Further Biogeochemical Characterization of a Trichloroethene-Contaminated Fractured Dolomite Aquifer:  Electron Source and Microbial Communities Involved in Reductive Dechlorination

112

115

Sodium lactate additions to a trichloroethene (TCE) residual source area in deep, fractured basalt at a U.S. Department of Energy site have resulted in the enrichment of the indigenous microbial community, the complete dechlorination of nearly all aqueous-phase TCE to ethene, and the continued depletion of the residual source since 1999. The bacterial and archaeal consortia in groundwater obtained from the residual source were assessed by using PCR-amplified 16S rRNA genes. A clone library of bacterial amplicons was predominated by those from members of the class Clostridia (57 of 93 clones), of which a phylotype most similar to that of the homoacetogen Acetobacterium sp. strain HAAP-1 was most abundant (32 of 93 clones). The remaining Bacteria consisted of phylotypes affiliated with Sphingobacteria, Bacteroides, Spirochaetes, Mollicutes, and Proteobacteria and candidate divisions OP11 and OP3. The two proteobacterial phylotypes were most similar to those of the known dechlorinators Trichlorobacter thiogenes and Sulfurospirillum multivorans. Although not represented by the bacterial clones generated with broad-specificity bacterial primers, a Dehalococcoides-like phylotype was identified with genus-specific primers. Only four distinct phylotypes were detected in the groundwater archaeal library, including predominantly a clone affiliated with the strictly acetoclastic methanogen Methanosaeta concilii (24 of 43 clones). A mixed culture that completely dechlorinates TCE to ethene was enriched from this groundwater, and both communities were characterized by terminal restriction fragment length polymorphism (T-RFLP). According to T-RFLP, the laboratory enrichment community was less diverse overall than the groundwater community, with 22 unique phylotypes as opposed to 43 and a higher percentage of Clostridia, including the Acetobacterium population. Bioreactor archaeal structure was very similar to that of the groundwater community, suggesting that methane is generated primarily via the acetoclastic pathway, using acetate generated by lactate fermentation and acetogenesis in both systems.Inadequate disposal practices of chlorinated solvents have resulted in the widespread contamination of groundwater with trichloroethene (TCE), a known toxin and suspected carcinogen. In situ biostimulation via anaerobic microbial reductive dechlorination is an attractive remediation strategy for TCE due to low operation and maintenance costs and minimal secondary-waste production relative to traditional extraction methods. Direct addition of nutrients such as electron donors to the contaminated subsurface can stimulate the activity of indigenous microbial communities. After oxygen is consumed, anaerobic populations reduce alternative electron acceptors such as nitrate, ferric iron, sulfate, and carbon dioxide (10). Chloroethenes can also be used as terminal electron acceptors (37,41,48,49), a process known as dehalorespiration, whereby hydrogen atoms sequentially replace chlorine atoms, leading to detoxification of the chloroethene...

Section: Discussionmentioning

confidence: 99%

Molecular Characterization of a Dechlorinating Community Resulting from In Situ Biostimulation in a Trichloroethene-Contaminated Deep, Fractured Basalt Aquifer and Comparison to a Derivative Laboratory Culture

Macbeth

Cummings

Spring

et al. 2004

112

115

“…Among the motivations for this study was a desire to understand the mechanism by which naturally occurring microbial populations at a coal tar-contaminated field site (2,20,23,33) adapt to introduced organic substrates. Previously it has been shown that Cg1-like plasmids, involved in horizontal transfer of naphthalene catabolic genes between bacteria at the study site, are homologous to the archetypal naphthalene catabolic plasmid pDTG1 from P. putida NCIB 9816-4 (despite some variability in overall plasmid sizes [e.g., pDTG1, 81 kb; and pCg1, 86 kb {52}]).…”

Section: Discussionmentioning

confidence: 99%

Identification and Characterization of the Conjugal Transfer Region of the pCg1 plasmid from Naphthalene-Degrading Pseudomonas putida Cg1

Park

Jeon

Hohnstock-Ashe

et al. 2003

Appl. Environ. Microbiol. 64:3633-3640, 1998) have shown that pCg1, a naphthalene catabolic plasmid carried by Pseudomonas putida Cg1, is homologous to the archetypal naphthalene catabolic plasmid, pDTG1, in P. putida NCIB 9816-4. Sequencing of the latter plasmid allowed PCR primers to be designed for amplifying and sequencing the conjugal transfer region in pCg1. The mating pair formation (mpf) gene, mpfA encoding the putative precursor of the conjugative pilin subunit from pCg1, was identified along with other trb-like mpf genes. Sequence comparison revealed that the 10 mpf genes in pCg1 and pDTG1 are closely related (61 to 84% identity) in sequence and operon structure to the putative mpf genes of catabolic plasmid pWW0 (TOL plasmid of P. putida) and pM3 (antibiotic resistance plasmid of Pseudomonas. spp). A polar mutation caused by insertional inactivation in mpfA of pCg1 and reverse transcriptase PCR analysis of mRNA showed that this mpf region was involved in conjugation and was transcribed from a promoter located upstream of an open reading frame adjacent to mpfA. lacZ transcriptional fusions revealed that mpf genes of pCg1 were expressed constitutively both in liquid and on solid media. This expression did not respond to host exposure to naphthalene. Conjugation frequency on semisolid media was consistently 10-to 100-fold higher than that in liquid media. Thus, conjugation of pCg1 in P. putida Cg1 was enhanced by expression of genes in the mpf region and by surfaces where conditions fostering stable, high-density cell-to-cell contact are manifest.

“…Groundwater was obtained from well 36 in October 2001 and used for biodegradation assays. To prevent contact between water samples and air (25,78), the receiving glass canning jars (2 liters for water samples and 0.5 liter for sediments) were flushed on site with nitrogen gas and, after they were filled, sealed without air bubbles. Freshly gathered, oxygen-free subsurface material (depth, 5 m) from an area adjacent to well 36 was obtained with a commercially operated Geoprobe hydraulic coring machine.…”

Section: Methodsmentioning

confidence: 99%

“…The sediment was immersed in anaerobic groundwater before it was sealed in a canning jar with no headspace air. The groundwater and sediments were placed on ice and maintained at 4°C until they were dispensed into serum bottles with an N 2 headspace containing a trace of H 2 in an anaerobic hood (Coy Laboratory Products, Grass Lake, MI) within 2 days, as previously described (25,78). Triplicate sterile 125-ml serum bottles received 72 ml of well 36 groundwater plus 10% (wt/vol) well 36 sediment from 2 m below the water table.…”

Section: Methodsmentioning

confidence: 99%

Subsurface Cycling of Nitrogen and Anaerobic Aromatic Hydrocarbon Biodegradation Revealed by Nucleic Acid and Metabolic Biomarkers

Yagi

Suflita

Gieg

et al. 2010

Microbial processes are crucial for ecosystem maintenance, yet documentation of these processes in complex open field sites is challenging. Here we used a multidisciplinary strategy (site geochemistry, laboratory biodegradation assays, and field extraction of molecular biomarkers) to deduce an ongoing linkage between aromatic hydrocarbon biodegradation and nitrogen cycling in a contaminated subsurface site. Three site wells were monitored over a 10-month period, which revealed fluctuating concentrations of nitrate, ammonia, sulfate, sulfide, methane, and other constituents. Biodegradation assays performed under multiple redox conditions indicated that naphthalene metabolism was favored under aerobic conditions. To explore in situ field processes, we measured metabolites of anaerobic naphthalene metabolism and expressed mRNA transcripts selected to document aerobic and anaerobic microbial transformations of ammonia, nitrate, and methylated aromatic contaminants. Gas chromatography-mass spectrometry detection of two carboxylated naphthalene metabolites and transcribed benzylsuccinate synthase, cytochrome c nitrite reductase, and ammonia monooxygenase genes indicated that anaerobic metabolism of aromatic compounds and both dissimilatory nitrate reduction to ammonia (DNRA) and nitrification occurred in situ. These data link formation (via DNRA) and destruction (via nitrification) of ammonia to in situ cycling of nitrogen in this subsurface habitat, where metabolism of aromatic pollutants has led to accumulation of reduced metabolic end products (e.g., ammonia and methane).Nonphotosynthetic microorganisms (particularly members of the Archaea and Bacteria) colonizing natural habitats generate metabolic energy by linking the transfer of electrons from reduced substrates (electron donors; e.g., ammonia, methane, sulfide, carbohydrates, and hydrocarbons) to oxidized substrates (electron acceptors; e.g., O 2 , nitrate, Fe 3ϩ , and sulfate) (18,40,62,73). Whenever possible, strategies for documenting biogeochemical change involve mass balance approaches that quantitatively link materials subject to a given metabolic process (e.g., consumption of carbon substrates) to formation of metabolic by-products (e.g., CO 2 ). However, owing to the open nature of many natural systems (including ocean water, rivers, and soils), convergent lines of evidence obtained using a variety of approaches (e.g., model incubations, analytical chemistry of metabolites, and molecular biology of genes and mRNA) are often needed to understand site biogeochemistry (40,46,71). Direct detection of mRNA in environmental samples has increasingly become an effective approach for documenting the in situ biogeochemical activity of microbial communities in field sites (33,34,42). This approach has included at least three techniques: (i) reverse transcription-PCR (RT-PCR)-based targeting of expression of specific functional genes (e.g., genes encoding naphthalene dioxygenase [74], Fe(II) uptake protein [47], RubisCo [70], or the anammox and denitrification pro...