This research demonstrates that groundwater contaminated by a relatively dilute but persistent concentration of 1,4-dioxane (1,4-D), approximately 60 mu g/L, and chlorinated aliphatic co-contaminants (1.4 to 10 mu g/L) can be efficiently and reliably treated by in situ aerobic cometabolic biodegradation (ACB). A field trial lasting 265 days was conducted at Operable Unit D at the former McClellan Air Force Base and involved establishing an in situ ACB reactor through amending recirculated groundwater with propane and oxygen. The stimulated indigenous microbial population was able to consistently degrade 1,4-D to below 3 mu g/L while the co-contaminants trichloroethene (TCE) and 1,2-dichloroethane (1,2-DCA) were decreased to below 1 mu g/L and 0.18 mu g/L, respectively. A stable treatment efficiency of more than 95% removal for 1,4-D and 1,2-DCA and of more than 90% removal for TCE was achieved. High treatment efficiencies for 1,4-D and all co-contaminants were sustained even without propane and oxygen addition for a 2-week period.

Hydrophobicity is a vital parameter for initial cell adhesion that ultimately leads to biofouling of surfaces and loss of system performance and health issues. The efficiency of a number of biological systems could be improved by increasing the hydrophobicity of concerned bacteria. Here we used ammonium sulfate (salt) to enhance the bacterial hydrophobicity, as measured by a commonly used liquid-liquid partitioning based hydrophobicity assessment assay - the MATH test. We observed successive increases in bacterial hydrophobicity with incremental increase in salt concentration for Gram-negative bacteria. Upon addition of 2 M salt, three closely related E. coli strains were easily distinguishable from one another. Gram-positive bacteria exhibited different trends than Gram-negative strains, with no change in the hydrophobicity of S. salivarius HB cells and a sharp decline followed by an increase in hydrophobicity for D. radiodurans. Cell size measurements revealed that Gram-positive cells exhibited a change in cell size on hydrocarbon exposure, while the Gram-negative cultures remained mostly unaffected. Overall, salt addition was observed to enhance the hydrophobicity of different test strains, especially at the higher concentrations used here of 1.5 and 2 M. Salt addition in conjunction with the MATH test successfully differentiated and quantified otherwise weakly hydrophobic bacteria, thus enhancing the range of this laboratory assay. Our results demonstrate the effectiveness of salt addition in increasing the bacterial hydrophobicity, which could potentially be used in diverse areas, ranging from applied microbiology and engineering to oral care. (C) Koninklijke Brill NV, Leiden, 2011

The process of nitrification has the potential for the in situ bioremediation of halogenated compounds provided a number of challenges can be overcome. In nitrification, the microbial process where ammonia is oxidized to nitrate, ammonia-oxidizing bacteria (AOB) are key players and are capable of carrying out the biodegradation of recalcitrant halogenated compounds. Through industrial uses, halogenated compounds often find their way into wastewater, contaminating the environment and bodies of water that supply drinking water. In the reclamation of wastewater, halogenated compounds can be degraded by AOB but can also be detrimental to the process of nitrification. This minireview considers the ability of AOB to carry out cometabolism of halogenated compounds and the consequent inhibition of nitrification. Possible cometabolism monitoring methods that were derived from current information about AOB genomes are also discussed. AOB expression microarrays have detected mRNA of genes that are expressed at higher levels during stress and are deemed "sentinel" genes. Promoters of selected "sentinel" genes have been cloned and used to drive the expression of gene-reporter constructs. The latter are being tested as early warning biosensors of cometabolism-induced damage in Nitrosomonas europaea with promising results. These and other biosensors may help to preserve the tenuous balance that exists when nitrification occurs in waste streams containing alternative AOB substrates such as halogenated hydrocarbons.

The effects of CdSO(4) additions on the gene expressions of a mercury reductase, merA, an oxidative stress protein, trxA, the ammonia-monooxygenase enzyme (AMO), amoA, and the hydroxylamine oxidoreductase enzyme (HAO), hao, were examined in continuously cultured N. europaea cells. The reactor was fed 50 mM NH(4)+ and was operated for 78 days with a 6.9 days hydraulic retention time. Over this period, six successive batch additions of CdSO(4) were made with increasing maximum concentrations ranging from 1 to 60 microM Cd(2+). The expression of merA was highly correlated with the level of Cd(2+) within the reactor (Rs = 0.90) with significant up-regulation measured at non-inhibitory Cd(2+) concentrations. Cd(2+) appears to target AMO specifically at lower concentrations and caused oxidative stress at higher concentrations, as indicated by the SOURs (specific oxygen uptake rates) and the up-regulation of trxA. Since Cd(2+) inhibition is irreversible and amoA was up-regulated in response to Cd(2+) inhibition, it is hypothesized that de novo synthesis of the AMO enzyme occurred and was responsible for the observed recovery in activity. Continuously cultured N. europaea cells were more resistant to Cd(2+) inhibition than previously examined batch cultured cells due to the presence of Mg(2+) and Ca(2+) in the growth media, suggesting that Cd(2+) enters the cell through Mg(2+) and Ca(2+) import channels. The up-regulation of merA during exposure to non-inhibitory Cd(2+) levels indicates that merA is an excellent early warning signal for Cd(2+) inhibition.

Bioaugmentation to enhance the rate and extent of reductive dechlorination of chlorinated ethenes was investigated in intermediate ( approximately 1 m) scale physical aquifer models (PAMs) designed to simulate the groundwater flow field near an injection well. Push-pull tests were used to quantify the reductive dechlorination of injected trichloroethene (TCE) and trichlorofluoroethene (TCFE) in prepared sediment packs with and without an added dechlorinating culture containing Dehalococcoides spp. Distribution of the added culture throughout the sediment pack was confirmed by microscopic observation. Repeated additions of TCE (100-350 microM) were completely transformed to ethene in 14 days and a subsequent TCFE addition (114 microM) was completely transformed to fluoroethene (FE) in 24 days. Similar transformation rates, product distributions, and time courses for TCE and TCFE transformation were observed when these compounds were added together at similar initial concentrations. In the control PAM (nonbioaugmented), TCE and TCFE were transformed to only cis-DCE and cis-DCFE, respectively, and transformation rates were 6-12 times slower than those in the bioaugmented PAM. The use of TCFE for push-pull tests is shown to be an effective tool for detecting and quantifying the effects of bioaugmentation on TCE transformation.

The effects of ZnCl 2 additions on a mercuric reductase, merA , ammonia monooxygenase, amoA , and hydroxylamine (NH 2 OH) oxidoreductase, hao , gene expression were examined in continuously cultured Nitrosomonas europaea cells. The reactor was operated for 85 days with a 6.9 d hydraulic retention time and with four successive additions of ZnCl 2 achieving maximum concentrations from 3 to 90 µM Zn 2+ . Continuously cultured N. europaea cells were more resistant to Zn 2+ inhibition than previously examined batch cultured cells due to the presence of Mg 2+ in the growth media, suggesting that Zn 2+ enters the cell through Mg 2+ import channels. The maximum merA up‐regulation was 45‐fold and expression increased with increases in Zn 2+ concentration and decreased as Zn 2+ concentrations decreased. Although Zn 2+ irreversibly inactivated ammonia oxidation in N. europaea , the addition of either 600 µM CuSO 4 or 2250 µM MgSO 4 protected N. europaea from ZnCl 2 inhibition, indicating a competition between Zn 2+ and Cu 2+ /Mg 2+ for uptake and/or AMO active sites. Since ZnCl 2 inhibition is irreversible and amoA was up‐regulated at 30 and 90 µM additions, it is hypothesized that de novo synthesis of the AMO enzyme is needed to overcome inhibition. The up‐regulation of merA during exposure to non‐inhibitory Zn 2+ levels indicates that merA is an excellent early warning signal for Zn 2+ inhibition. Biotechnol. Bioeng. 2009;102: 546–553. © 2008 Wiley Periodicals, Inc.

A field study was performed to evaluate the potential for in-situ aerobic cometabolism of 1,1,1-trichloroethane (1,1,1-TCA) through bioaugmentation with a butane enrichment culture containing predominantly two Rhodococcus sp. strains named 179BP and 183BP that could cometabolize 1,1,1-TCA and 1,1-dicholoroethene (1,1-DCE). Batch tests indicated that 1,1-DCE was more rapidly transformed than 1,1,1-TCA by both strains with 183BP being the most effective organism. This second in a series of bioaugmentation field studies was conducted in the saturated zone at the Moffett Field In Situ Test Facility in California. In the previous test, bioaugmentation with an enrichment culture containing the 183BP strain achieved short term in situ treatment of 1,1-DCE, 1,1,1-TCA, and 1,1-dichloroethane (1,1-DCA). However, transformation activity towards 1,1,1-TCA was lost over the course of the study. The goal of this second study was to determine if more effective and long-term treatment of 1,1,1-TCA could be achieved through bioaugmentation with a highly enriched culture containing 179BP and 183BP strains. Upon bioaugmentation and continuous addition of butane and dissolved oxygen and or hydrogen peroxide as sources of dissolved oxygen, about 70% removal of 1,1,1-TCA was initially achieved. 1,1-DCE that was present as a trace contaminant was also effectively removed (∼ 80%). No removal of 1,1,1-TCA resulted in a control test leg that was not bioaugmented, although butane and oxygen consumption by the indigenous populations was similar to that in the bioaugmented test leg. However, with prolonged treatment, removal of 1,1,1-TCA in the bioaugmented leg decreased to about 50 to 60%. Hydrogen pexoxide (H2O2) injection increased dissolved oxygen concentration, thus permitting more butane addition into the test zone, but more effective 1,1,1-TCA treatment did not result. The results showed bioaugmentation with the enrichment cultures was effective in enhancing the cometabolic treatment of 1,1,1-TCA and low concentrations of 1,1-DCE over the entire period of the 50-day test. Compared to the first season of testing, cometabolic treatment of 1,1,1-TCA was not lost. The better performance achieved in the second season of testing may be attributed to less 1,1-DCE transformation product toxicity, more effective addition of butane, and bioaugmentation with the highly enriched dual culture.

ABSTRACT We investigated the distribution and activity of chloroethene-degrading microorganisms and associated functional genes during reductive dehalogenation of tetrachloroethene to ethene in a laboratory continuous-flow column. Using real-time PCR, we quantified “Dehalococcoides” species 16S rRNA and chloroethene-reductive dehalogenase (RDase) genes (pceA, tceA, vcrA, and bvcA) in nucleic acid extracts from different sections of the column. Dehalococcoides 16S rRNA gene copies were highest at the inflow port [(3.6 ± 0.6) × 106 (mean ± standard deviation) per gram soil] where the electron donor and acceptor were introduced into the column. The highest transcript numbers for tceA, vcrA, and bvcA were detected 5 to 10 cm from the column inflow. bvcA was the most highly expressed of all RDase genes and the only vinyl chloride reductase-encoding transcript detectable close to the column outflow. Interestingly, no expression of pceA was detected in the column, despite the presence of the genes in the microbial community throughout the column. By comparing the 16S rRNA gene copy numbers to the sum of all four RDase genes, we found that 50% of the Dehalococcoides population in the first part of the column did not contain either one of the known chloroethene RDase genes. Analysis of 16S rRNA gene clone libraries from both ends of the flow column revealed a microbial community dominated by members of Firmicutes and Actinobacteria. Higher clone sequence diversity was observed near the column outflow. The results presented have implications for our understanding of the ecophysiology of reductively dehalogenating Dehalococcoides spp. and their role in bioremediation of chloroethenes.

Ammonia oxidizing bacteria (AOB) are inhibited by many compounds found in wastewater treatment plant (WWTP) influent, including aromatic hydrocarbons. The detection of "sentinel genes" to identify the presence of aromatic hydrocarbons could be useful to WWTP operators. In this study, the transcriptomic responses of Nitrosomonas europaea during the cometabolism of benzene to phenol and toluene to benzyl alcohol and benzaldehyde were evaluated using whole genome Affymetrix microarrays and qRT-PCR. Benzyl alcohol and benzaldehyde were found not to inhibit N. europaea. However, phenol concentrations as low as 5 microM directly inhibited ammonia oxidation. Surprisingly, there were no significant up- or down-regulation of genes in N. europaea cells exposed to 20 microM toluene, which caused 50% inhibition of ammonia oxidation. Exposing N. europaea to 40 microM benzene, which caused a similar degree of inhibition, resulted in the up-regulation of seven adjacent genes, including NE 1545 (a putative pirin protein) and NE 1546 (a putative membrane protein), that appear to be involved with fatty-acid metabolism, lipid biosynthesis, and membrane protein synthesis. qRT-PCR analysis revealed that NE 1545 and NE 1546 were significantly up-regulated upon exposure to benzene and phenol, but not upon exposure to toluene. Transmission electron microscope images revealed a shift in outer cell structure in response to benzene exposure.

A continuous-flow anaerobic column experiment was conducted to evaluate the reductive dechlorination of tetrachloroethene (PCE) in Hanford aquifer material after bioaugmentation with the Evanite (EV) culture. An influent PCE concentration of 0.09 mM was transformed to vinyl chloride (VC) and ethene (ETH) within a hydraulic residence time of 1.3 days. The experimental breakthrough curves were described by the one-dimensional two-site-nonequilibrium transport model. PCE dechlorination was observed after bioaugmentation and after the lactate concentration was increased from 0.35 to 0.67 mM. At the onset of reductive dehalogenation, cis-dichloroethene (c-DCE) concentrations in the column effluent exceeded the influent PCE concentration indicating enhanced PCE desorption and transformation. When the lactate concentration was increased to 1.34 mM, c-DCE reduction to vinyl chloride (VC) and ethene (ETH) occurred. Spatial rates of PCE and VC transformation were determined in batch-incubated microcosms constructed with aquifer samples obtained from the column. PCE transformation rates were highest in the first 5 cm from the column inlet and decreased towards the column effluent. Dehalococcoides cell numbers dropped from approximately 73.5% of the total Bacterial population in the original inocula, to about 0.5% to 4% throughout the column. The results were consistent with estimates of electron donor utilization, with 4% going towards dehalogenation reactions.

Fluoroethene (FE) is a stable molecule in aqueous solution and its aerobic transformation potentially yields F-. This work evaluated if FE is a suitable surrogate for monitoring aerobic vinyl chloride (VC) utilization or cometabolic transformation. Experiments were carried out with three isolates, Mycobacterium strain EE13a, Mycobacterium strain JS60, and Nocardioides strain JS614 to evaluate if their affinities for FE and VC and their rates of transformation were comparable and whether the transformation of FE and F- accumulation could be correlated with VC utilization. JS614 grew on FE in addition to VC, making it the first organism reported to use FE as a sole carbon and energy source. EE13a cometabolized VC and FE, and JS60 catabolized VC and cometabolized FE. There was little difference among the three strains in the Ks or kmax values for VC or FE. Competitive inhibition modeled the temporal responses of FE and VC transformations and Cl- and F- release when both substrates were present. Both the rate of FE transformation and rate of F-accumulation could be correlated with the rate of aerobic transformation of VC and showed promise for estimating VC rates in situ using FE as a reactive surrogate.

A series of laboratory, field, and modeling studies were performed evaluating the potential for in situ aerobic cometabolism of chlorinated aliphatic hydrocarbon (CAH) mixtures, including 1,1,1-trichloroethane (1,1,1-TCA), 1,1-dichloroethane (1,1-DCA) and 1,1-dichloroethene (1,1-DCE) by bioaugmented microorganisms that grew on butane. A butane-grown bioaugmentation culture, primarily comprised of a Rhodococcus sp., was developed that effectively transformed mixtures of the three CAHs, under subsurface nutrient conditions. Microcosm experiments and modeling studies showed rapid transformation of 1,1-DCE with high transformation product toxicity and weak inhibition by butane, while 1,1,1-TCA was much more slowly transformed and strongly inhibited by butane. Field studies were conducted in the saturated zone at the Moffett Field In-Situ Test Facility in California. In the bioaugmented test leg, 1,1-DCE was most effectively transformed, followed by 1,1-DCA, and 1,1,1-TCA, consistent with the results from the laboratory studies. A 1-D reactive/transport code simulated the field responses during the early stages of testing (first 20 days), with the following extents of removal achieved at the first monitoring well; 1,1-DCE (∼97%), 1,1-DCA (∼77%), and 1,1,1-TCA (∼36%), with little or no CAH transformation observed beyond the first monitoring well. As time proceeded, decreased performance was observed. The modeling analysis indicated that this loss of performance may have been associated with 1,1-DCE transformation toxicity combined with the limited addition of butane as a growth substrate with longer pulse cycles. When shorter pulse cycles were reinitiated after 40 days of operation, 1,1-DCE transformation was restored and the following transformation extents were achieved; 1,1-DCE (∼94%), 1,1-DCA (∼8%), and 1,1,1-TCA (∼0%), with some CAH transformation occurring past the first monitoring well. Modeling analysis of this period indicated that the bioaugmented culture was likely not the dominant butane-utilizing microorganism present. This was consistent with observations in the indigenous leg during this period that showed effective butane utilization and the following extents of transformation: 1,1-DCE (∼86 %), 1,1-DCA (∼5%), and 1,1,1-TCA (∼0%). The combination of lab and field scale studies and supporting modeling provide a means of evaluating the performance of bioaugmentation and the cometabolic treatment of CAH mixtures.

: Many sites in the DoD and DOE complex are contaminated with chlorinated solvent mixtures. Passive in-situ treatment via aerobic cometabolism is one means of potentially restoring the contaminated aquifers. The goal of the project was to develop a cometabolic culture that has potential for bioaugmentation and to evaluate its performance under laboratory conditions and under in-situ conditions in field demonstrations. The specific objectives of this study were to: 1) develop a butane utilizing culture for bioaugmentation in laboratory and field experiments; 2) characterize the bioaugmentation culture using molecular methods, including a clone library, sequencing, and PCR based methods; 3) develop kinetic information for substrate utilization and the transformation of the CAH mixtures; 4) develop molecular based methods for tracking the cultures that were bioaugmented and biostimulated in laboratory and field studies; 5) conduct laboratory microcosm and continuous flow column studies to evaluate the performance of the bioaugmented culture under geochemical conditions mimicking those present at the field site; 6) conduct field demonstrations to evaluate the bioaugmentation approach and to determine the effectiveness in treating problematic mixtures of 1,1,1-TCA, 1,1-DCE, and 1,1-DCA using butane as cometabolic substrate, and compare the results to those achieved by indigenous butane-utilizers; 7) track microbial community changes and quantify members of the bioaugmented culture in situ using molecular based methods; 8) simulate the results of laboratory and field studies using a transport code for these cometabolic transformations.

The focus of this study was to compare the behavior of different consortiums of aerobic propane-utilizing microorganisms, with respect to both the lag time for growth after exposure to propane, and their ability to transform three chlorinated aliphatic hydrocarbons (CAHs): chloroform (CF), cis-dichloroethylene (c-DCE) and trichloroethylene (TCE). Thirty-three slurry microcosms, representing seven combinations of aquifer solids and groundwater were constructed for this study. The lag time required for establishing propane-utilizing consortiums ranged between 24 and 29 days in 6 of the 7 combinations. Kinetic tests were performed with respect to propane utilization and CAH transformation. After CAH exposure, the ability of the microorganisms to metabolize propane was significantly reduced. CF and TCE were transformed more slowly than c-DCE, the average values of the initial transformation rates being equal to 0.10 ± 0.04, 0.09 ± 0.05 and 0.98 ± 0.18 μmol/(L h),respectively. CF caused the greatest reduction in propane uptake rates, whereas c-DCE exhibited an apparently reversible negative effect on propane uptake rates. The estimates of the Monod half-saturation constants relative to CF, TCE and c-DCE resulted in the 2–3 μmol/L range, but were characterized by a high degree of uncertainty.