Influence of growth substrate and contaminant mixtures on the degradation of BTEX and MTBE by Rhodococcus rhodochrous ATCC strain 21198.

The degradation of the prevalent environmental contaminants benzene, toluene, ethylbenzene, and xylenes (BTEX) along with a common co-contaminant methyl tert-butyl ether (MTBE) by Rhodococcus rhodochrous ATCC Strain 21198 was investigated. The ability of 21198 to degrade these contaminants individually and in mixtures was evaluated with resting cells grown on isobutane, 1-butanol, and 2-butanol. Growth of 21198 in the presence of BTEX and MTBE was also studied to determine the growth substrate that best supports simultaneous microbial growth and contaminants degradation. Cells grown on isobutane, 1-butanol, and 2-butanol were all capable of degrading the contaminants, with isobutane grown cells exhibiting the most rapid degradation rates and 1-butanol grown cells exhibiting the slowest. However, in conditions where BTEX and MTBE were present during microbial growth, 1-butanol was determined to be an effective substrate for supporting concurrent growth and contaminant degradation. Contaminant degradation was found to be a combination of metabolic and cometabolic processes. Evidence for growth of 21198 on benzene and toluene is presented along with a possible transformation pathway. MTBE was cometabolically transformed to tertiary butyl alcohol, which was also observed to be transformed by 21198. This work demonstrates the possible utility of primary and secondary alcohols to support biodegradation of monoaromatic hydrocarbons and MTBE. Furthermore, the utility of 21198 for bioremediation applications has been expanded to include BTEX and MTBE.

Effectiveness of the tropical plants Rhynchospora corymbosa and Coix lacryma-jobi in vertical flow constructed wetlands for municipal primary sewage effluent treatment.

The performance of two tropical plants, Rhynchospora corymbosa L. (RC) and Coix lacryma-jobi, L (CL) in treatment of primary sewage effluent in lab-scale vertical-flow constructed wetlands (VFCW) along with no plant control wetland was investigated. A batch-flow VFCWs were operated under batch fill and drain hydraulic loading system with hydraulic retention times (HRT) of 0.5, 1, and 2 days and fill rate of 8 L/day. Removal of solids, organics, nutrients, and pathogens were monitored. The volumetric contaminant removal rates were best described by 1st order kinetics except for ammonia and phosphate, which was best described by Stover-Kincannon kinetics. Influent TSS, PO43-, COD, BOD5, and total coliform concentration were low but high in NH4+ concentration. CL was better in nutrient removal as HRT increases compared to RC. RC was more efficient at TSS, turbidity, and organics removal. Pathogen removal was independent of plant type but HRT. Solids and organic removal were lower in CL planted CWs due to preferential flow paths created by their bulky root. CL planted CWs removed more nutrients followed by RC planted CWs and then no-plant control CWs. The results of these tests demonstrate that both CL and RC are suitable for the treatment of municipal wastewater in VFCW system.

Single-well push-pull tests evaluating isobutane as a primary substrate for promoting in situ cometabolic biotransformation reactions.

A series of single-well push-pull tests (SWPPTs) were performed to investigate the efficacy of isobutane (2-methylpropane) as a primary substrate for in situ stimulation of microorganisms able to cometabolically transform common groundwater contaminants, such as chlorinated aliphatic hydrocarbons and 1,4-dioxane (1,4-D). In biostimulation tests, the disappearance of isobutane relative to a nonreactive bromide tracer indicated an isobutane-utilizing microbial community rapidly developed in the aquifer around the test well. SWPPTs were performed as natural drift tests with first-order rates of isobutane consumption ranging from 0.4 to 1.4 day-1. Because groundwater contaminants were not present at the demonstration site, isobutene (2-methylpropene) was used as a nontoxic surrogate to demonstrate cometabolic activity in the subsurface after biostimulation. The transformation of isobutene to isobutene epoxide (2-methyl-1,2-epoxypropane) illustrates the epoxidation process previously shown for common groundwater contaminants after cometabolic transformation by alkane-utilizing bacteria. The rate and extent of isobutene consumption and the formation and transformation of isobutene epoxide were greater in the presence of isobutane, with no evidence of primary substrate inhibition. Modeled concentrations of isobutane-utilizing biomass in microcosms constructed with groundwater collected before and after each SWPPT offered additional evidence that the isobutane-utilizing microbial community was stimulated in the aquifer. Experiments in groundwater microcosms also demonstrated that the isobutane-utilizing bacteria stimulated in the subsurface could cometabolically transform a mixture of co-substrates including isobutene, 1,1-dichloroethene, cis-1,2-dichloroethene, and 1,4-D with the same co-substrate preferences as the bacterium Rhodococcus rhodochrous ATCC strain 21198 after growth on isobutane. This study demonstrated the effectiveness of isobutane as primary substrate for stimulating in situ cometabolic activity and the use of isobutene as surrogate to investigate in situ cometabolic reactions catalyzed by isobutane-stimulated bacteria.

Contrasting growth properties of Nocardioides JS614 on threedifferent vinyl halides.

Ethene (ETH)-grown inocula of Nocardioides JS614 grow on vinyl chloride (VC), vinyl fluoride (VF), or vinyl bromide (VB) as the sole carbon and energy source, with faster growth rates and higher cell yields on VC and VF than on VB. However, whereas acetate-grown inocula of JS614 grow on VC and VF after a lag period, growth on VB did not occur unless supplemental ethene oxide (EtO) was present in the medium. Despite inferior growth on VB, the maximum rate of VB consumption by ETH-grown cells was ~ 50% greater than the rates of VC and VF consumption, but Br- release during VB consumption was non-stoichiometric with VB consumption (~ 66%) compared to 100% release of Cl- and F- during VC and VF consumption. Evidence was obtained for VB turnover-dependent toxicity of cell metabolism in JS614 with both acetate-dependent respiration and growth being significantly reduced by VB turnover, but no VC or VF turnover-dependent toxicity of growth was detected. Reduced growth rate and cell yield of JS614 on VB probably resulted from a combination of inefficient metabolic processing of the highly unstable VB epoxide (t0.5 = 45 s), accompanied by growth inhibitory effects of VB metabolites on acetate-dependent metabolism. The exact role(s) of EtO in promoting growth of alkene repressed JS614 on VB remains unresolved, with evidence of EtO inducing epoxide consuming activity prior to an increase in alkene oxidizing activity and supplementing reductant supply when VB is the growth substrate.

Immobilization of Methylosinus trichosporium OB3b for methanol production.

Due to the natural gas boom in North America, there is renewed interest in the production of other chemical products from methane. We investigated the feasibility of immobilizing the obligate methanotrophic bacterium Methylosinus trichosporium OB3b in alginate beads, and selectively inactivating methanol dehydrogenase (MDH) with cyclopropane to produce methanol. In batch cultures and in semi-continuous flow columns, the exposure of alginate-immobilized cells to cyclopropane or cyclopropanol resulted in the loss of the majority of MDH activity (> 80%), allowing methanol to accumulate to significant concentrations while retaining all of M. trichosporium OB3b's methane monooxygenase capacity. Thereafter, the efficiency of methanol production fell due to recovery of most of the MDH activity; however, subsequent inhibition periods resulted in renewed methanol production efficiency, and immobilized cells retained methane-oxidizing activity for at least 14 days.

Survival of Vinyl Chloride Respiring Dehalococcoides mccartyi under Long-Term Electron Donor Limitation.

In anoxic groundwater aquifers, the long-term survival of Dehalococcoides mccartyi populations expressing the gene vcrA (or bvcA) encoding reductive vinyl chloride dehalogenases are important to achieve complete dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE) to nonchlorinated ethene. The absence or inactivity of vcrA-containing Dehalococcoides results in the accumulation of the harmful chlorinated intermediates dichloroethene (DCE) and vinyl chloride (VC). Although vcrA-containing Dehalococcoides subpopulations depend on synergistic interaction with other organohalide-respiring populations generating their metabolic electron acceptors (DCE and VC), their survival requires successful competition for electron donor within the entire organohalide-respiring microbial community. To understand this dualism of synergy and competition under growth conditions relevant in contaminated aquifers, we investigated Dehalococcoides-level population structure when subjected to a change in the ratio of electron donor to chlorinated electron acceptor in continuously stirred tank reactors (CSTRs) operated over 7 years. When the electron donor formate was supplied in stoichiometric excess to TCE, both tceA-containing and vcrA-containing Dehalococcoides populations persisted, and near-complete dechlorination to ethene was stably maintained. When the electron donor formate was supplied at substoichiometric concentrations, the interactions between tceA-containing and vcrA-containing populations shifted toward direct competition for the same limiting catabolic electron donor substrate with subsequent niche exclusion of the vcrA-containing population. After more than 2000 days of operation under electron donor limitation, increasing the electron donor to TCE ratio facilitated a recovery of the vcrA-containing Dehalococoides population to its original frequency. We demonstrate that electron donor scarcity alone, in the absence of competing metabolic processes or inhibitory dechlorination intermediate products, is sufficient to alter the Dehalococcoides population structure. These results underscore the importance of electron donor and chloroethene stoichiometry in maintaining balanced functional performance within consortia composed of multiple D. mccartyi subpopulations, even when other competing electron acceptor processes are absent.

Formation of Developmentally Toxic Phenanthrene Metabolite Mixtures by Mycobacterium sp. ELW1.

Mycobacterium sp. ELW1 co-metabolically degraded up to 1.8 µmol of phenanthrene (PHE) in ∼48 h, and hydroxyphenanthrene (OHPHE) metabolites, including 1-hydroxyphenanthrene (1-OHPHE), 3-hydroxyphenanthrene (3-OHPHE), 4-hydroxyphenanthrene (4-OHPHE), 9-hydroxyphenanthrene (9-OHPHE), 9,10-dihydroxyphenanthrene (1,9-OHPHE), and trans-9,10-dihydroxy-9,10-dihydrophenanthrene (trans-9,10-OHPHE), were identified and quantified over time. The monooxygenase responsible for co-metabolic transformation of PHE was inhibited by 1-octyne. First-order PHE transformation rates, kPHE, and half-lives, t1/2, for PHE-exposed cells were 0.16-0.51 h-1 and 1.4-4.3 h, respectively, and the 1-octyne controls ranged from 0.015-0.10 h-1 to 7.0-47 h, respectively. While single compound standards of PHE and trans-9,10-OHPHE, the major OHPHE metabolite formed by ELW1, were not toxic to embryonic zebrafish (Danio rerio), single compound standards of minor OHPHE metabolites, 1-OHPHE, 3-OHPHE, 4-OHPHE, 9-OHPHE, and 1,9-OHPHE, were toxic, with effective concentrations (EC50's) ranging from 0.5 to 5.5 µM. The metabolite mixtures formed by ELW1, and the reconstructed standard mixtures of the identified OHPHE metabolites, elicited a toxic response in zebrafish for the same three time points. EC50s for the metabolite mixtures formed by ELW1 were lower (more toxic) than those for the reconstructed standard mixtures of the identified OHPHE metabolites. Ten unidentified hydroxy PHE metabolites were measured in the derivatized mixtures formed by ELW1 and may explain the increased toxicity of the ELW1 metabolites mixture relative to the reconstructed standard mixtures of the identified OHPHE metabolites.

Kinetic parameter estimation in N. europaea biofilms using a 2-D reactive transport model.

Biofilms of the ammonia oxidizing bacterium Nitrosomonas europaea were cultivated to study microbial processes associated with ammonia oxidation in pure culture. We explored the hypothesis that the kinetic parameters of ammonia oxidation in N. europaea biofilms were in the range of those determined with batch suspended cells. Oxygen and pH microelectrodes were used to measure dissolved oxygen (DO) concentrations and pH above and inside biofilms and reactive transport modeling was performed to simulate the measured DO and pH profiles. A two dimensional (2-D) model was used to simulate advection parallel to the biofilm surface and diffusion through the overlying fluid while reaction and diffusion were simulated in the biofilm. Three experimental studies of microsensor measurements were performed with biofilms: i) NH3 concentrations near the Ksn value of 40 µM determined in suspended cell tests ii) Limited buffering capacity which resulted in a pH gradient within the biofilms and iii) NH3 concentrations well below the Ksn value. Very good fits to the DO concentration profiles both in the fluid above and in the biofilms were achieved using the 2-D model. The modeling study revealed that the half-saturation coefficient for NH3 in N. europaea biofilms was close to the value measured in suspended cells. However, the third study of biofilms with low availability of NH3 deviated from the model prediction. The model also predicted shifts in the DO profiles and the gradient in pH that resulted for the case of limited buffering capacity. The results illustrate the importance of incorporating both key transport and chemical processes in a biofilm reactive transport model.

Nanoliter qPCR platform for highly parallel, quantitative assessment of reductive dehalogenase genes and populations of dehalogenating microorganisms in complex environments.

Idiosyncratic combinations of reductive dehalogenase (rdh) genes are a distinguishing genomic feature of closely related organohalogen-respiring bacteria. This feature can be used to deconvolute the population structure of organohalogen-respiring bacteria in complex environments and to identify relevant subpopulations, which is important for tracking interspecies dynamics needed for successful site remediation. Here we report the development of a nanoliter qPCR platform to identify organohalogen-respiring bacteria and populations by quantifying major orthologous reductive dehalogenase gene groups. The qPCR assays can be operated in parallel within a 5184-well nanoliter qPCR (nL-qPCR) chip at a single annealing temperature and buffer condition. We developed a robust bioinformatics approach to select from thousands of computationally proposed primer pairs those that are specific to individual rdh gene groups and compatible with a single amplification condition. We validated hundreds of the most selective qPCR assays and examined their performance in a trichloroethene-degrading bioreactor, revealing population structures as well as their unexpected shifts in abundance and community dynamics.

Influence of Water Hardness on Silver Ion and Silver Nanoparticle Fate and Toxicity Toward Nitrosomonas europaea.

This study investigated the influence of water hardness (Mg2+ and Ca2+) on the fate and toxicity of 20 nm citrate silver nanoparticles (AgNPs) and Ag+ toward Nitrosomonas europaea, a model ammonia-oxidizing bacterium. Nitrification inhibition of N. europaea by 1 ppm AgNPs and 0.5 ppm Ag+ was reduced from 80% and 83%, respectively, in the absence of Mg2+ to 2% and 33%, respectively, in the presence of 730 µM Mg2+. Introduction of Mg2+ resulted in the rapid aggregation of the AgNP suspensions and reduced the 3 h Ag+ dissolution rates from 30%, in the absence of Mg2+, to 9%, in the presence of 730 µM Mg2+. Reduced AgNP dissolution rates resulted in decreased concentrations of silver that were found adsorbed to N. europaea cells. Increasing AgNP concentrations in the presence of Mg2+ increased the observed inhibition of nitrification, but was always less than what was observed in the absence of Mg2+. The presence of Mg2+ also reduced the adsorption of Ag+ to cells, possibly due to multiple mechanisms, including a reduction in the negative surface charge of the N. europaea membrane and a competition between Mg2+ and Ag+ for membrane binding sites and transport into the cells. Ca2+ demonstrated similar protection mechanisms, as Ag+ toxicity was reduced and AgNP suspensions aggregated and decreased their dissolution rates. These results indicate that the toxicity of Ag+ and AgNPs to nitrifying bacteria in wastewater treatment would be less pronounced in systems with hard water.

The optimization of poly(vinyl)-alcohol-alginate beads with a slow-release compound for the aerobic cometabolism of chlorinated aliphatic hydrocarbons.

Chlorinated aliphatic hydrocarbons (CAHs), such as cis-1,2-dichloroethylene (cDCE), are prevalent in groundwater at many locations throughout the United States. When immobilized in hydrogel beads with slow-release compounds, the bacteria strain Rhodococcus rhodochrous ATCC 21198 can be used for the in situ bioremediation of cDCE. These hydrogel beads must exhibit high mechanical strength and resist degradation to extend the lifetime of slow-release compounds and bioremediation. We engineered poly(vinyl)-alcohol - alginate (PVA-AG) beads to immobilize ATCC 21198 with the slow-release compound, tetrabutoxysilane (TBOS) that produces 1-butanol as a growth substrate, for high mechanical strength. We optimized three inputs (concentration of PVA, concentration of AG, and the crosslinking time) on two responses (compressive modulus and rate of oxygen utilization) for batch incubation experiments between 1 and 30 days using a design of experiments approach. The predictive models generated from design of experiments were then tested by measuring the compressive strength, oxygen utilization, and abiotic rates of hydrolysis for a predicted optimal bead formulation. The result of this study generated a hydrogel bead with immobilized R. rhodochrous ATCC 21198 and TBOS that exhibited a high compressive modulus on day 1 and day 30, which was accurately predicted by models. These hydrogel beads exhibited low metabolic activity based on oxygen rates on day 1 and day 30 but were not accurately predicted by the models. In addition, the ratio between oxygen utilization and abiotic rates of hydrolysis were observed to be roughly half of what was expected stoichiometrically. Lastly, we demonstrated the capability to use these beads as a bioremediation technology for cDCE as we found that, for all bead formulations, cDCE was significantly reduced after 30 days. Altogether, this work demonstrates the capability to capture and enhance the material properties of the complex hydrogel beads with predictive models yet signals the need for more robust methods to understand the metabolic activity that occurs in the hydrogel beads.

PAH bioremediation with Rhodococcus rhodochrous ATCC 21198: Impact of cell immobilization and surfactant use on PAH treatment and post-remediation toxicity.

Polycyclic aromatic hydrocarbons (PAHs) are prevalent environmental contaminants that are harmful to ecological and human health. Bioremediation is a promising technique for remediating PAHs in the environment, however bioremediation often results in the accumulation of toxic PAH metabolites. The objectives of this research were to demonstrate the cometabolic treatment of a mixture of PAHs by a pure bacterial culture, Rhodococcus rhodochrous ATCC 21198, and investigate PAH metabolites and toxicity. Additionally, the surfactant Tween ® 80 and cell immobilization techniques were used to enhance bioremediation. Total PAH removal ranged from 70-95% for fluorene, 44-89% for phenanthrene, 86-97% for anthracene, and 6.5-78% for pyrene. Maximum removal was achieved with immobilized cells in the presence of Tween ® 80. Investigation of PAH metabolites produced by 21198 revealed a complex mixture of hydroxylated compounds, quinones, and ring-fission products. Toxicity appeared to increase after bioremediation, manifesting as mortality and developmental effects in embryonic zebrafish. 21198's ability to rapidly transform PAHs of a variety of molecular structures and sizes suggests that 21198 can be a valuable microorganism for catalyzing PAH remediation. However, implementing further treatment processes to address toxic PAH metabolites should be pursued to help lower post-remediation toxicity in future studies.

Influence of bovine serum albumin and alginate on silver nanoparticle dissolution and toxicity to Nitrosomonas europaea.

Bovine serum albumin (BSA), a model protein, reduced the toxicity of 20 nm citrate silver nanoparticles (AgNP) toward Nitrosomonas europaea, a model ammonia oxidizing bacteria, through a dual-mode protection mechanism. BSA reduced AgNP toxicity by chelating the silver ions (Ag(+)) released from the AgNPs. BSA further reduced AgNP toxicity by binding to the AgNP surface thus preventing NH3-dependent dissolution from occurring. Due to BSA's affinity toward Ag(+) chemisorbed on the AgNP surface, increased concentrations of BSA lead to increased AgNP dissolution rates. This, however, did not increase AgNP toxicity as the dissolved Ag(+) were adsorbed onto the BSA molecules. Alginate, a model extracellular polysaccharide (EPS), lacks strong Ag(+) ligands and was unable to protect N. europaea from Ag(+) toxicity. However, at high concentrations, alginate reduced AgNP toxicity by binding to the AgNP surface and reducing AgNP dissolution rates. Unlike BSA, alginate only weakly interacted with the AgNP surface and was unable to completely prevent NH3-dependent AgNP dissolution from occurring. Based on these results, AgNP toxicity in high protein environments (e.g., wastewater) is expected to be muted while the EPS layers of wastewater biofilms may provide additional protection from AgNPs, but not from Ag(+) that have already been released.

Effects of sulfate reduction on the bacterial community and kinetic parameters of a dechlorinating culture under chemostat growth conditions.

Results are presented from a chemostat study where the reductive dehalogenation of PCE was evaluated in the absence and presence of sulfate. Two chemostats inoculated with the Point Mugu culture, which contains strains of Dehalococcoides mccartyi, were operated at a 50 day HRT and fed PCE (1.12 mM) and lactate (4.3 mM). The control chemostat (PM-5L, no sulfate), achieved pseudo-steady-state transformation of PCE to ethene (98%) and VC (2%) at 2.4 nM of H(2). Batch kinetic tests with chemostat harvested cells showed the maximum rate (k(max)X) value for each dehalogenation step remained fairly constant, while hupL clone library analyses showed maintenance of a diverse D. mccartyi community. Sulfate (1 mM) was introduced to the second chemostat, PM-2L. Effective sulfate reduction was achieved 110 days later, resulting in 600 µM of total sulfide. PCE dechlorination efficiency decreased following complete sulfate reduction, yielding ethene (25%), VC (67%), and cis-DCE (8%). VC dechlorination was most affected, with k(max)X values decreasing by a factor of 50. The decrease was associated with the enrichment of the Cornell group of D. mccartyi and decline of the Pinellas group. Long-term exposure to sulfides and/or competition for H(2) may have been responsible for the community shift.

Fluorescent spectroscopy paired with parallel factor analysis for quantitative monitoring of phenanthrene biodegradation and metabolite formation.

Polycyclic aromatic hydrocarbons (PAHs) are a class of environmental contaminants released into the environment from both natural and anthropogenic sources that are associated with carcinogenic, mutagenic, and teratogenic health effects. Many remediation strategies for the treatment of PAH contaminated material, including bioremediation, can lead to the formation of toxic transformation products. Analytical techniques for PAHs and PAH transformation products often require extensive sample preparation including solvent extraction and concentration, chromatographic separation, and mass spectrometry to identify and quantify compounds of interest. Excitation-emission matrix (EEM) fluorescent spectroscopy paired with parallel factor analysis (PARAFAC) is an approach for analyzing PAHs that eliminates the need for extensive sample preparation and separation techniques before analysis. However, this technique has rarely been applied to monitoring PAH biotransformation and formation of PAH metabolites. The objectives of this research were to compare an established targeted analytical method to two-dimensional fluorescent spectroscopy and combined EEM-PARAFAC methods to monitor phenanthrene degradation by a bacterial pure culture, Mycobacterium Strain ELW1, identify and quantify phenanthrene transformation products, and derive kinetic constants for phenanthrene degradation and metabolite formation. Both phenanthrene and its primary transformation product, trans-9,10-dihydroxy-9,10-dihydrophenanthrene, were identified and quantified with the EEM-PARAFAC method. The value of the EEM-PARAFAC method was demonstrated in the superiority of sensitivity and accuracy of quantification to two-dimensional fluorescent spectroscopy. Quantification of targets and derivation of kinetic constants using the EEM-PARAFAC method were validated with an established gas chromatography-mass spectrometry (GC-MS) method. To the authors' knowledge, this is the first study to use an EEM-PARAFAC method to monitor, identify, and quantify both PAH biodegradation and PAH metabolite formation by a bacterial pure culture.

Inhibition and gene expression of Nitrosomonas europaea biofilms exposed to phenol and toluene.

Pure culture biofilms of the ammonia-oxidizing bacterium Nitrosomonas europaea were grown in a Drip Flow Biofilm Reactor and exposed to the aromatic hydrocarbons phenol and toluene. Ammonia oxidation rates, as measured by nitrite production in the biofilms, were inhibited 50% when exposed to 56 µM phenol or 100 µM toluene, while 50% inhibition of suspended cells occurred at 8 µM phenol or 20 µM toluene. Biofilm-grown cells dispersed into liquid medium and immediately exposed to phenol or toluene experienced similar inhibition levels as batch grown cells, indicating that mass transfer may be a factor in N. europaea biofilm resistance. Whole genome microarray analysis of gene expression was used to detect genes up-regulated in biofilms during toluene and phenol exposure. Two genes, a putative pirin protein (NE1545) and a putative inner membrane protein (NE1546) were up-regulated during phenol exposure, but no genes were up-regulated during toluene exposure. Using qRT-PCR, up-regulation of NE1545 was detected in biofilms and suspended cells exposed to a range of phenol concentrations and levels of inhibition. In the biofilms, NE1545 expression was up-regulated an average of 13-fold over the range of phenol concentrations tested, and was essentially independent of phenol concentration. However, the expression of NE1545 in suspended cells increased from 20-fold at 7 µM phenol up to 80-fold at 30 µM phenol. This study demonstrates that biofilms of N. europaea are more resistant than suspended cells to inhibition of ammonia oxidation by phenol and toluene, even though the global transcriptional responses to the inhibitors do not differ in N. europaea between the suspended and attached growth states.

Long-term cometabolic transformation of 1,1,1-trichloroethane and 1,4-dioxane by Rhodococcus rhodochrous ATCC 21198 grown on alcohols slowly produced by orthosilicates.

Long-term cometabolic transformation of 1,1,1-trichlorethane (1,1,1-TCA) and 1,4-dioxane (1,4-D) was achieved using slow release compounds (SRCs) as growth substrates for pure cultures of Rhodococcus rhodochrous ATCC 21198 (ATCC strain 21198). Resting cell transformation tests showed 1,4-D transformation occurred without a lag phase for cells grown on 2-butanol, while an induction period of several hours was required for 1-butanol grown cells. These observations were consistent with activity-based labeling patterns for monooxygenase hydroxylase components and specific rates of tetrahydrofuran (THF) degradation. 1,1,1-TCA and 1,4-D degradation rates for alcohol-grown cells were slower than those for cells grown on gaseous alkanes such as isobutane. Batch metabolism and degradation tests were performed, in the presence of 1,1,1-TCA and 1,4-D, with the growth of ATCC strain 21198 on alcohols produced by the hydrolysis of orthosilicates. Three orthosilicates were tested: tetrabutylorthosilicate (TBOS), tetra-s-butylorthosilicate (T2BOS), and tetraisopropoxysilane (T2POS). The measured rates of alcohol release in poisoned controls depended on the orthosilicate structure with TBOS, which produced a 1° alcohol (1-butanol), hydrolyzing more rapidly than T2POS and T2BOS, that produced the 2° alcohols 2-butanol and 2-propanol, respectively. The orthosilicates were added as light non-aqueous phase liquids (LNAPLs) with ATCC strain 21198 and formed dispersed droplets when continuously mixed. Continuous rates of oxygen (O2) consumption and carbon dioxide (CO2) production confirmed alcohol metabolism by ATCC strain 21198 was occurring. The rates of metabolism (TBOS > T2POS > T2BOS) were consistent with the rates of alcohol release via abiotic hydrolysis. 1,4-D and 1,1,1-TCA were continuously transformed in successive additions by ATCC strain 21198 over 125 days, with the rates highly correlated with the rates of metabolism. The metabolism of the alcohols was not inhibited by acetylene, while transformation of 1,4-D and 1,1,1-TCA was inhibited by this gas. As acetylene is a potent inactivator of diverse bacterial monooxygenases, these results suggest that monooxygenase activity was required for the observed cometabolic transformations but not for alcohol utilization. Alcohol concentrations in the biologically active reactors were maintained below the levels of detection, indicating they were metabolized rapidly after being produced. Much lower rates of O2 consumption were observed in the reactors containing T2BOS, which has benefits for in-situ bioremediation. The results illustrate the importance of the structure of the SRC when developing passive aerobic cometabolic treatment systems.

Trichloroethene and cis-1,2-dichloroethene concentration-dependent toxicity model simulates anaerobic dechlorination at high concentrations: I. batch-fed reactors.

A model was developed to describe toxicity from high concentrations of chlorinated aliphatic hydrocarbons (CAHs) on reductively dechlorinating cultures under batch-growth conditions. A reductively dechlorinating anaerobic Evanite subculture (EV-cDCE) was fed trichloroethene (TCE) and excess electron donor to accumulate cis-1,2-dichloroethene (cDCE) in batch-fed reactors. A second Point Mugu (PM) culture was also studied in the cDCE accumulating batch-fed experiment, as well as in a time- and concentration-dependent cDCE exposure experiment. Both cultures accumulated cDCE to concentrations ranging from 9,000 to 12,000 microM before cDCE production from TCE ceased. Exposure to approximately 3,000 and 6,000 microM cDCE concentrations for 5 days during continuous TCE dechlorination exhibited greater loss in activity proportional to both time and concentration of exposure than simple endogenous decay. Various inhibition models were analyzed for the two cultures, including the previously proposed Haldane inhibition model and a maximum threshold inhibition model, but neither adequately fit all experimental observations. A concentration-dependent toxicity model is proposed, which simulated all the experimental observations well. The toxicity model incorporates CAH toxicity terms that directly increase the cell decay coefficient in proportion with CAH concentrations. We also consider previously proposed models relating toxicity to partitioning in the cell wall (K(M/B)), proportional to octanol-water partitioning (K(OW)) coefficients. A reanalysis of previously reported modeling of batch tests using the Haldane model of Yu and Semprini, could be fit equally well using the toxicity model presented here, combined with toxicity proportioned to cell wall partitioning. A companion paper extends the experimental analysis and our modeling approach to a completely mixed reactor and a fixed film reactor.

Trichloroethene and cis-1,2-dichloroethene concentration-dependent toxicity model simulates anaerobic dechlorination at high concentrations. II: continuous flow and attached growth reactors.

A model that was used to describe toxicity from high concentrations of chlorinated aliphatic hydrocarbons (CAHs) on reductively dechlorinating cultures in batch reactors (Sabalowsky and Semprini (in press)) was extended here to simulate observations in continuous flow suspended and attached growth reactors. The reductively dechlorinating anaerobic Evanite subculture (EV-cDCE) was fed trichloroethene (TCE) and excess electron donor to accumulate cis-1,2-dichloroethene (cDCE) in a continuous flow stirred tank reactor (CFSTR); and an attached growth recirculating packed column (RPC). A concentration-dependent toxicity model used to simulate the results of batch reactors in part I (Sabalowsky and Semprini (in press) Biotechnol Bioeng) also simulated well the observations for the CFSTR and RPC growth modes. The toxicity model incorporates cDCE and TCE toxicity coefficients that directly increase the cell decay coefficient in proportion with cDCE and TCE concentrations. Simulated estimates of the cDCE and TCE toxicity coefficients indicate reductively dechlorinating cells are most sensitive to high concentrations of cDCE and TCE in batch-fed growth, followed by CFSTR, with attached growth being least sensitive. The greater toxicity of TCE than cDCE, and ratio of the modeled toxicity coefficients, agrees with previously proposed models relating toxicity to partitioning in the cell wall (K(M/B)), proportional to octanol-water partitioning (K(OW)) coefficients.

Extending the alkene substrate range of vinyl chloride utilizing Nocardioides sp. strain JS614 with ethene oxide.

Nocardioides sp. strain JS614 grows on the C(2) alkenes ethene (Eth), vinyl chloride, and vinyl fluoride as sole carbon sources. The presence of 400-800 microM ethene oxide (EtO) extended the growth substrate range to propene (C(3)) and butene (C(4)). Propene-dependent growth of JS614 was CO(2) dependent and was prevented by the carboxylase/reductase inhibitor 2-bromoethanesulfonic acid, sodium salt (BES), while growth on Eth was not CO(2) dependent or BES sensitive. Although unable to promote growth, both propene and propene oxide (PrO)-induced expression of the genes encoding the alpha subunit of alkene monooxygenase (etnC) and epoxyethane CoM transferase (etnE) to similar levels as did Eth and EtO. Propene was transformed by Eth-grown and propene-grown/EtO-induced JS614 to PrO at a rate 4.2 times faster than PrO was consumed. As a result PrO accumulated in growth medium to 900 microM during EtO-induced growth on propene. PrO (50-100 microM) exerted inhibitory effects on growth of JS614 on both acetate and Eth, and on EtO-induced growth on Eth. However, higher EtO concentrations (300-400 microM) overcame the negative effects of PrO on Eth-dependent growth.