Distinct Dual C-Cl Isotope Fractionation Patterns during Anaerobic Biodegradation of 1,2-Dichloroethane: Potential To Characterize Microbial Degradation in the Field.

This study investigates, for the first time, dual C-Cl isotope fractionation during anaerobic biodegradation of 1,2-dichloroethane (1,2-DCA) via dihaloelimination by Dehalococcoides and Dehalogenimonas-containing enrichment cultures. Isotopic fractionation of 1,2-DCA (εbulkC and εbulkCl) for Dehalococcoides (-33.0 ± 0.4‰ and -5.1 ± 0.1‰) and Dehalogenimonas-containing microcosms (-23 ± 2‰ and -12.0 ± 0.8‰) resulted in distinctly different dual element C-Cl isotope correlations (Λ = Δδ13C/Δδ37Cl ≈ εbulkC/εbulkCl), 6.8 ± 0.2 and 1.89 ± 0.02, respectively. Determined isotope effects and detected products suggest that the difference on the obtained Λ values for biodihaloelimination could be associated with a different mode of concerted bond cleavage rather than two different reaction pathways (i.e., stepwise vs concerted). Λ values of 1,2-DCA were, for the first time, determined in two field sites under reducing conditions (2.1 ± 0.1 and 2.2 ± 2.9). They were similar to the one obtained for the Dehalogenimonas-containing microcosms (1.89 ± 0.02) and very different from those reported for aerobic degradation pathways in a previous laboratory study (7.6 ± 0.1 and 0.78 ± 0.03). Thus, this study illustrates the potential of a dual isotope analysis to differentiate between aerobic and anaerobic biodegradation pathways of 1,2-DCA in the field and suggests that this approach might also be used to characterize dihaloelimination of 1,2-DCA by different bacteria, which needs to be confirmed in future studies.

Comparative proteomics of Dehalococcoides spp. reveals strain-specific peptides associated with activity.

Anaerobic reductive dehalogenation by Dehalococcoides spp. is an ideal system for studying functional diversity of closely related strains of bacteria. In Dehalococcoides spp., reductive dehalogenases (RDases) are key respiratory enzymes involved in the anaerobic detoxification of halogenated compounds at contaminated sites globally. Although housekeeping genes sequenced from Dehalococcoides spp. are >85% identical at the amino acid level, different strains are capable of dehalogenating diverse ranges of compounds, depending largely on the suite of RDase genes that each strain harbors and expresses. We identified RDase proteins that corresponded to known functions in four characterized cultures and predicted functions in an uncharacterized Dehalococcoides-containing mixed culture. Homologues within RDase subclusters containing PceA, TceA, and VcrA were among the most frequently identified proteins. Several additional proteins, including a formate dehydrogenase-like protein (Fdh), had high coverage in all strains and under all growth conditions.

The effect of nutrient deficiency on removal of organic solvents from textile manufacturing wastewater during activated sludge treatment.

Textile manufacturing wastewater is often deficient in nitrogen and phosphorus and contains hazardous solvents, including methyl ethyl ketone (MEK), methyl isobutyl ketone (MIBK), toluene (TOL), and xylenes (XYL). The objectives of this study were to evaluate the effectiveness of a short-term batch assay for predicting when a nutrient deficient condition exists in textile wastewater activated sludge, and to determine if nutrient deficiency affects biodegradation of MEK, MIBK, TOL,and p-XYL to a greater or lesser extent than bulk soluble chemical oxygen demand (sCOD). Addition of N + P significantly improved sCOD removal during treatment of textile wastewater in laboratory-scale sequencing batch reactors (SBRs). Batch tests using mixed liquor suspended solids (MLSS) from the SBRs correctly predicted the nutrient deficiency in the reactors that received unamended wastewater. During batch tests in sealed containers (to prevent volatilization) when N + P were added, the solvents biodegraded faster and to a greater extent than the bulk wastewater sCOD. MEK and MIBK were also completely consumed in MLSS from the SBR that received unamended wastewater, indicating that a shortage of nutrients did not significantly impact biodegradation of these ketones. However, nutrient deficient conditions significantly decreased the rate of TOL and p-XYL biodegradation. The difference in biodegradability of the ketones and monoaromatics under nutrient deficient conditions may be related to loss of plasmids required for aerobic catabolism of TOL and p-XYL. These results demonstrate that N + P addition to nutrient-deficient textile wastewater improves bulk sCOD removal and also significantly improves the biodegradability of TOL and p-XYL, thereby reducing the amount released to the atmosphere by volatilization.

Phytoremediation of pyrene in a Cecil soil under field conditions.

We evaluated the effects of annual ryegrass (Lolium multiflorum Lam.) and phosphorus (P) availability on the dissipation of pyrene added at a concentration of approximately 600 mg kg-1 dry soil in the top 7.5 cm of a Cecil loamy sand (fine, kaolinitic, thermic Typic Kanhapludults) in a 10-month experiment under field conditions in Clemson, South Carolina. Plastic canopies were installed to prevent flooding of plots and raindrop dispersion of pyrene. Treatment factors were pyrene, vegetation, and available P levels. Each of the eight treatments had four replicates. The soil was adjusted to low and high P concentrations (an average of 41 and 66 kg extractable P ha-1, respectively). After a 175-d lag period for all treatments, the rate of pyrene removal followed first-order kinetics. The first-order rate constant was significantly higher in nonvegetated (0.098 d-1) than vegetated treatments (0.034 d-1). These data suggest that the presence of easily biodegradable organic matter from plant roots slowed the removal rate of pyrene. The levels of available P did not affect the rate of pyrene dissipation. Pyrene decreased below the detection limit of 6.25 mg kg-1 dry soil in all treatments after 301 d.

Transition from cometabolic to growth-linked biodegradation of vinyl chloride by a Pseudomonas sp. isolated on ethene.

Pseudomonas aeruginosa strain DL1 was isolated on ethene as a sole carbon and energy source. When ethene-grown DL1 was first exposed to vinyl chloride (VC), the rate of VC consumption was very rapid and then declined sharply, indicative of a cometabolic process. A lack of growth and significant release of soluble products during this interval also indicates that the initial activity on VC was cometabolic. Following the rapid initial rate of VC cometabolism, a slow rate of VC utilization continued. After an extended period of incubation (>40 days), a transition occurred that allowed DL1 to begin using VC as a primary growth substrate, with an observed yield, maximum growth rate, and Monod half saturation coefficient of 0.21 mg of total suspended solids/mg VC, 0.046 d(-1), and 1.17 microM VC, respectively, at 22 degrees C. Acetylene inhibits consumption of ethene and VC by ethene-grown cells, suggesting a monooxygenase is responsible for initiating metabolism of these alkenes. Resting cells grown on ethene cometabolized VC with an observed transformation capacity of 9.1 micromol VC/mg total suspended solids and a transformation yield of 0.22 mol VC/mol ethene. The presence of 40 microM ethene increased the rate and amount of VC cometabolized. However, consumption of higher concentrations of ethene decreased the total amount of VC consumed, and VC inhibited ethene utilization. A kinetic model was developed that describes substrate interactions during batch depletion of ethene and VC for a range of initial concentrations. The results suggest that ethene may stimulate in situ biodegradation of VC either by functioning as a primary substrate to support cometabolism of VC or by selecting for organisms that can utilize VC as a primary substrate.

Substrate interactions during aerobic biodegradation of methane, ethene, vinyl chloride and 1,2-dichloroethenes.

Intrinsic biodegradation of trichloroethene and 1,1,1-trichloroethane in groundwater at a Superfund site in California has been observed. An anaerobic zone exists in the area closest to the source location, yielding the expected complement of reductive dechlorination daughter products, including cis-1,2-dichloroethene (cis-DCE) and vinyl chloride (VC). Significant levels of methane and ethene were also generated in the anaerobic zone. The groundwater returns to aerobic conditions downgradient of the source, with methane, ethene, VC, and several other compounds still present. Attenuation of VC in the aerobic zone suggests that it is being biodegraded. In this study microcosms were used to evaluate the role of methane and ethene as primary substrates for aerobic biodegradation of VC. Biodegradation of VC was fastest in the bottles containing ethene, with 40 mumol of VC consumed over a 150 day period, compared to approximately 15-20 mumol with methane or a mixture of methane and ethene. VC did not noticeably inhibit ethene biodegradation but did slow the rate of methane use. Methane inhibited ethene metabolism, which apparently caused a reduction in VC biodegradation when methane was present with ethene. These results suggest that ethene plays an important role during in situ natural attenuation of VC under aerobic conditions. Microcosms were also set up with VC alone. Following a 75 day lag period. VC consumption began and subsequent additions were consumed without a lag, suggesting the presence of organisms capable of using VC as a growth substrate. After providing VC alone for nearly 400 days, aliquots of the enrichment culture were used to evaluate its ability to biodegrade cis- and trans-DCE. Both compounds were readily consumed, although addition of VC as the primary substrate was needed to sustain biodegradation of repeated additions. This result suggests that organisms capable of using VC as a sole substrate may play an active role in aerobic natural attenuation of DCEs.

Characterization of an isolate that uses vinyl chloride as a growth substrate under aerobic conditions.

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 as Pseudomonas 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 micromol of VC/mg of TSS/day, the maximum specific growth rate was 0.0048/day, and the Monod half-saturation coefficient was 0.26 microM. 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.

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.

Physical map and gene survey of the Ochrobactrum anthropi genome using bacterial artificial chromosome contigs.

Bacterial artificial chromosome (BAC) clones are effective mapping and sequencing reagents for use with a wide variety of small and large genomes. This report describes research aimed at determining the genome structure of Ochrobactrum anthropi, an opportunistic human pathogen that has potential applications in biodegradation of hazardous organic compounds. A BAC library for O. anthropi was constructed that provides a 70-fold genome coverage based on an estimated genome size of 4.8 Mb. The library contains 3072 clones with an average insert size of 112 kb. High-density colony filters of the library were made, and a physical map of the genome was constructed using a hybridization without replacement strategy. In addition, 1536 BAC clones were fingerprinted with HindIII and analyzed using IMAGE and Fingerprint Contig software (FPC, Sanger Centre, U.K.). The FPC results supported the hybridization data, resulting in the formation of two major contigs representing the two major replicons of the O. anthropi genome. After determining a reduced tiling path, 138 BAC ends from the reduced tile were sequenced for a preliminary gene survey. A search of the public databases with the BLASTX algorithm resulted in 77 strong hits (E-value < 0.001), of which 89% showed similarity to a wide variety of prokaryotic genes. These results provide a contig-based physical map to assist the cloning of important genomic regions and the potential sequencing of the O. anthropi genome.

Enhanced biotransformation of carbon tetrachloride by Acetobacterium woodii upon addition of hydroxocobalamin and fructose.

The objective of this study was to evaluate the effect of hydroxocobalamin (OH-Cbl) on transformation of high concentrations of carbon tetrachloride (CT) by Acetobacterium woodii (ATCC 29683). Complete transformation of 470 microM (72 mg/liter [aqueous]) CT was achieved by A. woodii within 2.5 days, when 10 microM OH-Cbl was added along with 25.2 mM fructose. This was approximately 30 times faster than A. woodii cultures (live or autoclaved) and medium that did not receive OH-Cbl and 5 times faster than those controls that did receive OH-Cbl, but either live A. woodii or fructose was missing. CT transformation in treatments with only OH-Cbl was indicative of the important contribution of nonenzymatic reactions. Besides increasing the rate of CT transformation, addition of fructose and OH-Cbl to live cultures increased the percentage of [(14)C]CT transformed to (14)CO(2) (up to 31%) and (14)C-labeled soluble materials (principally L-lactate and acetate), while decreasing the percentage of CT reduced to chloroform and abiotically transformed to carbon disulfide. (14)CS(2) represented more than 35% of the [(14)C]CT in the presence of reduced medium and OH-Cbl. Conversion of CT to CO was a predominant pathway in formation of CO(2) in the presence of live cells and added fructose and OH-Cbl. These results indicate that the rate and distribution of products during cometabolic transformation of CT by A. woodii can be improved by the addition of fructose and OH-Cbl.

Helcococcus kunzii as sole isolate from an infected sebaceous cyst.

Helcococcus kunzii was isolated in pure culture from pus drained from an infected sebaceous cyst associated with marked cellulitis. The cyst was excised one month later after the inflammation had subsided with flucloxacillin treatment. This is the first report of the isolation of H. kunzii as the sole pathogen from an infected site.

Reduction and Acetylation of 2,4-Dinitrotoluene by a Pseudomonas aeruginosa Strain.

Aerobic and anoxic biotransformation of 2,4-dinitrotoluene (DNT) was examined by using a Pseudomonas aeruginosa strain isolated from a plant treating propellant manufacturing wastewater. DNT biotransformation in the presence and absence of oxygen was mostly reductive and was representative of the type of cometabolic transformations that occur when a high concentration of an easily degradable carbon source is present. P. aeruginosa reduced both nitro groups on DNT, with the formation of mainly 4-amino-2-nitrotoluene and 2-amino-4-nitrotoluene and small quantities of 2,4-diaminotoluene. Acetylation of the arylamines was a significant reaction. 4-Acetamide-2-nitrotoluene and the novel compounds 2-acetamide-4-nitrotoluene, 4-acetamide-2-aminotoluene, and 2,4-diacetamidetoluene were identified as DNT metabolites. The biotransformation of 2,4-diaminotoluene to 4-acetamide-2-aminotoluene was 24 times faster than abiotic transformation. 2-Nitrotoluene and 4-nitrotoluene were also reduced to their corresponding toluidines and then acetylated. However, the yield of 4-acetamidetoluene was much higher than that of 2-acetamidetoluene, demonstrating that acetylation at the position para to the methyl group was favored.

Biodegradation of dichloromethane and its utilization as a growth substrate under methanogenic conditions.

Biodegradation of dichloromethane (DCM) to environmentally acceptable products was demonstrated under methanogenic conditions (35 degrees C). When DCM was supplied to enrichment cultures as the sole organic compound at a low enough concentration to avoid inhibition of methanogenesis, the molar ratio of CH4 formed to DCM consumed (0.473) was very close to the amount predicted by stoichiometric conservation of electrons. DCM degradation was also demonstrated when methanogenesis was partially inhibited (with 0.5 to 1.5 mM 2-bromoethanesulfonate or approximately 2 mM DCM) or completely stopped (with 50 to 55.5 mM 2-bromoethanesulfonate). Addition of a eubacterial inhibitor (vancomycin, 100 mg/liter) greatly reduced the rate of DCM degradation. 14CO2 was the principal product of [14C]DCM degradation, followed by 14CH4 (when methanogenesis was uninhibited) or 14CH3COOH (when methanogenesis was partially or completely inhibited). Hydrogen accumulated during DCM degradation and then returned to background levels when DCM was consumed. These results suggested that nonmethanogenic organisms mediated DCM degradation, oxidizing a portion to CO2 and fermenting the remainder to acetate; acetate formation suggested involvement of an acetogen. Methanogens in the enrichment culture then converted the products of DCM degradation to CH4. Aceticlastic methanogens were more easily inhibited by 2-bromoethanesulfonate and DCM than were CO2-reducing methanogens. When DCM was the sole organic-carbon and electron donor source supplied, its use as a growth substrate was demonstrated. The highest observed yield was 0.085 g of suspended organic carbon formed per g of DCM carbon consumed. Approximately 85% of the biomass formed was attributable to the growth of nonmethanogens, and 15% was attributable to methanogens.

Biological reductive dechlorination of tetrachloroethylene and trichloroethylene to ethylene under methanogenic conditions.

A biological process for remediation of groundwater contaminated with tetrachloroethylene (PCE) and trichloroethylene (TCE) can only be applied if the transformation products are environmentally acceptable. Studies with enrichment cultures of PCE- and TCE-degrading microorganisms provide evidence that, under methanogenic conditions, mixed cultures are able to completely dechlorinate PCE and TCE to ethylene, a product which is environmentally acceptable. Radiotracer studies with [14C]PCE indicated that [14C]ethylene was the terminal product; significant conversion to 14CO2 or 14CH4 was not observed. The rate-limiting step in the pathway appeared to be conversion of vinyl chloride to ethylene. To sustain reductive dechlorination of PCE and TCE, it was necessary to supply an electron donor; methanol was the most effective, although hydrogen, formate, acetate, and glucose also served. Studies with the inhibitor 2-bromoethanesulfonate suggested that methanogens played a key role in the observed biotransformations of PCE and TCE.

Treatment of prostatic carcinoma by gonadotropin inhibition with danazol. A preliminary report.

The effect of an oral gonadotropin agent--Danazol--on 5 patients with prostatic carcinoma was investigated. A phase 2 pilot study, using a combination of repeated clinical findings, hormone analyses and cytological examinations, allowed some assessment of results after 16 weeks. There was a marked fall in gonadotropin and testosterone serum levels in all patients. Despite a minor rate of cancer remission, results of clinical and cytological examinations were encouraging enough to stimulate further investigations.

Inguinal herniorrhaphy in a health center.

39 selected patients had their inguinal herniae repaired on 41 separate occasions in a health center. Local anaesthesia allowed adequate painfree surgery when supplemented with Diazepam and Pentazocin. All patients could return to their own homes within 6 hours. With an observation time of between one month and six years no recurrences have so far been observed. Apart from a mild post-operative pain, there were no complications. All returned to their normal employment after 5 to 32 days (average 20 days) depending on occupational demands. The pros and cons of out-patient surgery under local anaesthesia are discussed.