Efficient removal of residual lignin from eucalyptus pulp via high-concentration chlorine dioxide treatment and its effect on the properties of residual solids.

In fact, effectively removing lignin from pulp fibers facilitates the conversion and utilization of cellulose. In this study, the residual lignin in eucalyptus pulp was separated using a high concentration of chlorine dioxide. The effects of chlorine dioxide dosage, temperature, and time on lignin removal were investigated. The optimal conditions are chlorine dioxide dosage 5.0%, reaction temperature 40 °C, and reaction time 30 min. The lignin removal yield is 88.21%. The removal yields of cellulose and hemicellulose are 2.28 and 17.00%, respectively. The treated eucalyptus pulp has higher fiber crystallinity and thermal stability. The carbon content on the fiber surface is significantly reduced. The results show that lignin is removed by efficient oxidation, and the degradation of carbohydrates is inhibited using high concentrations of chlorine dioxide at low temperatures and short reaction times. This provides theoretical support for high value conversion of cellulose.

Use of chlorine dioxide to sterilize medium for tissue culture of potato.

In vitro cultured seedlings or microtubers are the major starting materials for the production of potato. Currently, seedlings are cultured in media sterilized by autoclaving, which, however, consumes more electricity and takes longer for sterilization, and also requires high temperature-tolerant vessel materials. In order to identify alternative methods of sterilizing culture conditions, the disinfection effects of chlorine dioxide (CD) at 88.0, 29.3, 17.6, 12.6 and 8.8 µM were evaluated in potato medium and vessels. The ≥12.6 µM gaseous CD effectively disinfected vessel through a 30-min fumigation process, and its aqueous solution disinfected potato medium efficiently as well. In presence of 12.6 µM CD in the medium, the potato seedlings had similar morphological features as those grown on autoclaved medium, with some exceptions. The use of 12.6-29.3 µM aqueous CD to sterilize the medium increased antioxidant enzyme activities in potato seedlings, while the use of higher concentration decreased antioxidant enzyme activity levels. SSR analysis did not reveal significant molecular differences in potato seedlings cultured between autoclaved and CD-sterilized medium. In addition to this, CD-sterilized medium induced potato microtuber formation at a similar rate as autoclaved medium. In summary, using CD to sterilize potato medium and vessels did not compromise the growth of seedlings and microtuber induction. This study provides an economical and simplified sterilization method for media used to culture potato plantlets, and this can improve energy use of the large-scale tissue culture industry.

Vanadium Chloroperoxidases: The Missing Link in the Formation of Chlorinated Compounds and Chloroform in the Terrestrial Environment?

It is well established that the majority of chlorinated organic substances found in the terrestrial environment are produced naturally. The presence of these compounds in soils is not limited to a single ecosystem. Natural chlorination is also a widespread phenomenon in grasslands and agricultural soils typical for unforested areas. These chlorinated compounds are formed from chlorination of natural organic matter consisting of very complex chemical structures, such as lignin. Chlorination of several lignin model compounds results in the intermediate formation of trichloroacetyl-containing compounds, which are also found in soils. These decay, in general, through a haloform-type reaction mechanism to CHCl3 . Upon release into the atmosphere, CHCl3 will produce chlorine radicals through photolysis, which will, in turn, lead to natural depletion of ozone. There is evidence that fungal chloroperoxidases able to produce HOCl are involved in the chlorination of natural organic matter. The objective of this review is to clarify the role and source of the various chloroperoxidases involved in the natural formation of CHCl3 .

Effect of chlorine dioxide on cyanobacterial cell integrity, toxin degradation and disinfection by-product formation.

Bench scale tests were conducted to study the effect of chlorine dioxide (ClO2) oxidation on cell integrity, toxin degradation and disinfection by-product formation of Microcystis aeruginosa. The simulated cyanobacterial suspension was prepared at a concentration of 1.0×10(6)cells/mL and the cell integrity was measured with flow cytometry. Results indicated that ClO2 can inhibit the photosynthetic capacity of M. aeruginosa cells and almost no integral cells were left after oxidation at a ClO2 dose of 1.0mg/L. The total toxin was degraded more rapidly with the ClO2 dosage increasing from 0.1mg/L to 1.0mg/L. Moreover, the damage on cell structure after oxidation resulted in released intracellular organic matter, which contributed to the formation of trihalomethanes (THMs) and haloacetic acids (HAAs) as disinfection by-products. Therefore, the use of ClO2 as an oxidant for treating algal-rich water should be carefully considered.

Hydrolase stabilization via entanglement in poly(propylene sulfide) nanoparticles: stability towards reactive oxygen species.

In the advancement of green syntheses and sustainable reactions, enzymatic biocatalysis offers extremely high reaction rates and selectivity that goes far beyond the reach of chemical catalysts; however, these enzymes suffer from typical environmental constraints, e.g. operational temperature, pH and tolerance to oxidative environments. A common hydrolase enzyme, diisopropylfluorophosphatase (DFPase, EC 3.1.8.2), has demonstrated a pronounced efficacy for the hydrolysis of a variety of substrates for potential toxin remediation, but suffers from the aforementioned limitations. As a means to enhance DFPase's stability in oxidative environments, enzymatic covalent immobilization within the polymeric matrix of poly(propylene sulfide) (PPS) nanoparticles was performed. By modifying the enzyme's exposed lysine residues via thiolation, DFPase is utilized as a comonomer/crosslinker in a mild emulsion polymerization. The resultant polymeric polysulfide shell acts as a 'sacrificial barrier' by first oxidizing to polysulfoxides and polysulfones, rendering DFPase in an active state. DFPase-PPS nanoparticles thus retain activity upon exposure to as high as 50 parts per million (ppm) of hypochlorous acid (HOCl), while native DFPase is observed as inactive at 500 parts per billion (ppb). This trend is also confirmed by enzyme-generated (chloroperoxidase (CPO), EC 1.11.1.10) reactive oxygen species (ROS) including both HOCl (3 ppm) and ClO(2) (100 ppm).

Microbial diversity, tolerance, and biodegradation potential of urban wetlands with different input regimes.

Though microbial transformations are the primary mechanism of contaminant attenuation in wetlands, much remains to be known about microbial communities in urban wetlands. In this study, the microbial communities from urban wetlands with different runoff regimes (i.e., a contaminated remnant wetland, a constructed wetland, and a remnant wetland) were assessed for their capacity to attenuate and tolerate typical urban runoff pollutants. Results from denaturing gradient gel electrophoresis of 16S rRNA genes showed relatively high similarity in community composition among the wetlands. Community-level physiological profiles had similar results but exhibited within-site variation in both the contaminated remnant and remnant wetlands. All wetland communities were less tolerant to copper than 2,4-dichlorophenoxyacetic acid; however, the contaminated remnant wetland had the highest tolerance. All study wetlands had a limited capacity to biodegrade model chlorinated aromatic compounds (e.g., 2,4-dichlorophenoxyacetic acid and 3-chlorobenzoate). Though having different input regimes and contaminant exposure histories, the study wetlands were generally similar with respect to microbial community diversity and function. Additionally, the generally low capacity for these wetlands to biodegrade mobile chlorinated organic contaminants offers preliminary insight into the limited ecosystem services these wetlands may provide in urban environments.

Complementing approaches to demonstrate chlorinated solvent biodegradation in a complex pollution plume: Mass balance, PCR and compound-specific stable isotope analysis.

This work describes the use of different complementing methods (mass balance, polymerase chain reaction assays and compound-specific stable isotope analysis) to demonstrate the existence and effectiveness of biodegradation of chlorinated solvents in an alluvial aquifer. The solvent-contaminated site is an old chemical factory located in an alluvial plain in France. As most of the chlorinated contaminants currently found in the groundwater at this site were produced by local industries at various times in the past, it is not enough to analyze chlorinated solvent concentrations along a flow path to convincingly demonstrate biodegradation. Moreover, only a few data were initially available to characterize the geochemical conditions at this site, which were apparently complex at the source zone due to (i) the presence of a steady oxygen supply to the groundwater by irrigation canal losses and river infiltration and (ii) an alkaline pH higher than 10 due to former underground lime disposal. A demonstration of the existence of biodegradation processes was however required by the regulatory authority within a timeframe that did not allow a full geochemical characterization of such a complex site. Thus a combination of different fast methods was used to obtain a proof of the biodegradation occurrence. First, a mass balance analysis was performed which revealed the existence of a strong natural attenuation process (biodegradation, volatilization or dilution), despite the huge uncertainty on these calculations. Second, a good agreement was found between carbon isotopic measurements and PCR assays (based on 16S RNA gene sequences and functional genes), which clearly indicated reductive dechlorination of different hydrocarbons (Tetrachloroethene--PCE-, Trichloroethene--TCE-, 1,2-cisDichloroethene--cis-1,2-DCE-, 1,2-transDichloroethene-trans--1,2-DCE-, 1,1-Dichloroethene--1,1-DCE-, and Vinyl Chloride--VC) to ethene. According to these carbon isotope measurements, although TCE biodegradation seems to occur only in the upgradient part of the studied zone, DCE and VC dechlorination (originating from the initial TCE dechlorination) occurs along the entire flowpath. TCE reductase was not detected among the Dehalococcoides bacteria identified by quantitative PCR (qPCR), while DCE and VC reductases were present in the majority of the population. Reverse transcriptase PCR assays (rt-PCR) also indicated that bacteria and their DCE and VC reductases were active. Mass balance calculations showed moreover that 1,1-DCE was the predominant DCE isomer produced by TCE dechlorination in the upgradient part of the site. Consequently, coupling rt-PCR assays with isotope measurements removes the uncertainties inherent in a simple mass balance approach, so that when the three methods are used jointly, they allow the identification and quantification of natural biodegradation, even under apparently complex geochemical and hydraulic conditions.

Regional contamination versus regional dietary differences: understanding geographic variation in brominated and chlorinated contaminant levels in polar bears.

The relative contribution of regional contamination versus dietary differences to geographic variation in polar bear (Ursus maritimus) contaminant levels is unknown. Dietary variation between Alaska, Canada, East Greenland, and Svalbard subpopulations was assessed by muscle nitrogen and carbon stable isotope (δ(15)N, δ(13)C) and adipose fatty acid (FA) signatures relative to their main prey (ringed seals). Western and southern Hudson Bay signatures were characterized by depleted δ(15)N and δ(13)C, lower proportions of C(20) and C(22) monounsaturated FAs and higher proportions of C(18) and longer chain polyunsaturated FAs. East Greenland and Svalbard signatures were reversed relative to Hudson Bay. Alaskan and Canadian Arctic signatures were intermediate. Between-subpopulation dietary differences predominated over interannual, seasonal, sex, or age variation. Among various brominated and chlorinated contaminants, diet signatures significantly explained variation in adipose levels of polybrominated diphenyl ether (PBDE) flame retardants (14-15%) and legacy PCBs (18-21%). However, dietary influence was contaminant class-specific, since only low or nonsignificant proportions of variation in organochlorine pesticide (e.g., chlordane) levels were explained by diet. Hudson Bay diet signatures were associated with lower PCB and PBDE levels, whereas East Greenland and Svalbard signatures were associated with higher levels. Understanding diet/food web factors is important to accurately interpret contaminant trends, particularly in a changing Arctic.

Identification of molecular markers to follow up the bioremediation of sites contaminated with chlorinated compounds.

The use of microorganisms to clean up xenobiotics from polluted ecosystems (soil and water) represents an ecosustainable and powerful alternative to traditional remediation processes. Recent developments in molecular-biology-based techniques have led to rapid and sensitive strategies for monitoring and identifying bacteria and catabolic genes involved in the degradation of xenobiotics. This chapter provides a description of recently developed molecular-biology-based techniques, such as PCR with degenerate primers set, real-time quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), southern blot hybridization, and long-range PCR, used to give a picture of the catabolically relevant microorganisms and of the functional genes present in a polluted system. By using a case study of a groundwater aquifer contaminated with 1,2-dichloroethane (1,2-DCA), we describe the identification of microorganisms potentially involved in the 1,2-DCA dehalorespiration (Dehalobacter sp. and Desulfitobacterium sp.) and a complete new gene cluster encoding for a 1,2-DCA reductive dehalogenase. The application of these techniques to bioremediation can improve our understanding of the inner mechanisms to evaluate the feasibility of a given treatment and provide us with a method to follow up bacteria and catabolic genes involved in the degradation of contaminants during the activities in situ.

Catalytic ozonation-biological coupled processes for the treatment of industrial wastewater containing refractory chlorinated nitroaromatic compounds.

A treatability study of industrial wastewater containing chlorinated nitroaromatic compounds (CNACs) by a catalytic ozonation process (COP) with a modified Mn/Co ceramic catalyst and an aerobic sequencing batch reactor (SBR) was investigated. A preliminary attempt to treat the diluted wastewater with a single SBR resulted in ineffective removal of the color, ammonia, total organic carbon (TOC) and chemical oxygen demand (COD). Next, COP was applied as a pretreatment in order to obtain a bio-compatible wastewater for SBR treatment in a second step. The effectiveness of the COP pretreatment was assessed by evaluating wastewater biodegradability enhancement (the ratio of biology oxygen demand after 5 d (BOD(5)) to COD), as well as monitoring the evolution of TOC, carbon oxidation state (COS), average oxidation state (AOS), color, and major pollutant concentrations with reaction time. In the COP, the catalyst preserved its catalytic properties even after 70 reuse cycles, exhibiting good durability and stability. The performance of SBR to treat COP effluent was also examined. At an organic loading rate of 2.0 kg COD/(m(3)xd), with hydraulic retention time (HRT)=10 h and temperature (30+/-2) degrees C, the average removal efficiencies of NH(3)-N, COD, BOD(5), TOC, and color in a coupled COP/SBR process were about 80%, 95.8%, 93.8%, 97.6% and 99.3%, respectively, with average effluent concentrations of 10 mg/L, 128 mg/L, 27.5 mg/L, 25.0 mg/L, and 20 multiples, respectively, which were all consistent with the national standards for secondary discharge of industrial wastewater into a public sewerage system (GB 8978-1996). The results indicated that the coupling of COP with a biological process was proved to be a technically and economically effective method for treating industrial wastewater containing recalcitrant CNACs.

QSAR study on melanocortin-4 receptors by support vector machine.

Quantitative structure activity relationship (QSAR) of the melanocortin-4 receptor (MC4R) binding affinities (K(i)) of trans-4-(4-chlorophenyl) pyrrolidine-3-carboxamides of piperazinecyclohexanes was studied. A suitable set of molecular descriptors was calculated and the genetic algorithm (GA) was employed to select those descriptors that resulted in the best-fit models. The multiple linear regression (MLR), and the support vector machine (SVM) were utilized to construct the linear and nonlinear QSAR models. The models were validated using Leave-One-Out (LOO) and Leave-Group-Out (LGO) cross-validation, external test set, and chance correlation. The SVM model generalizes better than the MLR model. The SVM model, with high statistical significance (R(2)(train)=0.908, Q(2)(LOO)=0.781, Q(2)(LGO)=0.872), could be used to predict melanocortin-4 receptor binding affinities of piperazinecyclohexanes.

Assessment of in situ reductive dechlorination using compound-specific stable isotopes, functional gene PCR, and geochemical data.

Isotopic analysis and molecular-based bioassay methods were used in conjunction with geochemical data to assess intrinsic reductive dechlorination processes for a chlorinated solvent-contaminated site in Tucson, Arizona. Groundwater samples were obtained from monitoring wells within a contaminant plume comprising tetrachloroethene and its metabolites, trichloroethene, cis-1,2-dichloroethene, vinyl chloride, and ethene, as well as compounds associated with free phase diesel present at the site. Compound-specific isotope analysis was performed to characterize biotransformation processes influencing the transport and fate of the chlorinated contaminants. Polymerase chain reaction (PCR) analysis was used to assess the presence of indigenous reductive dechlorinators. The target regions employed were the 16s rRNA gene sequences of Dehalococcoides sp. and Desulfuromonas sp. and DNA sequences of genes pceA, tceA, bvcA, and vcrA, which encode reductive dehalogenases. The results of the analyses indicate that relevant microbial populations are present and that reductive dechlorination is presently occurring at the site. The results further show that potential degrader populations as well as biotransformation activity is nonuniformly distributed within the site. The results of laboratory microcosm studies conducted using groundwater collected from the field site confirmed the reductive dechlorination of tetrachloroethene to dichloroethene. This study illustrates the use of an integrated, multiple-method approach for assessing natural attenuation at a complex chlorinated solvent-contaminated site.

'Single lay out' and 'mixed lay out' enzymatic processes for bio-bleaching of kraft pulp.

Xylanase alone as 'single lay out' (strategy I) and in combination with pectinase as 'mixed lay out' (strategy II) was used to investigate their bio-bleaching potentials. Strategy I was carried at 70 degrees C using 5 U/g of xylanase at pH 9.5 and 12.5 whereas strategy II was carried out at 70 degrees C using 5 U/g of each of the enzyme, respectively at pH 9.5. Bio-bleaching caused 15% and 20% less Cl(2) consumption though strategy I and II, respectively over chemical bleaching. Strategy II was proved to be 35.71% more efficient in ClO(2) saving than conventional method. Significant improvement in various pulp properties viz. tensile strength 25.70%, breaking length 21.80%, burst factor 20.00%, burstness 13.86%, tear factor 6.61% and tearness 18.88%, was also observed through 'mixed lay out' strategy.

Mechanism of reaction of horseradish peroxidase with chlorite and chlorine dioxide.

It is demonstrated that horseradish peroxidase (HRP) mixed with chlorite follows the whole peroxidase cycle. Chlorite mediates the two-electron oxidation of ferric HRP to compound I (k(1)) thereby releasing hypochlorous acid. Furthermore, chlorite acts as one-electron reductant of both compound I (k(2)) and compound II (k(3)) forming chlorine dioxide. The strong pH-dependence of all three reactions clearly suggests that chlorous acid is the reactive species. Typical apparent bimolecular rate constants at pH 5.6 are 1.4 x 10(5)M(-1)s(-1) (k(1)), 2.25 x 10(5)M(-1)s(-1) (k(2)), and 2.4 x 10(4)M(-1)s(-1) (k(3)), respectively. Moreover, the reaction products hypochlorous acid and chlorine dioxide, which are known to induce heme bleaching and amino acid modification upon longer incubation times, also mediate the oxidation of ferric HRP to compound I (2.4 x 10(7)M(-1)s(-1) and 2.7 x 10(4)M(-1)s(-1), respectively, pH 5.6) but do not react with compounds I and II. A reaction scheme is presented and discussed from both a mechanistic and thermodynamic point of view. It helps to explain the origin of contradictory data so far found in the literature on this topic.

Effects of inhibitors on anaerobic microbial consortium with enhanced dechlorination activity in polychlorinated biphenyl mixture.

Characterization was carried out on the anaerobic microbial consortium with enhanced degradation activity toward polychlorinated biphenyls in Kanechlor-300 and Kanechlor-400 mixtures in a burnt soil (BS) culture. The addition of molybdate to the BS culture resulted in the accumulation of less-chlorinated biphenyls such as 4,4'-dichlorinated biphenyl and 2,3',4-trichlorinated biphenyl; however, no such accumulation occurred without molybdate supplementation. No significant effect was observed in individual congeners in the BS culture supplemented with 2-bromoethane sulfonic acid. Analyses involving both the polymerase chain reaction-denaturing gradient gel electrophoresis of partial 16S rRNA genes and respiratory quinones showed that the predominant microorganisms in the BS culture were anaerobic Firmicutes, while sulfate reducers of the phyla Deltaproteobacteria, Firmicutes and Chloroflexi were absent in the culture amended with the inhibitors. No positive correlation was observed between the dechlorination activity and a PCR-based detection of gene fragments of known dechlorinating bacteria. These results suggest that sulfate reducers played an important role in the enhanced anaerobic dechlorination of PCBs in the BS culture.

Comparison of anaerobic microbial communities from Estuarine sediments amended with halogenated compounds to enhance dechlorination of 1,2,3,4-tetrachlorodibenzo-p-dioxin.

A suite of experiments were conducted to ascertain whether dehalogenation of a model dioxin compound could be stimulated in marine sediments by supplementation with halogenated analogues to enrich for dehalogenating bacteria and if growth by members of the Chloroflexi-like group was associated with dioxin removal. Five halogenated compounds (tetrachlorobenzene, tetrachloroanisole, tetrachlorophenol, tetrachlorobenzoic acid and trichloroacetophenone) were added with 1,2,3,4-tetrachlorodibenzo-p-dioxin (TeCDD) to estuarine sediments from four sites in San Diego Bay and the coast of southern New Jersey to test for dioxin dehalogenation. Most of the halogenated additives were found to stimulate dechlorination of the model dioxin. Molecular analysis of the bacterial population using 16S rRNA and reductive dehalogenase genes indicated that distinct microbial populations were enriched with each halogenated co-amendment. Additionally, Chloroflexi-like ribosomal genes associated with dehalogenation were detected. For example, quantitative real-time PCR analysis of 16S rRNA and reductive dehalogenase gene copy number in the microcosms showed a positive correlation with 1,2,3,4-TeCDD reductive dechlorination in coastal sediments amended with different halogenated additives. These results suggest that specific Chloroflexi-like microorganisms related to Dehalococcoides are involved in 1,2,3,4-TeCDD reductive dechlorination.

Dechlorination of polychlorinated biphenyl congeners by anaerobic microorganisms from river sediment.

The microbial dechlorination of seven kinds of polychlorinated biphenyls (PCBs) by anaerobic microorganisms from river sediment was investigated. Dechlorination rates were found to be affected by the chlorine level of PCB congeners; dechlorination rates decreased as chlorine levels increased. Dechlorination rates were fastest under methanogenic conditions and slowest under nitrate-reducing conditions. The addition of individual electron donors (acetate, pyruvate, and lactate) enhanced the dechlorination of PCB congeners under methanogenic and sulfate-reducing conditions but delayed the dechlorination of PCB congeners under nitrate-reducing conditions. PCB congener dechlorination also was delayed by the addition of various polycyclic aromatic hydrocarbons (PAHs) under three reducing conditions and by surfactants, such as brij30, triton SN70, and triton N101. The results suggest that methanogen, sulfate-reducing bacteria, and nitrate-reducing bacteria all are involved in the dechlorination of PCB congeners.

Identification and formation pathway of laccase-mediated oxidation products formed from hydroxyphenylureas.

Hydroxyphenylureas are the first main metabolites formed in the environment from pesticide and biocide urea compounds. Because fungi release potent exocellular oxidases, we studied the ability of laccases produced by the white rot fungus, T. versicolor, to catalyze in vitro the transformation of five hydroxyphenylureas, to identify transformation pathways and mechanisms. Our results establish that the pH of the reaction has a strong influence on both the kinetics of the reaction and the nature of the transformation products. Structural characterization by spectroscopic methods (NMR, mass spectrometry) of eleven transformation products shows that laccase oxidizes the substrates to quinones or to polyaromatic oligomers. Slightly acidic conditions favor the formation of quinones as final transformation products. In contrast, at pH 5-6, the quinones further react with the remaining substrate in solution to give hetero-oligomers via carbon-carbon or carbon-oxygen bond formation. A reaction pathway is proposed for each of the identified products. These results demonstrate that fungal laccases could assist the transformation of hydroxyphenylureas.

Stable isotope evidence for biodegradation of chlorinated ethenes at a fractured bedrock site.

Stable carbon isotope analysis of chlorinated ethenes and ethene was performed at a site contaminated with trichloroethene (TCE), a dense non-aqueous phase liquid (DNAPL). The site is located in fractured bedrock and had variable groundwater hydraulic gradients during the study due to a local excavation project. Previous attempts to biostimulate a pilot treatment area at the site resulted in the production of cis-1,2-dichloroethene (cis-DCE), the first product of reductive dechlorination of TCE. Cis-DCE concentrations accumulated however, and there was no appreciable production of the breakdown products from further reductive dechlorination, vinyl chloride (VC) and ethene (ETH). Consequently, the pilot treatment area was bioaugmented with a culture of KB-1, a natural microbial consortium known to completely reduce TCE to nontoxic ETH. Due to ongoing dissolution of TCE from DNAPL in the fractured bedrock, and to variable hydraulic gradients, concentration profiles of dissolved TCE and its degradation products cis-DCE, VC, and ETH could not convincingly confirm biodegradation of the chlorinated ethenes. Isotopic analysis of cis-DCE and VC, however, demonstrated that biodegradation was occurring in the pilot treatment area. The isotope values of cis-DCE and VC became significantly more enriched in 13C over the last two sampling dates (in one well from -17.6%o to -12.8%o and from -22.5%o to -18.2%o for cis-DCE and VC, respectively). Quantification of the extent of biodegradation in the pilot treatment area using the Rayleigh model indicated that, depending on the well, between 21.3% and 40.7% of the decrease in cis-DCE and between 15.2% and 36.7% of the decrease in VC concentrations can be attributed to the effects of biodegradation during this time period. Within each well, the isotope profile of TCE remained relatively constant due to the continuous input of undegraded TCE due to DNAPL dissolution.