Chlorine isotope fractionation during reductive dechlorination of chlorinated ethenes by anaerobic bacteria

Chlorine isotope fractionation during reductive dechlorination of trichloroethene (TCE) and tetrachloroethene (PCE) to cis-1,2-dichloroethene (cDCE) by anaerobic bacteria was investigated. The changes in the 37Cl/35Cl ratio observed during the one-step reaction (TCE to cDCE) can be explained by the regioselective elimination of chlorine accompanied by the Rayleigh fractionation. The fractionation factors (alpha) of the TCE dechlorination by three kinds of anaerobic cultures were approximately 0.994-0.995 at 30 degrees C. The enrichment of 37Cl in the organic chlorine during the two-step reaction (PCE to cDCE) can be explained by the random elimination of one chlorine atom in the PCE molecule followed by the regioselective elimination of one chlorine atom in the TCE molecule. The fractionation factors for the first step of the PCE dechlorination with three kinds of anaerobic cultures were estimated to be 0.987-0.991 at 30 degrees C using a mathematical model. Isotope fractionation during the first step would be the primary factor for the chlorine isotope fractionation during the PCE dechorination to cDCE. The developed models can be utilized to evaluate the fractionation factors of regioselective and multistep reactions.     

Experimental evidence for a lack of thermodynamic control on hydrogen concentrations during anaerobic degradation of chlorinated ethenes

Hydrogen (H2) concentrations during reductive dechlorination of cis-dichloroethene (cDCE) and vinyl chloride (VC) were investigated with respectto the influence of parameters entering the Gibbs free energy expression of the reactions. A series of laboratory experiments was conducted employing a mixed, Dehalococcoides-containing enrichment culture capable of complete dechlorination of chlorinated ethenes. The objective was to investigate whether a constant energy gain controls H2 levels in dechlorinating systems, thereby evaluating the applicability of the partial equilibrium approach to microbial dechlorination at contaminated sites. Variations in the temperature between 10 and 30 degrees C did not affect the H2 concentration in a fashion that suggested thermodynamic control through a constant energy gain. In another set of experiments, H2 levels at constant ionic strength were independent of the chloride concentration between 10 and 110 mmol chloride per liter. These findings demonstrate that the partial equilibrium approach is not directly applicable to the interpretation of reductive degradation of chlorinated ethenes. We also present recalculated thermodynamic properties of aqueous chlorinated ethene species that allow for calculation of in-situ Gibbs free energy of dechlorination reactions at different temperatures.     

Oxidation of chlorinated ethenes by heat-activated persulfate: kinetics and products

In situ chemical oxidation (ISCO) and in situ thermal remediation (ISTR) are applicable to treatment of groundwater contaminated with chlorinated ethenes. ISCO with persulfate (S2O8(2-)) requires activation, and this can be achieved with the heat from ISTR, so there may be advantages to combining these technologies. To explore this possibility, we determined the kinetics and products of chlorinated ethene oxidation with heat-activated persulfate and compared them to the temperature dependence of other degradation pathways. The kinetics of chlorinated ethene disappearance were pseudo-first-order for 1-2 half-lives, and the resulting rate constants-measured from 30 to 70 degrees C--fit the Arrhenius equation, yielding apparent activation energies of 101 +/- 4 kJ mol(-1) for tetrachloroethene (PCE), 108 +/- 3 kJ mol(-1) for trichloroethene (TCE), 144 +/- 5 kJ mol(-1) for cis-1,2-dichloroethene (cis-DCE), and 141 +/- 2 kJ mol(-1) for trans-1,2-dichloroethene (trans-DCE). Chlorinated byproducts were observed, but most of the parent material was completely dechlorinated. Arrhenius parameters for hydrolysis and oxidation by persulfate or permanganate were used to calculate rates of chlorinated ethene degradation by these processes over the range of temperatures relevant to ISTR and the range of oxidant concentrations and pH relevant to ISCO.     

Competition for sorption and degradation of chlorinated ethenes in batch zero-valent iron systems

The sorption and degradation of the chlorinated ethenes tetrachloroethene (PCE, 5 mg L(-1)) and trichloroethene (TCE, 10 mg L(-1)) were investigated in zero-valent iron systems (ZVI, 100 g L(-1)) in the presence of compounds common to contaminated groundwater with varying physicochemical properties. The potential competitors were chlorinated ethenes, monocyclic aromatic hydrocarbons, and humic acids. The effect of a complex matrix was tested with landfill contaminated groundwater. Nonlinear Freundlich isotherms adequately described chloroethene sorption to ZVI. In the presence of the more hydrophobic PCE (5 mg L(-1)), TCE sorption and degradation decreased by 33% and 30%, respectively, while TCE (10 mg L(-1)) decreased PCE degradation by 30%. In the presence of nonreactive hydrophobic hydrocarbons (i.e., benzene, toluene, and m-xylene at 100 mg L(-1)), TCE and PCE sorption decreased by 73% and 55%, respectively. The presence of the hydrocarbons had no effect on TCE degradation and increased PCE reduction rates by 50%, suggesting that the displacement of the chloroethenes from the sorption sites by the aromatic hydrocarbons enhanced the degradation rates. Humic acids did not interfere significantly with chloroethene sorption or with TCE degradation but lowered PCE degradation kinetics by 36% when present at high concentrations (100 mg L(-1)). The landfill groundwater with an organic carbon content of 109 mg L(-1) C had no effect on chloroethene sorption but inhibited TCE and PCE degradation by 60% and 70%, respectively.     

TBA biodegradation in surface-water sediments under aerobic and anaerobic conditions

The potential for [U-14C] TBA biodegradation was examined in laboratory microcosms under a range of terminal electron accepting conditions. TBA mineralization to CO2 was substantial in surface-water sediments under oxic, denitrifying, or Mn(IV)-reducing conditions and statistically significant but low under SO4-reducing conditions. Thus, anaerobic TBA biodegradation may be a significant natural attenuation mechanism for TBA in the environment, and stimulation of in situ TBA bioremediation by addition of suitable terminal electron acceptors may be feasible. No degradation of [U-14C] TBA was observed under methanogenic or Fe(III)-reducing conditions.     

Monitoring oxidation of chlorinated ethenes by permanganate in groundwater using stable isotopes: laboratory and field studies

Permanganate injection is increasingly applied for in situ destruction of chlorinated ethenes in groundwater. This laboratory and field study demonstrates the roles that carbon isotope analysis can play in the assessment of oxidation of trichloroethene (TCE) by permanganate. In laboratory experiments a strong carbon isotope fractionation was observed during oxidation of TCE with similar isotopic enrichment factors (-25.1 to -26.8 per thousand) for initial KMnO4 concentrations between 67 and 1,250 mg/L. At the field site, a single permanganate injection episode was conducted in a sandy aquifer contaminated with TCE as dense nonaqueous liquid (DNAPL). After injection, enriched delta13C values of up to +204% and elevated Cl- concentrations were observed at distances of up to 4 m from the injection point. Farther away, the Cl- increased without any change in delta13C of TCE suggesting that Cl- was not produced locally but migrated to the sampling point Except for the closest sampling location to the injection point, the delta13C rebounded to the initial 613C again, likely due to dissolution of DNAPL Isotope mass balance calculations made it possible to identify zones where TCE oxidation continued to occur during the rebound phase. The study indicates that delta13C values can be used to assess the dynamics between TCE oxidation and dissolution and to locate zones of oxidation of chlorinated ethenes that cannot be identified from the Cl- distribution alone.     

Variability in carbon isotopic fractionation during biodegradation of chlorinated ethenes: implications for field applications

Stable carbon isotopic analysis has the potential to assess biodegradation of chlorinated ethenes. Significant isotopic shifts, which can be described by Rayleigh enrichment factors, have been observed for the biodegradation of trichloroethlyene (TCE), cis-dichloroethylene (cDCE), and vinyl chloride (VC). However, until this time, no systematic investigation of isotopic fractionation during perchloroethylene (PCE) degradation has been undertaken. In addition, there has been no comparison of isotopic fractionation by different microbial consortia, nor has there been a comparison of isotopic fractionation by consortia generated from the same source, but growing under different conditions. This study characterized carbon isotopic fractionation during reductive dechlorination of the chlorinated ethenes, PCE in particular, for microbial consortia from two different sources growing under different environmental conditions in order to assess the extent to which different microbial consortia result in different fractionation factors. Rayleigh enrichment factors of -13.8@1000, -20.4@1000, and -22.4@1000 were observed for TCE, cDCE, and VC, respectively, for dechlorination by the KB-1 consortium. In contrast, isotopic fractionation during reductive dechlorination of perchloroethylene (PCE) could not always be approximated by a Rayleigh model. Dechlorination by one consortium followed Rayleigh behavior (epsilon = -5.2), while a systematic change in the enrichment factor was observed over the course of PCE degradation by two other consortia. Comparison of all reported enrichment factors for reductive dechlorination of the chlorinated ethenes shows significant variation between experiments. Despite this variability, these results demonstrate that carbon isotopic analysis can provide qualitative evidence of the occurrence and relative extent of microbial reductive dechlorination of the chlorinated ethenes.     

Combined removal of chlorinated ethenes and heavy metals by zerovalent iron in batch and continuous flow column systems

The combined removal of chlorinated ethenes and heavy metals from a simulated groundwater matrix by zerovalent iron (ZVI) was investigated. In batch, Ni (5-100 mg L(-1)) enhanced trichloroethene (TCE, 10 mg L(-1)) reduction by ZVI (100 g L(-1)) due to catalytic hydrodechlorination by bimetallic Fe0/Ni0. Cr(VI) or Zn (5-100 mg L(-1)) lowered TCE degradation rates by a factor of 2 to 13. Cr(VI) (100 mg L(-1)) in combination with Zn or Ni (50-100 mg L(-1)) inhibited TCE degradation. Addition of 20% H2(g) in the headspace, or of Zn (50-100 mg L(-1)), enhanced TCE removal in the presence of Ni and Cr(VI). Sorption of Zn to ZVI alleviated the Cr(VI) induced inhibition of bimetallic Fe0/Ni0 apparently due to release of protons necessary for TCE hydrodechlorination. In continuous ZVI columns treating tetrachloroethene (PCE, 1-2 mg L(-1)) and TCE (10 mg L(-1)), and a mixture of the metals Cr(VI), Zn(II), and Ni(II) (5 mg (L-1)), the PCE removal efficiency decreased from 100% to 90% in columns operated without heavy metals. The PCE degradation efficiency remained above 99% in columns receiving heavy metals as long as Ni was present. The findings of this study indicate the feasibility and limitations of the combined treatment of mixtures of organic and inorganic pollutants by ZVI.     

Inactivation of Ascaris suum and poliovirus in biosolids under thermophilic anaerobic digestion conditions

There is considerable interest in the United States in production of Class A (low pathogen content) biosolids from the treatment of municipal wastewater sludge. Current requirements imposed by the U.S. Environmental Protection Agency make it difficult for thermophilic anaerobic digestion, in its simplest process configurations, to achieve Class A status. In particular, the time-temperature requirements necessitate long batch treatment times at temperatures associated with thermophilic anaerobic digestion. The time-temperature requirements are meant to ensure extensive inactivation of helminth eggs and enteric viruses, considered to be the most heat-resistant of the relevant pathogen classes. However, data on inactivation kinetics of these pathogens at precisely controlled and well-characterized temperatures are scarce. We measured inactivation of vaccine-strain poliovirus and eggs from the helminth Ascaris suum at temperatures from 49 to 55 degrees C in a lab-scale batch reactor containing biosolids from a continuous-flow thermophilic anaerobic digester. Both microbes were inactivated rapidly, with Ascaris more resistant to inactivation than poliovirus, and the relationships between inactivation rate and temperature were steep. The Arrhenius correlation between inactivation rate and temperature over the range 49-53 degrees C is consistent with protein denaturation as the inactivation mechanism for both microbes. The least stringent of the EPA time-temperature equations for thermal processes requires batch treatment times more than 2 orders of magnitude greater than would be required for three-log reduction of Ascaris at the rates we measured, suggesting an overly conservative regulatory approach. Such a grossly conservative approach can hinder full-scale implementation of thermophilic anaerobic digestion.     

Enhanced dechlorination of chlorinated methanes and ethenes by chloride green rust in the presence of copper(II)

The enhanced removal of carbon tetrachloride (CCl4), tetrachloroethene (C2Cl4), and trichloroethene (C2HCl3) by chloride green rust (GR(Cl)) in the presence of copper ions was investigated. X-ray powder diffraction (XRPD) and X-ray photoelectron spectroscopy (XPS) were used to characterize the crystallization and chemical speciation, respectively, of the secondary mineral phases produced in the GR(Cl)-Cu(II) system. The addition of Cu(II) to GR(Cl) suspensions resulted in enhanced dechlorination of the chlorinated hydrocarbons examined in this study. The degradation reactions followed pseudo-first-order kinetics and the pseudo-first-order rate constant (k(obs)) for CCl4 (20 microM) removal by GR(CI) at pH 7.2 was 0.0808 h(-1). Addition of 0.5 mM Cu(II) completely dechlorinated CCl4 within 35 min, and the k(obs) was 84 times greater than that in the absence of Cu(II). Chloroform (CHCl3), the major chlorinated product in CCl4 dechlorination, accumulated at a concentration up to 13 microM in the GR(Cl) system alone, but was completely dechlorinated within 9 h in the GR(Cl)-Cu(II) suspension. Also, rapid removal of C2Cl4 and C2HCl3 by GR(Cl) was observed when Cu(II) was added. The k(obs) values for the removal of chlorinated ethenes were 4.7-7 times higher than that obtained in the absence of Cu(II). In addition, the k(obs) for PCE removal increased linearly with respect to Cu(II) concentrations in the range from 0.1 to 1.0 mM. Addition of Cu(II) at a concentration higher than 1.0 mM decreased the k(obs) for the removal of both C2Cl4 and C2HCl3 due to the decrease in structural Fe(II) concentration in GR(Cl) and the changes in redox potentials and pH values. Moreover, the highest removal efficiency and rate of C2Cl4 was obtained at near-neutral pH when Cu(II) was added into the GR(Cl) suspension. XPS and XRPD results showed that the Fe(II) in the GR(Cl) suspension could reduce Cu(II) to both Cu(I) and metallic Cu. These findings are relevant to the better understanding of the role of abiotic removal of chlorinated hydrocarbons during remediation and/or natural attenuation in iron-reducing environments.     

Apparent thermal resistance of Bacillus stearothermophilus spores recovered under anaerobic conditions

The effect of recovering Bacillus stearothermophilus spores under anaerobic conditions on their apparent thermal resistance was studied. Spores were suspended in bidistilled water as a reference medium, heated at 115, 117, 119, 121, 123 and 125°C and recovered under aerobic and anaerobic conditions. D values (decimal reduction time) obtained following recovery under anaerobic conditions were lower than those obtained under aerobic conditions. Reductions of between 31 and 48% were found for all the temperatures studied. When spores were suspended in mushroom extract and recovered under anaerobic conditions the apparent heat resistance was much lower than that obtained under aerobic conditions (D _121°C was 4.3 min and 1.7 min, under aerobic and anaerobic conditions, respectively). Heating the spores in mushroom extract and recovering the spores under anaerobic conditions produced an additive effect, decreasing the apparent heat resistance of the B. stearothermophilus spores.     

Apparent thermal resistance of Bacillus stearothermophilus spores recovered under anaerobic conditions

 The effect of recovering Bacillus stearothermophilus spores under anaerobic conditions on their apparent thermal resistance was studied. Spores were suspended in bidistilled water as a reference medium, heated at 115, 117, 119, 121, 123 and 125°C and recovered under aerobic and anaerobic conditions. D values (decimal reduction time) obtained following recovery under anaerobic conditions were lower than those obtained under aerobic conditions. Reductions of between 31 and 48% were found for all the temperatures studied. When spores were suspended in mushroom extract and recovered under anaerobic conditions the apparent heat resistance was much lower than that obtained under aerobic conditions (D _121°C was 4.3 min and 1.7 min, under aerobic and anaerobic conditions, respectively). Heating the spores in mushroom extract and recovering the spores under anaerobic conditions produced an additive effect, decreasing the apparent heat resistance of the B. stearothermophilus spores. Received: 1 July 1997     

Carbon and chlorine isotope ratios of chlorinated ethenes migrating through a thick unsaturated zone of a sandy aquifer

Compound-specific isotope analysis (CSIA) can potentially be used to relate vapor phase contamination by volatile organic compounds (VOCs) to their subsurface sources. This field and modeling study investigated how isotope ratios evolve during migration of gaseous chlorinated ethenes across a 18 m thick unsaturated zone of a sandy coastal plain aquifer. At the site, high concentrations of tetrachloroethene (PCE up to 380 μg/L), trichloroethene (TCE up to 31,600 μg/L), and cis-1,2-dichloroethene (cDCE up to 680 μg/L) were detected in groundwater. Chlorinated ethene concentrations were highest at the water table and steadily decreased upward toward the land surface and downward below the water table. Although isotopologues have different diffusion coefficients, constant carbon and chlorine isotope ratios were observed throughout the unsaturated zone, which corresponded to the isotope ratios measured at the water table. In the saturated zone, TCE became increasingly depleted along a concentration gradient, possibly due to isotope fractionation associated with aqueous phase diffusion. These results indicate that carbon and chlorine isotopes can be used to link vapor phase contamination to their source even if extensive migration of the vapors occurs. However, the numerical model revealed that constant isotope ratios are only expected for systems close to steady state.     

The anaerobic conversion of methanol under thermophilic conditions: pH and bicarbonate dependence

The thermophilic (55 degrees C) anaerobic conversion of methanol was studied in an unbuffered medium (pH 4+/-0.2) and in a phosphate buffered medium (pH 6.4+/-0.1), in both cases without bicarbonate addition. Our cultivated sludge consortium was unable to degrade methanol under acidic conditions. During the 160 d of continuous operation of an up-flow anaerobic sludge blanket (UASB) reactor (R1), at an organic loading rate (ORL) of 6 gCOD/(l.d) and pH around 4, only 5% of the applied methanol load was consumed and no methane (CH4) was detected. However, hydrogenotrophic methanogens were found to be resistant to exposure to such conditions. At the end of the trial, the hydrogenotrophic methanogenic activity of the sludge was 1.23+/-0.16 gCOD/(gVSS.d) at neutral pH. With methanol as the test substrate, the addition of bicarbonate led to acetate accumulation. A second reactor (R2) was operated for 303 d at OLRs ranging from 5.5 to 25.4 gCOD/(l.d) in order to assess the conversion of methanol at neutral pH (phosphate buffered) in a bicarbonate deprived medium. The reactor performance was poor with a methanol-COD removal capacity limited to about 9.5 gCOD/(l.d). The system appeared to be quite susceptible to any type of disturbance, even at low OLR. The fraction of methanol-COD converted to CH4 and acetate was found to be unaffected by the OLR applied. At the end of the trial, the outcome of the competition was about 50% methanogenesis and 50% homoacetogenesis.     

Decolorization of molasses wastewater by Bacillus sp. under thermophilic and anaerobic conditions

Various microorganisms were screened for their ability to decolorize molasses wastewater under thermophilic and anaerobic conditions. Strain MD-32, which was newly isolated from a soil sample, was selected as the best strain. From taxonomical studies, the strain was concluded to belong to the genus Bacillus, most closely resembling B. smithii. The strain decolorized 35.5% of molasses pigment within 20 d at 55 degrees C under anaerobic conditions, but no decolorization activity was observed when it was cultivated aerobically. At all the concentrations tested molasses pigment was effectively decolorized by MD-32, with decolorization yields of around 15% within 2 d. The molecular weight distribution as determined by gel filtration chromatography revealed that the decolorization of molasses pigment by the isolated strain is accompanied by a decrease in not only small molecules but also large ones.     

Preservation of Ceriporiopsis subvermispora and Lentinula edodes treated wheat straw under anaerobic conditions

BACKGROUND

No attention has been paid so far to the preservation of fungal‐treated lignocellulose for longer periods. In the present study, we treated wheat straw (WS) with the white‐rot fungi Ceriporiopsis subvermispora and Lentinula edodes for 8 weeks and assessed changes in pH, chemical composition and in vitro gas production (IVGP) weekly. Fungal‐treated WS was also stored for 64 days ‘as is’, with the addition of lactic acid bacteria (LAB) or with a combination of LAB and molasses in airtight glass jars mimicking ensiling conditions.

RESULTS

Both fungi significantly reduced the lignin and hemicellulose content of WS, and increased the cellulose content. The IVGP increased with increasing time of incubation, indicating the increase in digestibility. Both fungi lowered the pH of WS under 4.3, which guarantees an initial and stable low pH during anaerobic storage. Minor changes in fibre composition and IVGP were observed for stored L. edodes treated WS, whereas no change occurred for C. subvermispora.

CONCLUSION

It is possible to conserve C. subvermispora and L. edodes treated straw under anaerobic condition without additives up to 64 days. This finding is important for practical application to supply fungi‐treated feed to ruminant animals for a prolonged period. © 2017 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Physiological requirements for growth and competitiveness of Dekkera bruxellensis under oxygen-limited or anaerobic conditions

The effect of glucose and oxygen limitation on the growth and fermentation performances of Dekkera bruxellensis was investigated in order to understand which factors favour its propagation in ethanol or wine plants. Although D. bruxellensis has been described as a facultative anaerobe, no growth was observed in mineral medium under complete anaerobiosis while growth was retarded under severe oxygen limitation. In a continuous culture with no gas inflow, glucose was not completely consumed, most probably due to oxygen limitation. When an air/nitrogen mixture (O₂‐content ca. 5%) was sparged to the culture, growth became glucose‐limited. In co‐cultivations with Saccharomyces cerevisiae, ethanol yields/g consumed sugar were not affected by the co‐cultures as compared to the pure cultures. However, different population responses were observed in both systems. In oxygen‐limited cultivation, glucose was depleted within 24 h after challenging with S. cerevisiae and both yeast populations were maintained at a stable level. In contrast, the S. cerevisiae population constantly decreased to about 1% of its initial cell number in the sparged glucose‐limited fermentation, whereas the D. bruxellensis population remained constant. To identify the requirements of D. bruxellensis for anaerobic growth, the yeast was cultivated in several nitrogen sources and with the addition of amino acids. Yeast extract and most of the supplied amino acids supported anaerobic growth, which points towards a higher nutrient demand for D. bruxellensis compared to S. cerevisiae in anaerobic conditions. Copyright © 2012 John Wiley & Sons, Ltd.