFA) profiles showed dominance of type II methanotrophs (83–92% of biomass). Phylogeny based on a 16S-rRNA gene clone library revealed that the dominating methanotrophs belonged to Methylosinus/Methylocystis spp. and that members of at least 4 different non-methanotrophic genera were present (Pseudomonas, Flavobacterium, Janthinobacterium and Rubivivax).     

Cr(VI) reduction by sulfidogenic and nonsulfidogenic microbial consortia

In time course experiments, bacterial community compositions were compared between a sulfidogenic and two nonsulfidogenic Cr(VI)-reducing consortia enriched from metal-contaminated sediments. The consortia were subjected to 0 and 0.85 mM or 1.35 mM Cr(VI), and Cr(VI) reduction, growth, and denaturing gradient gel electrophoresis profiles of PCR products of small-subunit (16S) ribosomal genes were compared. Results showed that although Cr(VI) was completely reduced by the three consortia, Cr(VI) inhibited cell growth, with sulfate-reducing bacteria being particularly sensitive to Cr(VI) toxicity relative to other bacteria in the consortia.     

Anaerobic biodegradation of BTEX and gasoline in various aquatic sediments

We examined the extent of biodegradation of benzene, toluene, ethylbenzene and the three isomers of xylene (BTEX) as a mixture and from gasoline in four different sediments: the New York/New Jersey Harbor estuary (polluted); Tuckerton, N.J. (pristine); Onondaga Lake, N.Y. (polluted) and Blue Mtn. Lake, N.Y. (pristine). Enrichment cultures were established with each sediment using denitrifying, sulfidogenic, methanogenic and iron reducing media, as well as site water. BTEX loss, as measured by GC-FID, was extensive in the sediments which had a long history of pollution, with all compounds being utilized within 21–91 days in the most active cultures, and was very slight or non-existent in the pristine sediments. Also, the pattern of loss was different under the various reducing conditions within each sediment and between sediments. For example benzene loss was only observed in sulfidogenic cultures from the NY/NJ Harbor sediments while toluene was degraded under all redox conditions. The loss of BTEX was correlated to the reduction of the various electron acceptors. In cultures amended with gasoline the degradation was much slower and incomplete. These results show that the fate of the different BTEX components in anoxic sediments is dependent on the prevailing redox conditions as well as on the characteristics and pollution history of the sediment.     

Epiphytic microorganisms degrading aromatic hydrocarbons from the phyllosphere of urban woody plants

From the leaves of three urban trees (Tilia sp., Acer sp., and Fraxinus sp.), 180 strains degrading phenanthrene, naphthalene, and salicylate were isolated by direct plating and enrichment cultures. The leaves of each tree species were characterized by a specific profile of aromatic hydrocarbon-degrading microflora. Members of the type Actinobacteria were predominant in the case of direct plating on media with phenanthrene and naphthalene. Enrichment cultures with phenanthrene and salicylate were shown to yield microbial consortia, the composition of which changed with time. Members of the type Proteobacteria were predominant in these consortia. No plasmids of polycyclic aromatic hydrocarbon degradation of the P-7 and P-9 incompatibility groups were revealed in the studied strains.     

Effect of model sorptive phases on phenanthrene biodegradation: different enrichment conditions influence bioavailability and selection of phenanthrene-degrading isolates

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.     

Multiple lateral transfers of dissimilatory sulfite reductase genes between major lineages of sulfate-reducing prokaryotes

A large fragment of the dissimilatory sulfite reductase genes (dsrAB) was PCR amplified and fully sequenced from 30 reference strains representing all recognized lineages of sulfate-reducing bacteria. In addition, the sequence of the dsrAB gene homologs of the sulfite reducer Desulfitobacterium dehalogenans was determined. In contrast to previous reports, comparative analysis of all available DsrAB sequences produced a tree topology partially inconsistent with the corresponding 16S rRNA phylogeny. For example, the DsrAB sequences of several Desulfotomaculum species (low G+C gram-positive division) and two members of the genus Thermodesulfobacterium (a separate bacterial division) were monophyletic with delta-proteobacterial DsrAB sequences. The most parsimonious interpretation of these data is that dsrAB genes from ancestors of as-yet-unrecognized sulfate reducers within the delta-Proteobacteria were laterally transferred across divisions. A number of insertions and deletions in the DsrAB alignment independently support these inferred lateral acquisitions of dsrAB genes. Evidence for a dsrAB lateral gene transfer event also was found within the delta-Proteobacteria, affecting Desulfobacula toluolica. The root of the dsr tree was inferred to be within the Thermodesulfovibrio lineage by paralogous rooting of the alpha and beta subunits. This rooting suggests that the dsrAB genes in Archaeoglobus species also are the result of an ancient lateral transfer from a bacterial donor. Although these findings complicate the use of dsrAB genes to infer phylogenetic relationships among sulfate reducers in molecular diversity studies, they establish a framework to resolve the origins and diversification of this ancient respiratory lifestyle among organisms mediating a key step in the biogeochemical cycling of sulfur.     

Detection and enumeration of haloacetic acid-degrading bacteria in drinking water distribution systems using dehalogenase genes

Aims:  To develop a PCR-based tracking method for the detection of a subset of bacteria in drinking water distribution systems capable of degrading haloacetic acids (HAAs).
Methods and Results:  Published degenerate PCR primers were used to determine that 54% of tap water samples (7/13) were positive for a deh gene, indicating that drinking water distribution systems may harbour bacteria capable of HAA degradation. As the published primer sets were not sufficiently specific for quantitative PCR, new primers were designed to amplify deh II genes from selected indicator strains. The developed primer sets were effective in directly amplifying deh II genes from enriched consortia samples, and the DNA extracted from tap water provided that an additional nested PCR step for detection of the deh II gene was used.
Conclusions:  This study demonstrates that drinking water distribution systems harbour microbes capable of degrading HAAs. In addition, a quantitative PCR method was developed to detect and quantify deh II genes in drinking water systems.
Significance and Impact of the Study:  The development of a technique to rapidly screen for the presence of dehalogenase genes in drinking water distribution systems could help water utilities determine if HAA biodegradation is occurring in the distribution system.     

Use of bromodeoxyuridine immunocapture to identify psychrotolerant phenanthrene-degrading bacteria in phenanthrene-enriched polluted Baltic Sea sediments

The aim of this study was to enrich and identify psychrotolerant phenanthrene-degrading bacteria from polluted Baltic Sea sediments. Polyaromatic hydrocarbon (PAH)-contaminated sediments were spiked with phenanthrene and incubated for 2 months in the presence of bromodeoxyuridine that is incorporated into the DNA of replicating cells. The bromodeoxyuridine-incorporated DNA was extracted by immunocapture and analyzed by terminal-restriction fragment length polymorphism and 16S rRNA gene cloning and sequencing to identify bacterial populations that were growing. In addition, degradation genes were quantified in the bromodeoxyuridine-incorporated DNA by real-time PCR. Phenanthrene concentrations decreased after 2 months of incubation in the phenanthrene-enriched sediments and this reduction correlated to increases in copy numbers of xylE and phnAc dioxygenase genes. Representatives of Exiguobacterium , Schewanella , Methylomonas, Pseudomonas , Bacteroides and an uncultured Deltaproteobacterium and a Gammaproteobacterium dominated the growing community in the phenanthrene-spiked sediments. Isolates that were closely related to three of these bacteria (two pseudomonads and an Exiguobacterium sp.) could reduce phenanthrene concentrations in pure cultures and they all harbored phnAc dioxygenase genes. These results confirm that this combination of culture-based and molecular approaches was useful for identification of actively growing bacterial species with a high potential for phenanthrene degradation.     

Characterisation of bacterial cultures enriched on the chlorophenoxyalkanoic acid herbicides 4-(2,4-dichlorophenoxy) butyric acid and 4-(4-chloro-2-methylphenoxy) butyric acid

The aim of this study was to enrich and characterise bacterial consortia from soils around a herbicide production plant through their capability to degrade the herbicides 4-(2,4-dichlorophenoxy) butyric acid (2,4-DB) and 4-(4-chloro-2-methylphenoxy) butyric acid (MCPB). Partial 16S rRNA gene sequencing revealed members of the genera Stenotrophomonas, Brevundimonas, Pseudomonas, and Ochrobactrum in the 2,4-DB- and MCPB-degrading communities. The degradation of 2,4-DB and MCPB was facilitated by the combined activities of the community members. Some of the members were able to utilise other herbicides from the family of chlorophenoxyalkanoic acids. During degradation of 2,4-DB and MCPB, phenol intermediates were detected, indicating ether cleavage of the side chain as the initial step responsible for the breakdown. This was also verified using an indicator medium. Repeated attempts to amplify putatively conserved tfd genes by PCR indicated the absence of tfd genes among the consortia members. First step cleavage of the chlorophenoxybutyric acid herbicides is by ether cleavage in bacteria and is encoded by divergent or different tfd gene types. The isolation of mixed cultures capable of degrading 2,4-DB and MCPB will aid future investigations to determine both the metabolic route for dissimilation and the fate of these herbicides in natural environments.     

Isolation and Characterization of Novel Hydrocarbon-Degrading Euryhaline Consortia from Crude Oil and Mangrove Sediments

Two novel and versatile bacterial consortia were developed for the biodegradation of hydrocarbons. They were isolated from crude oil from the Cormorant Field in the North Sea (MPD-7) and from sediment associated with mangrove roots (MPD-M). The bacterial consortia were able to degrade both aliphatic and aromatic hydrocarbons in crude oils very effectively in seawater (35 g/L NaCl) and synthetic media containing 0 to 100 g/L NaCl (1.7 M). Salinities over twice that of normal seawater decreased the biodegradation rates. However, even at the highest salinity biodegradation was significant. Ratios of nC17 to pristane and nC18 to phytane were significantly lowered across the range of salinity. The lowest values were at 0 and 20 g/L (0.34 M). Phytane was degraded in preference to pristane. The degradation of these compounds was constant over the salinity range, with evidence of a slight increase for consortium MPD-M with increasing salinity. In general, the consortium isolated from mangrove root sediments was more efficient in metabolizing North Sea crude oil than the consortium isolated from Cormorant crude oil. The 5 strains that comprise MPD-M have been tentatively identified as species of the genera Marinobacter, Bacillus, and Erwinia. This is the first report of hydrocarbon-degrading consortia isolated from crude oil and mangrove sediments that are capable of treating oily wastes over such a wide range of salinity. Received June 30, 1999; accepted May 29, 2000.     

Microbial Dioxygenase Gene Population Shifts during Polycyclic Aromatic Hydrocarbon Biodegradation

The degradation of polycyclic aromatic hydrocarbons (PAHs) by bacteria has been widely studied. While many pure cultures have been isolated and characterized for their ability to grow on PAHs, limited information is available on the diversity of microbes involved in PAH degradation in the environment. We have designed generic PCR primers targeting the gene fragment encoding the Rieske iron sulfur center common to all PAH dioxygenase enzymes. These Rieske primers were employed to track dioxygenase gene population shifts in soil enrichment cultures following exposure to naphthalene, phenanthrene, or pyrene. PAH degradation was monitored by gas chromatograph with flame ionization detection. DNA was extracted from the enrichment cultures following PAH degradation. 16S rRNA and Rieske gene fragments were PCR amplified from DNA extracted from each enrichment culture and an unamended treatment. The PCR products were cloned and sequenced. Molecular monitoring of the enrichment cultures before and after PAH degradation using denaturing gradient gel electrophoresis and 16S rRNA gene libraries suggests that specific phylotypes of bacteria were associated with the degradation of each PAH. Sequencing of the cloned Rieske gene fragments showed that different suites of genes were present in soil microbe populations under each enrichment culture condition. Many of the Rieske gene fragment sequences fell into clades which are distinct from the reference dioxygenase gene sequences used to design the PCR primers. The ability to profile not only the bacterial community but also the dioxygenases which they encode provides a powerful tool for both assessing bioremediation potential in the environment and for the discovery of novel dioxygenase genes.     

Microbial dioxygenase gene population shifts during polycyclic aromatic hydrocarbon biodegradation

The degradation of polycyclic aromatic hydrocarbons (PAHs) by bacteria has been widely studied. While many pure cultures have been isolated and characterized for their ability to grow on PAHs, limited information is available on the diversity of microbes involved in PAH degradation in the environment. We have designed generic PCR primers targeting the gene fragment encoding the Rieske iron sulfur center common to all PAH dioxygenase enzymes. These Rieske primers were employed to track dioxygenase gene population shifts in soil enrichment cultures following exposure to naphthalene, phenanthrene, or pyrene. PAH degradation was monitored by gas chromatograph with flame ionization detection. DNA was extracted from the enrichment cultures following PAH degradation. 16S rRNA and Rieske gene fragments were PCR amplified from DNA extracted from each enrichment culture and an unamended treatment. The PCR products were cloned and sequenced. Molecular monitoring of the enrichment cultures before and after PAH degradation using denaturing gradient gel electrophoresis and 16S rRNA gene libraries suggests that specific phylotypes of bacteria were associated with the degradation of each PAH. Sequencing of the cloned Rieske gene fragments showed that different suites of genes were present in soil microbe populations under each enrichment culture condition. Many of the Rieske gene fragment sequences fell into clades which are distinct from the reference dioxygenase gene sequences used to design the PCR primers. The ability to profile not only the bacterial community but also the dioxygenases which they encode provides a powerful tool for both assessing bioremediation potential in the environment and for the discovery of novel dioxygenase genes.     

Characterization of monooxygenase gene diversity in benzene‐amended soils

Aim: To understand soil benzene monooxygenase gene diversity by clone library construction and microarray profiling. Methods and Results: A primer set was designed, and benzene monooxygenase gene diversity was characterized in two benzene‐amended soils. The dominant sequence types in the clone libraries were distinct between the two soils, and both sequences were assigned to novel clusters. Monooxygenase gene richness and diversity increased after benzene degradation. Oligonucleotide probes for microarray analysis were designed to detect a number of sequenced clones and reported monooxygenase genes. The microarray detected several genes that were not detected in the clone libraries of the same samples. Six probes were detected in more than one soil. Conclusions: The primer set designed in this study successfully detected diverse benzene monooxygenase genes. The level of diversity may have increased because the degradation of benzene differed from soil to soil. Microarrays have great potential in the comprehensive detection of gene richness as well as the elucidation of key genes for degradation. Significance and Impact of the Study: This study introduces a new primer set that may be used to identify diverse benzene monooxygenase genes in the environment; moreover, it demonstrates the potential of microarray technology in the profiling of environmental samples.     

Molecular characterization of sulfate-reducing bacteria in a New England salt marsh

Sulfate reduction, mediated by sulfate-reducing bacteria (SRB), is the dominant remineralization pathway in sediments of New England salt marshes. High sulfate reduction rates are associated with the rhizosphere of Spartina alterniflora when plants elongate aboveground. The growth process concurrently produces significant amounts of new rhizome material belowground and the plants leak dissolved organic compounds. This study investigated the diversity of SRB in a salt marsh over an annual growth cycle of S. alterniflora by exploring the diversity of a functional gene, dissimilatory sulfite reductase (dsrAB). Because the dsrAB gene is a key gene in the anaerobic sulfate-respiration pathway, it allows the identification of microorganisms responsible for sulfate reduction. Conserved dsrAB primers in polymerase chain reaction (PCR) generated full-length dsrAB amplicons for cloning and DNA sequence analysis. Nearly 80% of 380 clone sequences were similar to genes from Desulfosarcina and Desulfobacterium species within Desulfobacteraceae. This reinforces the hypothesis that complete oxidizers with high substrate versatility dominate the marsh. However, the phylotypes formed several clades that were distinct from cultured representatives, indicating a greater diversity of SRB than previously appreciated. Several dsrAB sequences were related to homologues from gram-positive, thermophilic and non-thermophilic Desulfotomaculum species. One dsrAB lineage formed a sister group to cultured members of the delta-proteobacterial group Syntrophobacteraceae. A deeply branching dsrAB lineage was not affiliated with genes from any cultured SRB. The sequence data from this study will allow for the design of probes or primers that can quantitatively assess the diverse range of sulfate reducers present in the environment.     

Assessment of anaerobic benzene degradation potential using 16S rRNA gene-targeted real-time PCR

Benzene is a common groundwater pollutant that is often recalcitrant under the anaerobic conditions that prevail at hydrocarbon-contaminated aquifers. Thus, determining the potential for anaerobic benzene degradation is important to assess the feasibility of intrinsic bioremediation. In this work we developed a 16S rRNA biomarker to estimate the concentration of putative benzene degraders in a methanogenic consortium that has been enriched on benzene for several years. Primers were designed based on phylogenetic information from this consortium. The primers and probe were obtained by sequencing the dominant denaturing gradient gel electrophoresis band of this consortium, which corresponded to Desulfobacterium sp. clone OR-M2. No hybridization was observed with DNA samples from negative controls (i.e. toluene-degrading and dehalorespiring methanogenic consortia that do not degrade benzene). Samples from an anaerobic aquifer column that was bioaugmented with this benzene-degrading consortium showed a strong correlation between benzene degradation activity and the concentration of the target organism. Although our data do not prove that Desulfobacterium sp. is a benzene degrader, its enrichment as a result of benzene consumption and its correlation to anaerobic benzene degradation activity suggest that it either initiates benzene degradation or is a critical (commensal) partner. Therefore, the utility of this primers and probe set to assess anaerobic benzene degradation potential was demonstrated. This is the first report of the use of real-time quantitative PCR for forensic analysis of anaerobic benzene degradation. Whether this biomarker will be adequately selective and broadly applicable to assess benzene degradation potential under strongly anaerobic (sulfate reducing and methanogenic) conditions will require further research.