Benjamin K. Amos

Quantitative PCR Targeting 16S rRNA and Reductive Dehalogenase Genes Simultaneously Monitors Multiple Dehalococcoides Strains

ABSTRACT The 16S rRNA gene provides insufficient information to infer the range of chloroorganic electron acceptors used by different Dehalococcoides organisms. To overcome this limitation and provide enhanced diagnostic tools for growth measurements, site assessment, and bioremediation monitoring, a quantitative real-time PCR (qPCR) approach targeting 16S rRNA genes and three Dehalococcoides reductive dehalogenase (RDase) genes with assigned function (i.e., tceA, bvcA, and vcrA) was designed and evaluated. qPCR standard curves generated for the RDase genes by use of genomic DNA from Dehalococcoides pure cultures correlated with standard curves obtained for both Bacteria- and Dehalococcoides-targeted 16S rRNA genes, suggesting that the RDase genes are useful targets for quantitative assessment of Dehalococcoides organisms. RDase gene probe/primer pairs were specific for the Dehalococcoides strains known to carry the diagnostic RDase gene sequences, and the qPCR method allowed the detection of as few as 1 to 20 and quantification of as few as 50 to 100 tceA, bvcA, or vcrA gene targets per PCR volume. The qPCR approach was applied to dechlorinating enrichment cultures, microcosms, and samples from a contaminated site. In characterized enrichment cultures where known Dehalococcoides strains were enumerated, the sum of the three RDase genes equaled the total Dehalococcoides cell numbers. In site samples and chloroethane-dechlorinating microcosms, the sum of the three RDase genes was much less than that predicted by Dehalococcoides-targeted qPCR, totaling 10 to 30% of the total Dehalococcoides cell numbers. Hence, a large number of Dehalococcoides spp. contain as-yet-unidentified RDase genes, indicating that our current understanding of the dechlorinating Dehalococcoides community is incomplete.

Microbial activity and distribution during enhanced contaminant dissolution from a NAPL source zone

Laboratory experiments were conducted to assess microbial reductive dechlorination in one-dimensional sand columns containing a 10 cm long source zone of uniformly distributed residual tetrachloroethene (PCE) nonaqueous phase liquid (NAPL), a 10 cm long transition zone directly down-gradient of the source zone containing some nonuniformly distributed NAPL ganglia, and a 40 cm long plume region down-gradient of the transition zone. The activity and distribution of Sulfurospirillum multivorans, a PCE-to-1,2-cis-dichloroethene (cis-DCE) dechlorinating bacterium, was evaluated in columns containing either a mixed-NAPL (0.25 mol/mol PCE in hexadecane) or pure PCE-NAPL. Significant dechlorination of PCE to cis-DCE was observed in the mixed-NAPL column, resulting in 53% PCE-NAPL mass recovery in the effluent with PCE-NAPL dissolution enhanced by up to 13.6-fold (maximum) and 4.6-fold (cumulative) relative to abiotic dissolution. Quantitative real-time PCR targeting pceA, the PCE reductive dehalogenase gene of S. multivorans, revealed that S. multivorans cells were present in the NAPL source zone, and increased in numbers (i.e., grew) throughout the source and transition zones. In contrast, minimal reductive dechlorination and microbial growth were observed in the column containing pure PCE-NAPL, where aqueous-phase PCE concentrations reached saturation. These results demonstrate that microbial growth within NAPL source zones is possible, provided that contaminant concentrations remain below levels toxic to the dechlorinating organisms, and that microbial growth can result in significant bioenhanced NAPL dissolution.

Detection and Quantification of Geobacter lovleyi Strain SZ: Implications for Bioremediation at Tetrachloroethene- and Uranium-Impacted Sites

ABSTRACT Geobacter lovleyi strain SZ reduces hexavalent uranium, U(VI), to U(IV) and is the first member of the metal-reducing Geobacter group capable of using tetrachloroethene (PCE) as a growth-supporting electron acceptor. Direct and nested PCR with specific 16S rRNA gene-targeted primer pairs distinguished strain SZ from other known chlorinated ethene-dechlorinating bacteria and closely related Geobacter isolates, including its closest cultured relative, G. thiogenes. Detection limits for direct and nested PCR were approximately 1 × 106 and 1 × 104 16S rRNA gene copies per μl of template DNA, respectively. A quantitative real-time PCR (qPCR) approach increased the sensitivity to as few as 30 16S rRNA gene copies per μl of template DNA but was less specific. Melting curve analysis and comparison of the shapes of amplification plots identified false-positive signals and distinguished strain SZ from G. thiogenes when analyzed separately. These indicators were less reliable when target (strain SZ) DNA and nontarget (G. thiogenes) DNA with high sequence similarity were mixed, indicating that the development of qPCR protocols should not only evaluate specificity but also explore the effects of nontarget DNA on the accuracy of quantification. Application of specific tools detected strain SZ-like amplicons in PCE-dechlorinating consortia, including the bioaugmentation consortium KB-1, and two chlorinated ethene-impacted groundwater samples. Strain SZ-like amplicons were also detected in 13 of 22 groundwater samples following biostimulation at the uranium- and chlorinated solvent-contaminated Integrated Field-Scale Subsurface Research Challenge (IFC) site in Oak Ridge, TN. The numbers of strain SZ-like cells increased from below detection to 2.3 × 107 ± 0.1 × 107 per liter groundwater, suggesting that strain SZ-like organisms contribute to contaminant transformation. The G. lovleyi strain SZ-specific tools will be useful for monitoring bioremediation efforts at uranium- and/or chlorinated solvent-impacted sites such as the Oak Ridge IFC site.

Unique ecophysiology among U(VI)-reducing bacteria as revealed by evaluation of oxygen metabolism in Anaeromyxobacter dehalogenans strain 2CP-C.

ABSTRACT Anaeromyxobacter spp. respire soluble hexavalent uranium, U(VI), leading to the formation of insoluble U(IV), and are present at the uranium-contaminated Oak Ridge Integrated Field Research Challenge (IFC) site. Pilot-scale in situ bioreduction of U(VI) has been accomplished in area 3 of the Oak Ridge IFC site following biostimulation, but the susceptibility of the reduced material to oxidants (i.e., oxygen) compromises long-term U immobilization. Following oxygen intrusion, attached Anaeromyxobacter dehalogenans cells increased approximately 5-fold from 2.2 × 107 ± 8.6 × 106 to 1.0 × 108 ± 2.2 × 107 cells per g of sediment collected from well FW101-2. In the same samples, the numbers of cells of Geobacter lovleyi, a population native to area 3 and also capable of U(VI) reduction, decreased or did not change. A. dehalogenans cells captured via groundwater sampling (i.e., not attached to sediment) were present in much lower numbers (<1.3 × 104 ± 1.1 × 104 cells per liter) than sediment-associated cells, suggesting that A. dehalogenans cells occur predominantly in association with soil particles. Laboratory studies confirmed aerobic growth of A. dehalogenans strain 2CP-C at initial oxygen partial pressures (pO2) at and below 0.18 atm. A negative linear correlation [μ = (−0.09 × pO2) + 0.051; R2 = 0.923] was observed between the instantaneous specific growth rate μ and pO2, indicating that this organism should be classified as a microaerophile. Quantification of cells during aerobic growth revealed that the fraction of electrons released in electron donor oxidation and used for biomass production (fs) decreased from 0.52 at a pO2 of 0.02 atm to 0.19 at a pO2 of 0.18 atm. Hence, the apparent fraction of electrons utilized for energy generation (i.e., oxygen reduction) (fe) increased from 0.48 to 0.81 with increasing pO2, suggesting that oxygen is consumed in a nonrespiratory process at a high pO2. The ability to tolerate high oxygen concentrations, perform microaerophilic oxygen respiration, and preferentially associate with soil particles represents an ecophysiology that distinguishes A. dehalogenans from other known U(VI)-reducing bacteria in area 3, and these features may play roles for stabilizing immobilized radionuclides in situ.

Microbially enhanced dissolution and reductive dechlorination of PCE by a mixed culture: Model validation and sensitivity analysis

Reductive dechlorination catalyzed by organohalide-respiring bacteria is often considered for remediation of non-aqueous phase liquid (NAPL) source zones due to cost savings, ease of implementation, regulatory acceptance, and sustainability. Despite knowledge of the key dechlorinators, an understanding of the processes and factors that control NAPL dissolution rates and detoxification (i.e., ethene formation) is lacking. A recent column study demonstrated a 5-fold cumulative enhancement in tetrachloroethene (PCE) dissolution and ethene formation (Amos et al., 2009). Spatial and temporal monitoring of key geochemical and microbial (i.e., Geobacter lovleyi and Dehalococcoides mccartyi strains) parameters in the column generated a data set used herein as the basis for refinement and testing of a multiphase, compositional transport model. The refined model is capable of simulating the reactive transport of multiple chemical constituents produced and consumed by organohalide-respiring bacteria and accounts for substrate limitations and competitive inhibition. Parameter estimation techniques were used to optimize the values of sensitive microbial kinetic parameters, including maximum utilization rates, biomass yield coefficients, and endogenous decay rates. Comparison and calibration of model simulations with the experimental data demonstrate that the model is able to accurately reproduce measured effluent concentrations, while delineating trends in dechlorinator growth and reductive dechlorination kinetics along the column. Sensitivity analyses performed on the optimized model parameters indicate that the rates of PCE and cis-1,2-dichloroethene (cis-DCE) transformation and Dehalococcoides growth govern bioenhanced dissolution, as long as electron donor (i.e., hydrogen flux) is not limiting. Dissolution enhancements were shown to be independent of cis-DCE accumulation; however, accumulation of cis-DCE, as well as column length and flow rate (i.e., column residence time), strongly influenced the extent of reductive dechlorination. When cis-DCE inhibition was neglected, the model over-predicted ethene production ten-fold, while reductions in residence time (i.e., a two-fold decrease in column length or two-fold increase in flow rate) resulted in a more than 70% decline in ethene production. These results suggest that spatial and temporal variations in microbial community composition and activity must be understood to model, predict, and manage bioenhanced NAPL dissolution.