David L. Freedman

Characterization of an Isolate That Uses Vinyl Chloride as a Growth Substrate under Aerobic Conditions

ABSTRACT An aerobic enrichment culture was developed by using vinyl chloride (VC) as the sole organic carbon and electron donor source. VC concentrations as high as 7.3 mM were biodegraded without apparent inhibition. VC use did not occur when nitrate was provided as the electron acceptor. A gram-negative, rod-shaped, motile isolate was obtained from the enrichment culture and identified based on biochemical characteristics and the sequence of its 16S rRNA gene asPseudomonas aeruginosa, designated strain MF1. The observed yield of MF1 when it was grown on VC was 0.20 mg of total suspended solids (TSS)/mg of VC. Ethene, acetate, glyoxylate, and glycolate also served as growth substrates, while ethane, chloroacetate, glycolaldehyde, and phenol did not. Stoichiometric release of chloride and minimal accumulation of soluble metabolites following VC consumption indicated that the predominant fate for VC is mineralization and incorporation into cell material. MF1 resumed consumption of VC after at least 24 days when none was provided, unlike various mycobacteria that lost their VC-degrading ability after brief periods in the absence of VC. When deprived of oxygen for 2.5 days, MF1 did not regain the ability to grow on VC, and a portion of the VC was transformed into VC-epoxide. Acetylene inhibited VC consumption by MF1, suggesting the involvement of a monooxygenase in the initial step of VC metabolism. The maximum specific VC utilization rate for MF1 was 0.41 μmol of VC/mg of TSS/day, the maximum specific growth rate was 0.0048/day, and the Monod half-saturation coefficient was 0.26 μM. A higher yield and faster kinetics occurred when MF1 grew on ethene. When grown on ethene, MF1 was able to switch to VC as a substrate without a lag. It therefore appears feasible to grow MF1 on a nontoxic substrate and then apply it to environments that do not exhibit a capacity for aerobic biodegradation of VC.

Involvement of Linear Plasmids in Aerobic Biodegradation of Vinyl Chloride

ABSTRACT Pseudomonas putida strain AJ and Ochrobactrum strain TD were isolated from hazardous waste sites based on their ability to use vinyl chloride (VC) as the sole source of carbon and energy under aerobic conditions. Strains AJ and TD also use ethene and ethylene oxide as growth substrates. Strain AJ contained a linear megaplasmid (approximately 260 kb) when grown on VC or ethene, but it contained no circular plasmids. While strain AJ was growing on ethylene oxide, it was observed to contain a 100-kb linear plasmid, and its ability to use VC as a substrate was retained. The linear plasmids in strain AJ were cured, and the ability of strain AJ to consume VC, ethene, and ethylene oxide was lost following growth on a rich substrate (Luria-Bertani broth) through at least three transfers. Strain TD contained three linear plasmids, ranging in size from approximately 90 kb to 320 kb, when growing on VC or ethene. As with strain AJ, the linear plasmids in strain TD were cured following growth on Luria-Bertani broth and its ability to consume VC and ethene was lost. Further analysis of these linear plasmids may help reveal the pathway for VC biodegradation in strains AJ and TD and explain why this process occurs at many but not all sites where groundwater is contaminated with chloroethenes. Metabolism of VC and ethene by strains AJ and TD is initiated by an alkene monooxygenase. Their yields during growth on VC (0.15 to 0.20 mg of total suspended solids per mg of VC) are similar to the yields reported for other isolates (i.e., Mycobacterium sp., Nocardioides sp., and Pseudomonas sp.).

Use of Cyanocobalamin To Enhance Anaerobic Biodegradation of Chloroform

Biodegradation of chloroform (CF) was examined in a methanogenic enrichment culture grown on dichloromethane (DCM) as the sole organic carbon and energy source, with and without the addition of supplemental cyanocobalamin. In the absence of cyanocobalamin, the principal products of P4C1 CF biodegradation were 14C02 and V4C1DCM. The extent of CF reduction to DCM increased significantly when CF was biodegraded in the presence of a large amount of DCM. The addition of cyanocobalamin enhanced CF biodegradation in two ways. First, the rate of CF biodegradation increased approximately 10-fold. Second, the metallocofactor increased the extent of CF oxidation to C02 and virtually eliminated the accumulation of DCM. These effects were not observed in autoclaved cultures supplemented with cyanocobalamin. When cyanocobalamin was added to viable cultures, as much as 10 % of the [l4C1CF transformed accumulated as 14C-labeled carbon monoxide. This suggested that the oxidation of CF to C02 proceeds via net hydrolysis to CO. CF levels as high as 2.2 mM were readily transformed, without accumulation of DCM, at cyanocobalamin to CF molar ratios of 3-596. Although the organism or consortium responsible for CF biodegradation was not identified, prior work with DCM suggests that acetogenic bacteria are involved.

Natural Attenuation of Trichloroethylene in Rhizosphere Soils at the Savannah River Site

Extensive trichloroethylene (TCE) groundwater contamination has resulted from discharges to a former seepage basin in the A/M Area at the Department of Energys Savannah River Site. The direction of groundwater flow has been determined and a seep line where the contaminated groundwater is estimated to emerge as surface water has been identified in a region of the Southern Sector of the A/M Area. This study was undertaken to estimate the potential of four rhizosphere soils along the seep line to naturally attenuate TCE. Microcosms were setup to evaluate both biotic and abiotic attenuation of TCE. Results demonstrated that sorption to soil was the dominant mechanism during the first week of incubation, with as much as 90% of the TCE removed from the aqueous phase. Linear partitioning coefficients (Kd) ranged from 0.83 to 7.4 mL/g, while organic carbon partition coefficients (Koc) ranged from 72 to 180 mL/gC. Diffu-sional losses from the microcosms appeared to be a dominant fate mechanism during the remainde...

Biotransformation of explosive-grade nitrocellulose under denitrifying and sulfidogenic conditions

Waste nitrocellulose (NC) is regulated as a hazardous material. The objective of this study was to determine if NC exposed to denitrifying and sulfidogenic conditions would undergo sufficient removal of the nitro groups to yield a material that is no longer explosive. Enrichment cultures were established with methanol as the electron donor for nitrate-reducing conditions and lactate for sulfate-reducing conditions. NC was added to the cultures at 10 g/l. A statistically significant decrease in the nitrogen (N) content of NC occurred in both enrichment cultures, from approximately 13.1-13.2% in virgin NC to 12.2-12.4%. This was accompanied by an increase in nitrogen gas formation. The presence of a primary substrate (methanol and lactate) was necessary to affect this change; NC itself did not serve as an electron donor. In cultures that were carrying out denitrification but were then depleted of nitrate, with methanol still present, a slightly greater removal of nitro groups from NC occurred along with additional formation of nitrogen gas. NC did not have an inhibitory affect on the denitrification process but it did significantly slow the rate of lactate consumption and sulfate reduction. Fourier Transform Infrared Spectroscopy (FTIR) results indicated that NC exposed to denitrifying conditions was enriched in hydroxyl groups, consistent with removal of some of the nitro groups by hydrolysis of the nitrate esters. NC exposed to nitrate- and sulfate-reducing conditions and virgin NC were also compared based on their explosive properties using a small-scale burning test. The biologically treated NC exhibited somewhat less reactivity, but was still rated as explosive. The decrease in%N, increase in N2, and FTIR results demonstrated that NC does undergo biotransformation in the presence of nitrate- and sulfate-reducing enrichment cultures, but the extent of denitration does not appear to be adequate to yield a nonhazardous product.

Henry’s law constants of chlorinated solvents at elevated temperatures

Henrys law constants for 12 chlorinated volatile organic compounds (CVOCs) were measured as a function of temperature ranging from 8 to 93°C, using the modified equilibrium partitioning in closed system (EPICS) method. The chlorinated compounds include tetrachloroethylene, trichloroethylene, cis-1,2-dichloroethylene, vinyl chloride, 1,1,1-trichloroethane, 1,1-dichloroethane, 1,2-dichloroethane, chloroethane, carbon tetrachloride, chloroform, dichloromethane, and chloromethane. The variation in Henrys constants for these compounds as a function of temperature ranged from around 3-fold (chloroethane) to 30-fold (1,2-dichloroethane). Aqueous solubilities of the pure compounds were measured over the temperature range of 8-75°C. The temperature dependence of Henrys constant was predicted using the ratio of pure vapor pressure to aqueous solubility, both of which are functions of temperature. The calculated Henrys constants are in a reasonable agreement with the measured results. With the improved data on Henrys law constants at high temperatures measured in this study, it will be possible to more accurately model subsurface remediation processes that operate near the boiling point of water.

High protein expression in fermentation of recombinant Pichia pastoris by a fed-batch process

Abstract A fed-batch fermentation process was developed to culture recombinant Pichia pastoris at high cell density and with high protein expression. High levels of the thrombomodulin fragment were obtained from an SMD1168 strain (pep4−) that had a methanol utilization slow (mut8) phenotype. Dissolved oxygen concentration (DO) was controlled by cascading DO with agitation and pure oxygen supplementation, thereby avoiding oxygen limitation during the entire process. Wet cell density reached 420 g/litre and the concentration of protein, an 86 amino acid thrombomodulin fragment, was 360 mg/litre.

Involvement of Coenzyme M during Aerobic Biodegradation of Vinyl Chloride and Ethene by Pseudomonas putida Strain AJ and Ochrobactrum sp. Strain TD

ABSTRACT The involvement of coenzyme M in aerobic biodegradation of vinyl chloride and ethene in Pseudomonas putida strain AJ and Ochrobactrum sp. strain TD was demonstrated using PCR, hybridization, and enzyme assays. The results of this study extend the range of eubacteria known to use epoxyalkane:coenzyme M transferase.

Modeling the kinetics of vinyl chloride cometabolism by an ethane-grown Pseudomonas sp.

Pseudomonas sp strain EA1 was isolated under aerobic conditions using ethane as the sole organic carbon and electron donor source, with an observed yield of 0.99 mg total suspended solids/mg ethane (0.85 mg volatile suspended solids / mg ethane) and a maximum specific growth rate of 0.015 d(-1). When grown on ethane, EA1 cometabolizes vinyl chloride (VC) at a maximum rate of 0.350 micromol/mg volatile suspended solids/d and with a half saturation constant of 0.62 microM VC. The rate of VC use by EA1 is twice as high when ethane is also provided, even though consumption of ethane is almost completely inhibited until VC is consumed. The presence of ethane also reduces the total amount of VC cometabolized. A model was developed that adequately describes the batch kinetics of VC cometabolism in the presence and absence of ethane, as well as ethane metabolism in the presence and absence of VC. Terms are included that increase the initial rate of VC use in the presence of ethane (according to the ratio of initial ethane concentration to the half saturation coefficient) but decrease the total amount of VC cometabolized. Parameter estimates for the model were obtained using a step-wise experimental approach, with varying initial concentrations of VC and ethane. Strain EA1 completely dechlorinates VC in the presence and absence of ethane. Measurements of soluble chemical oxygen demand indicate that approximately 50% of the VC consumed is mineralized, with the balance released as soluble, nonchlorinated products. Ethene is not used as a substrate by EA1 but it does inhibit ethane metabolism and VC cometabolism. In mixtures containing all three compounds, more VC is degraded and at a faster rate compared to VC plus ethene. The results suggest that ethane-enhanced biodegradation of VC may contribute to VC removal at the aerobic fringe of groundwater plumes undergoing reductive dechlorination.

Bioaugmentation of Sequencing Batch Reactors for Biological Phosphorus Removal: Comparative rRNA Sequence Analysis and Hybridization with Oligonucleotide Probes

Four laboratory-scale sequencing batch reactors (SBRs) were operated to evaluate whether bioaugmentation with Acinetobacter spp. can be used to improve start-up and performance of enhanced biological phosphorus removal (EBPR) systems. Two of the SBRs were bioaugmented during start-up by adding pure cultures of Acinetobacter spp., the third reactor received an amendment of activated sludge from a laboratory-scale EBPR system, and the fourth reactor, receiving no amendment, served as a control. Various chemical parameters were measured to monitor the performance of the four SBRS. Oligonucleotide probes of nested phylogenetic specificity were designed to quantify the contribution of Acinetobacter to EBPR. The probes were characterized for use in quantitative membrane hybridizations and fluorescent in situ hybridizations. Data from hybridizations with samples collected from the SBRs show declining levels of Acinetobacter spp. over the experiment. All four reactors achieved significant phosphorus removal and 90% nitrification after three days of operation. The results do not show a positive correlation between levels of Acinetobacter and successful EBPR.