Strain SZ Research Articles

Pathogenicity and genome changes in QX-like infectious bronchitis virus during continuous passaging in embryonated chicken eggs

Infectious bronchitis (IB) remains a major problem in the global poultry industry despite the many available vaccines. Live attenuated vaccines are the most effective means of preventing IB and are traditionally generated by serial passaging of a wild strain in embryonated chicken eggs. In this study, the SZ isolate of the QX-like infectious bronchitis virus (IBV) was continuously passaged in chicken embryos for 250 passages. We compared the pathogenicity of different passages (SZ50, SZ100, SZ150, SZ200 and SZ250) of strain SZ by clinical signs, gross lesions, viral load, tissue tropism, weight gain and tracheal ciliary activity. As the passaging increased in the chicken embryos, the strain lost its ability to infect many organs, and the viral pathogenicity gradually decreased. We also found 23 genomic variations of the QX-like strain SZ throughout the passaging process by further analyzing its complete genome sequence. This work offers valuable insight for IBV vaccine development and further research on the IBV attenuation mechanisms.

Nitrous Oxide Is a Potent Inhibitor of Bacterial Reductive Dechlorination

Organohalide-respiring bacteria are key players for the turnover of organohalogens. At sites impacted with chlorinated ethenes, bioremediation promotes reductive dechlorination; however, stoichiometric conversion to environmentally benign ethene is not always achieved. We demonstrate that nitrous oxide (N2O), a compound commonly present in groundwater, inhibits organohalide respiration. N2O concentrations in the low micromolar range decreased dechlorination rates and resulted in incomplete dechlorination of tetrachloroethene (PCE) in Geobacter lovleyi strain SZ and of cis-1,2-dichloroethene ( cDCE) and vinyl chloride (VC) in Dehalococcoides mccartyi strain BAV1 axenic cultures. Presumably, N2O interferes with reductive dechlorination by reacting with super-reduced Co(I)-corrinoids of reductive dehalogenases, which is supported by the finding that N2O did not inhibit corrinoid-independent fumarate-to-succinate reduction in strain SZ. Kinetic analyses revealed a best fit to the noncompetitive Michaelis-Menten inhibition model and determined N2O inhibitory constants, KI, for PCE and cDCE dechlorination of 40.8 ± 3.8 and 21.2 ± 3.5 μM in strain SZ and strain BAV1, respectively. The lowest KI value of 9.6 ± 0.4 μM was determined for VC to ethene reductive dechlorination in strain BAV1, suggesting that this crucial dechlorination step for achieving detoxification is most susceptible to N2O inhibition. Groundwater N2O concentrations exceeding 100 μM are not uncommon, especially in watersheds impacted by nitrate runoff from agricultural sources. Thus, dissolved N2O measurements can inform about cDCE and VC stalls at sites impacted with chlorinated ethenes.

Epoxidised natural rubber–polyaniline dodecylbenzenesulfonate (ENR-PAni.DBSA) blends with adjustable electrostrictive properties

Sulphur-vulcanised epoxidised natural rubber–polyaniline dodecylbenzenesulfonate (ENR-PAni.DBSA) blends with useful electrical conductivities (up to 10−1 S/cm) and adjustable electrostrictive behaviour were successfully produced via a thermomechanical mixing method. A reasonably high level of compatibility between ENR and PAni.DBSA was observed from both Fourier transform infrared spectroscopy and transmission electron microscopy studies. As found from both electrostrictive strain ( Sz) tests, ENR-PAni.DBSA blends with 10.0–30.0 wt% of PAni.DBSA content possessed the best electrostrictive response ( Sz up to 12.25%) with applied electric field strengths up to 50 V/µm. Apart from this, blends with very low and very high PAni.DBSA contents (i.e. ≤5.0 wt% and ≥40.0 wt%) had much poorer electrostrictive responses under the same level of applied electrical field, which is due to their lower dielectric properties and higher level of phase separation between ENR and PAni.DBSA.

Electrostrictive Properties of Poly(butadiene-co-acrylonitrile)-Polyaniline Dodecylbenzenesulfonate [NBR-PAni.DBSA] Blends: Effects of Conductive Filler Loading

Peroxide-vulcanised NBR-PAni. DBSA blends with useful electrical conductivities (up to 10−1 S. cm−1) were prepared and their electrostrictive behaviours under certain levels of applied electric field were also successfully assessed. A reasonable high level of compatibility between NBR and PAni. DBSA was observed from both FT-IR spectroscopy and TEM microscopy studies. As found from both electrostrictive strain (Sz) tests, NBR-PAni. DBSA blends with 10.0–30.0 wt% of PAni.DBSA content possessed the best electrostrictive response (Sz up to 11.8%) with applied electrical field strengths up to 50 V/μm. Besides this, blends consisting of very low and very high PAni.DBSA contents (i.e. ≤ 5. 0 wt % and ≥ 40. 0 wt %) possessed poorer electrostrictive responses under the same level of applied electrical field due to their lower dielectrical properties and higher phase separation problem between the two major constituent polymers.

Electrochemical characterization of Geobacter lovleyi identifies limitations of microbial fuel cell performance in constructed wetlands.

Power generation in microbial fuel cells implemented in constructed wetlands (CW-MFCs) is low despite the enrichment of anode electricigens most closely related to Geobacter lovleyi. Using the model representative G. lovleyi strain SZ, we show that acetate, but not formate or lactate, can be oxidized efficiently but growth is limited by the high sensitivity of the bacterium to oxygen. Acetate and highly reducing conditions also supported the growth of anode biofilms but only at optimal anode potentials (450 mV vs. standard hydrogen electrode). Still, electrode coverage was poor and current densities, low, consistent with the lack of key c-type cytochromes. The results suggest that the low oxygen tolerance of G. lovleyi and inability to efficiently colonize and form electroactive biofilms on the electrodes while oxidizing the range of electron donors available in constructed wetlands limits MFC performance. The implications of these findings for the optimization of CW-MFCs are discussed. [Int Microbiol 20(2):55-64 (2017)].

Spatial and temporal dynamics of organohalide-respiring bacteria in a heterogeneous PCE–DNAPL source zone

Effective treatment of sites contaminated with dense non-aqueous phase liquids (DNAPLs) requires detailed understanding of the microbial community responses to changes in source zone strength and architecture. Changes in the spatial and temporal distributions of the organohalide-respiring Dehalococcoides mccartyi (Dhc) strains and Geobacter lovleyi strain SZ (GeoSZ) were examined in a heterogeneous tetrachloroethene- (PCE-) DNAPL source zone within a two-dimensional laboratory-scale aquifer flow cell. As part of a combined remedy approach, flushing with 2.3 pore volumes (PVs) of 4% (w/w) solution of the nonionic, biodegradable surfactant Tween® 80 removed 55% of the initial contaminant mass, and resulted in a PCE–DNAPL distribution that contained 51% discrete ganglia and 49% pools (ganglia-to-pool ratio of 1.06). Subsequent bioaugmentation with the PCE-to-ethene-dechlorinating consortium BDI-SZ resulted in cis-1,2-dichloroethene (cis-DCE) formation after 1 PV (ca. 7days), while vinyl chloride (VC) and ethene were detected 10 PVs after bioaugmentation. Maximum ethene yields (ca. 90μM) within DNAPL pool and ganglia regions coincided with the detection of the vcrA reductive dehalogenase (RDase) gene that exceeded the Dhc 16S rRNA genes by 2.0±1.3 and 4.0±1.7 fold in the pool and ganglia regions, respectively. Dhc and GeoSZ cell abundance increased by up to 4 orders-of-magnitude after 28 PVs of steady-state operation, with 1 to 2 orders-of-magnitude increases observed in close proximity to residual PCE–DNAPL. These observations suggest the involvement of these dechlorinators the in observed PCE dissolution enhancements of up to 2.3 and 6.0-fold within pool and ganglia regions, respectively. Analysis of the solid and aqueous samples at the conclusion of the experiment revealed that the highest VC (≥155μM) and ethene (≥65μM) concentrations were measured in zones where Dhc and GeoSZ were predominately attached to the solids. These findings demonstrate dynamic responses of organohalide-respiring bacteria in a heterogeneous DNAPL source zone, and emphasize the influence of source zone architecture on bioremediation performance.

A Robust Multilayer Dielectric Elastomer Actuator

ABSTRACTWe recently fabricated and characterized a new class of multilayer dielectric elastomer films comprising alternating layers of two different polymers, at least one of which is an elastomer. The films discussed here contain THV (a terpolymer of poly(vinylidene fluoride)) and poly(ethylene octene) [EO] elastomer. The multilayer structure provides improved dielectric and electromechanical performance relative to monolithic films of THV or EO. These properties are controlled by the composition and the layer structure. For example, increasing the concentration of the elastomeric EO component increases the maximum axial strain (sz). Layering EO with THV also increases the breakdown strength (EB ∼ 265 - 300 V/µm) relative to monolithic EO (EB ∼ 150 V/µm) or THV (EB ∼ 245 V/µm) control films. This enhancement in breakdown strength is consistent with a barrier effect that is also observed in multilayer polymer capacitor films. The increase in breakdown strength allows 512-layer 75 vol% EO / 25 vol% THV films to achieve maximum axial strains of sz nearly 4%, higher than can be attained by either EO or THV films alone. In addition, layering reduces remnant strain and electromechanical hysteresis by limiting the effective field within the THV layers. The 75% EO/ 25% THV films show robust operational longevity with little loss in axial strain when subjected to repeated actuation at Emax = 225 V/µm (producing sz = 2.2%). Under these conditions, we observe 3,000 consecutive actuation cycles with no electrical breakdown. In comparison, single component EO control films undergo electrical breakdown at this field and THV control films survive only a few hundred actuation cycles under these conditions. The results demonstrate that multilayering is an effective technique to increase the dielectric strength of elastomer materials and in turn improve upon strain and operational longevity (repeated actuation cycles) characteristics.

Distribution of organohalide-respiring bacteria between solid and aqueous phases.

Contemporary microbial monitoring of aquifers relies on groundwater samples to enumerate nonattached cells of interest. One-dimensional column studies quantified the distribution of bacterial cells in solid and the aqueous phases as a function of microbial species, growth substrate availability and porous medium (i.e., Appling soil versus Federal Fine Ottawa sand with 0.75% and 0.01% [w/w] organic carbon, respectively). Without supplied growth substrates, effluent from columns inoculated with the tetrachloroethene- (PCE-) to-ethene-dechlorinating bacterial consortium BDI-SZ containing Dehalococcoides mccartyi (Dhc) strains and Geobacter lovleyi strain SZ (GeoSZ), or inoculated with Anaeromyxobacter dehalogenans strain W (AdehalW), captured 94-96, 81-99, and 73-84% of the Dhc, GeoSZ, and AdehalW cells, respectively. Cell retention was organism-specific and increased in the order Dhc < GeoSZ < AdehalW. When amended with 10 mM lactate and 0.11 mM PCE, aqueous samples accounted for 1.3-27 and 0.02-22% of the total Dhc and GeoSZ biomass, respectively. In Appling soil, up to three orders-of-magnitude more cells were associated with the solid phase, and attachment rate coefficients (katt) were consistently greater compared to Federal Fine sand. Cell-solid interaction energies ranged from -2.5 to 787 kT and were consistent with organism-specific deposition behavior, where GeoSZ and AdehalW exhibited greater attachment than Dhc cells. The observed disparities in microbial cell distributions between the aqueous and solid phases imply that groundwater analysis can underestimate the total cell abundance in the aquifer by orders-of-magnitude under conditions of growth and in porous media with elevated organic carbon content. The implications of these findings for monitoring chlorinated solvent sites are discussed.

Genome Sequence of Borrelia garinii Strain SZ, Isolated in China.

We announce the genome sequence of Borrelia garinii strain SZ, isolated from Dermacentor ticks collected in northeastern China. B. garinii strain SZ carries numerous plasmids, both 10 circular and 9 linear plasmids. The 902,487-bp linear chromosome (28.2% GC content) contains 820 open reading frames, 33 tRNAs, and 4 complete rRNAs. The plasmid cp32-10 contains one clustered regularly interspaced short palindromic repeat (CRISPR) with four repeats.

Chlorine isotope effects from isotope ratio mass spectrometry suggest intramolecular C-Cl bond competition in trichloroethene (TCE) reductive dehalogenation.

Chlorinated ethenes are prevalent groundwater contaminants. To better constrain (bio)chemical reaction mechanisms of reductive dechlorination, the position-specificity of reductive trichloroethene (TCE) dehalogenation was investigated. Selective biotransformation reactions (i) of tetrachloroethene (PCE) to TCE in cultures of Desulfitobacterium sp. strain Viet1; and (ii) of TCE to cis-1,2-dichloroethene (cis-DCE) in cultures of Geobacter lovleyi strain SZ were investigated. Compound-average carbon isotope effects were −19.0‰ ± 0.9‰ (PCE) and −12.2‰ ± 1.0‰ (TCE) (95% confidence intervals). Using instrumental advances in chlorine isotope analysis by continuous flow isotope ratio mass spectrometry, compound-average chorine isotope effects were measured for PCE (−5.0‰ ± 0.1‰) and TCE (−3.6‰ ± 0.2‰). In addition, position-specific kinetic chlorine isotope effects were determined from fits of reactant and product isotope ratios. In PCE biodegradation, primary chlorine isotope effects were substantially larger (by −16.3‰ ± 1.4‰ (standard error)) than secondary. In TCE biodegradation, in contrast, the product cis-DCE reflected an average isotope effect of −2.4‰ ± 0.3‰ and the product chloride an isotope effect of −6.5‰ ± 2.5‰, in the original positions of TCE from which the products were formed (95% confidence intervals). A greater difference would be expected for a position-specific reaction (chloride would exclusively reflect a primary isotope effect). These results therefore suggest that both vicinal chlorine substituents of TCE were reactive (intramolecular competition). This finding puts new constraints on mechanistic scenarios and favours either nucleophilic addition by Co(I) or single electron transfer as reductive dehalogenation mechanisms.

Electrostriction of Natural Rubber Latex/Carbon Black Nanocomposites

The purpose of this paper is to investigate an electrostrictive behavior of natural rubber (NR) and NR composites filled with carbon black (CB) nanopowders below percolation threshold. These NR elastomers present advantageous features such as a high productivity, elasticity, and ease of processing. In addition, such materials also exhibit the high induced strain and low young modulus for electrostrictive materials that can be used as actuators and energy harvesting. The NR and all composites were prepared by using solution casting method. The electrostrictive property of the composites was evaluated at low electric field (E 5 MV/m) by measuring the electric field induced strain Sz with the photonic displacement apparatus. The surface morphology of the samples was observed by the atomic force microscopy (AFM) and their electrical properties were analyzed as function of concentration and frequency in a range of 102105 Hz. The results show that the dielectric constant and the dielectric loss decrease when the frequency was increased. Moreover, the dielectric constant and the electrical conductivity strongly increase with increasing the CB contents, relate to interfacial charge distribution. While the dielectric loss slightly increases with increasing filler concentration. The electrostriction coefficient tended to increase with a higher CB loading. In comparison at CB 1 wt%, it was found that the electrostriction coefficient of NR composites is approximately 7 times larger than the pure NR. The NR nanocomposites thus seem to be very attractive for low frequency electromechanical applications.

Pathogenic analysis of Borrelia garinii strain SZ isolated from northeastern China

BackgroundVarious genospecies of Borrelia burgdorferi sensu lato (s.l.) have been identified from patients and animals worldwide. Genospecies-related dissemination of disease has been reported. The present study aimed to elucidate the pathogenicity of infections caused by B. garinii SZ isolated in China. B. burgdorferi B31 and B. afzelii BO23 were used for comparison.MethodsSpirochete load in blood and tissue samples of infected mice were measured by minor groove binder-based real-time polymerase chain reaction. The kinetics of spirochete dissemination and disease severity were assessed in BALB/c mice.ResultsThe pattern of bacterial load differed between the three genospecies. The B. garinii SZ strain is highly pathogenic and can trigger multi-system pathological damage in mice.ConclusionsSpirochete dissemination, persistence, tissue tropism and disease severity varied significantly, suggesting that different genospecies may play an important role in the pathogenicity and development of clinical diseases.

Reductive Dechlorination of TCE by Chemical Model Systems in Comparison to Dehalogenating Bacteria: Insights from Dual Element Isotope Analysis (13C/12C, 37Cl/35Cl)

Chloroethenes like trichloroethene (TCE) are prevalent environmental contaminants, which may be degraded through reductive dechlorination. Chemical models such as cobalamine (vitamin B12) and its simplified analogue cobaloxime have served to mimic microbial reductive dechlorination. To test whether in vitro and in vivo mechanisms agree, we combined carbon and chlorine isotope measurements of TCE. Degradation-associated enrichment factors ε(carbon) and ε(chlorine) (i.e., molecular-average isotope effects) were -12.2‰ ± 0.5‰ and -3.6‰ ± 0.1‰ with Geobacter lovleyi strain SZ; -9.1‰ ± 0.6‰ and -2.7‰ ± 0.6‰ with Desulfitobacterium hafniense Y51; -16.1‰ ± 0.9‰ and -4.0‰ ± 0.2‰ with the enzymatic cofactor cobalamin; -21.3‰ ± 0.5‰ and -3.5‰ ± 0.1‰ with cobaloxime. Dual element isotope slopes m = Δδ(13)C/ Δδ(37)Cl ≈ ε(carbon)/ε(chlorine) of TCE showed strong agreement between biotransformations (3.4 to 3.8) and cobalamin (3.9), but differed markedly for cobaloxime (6.1). These results (i) suggest a similar biodegradation mechanism despite different microbial strains, (ii) indicate that transformation with isolated cobalamin resembles in vivo transformation and (iii) suggest a different mechanism with cobaloxime. This model reactant should therefore be used with caution. Our results demonstrate the power of two-dimensional isotope analyses to characterize and distinguish between reaction mechanisms in whole cell experiments and in vitro model systems.

Genomic determinants of organohalide-respiration in Geobacter lovleyi, an unusual member of the Geobacteraceae

BackgroundGeobacter lovleyi is a unique member of the Geobacteraceae because strains of this species share the ability to couple tetrachloroethene (PCE) reductive dechlorination to cis-1,2-dichloroethene (cis-DCE) with energy conservation and growth (i.e., organohalide respiration). Strain SZ also reduces U(VI) to U(IV) and contributes to uranium immobilization, making G. lovleyi relevant for bioremediation at sites impacted with chlorinated ethenes and radionuclides. G. lovleyi is the only fully sequenced representative of this distinct Geobacter clade, and comparative genome analyses identified genetic elements associated with organohalide respiration and elucidated genome features that distinguish strain SZ from other members of the Geobacteraceae.ResultsSequencing the G. lovleyi strain SZ genome revealed a 3.9 Mbp chromosome with 54.7% GC content (i.e., the percent of the total guanines (Gs) and cytosines (Cs) among the four bases within the genome), and average amino acid identities of 53–56% compared to other sequenced Geobacter spp. Sequencing also revealed the presence of a 77 kbp plasmid, pSZ77 (53.0% GC), with nearly half of its encoded genes corresponding to chromosomal homologs in other Geobacteraceae genomes. Among these chromosome-derived features, pSZ77 encodes 15 out of the 24 genes required for de novo cobalamin biosynthesis, a required cofactor for organohalide respiration. A plasmid with 99% sequence identity to pSZ77 was subsequently detected in the PCE-dechlorinating G. lovleyi strain KB-1 present in the PCE-to-ethene-dechlorinating consortium KB-1. Additional PCE-to-cis-DCE-dechlorinating G. lovleyi strains obtained from the PCE-contaminated Fort Lewis, WA, site did not carry a plasmid indicating that pSZ77 is not a requirement (marker) for PCE respiration within this species. Chromosomal genomic islands found within the G. lovleyi strain SZ genome encode two reductive dehalogenase (RDase) homologs and a putative conjugative pilus system. Despite the loss of many c-type cytochrome and oxidative-stress-responsive genes, strain SZ retained the majority of Geobacter core metabolic capabilities, including U(VI) respiration.ConclusionsGene acquisitions have expanded strain SZ’s respiratory capabilities to include PCE and TCE as electron acceptors. Respiratory processes core to the Geobacter genus, such as metal reduction, were retained despite a substantially reduced number of c-type cytochrome genes. pSZ77 is stably maintained within its host strains SZ and KB-1, likely because the replicon carries essential genes including genes involved in cobalamin biosynthesis and possibly corrinoid transport. Lateral acquisition of the plasmid replicon and the RDase genomic island represent unique genome features of the PCE-respiring G. lovleyi strains SZ and KB-1, and at least the latter signifies adaptation to PCE contamination.

Spatial and Temporal Distributions of Geobacter lovleyi and Dehalococcoides spp. during Bioenhanced PCE-NAPL Dissolution

The spatial and temporal distributions of multiple reductively dechlorinating bacteria were simultaneously assessed in a one-dimensional sand column containing a tetrachloroethene (PCE) nonaqueous phase liquid (NAPL) source and associated plume zones. The column was uniformly inoculated with a PCE-to-ethene dechlorinating microbial consortium that contained Dehalococcoides spp., Dehalobacter spp., and Geobacter lovleyi strain SZ. Geobacter and Dehalococcoides populations grew and colonized the column material, including the mixed-NAPL (0.25 mol/mol PCE in hexadecane) source zone. In contrast, Dehalobacter cells did not colonize the porous column material, and planktonic Dehalobacter cell titers remained below the detection limit of ca. 2.6 x 10(2) cells/mL throughout the experiment. Significant PCE dechlorination was observed and resulted in bioenhanced NAPL dissolution up to 21-fold (maximum) and 5.2-fold (cumulative) relative to abiotic dissolution. cis-1,2-Dichloroethene (cis-DCE) wasthe primary dechlorination product although vinyl chloride (VC) was also formed throughout the experiment. Ethene production occurred after significant depletion of PCE from the NAPL and when cis-DCE concentrations dropped below 6 microM. Data obtained after increasing the column residence time from 1.1 to 2.8 days and introducing a VC pulse to the column indicated that both the residence time and cis-DCE inhibition limited significant VC and ethene production. Although both Geobacter and Dehalococcoides cells were present and active in the mixed-NAPL source zone and plume region, Geobacter cell numbers were typically more than 1 order of magnitude higher than Dehalococcoides cell numbers, which is consistent with the production of predominantly cis-DCE. Analysis of both liquid- and solid-phase samples indicated that Geobacter cells grew and remained attached to the porous medium within the source zone but were largely planktonic in the plume region. In contrast Dehalococcoides cell were attached throughoutthecolumn,and Dehalococcoides cell titers increased by 1 to 2 orders of magnitude over the length of the column, correlating to increases in VC concentrations. The results from this study highlight that bioenhanced dissolution is governed by a complex interplay between resident dechlorinators, contaminant concentrations, and other aquifer-specific characteristics (e.g., hydrology).

Variability in microbial carbon isotope fractionation of tetra- and trichloroethene upon reductive dechlorination

The variability of stable carbon isotope fractionation upon reductive dechlorination of tetra- and trichloroethene by several microbial strains was investigated to examine the uncertainties related to the in situ application of compound specific isotope analysis (CSIA) of chlorinated ethenes. Carbon isotope fractionation was investigated with a set of microorganisms representative for the currently known diversity of dehalorespirers: Dehalococcoides ethenogenes strain 195, Desulfitobacterium sp. strain Viet1, Desulfuromonas michiganensis and Geobacter lovleyi sp. strain SZ and compared to the previous reports using Sulfurospirillum spp. and Desulfitobacterium sp. strain PCE-S. Carbon isotope fractionation of tetrachloroethene (PCE) and trichlorethene (TCE) was highly variable ranging from the absence of significant fractionation to carbon isotope fractionation (εC) of 16.7 and 3.5–18.9 for PCE and TCE, respectively. Fractionation of both compounds by D. ethenogenes strain 195 (PCE: εC = 6.0; TCE: εC = 13.7) was similar to the literature data for mixed cultures containing Dehalococcoides spp. D. michiganensis (PCE: no significant fractionation; TCE: εC = 3.5) and G. lovleyi sp. strain SZ (PCE no significant fractionation; TCE: εC = 8.5) generated the lowest fractionation of all studied strains. Desulfitobacterium sp. strain Viet1 (PCE: εC = 16.7) gave the highest enrichment factor for PCE.

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

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 x 10(6) and 1 x 10(4) 16S rRNA gene copies per mul of template DNA, respectively. A quantitative real-time PCR (qPCR) approach increased the sensitivity to as few as 30 16S rRNA gene copies per mul 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 x 10(7) +/- 0.1 x 10(7) 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.

Experimental Evaluation and Mathematical Modeling of Microbially Enhanced Tetrachloroethene (PCE) Dissolution

Experiments to assess metabolic reductive dechlorination (chlororespiration) at high concentration levels consistent with the presence of free-phase tetrachloroethene (PCE) were performed using three PCE-to-cis-1,2-dichloroethene (cis-DCE) dechlorinating pure cultures (Sulfurospirillum multivorans, Desulfuromonas michiganensis strain BB1, and Geobacter lovleyi strain SZ) and Desulfitobacterium sp. strain Viet1, a PCE-to-trichloroethene (TCE) dechlorinating isolate. Despite recent evidence suggesting bacterial PCE-to-cis-DCE dechlorination occurs at or near PCE saturation (0.9-1.2 mM), all cultures tested ceased dechlorinating at approximately 0.54 mM PCE. In the presence of PCE dense nonaqueous phase liquid (DNAPL), strains BB1 and SZ initially dechlorinated, but TCE and cis-DCE production ceased when aqueous PCE concentrations reached inhibitory levels. For S. multivorans, dechlorination proceeded at a rate sufficient to maintain PCE concentrations below inhibitory levels, resulting in continuous cis-DCE production and complete dissolution of the PCE DNAPL. A novel mathematical model, which accounts for loss of dechlorinating activity at inhibitory PCE concentrations, was developed to simultaneously describe PCE-DNAPL dissolution and reductive dechlorination kinetics. The model predicted that conditions corresponding to a bioavailability number (Bn) less than 1.25 x 10(-2) will lead to dissolution enhancement with the tested cultures, while conditions corresponding to a Bn greater than this threshold value can result in accumulation of PCE to inhibitory dissolved-phase levels, limiting PCE transformation and dissolution enhancement. These results suggest that microorganisms incapable of dechlorinating at high PCE concentrations can enhance the dissolution and transformation of PCE from free-phase DNAPL.

Geobacter lovleyi sp. nov. Strain SZ, a Novel Metal-Reducing and Tetrachloroethene-Dechlorinating Bacterium

A bacterial isolate, designated strain SZ, was obtained from noncontaminated creek sediment microcosms based on its ability to derive energy from acetate oxidation coupled to tetrachloroethene (PCE)-to-cis-1,2-dichloroethene (cis-DCE) dechlorination (i.e., chlororespiration). Hydrogen and pyruvate served as alternate electron donors for strain SZ, and the range of electron acceptors included (reduced products are given in brackets) PCE and trichloroethene [cis-DCE], nitrate [ammonium], fumarate [succinate], Fe(III) [Fe(II)], malate [succinate], Mn(IV) [Mn(II)], U(VI) [U(IV)], and elemental sulfur [sulfide]. PCE and soluble Fe(III) (as ferric citrate) were reduced at rates of 56.5 and 164 nmol min(-1) mg of protein(-1), respectively, with acetate as the electron donor. Alternate electron acceptors, such as U(VI) and nitrate, did not inhibit PCE dechlorination and were consumed concomitantly. With PCE, Fe(III) (as ferric citrate), and nitrate as electron acceptors, H(2) was consumed to threshold concentrations of 0.08 +/- 0.03 nM, 0.16 +/- 0.07 nM, and 0.5 +/- 0.06 nM, respectively, and acetate was consumed to 3.0 +/- 2.1 nM, 1.2 +/- 0.5 nM, and 3.6 +/- 0.25 nM, respectively. Apparently, electron acceptor-specific acetate consumption threshold concentrations exist, suggesting that similar to the hydrogen threshold model, the measurement of acetate threshold concentrations offers an additional diagnostic tool to delineate terminal electron-accepting processes in anaerobic subsurface environments. Genetic and phenotypic analyses classify strain SZ as the type strain of the new species, Geobacter lovleyi sp. nov., with Geobacter (formerly Trichlorobacter) thiogenes as the closest relative. Furthermore, the analysis of 16S rRNA gene sequences recovered from PCE-dechlorinating consortia and chloroethene-contaminated subsurface environments suggests that Geobacter lovleyi belongs to a distinct, dechlorinating clade within the metal-reducing Geobacter group. Substrate versatility, consumption of electron donors to low threshold concentrations, and simultaneous reduction of electron acceptors suggest that strain SZ-type organisms have desirable characteristics for bioremediation applications.