Mark E. Dolan

Monitoring Abundance and Expression of “Dehalococcoides” Species Chloroethene-Reductive Dehalogenases in a Tetrachloroethene-Dechlorinating Flow Column

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.

Methanotrophic Chloroethene Transformation Capacities And 1,1-dichloroethene Transformation Product Toxicity

For a mixed methanotrophic culture grown under copper deficiency, the relative transformation capacities of the chloroethenes with 10 mM formate present was in the order from highest to lowest : 1,2-transdichloroethylene (t-DCE) ; 1,2-cis-dichloroethylene (c-DCE) ; vinyl chloride (VC) ; trichloroethylene (TCE) ; and 1,1-dichloroethylene (1,1-DOE). Respective values were 4.8, 3.6, 2.3, 0.85, and 0.13 μmol transformed per mg of cells. Chloroethenes with asymmetric chlorine distributions had lower transformation capacities, probably due to higher transformation product toxicity. While 1,1-DCE itself was not toxic at the concentrations evaluated (up to 1 mg/L), its transformation products were highly toxic. Aquifer microcosms transformed up to 4.8 mg/L VC with no apparent toxic effects, but when both VC and 1,1-DCE were present, about 75% less transformation of VC and a marked decrease in methane oxidation rate resulted because of 1,1-DCE transformation product toxicity. About 25 times more VC was transformed in the soil microcosms per unit of methane consumed than in aqueous batch tests.

Expression of merA, amoA and hao in continuously cultured Nitrosomonas europaea cells exposed to zinc chloride additions

The effects of ZnCl2 additions on a mercuric reductase, merA, ammonia monooxygenase, amoA, and hydroxylamine (NH2OH) 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 ZnCl2 achieving maximum concentrations from 3 to 90 µM Zn2+. Continuously cultured N. europaea cells were more resistant to Zn2+ inhibition than previously examined batch cultured cells due to the presence of Mg2+ in the growth media, suggesting that Zn2+ enters the cell through Mg2+ import channels. The maximum merA up‐regulation was 45‐fold and expression increased with increases in Zn2+ concentration and decreased as Zn2+ concentrations decreased. Although Zn2+ irreversibly inactivated ammonia oxidation in N. europaea, the addition of either 600 µM CuSO4 or 2250 µM MgSO4 protected N. europaea from ZnCl2 inhibition, indicating a competition between Zn2+ and Cu2+/Mg2+ for uptake and/or AMO active sites. Since ZnCl2 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 Zn2+ levels indicates that merA is an excellent early warning signal for Zn2+ inhibition. Biotechnol. Bioeng. 2009;102: 546–553.

Small-column microcosm for assessing methane-stimulated vinyl chloride transformation in aquifer samples.

A small-column microcosm was designed and operated to evaluate the potential for in-situ vinyl chloride (VC) biotransformation by a methane-stimulated culture. Microcosms consisted of 15-mL test tubes filled with aquifer material and fitted with a fluid exchange system to allow sequential batch testing. Aquifer material was aseptically obtained from two locations at a chloroethene-contaminated Superfund site in St. Joseph, Ml. In microcosm tests, influent VC concentrations ranged from 1 to 17 mg/L (15-275 μM) with methane concentrations of 0.5-4.0 mg/L (30-250 μM) and dissolved oxygen concentrations of about 29 mg/L (900 μM). Stimulation of an active methane-utilizing population occurred within 60 days, and VC transformation was observed in all of the methane-fed microcosms. A maximum amount of VC transformation, independent of influent VC concentration, was observed for each methane concentration used. Significant differences in VC transformation ability was found in the aquifer samples from the two locations. Transformation yields (T y ) of up to 3.5 mg of VC transformed/mg of methane utilized (0.9 mol of VC/mol of CH 4 ) were found atone location compared to a maximum of 1.0 mg of VC/mg of CH 4 (0.26 mol of VC/mol of CH 4 ) at the other. Up to 16 mg/L VC (250 μM) was transformed with an addition of 4 mg/L methane (250 μM) with no significant toxic effects observed. The microcosms performed well throughout the study, providing consistent repeatable results.

Continuous-flow column study of reductive dehalogenation of PCE upon bioaugmentation with the Evanite enrichment culture

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.

Nitrification and degradation of halogenated hydrocarbons—a tenuous balance for ammonia-oxidizing bacteria

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.

Expression of merA, trxA, amoA, and hao in continuously cultured Nitrosomonas europaea cells exposed to cadmium sulfate additions

The effects of CdSO4 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 NH4+ and was operated for 78 days with a 6.9 days hydraulic retention time. Over this period, six successive batch additions of CdSO4 were made with increasing maximum concentrations ranging from 1 to 60 µM Cd2+. The expression of merA was highly correlated with the level of Cd2+ within the reactor (Rs = 0.90) with significant up‐regulation measured at non‐inhibitory Cd2+ concentrations. Cd2+ 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 Cd2+ inhibition is irreversible and amoA was up‐regulated in response to Cd2+ 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 Cd2+ inhibition than previously examined batch cultured cells due to the presence of Mg2+ and Ca2+ in the growth media, suggesting that Cd2+ enters the cell through Mg2+ and Ca2+ import channels. The up‐regulation of merA during exposure to non‐inhibitory Cd2+ levels indicates that merA is an excellent early warning signal for Cd2+ inhibition. Biotechnol. Bioeng. 2009; 104: 1004–1011.

Bioaugmentation with butane-utilizing microorganisms to promote in situ cometabolic treatment of 1,1,1-trichloroethane and 1,1-dichloroethene.

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 (approximately 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.

A KINETIC STUDY OF AEROBIC PROPANE UPTAKE AND COMETABOLIC DEGRADATION OF CHLOROFORM, CIS-DICHLOROETHYLENE AND TRICHLOROETYLENE IN MICROCOSMS WITH GROUNDWATER/AQUIFER SOLIDS

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.

Application of Salting-Out Agent to Enhance the Hydrophobicity of Weakly Hydrophobic Bacterial Strains

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.