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. Biotechnol. Bioeng. 2010;107: 540–549. © 2010 Wiley Periodicals, Inc.     

Development and mathematical modeling of a two-stage reactor system for trichloroethylene degradation using <Emphasis Type="Italic">Methylosinus trichosporium</Emphasis> OB3b

A two-stage reactor system was developed for the continuous degradation of gas-phase trichloroethylene (TCE). Methylosinus trichosporium OB3b was immobilized on activated carbon in a TCE degradation reactor, trickling biofilter (TBF). The TBF was coupled with a continuous stirred tank reactor (CSTR) to allow recirculation of microbial cells from/to the TBF for the reactivation of inactivated cells during TCE degradation. The mass transfer aspect of the TBF was analyzed, and mass transfer coefficient of 3.9 h⁻¹ was estimated. The loss of soluble methane monooxygenase (sMMO) activity was modeled based on a material balance on the CSTR and TBF, and transformation capacity (T _c) was determined to be 20.2 mol mg⁻¹. Maximum TCE degradation rate of 525 mg 1⁻¹ d⁻¹ was obtained and reactor has been stably operated for more than 270 days.     

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 µM before cDCE production from TCE ceased. Exposure to approximately 3,000 and 6,000 µM 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. Biotechnol. Bioeng. 2010;107: 529–539. © 2010 Wiley Periodicals, Inc.     

Characterization of a Highly Enriched Dehalococcoides-Containing Culture That Grows on Vinyl Chloride and Trichloroethene

A highly enriched culture that reductively dechlorinates trichloroethene (TCE), cis-1,2-dichloroethene (cDCE), and vinyl chloride (VC) to ethene without methanogenesis is described. The Dehalococcoides strain in this enrichment culture had a yield of (5.6 ± 1.4) × 10⁸ 16S rRNA gene copies/μmol of Cl⁻ when grown on VC and hydrogen. Unlike the other VC-degrading cultures described in the literature, strains VS and BAV1, this culture maintained the ability to grow on TCE with a yield of (3.6 ± 1.3) × 10⁸ 16S rRNA gene copies/μmol of Cl⁻. The yields on an electron-equivalent basis measured for the culture grown on TCE and on VC were not significantly different, indicating that both substrates supported growth equally well. PCR followed by denaturing gradient gel electrophoresis, cloning, and phylogenetic analyses revealed that this culture contained one Dehalococcoides 16S rRNA gene sequence, designated KB-1/VC, that was identical (over 1,386 bp) to the sequences of previously described organisms FL2 and CBDB1. A second Dehalococcoides sequence found in separate KB-1 enrichment cultures maintained on cDCE, TCE, and tetrachloroethene was no longer present in the VC-H₂ enrichment culture. This second Dehalococcoides sequence was identical to that of BAV1. As neither FL2 nor CBDB1 can dechlorinate VC to ethene in a growth-related fashion, it is clear that current 16S rRNA gene-based analyses do not provide sufficient information to distinguish between metabolically diverse members of the Dehalococcoides group.     

Induction of hydroxyl radical production in Trametes versicolor to degrade recalcitrant chlorinated hydrocarbons

Extracellular hydroxyl radical (OH) production via quinone redox cycling in Trametes versicolor, grown in a chemically defined medium, was investigated to degrade trichloroethylene (TCE), perchloroethylene (PCE), 1,2,4- and 1,3,5-trichlorobenzene (TCB). The activity of the enzymes catalyzing the quinone redox cycle, quinone reductase and laccase, as well as the rate of OH production, estimated as the formation of thiobarbituric acid reactive substances (TBARS) from 2-deoxyribose, increased rapidly during the first 2–3 days and then remained at relatively constant levels. Under quinone redox cycling conditions, TCE degradation was concomitant to TBARS production and chloride release, reaching a plateau after 6 h of incubation. Similar results were obtained in PCE, 1,2,4- and 1,3,5-TCB time course degradation experiments. The mole balance of chloride release and 1,2,4-TCB and TCE degraded suggests that these chemicals were almost completely dechlorinated. Experiments using [¹³C]-TCE confirmed unequivocal transformation of TCE to ¹³CO₂. These results are of particular interest because PCE and 1,3,5-TCB degradation in aerobic conditions has been rarely reported to date in bacterial or fungal systems.     

Isopropylbenzene (cumene) — a new substrate for the isolation of trichloroethene-degrading bacteria

Various bacterial isolates from enrichments with isopropylbenzene (cumene), toluene or phenol as carbon and energy sources were tested as to their potential to oxidize trichloroethene (TCE). In contrast to toluene and phenol, all isolates enriched on isopropylbenzene were able to oxidize TCE. Two isolates, strain JR1 and strain BD1, were identified as Pseudomonas spec. and as Rhodococcus erythropolis, respectively. TCE oxidation was accompanied by the liberation of stoichiometric amounts of chloride. Initial TCE oxidation rate increased proportional to the substrate concentration from 25 to 200 M TCE. Maximal initial TCE-degradation rates found here were 4 to 5 nmol · min^-1 · mg protein^-1. The TCE degradation rate decreased with time. The two isolates showed a temperature optimum for TCE degradation between 10 and 20 °C. In addition to TCE, R. erythropolis BD1 degraded only cis- and trans-dichloroethene whereas Pseudomonas spec. JR1 was able to oxidize also 1,1-dichloroethene, vinyl chloride, trichloroethane, and 1,2-dichloroethane.Abbrevations DMF dimethylformamide - TCE trichloroethene     

Evaluation of enrichments of sulfate reducing bacteria from pristine hydrothermal vents sediments as potential inoculum for reducing trichloroethylene

The evaluation of enrichments from pristine hydrothermal vents sediments on its capability of reducing trichloroethylene (TCE) under sulfate reducing conditions with lactate and volatile fatty acids (VFAs) as substrates was performed. Effect of the possible TCE biodegradation intermediates cis and trans 1,2 dichloroethenes on sulfate reduction (SR) was also evaluated. The influence of cyanocobalamin (CNB₁₂) and riboflavin (RF) on the SR and biodegradation of TCE was also determined. Sediments from the vents were incubated at 37°C and supplemented with 4 g l⁻¹ SO₄ ²⁻, lactate or VFAs and amended in the corresponding treatments with either CNB₁₂ or RF in separated experiments. A percentage of TCE removal of 86 (150 μmol l⁻¹ initial concentration) was attained coupled to 48% sulfate depletion with lactate as substrate. Up to 93% removal of TCE (300 μmol l⁻¹ initial concentration) and 40% of sulfate was reached for VFAs as electron donor. A combination of lactate and CNB₁₂ yielded the best SR. The overall results suggest a syntrophic association in this microbial community in which sulfate reducers, dehalogenating, and probably halorespiring bacteria may be interacting and taking advantage of the fermentation of substrates differently, but without interruption of SR in spite of the fact that TCE was always present. It was also clear that sulfate reduction must be established in the cultures before any degradation can occur. The microbial community present in these hydrothermal vents sediments could be a new source of inoculum for bioreactors designed for dechlorination purposes.     

Tetrachloroethene metabolism of Dehalospirillum multivorans

Dehalospirillum multivorans is a strictly anaerobic bacterium that is able to dechlorinate tetrachloroethene (perchloroethylene; PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (DCE) as part of its energy metabolism. The present communication describes some features of the dechlorination reaction in growing cultures, cell suspensions, and cell extracts of D. multivorans. Cell suspensions catalyzed the reductive dechlorination of PCE with pyruvate as electron donor at specific rates of up to 150 nmol (chloride released) min^-1 (mg cell protein)^-1 (300 M PCE initially, pH 7.5, 25°C). The rate of dechlorination depended on the PCE concentration; concentrations higher than 300 M inhibited dehalogenation. The temperature optimum was between 25 and 30°C; the pH optimum at about 7.5. Dehalogenation was sensitive to potential alternative electron acceptors such as fumarate or sulfur; nitrate or sulfate had no significant effect on PCE reduction. Propyl iodide (50 M) almost completely inhibited the dehalogenation of PCE in cell suspensions. Cell extracts mediated the dehalogenation of PCE and of TCE with reduced methyl viologen as the electron donor at specific rates of up to 0.5 mol (chloride released) min^-1 (mg protein).^-1 An abiotic reductive dehalogenation could be excluded since cell extracts heated for 10 min at 95°C were inactive. The PCE dehalogenase was recovered in the soluble cell fraction after ultracentrifugation. The enzyme was not inactivated by oxygen.Abbreviations PCE Perchloroethylene or tetrachloroethene - TCE Trichloroethene - DCE cis-1,2-Dichloroethene - CHC Chlorinated hydrocarbon - MV Methyl viologen     

Growth of a Dehalococcoides-Like Microorganism on Vinyl Chloride and cis-Dichloroethene as Electron Acceptors as Determined by Competitive PCR

A competitive PCR (cPCR) assay targeting 16S ribosomal DNA was developed to enumerate growth of a Dehalococcoides-like microorganism, bacterium VS, from a mixed culture catalyzing the reductive dehalogenation of cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC), with hydrogen being used as an electron donor. The growth of bacterium VS was found to be coupled to the dehalogenation of VC and cDCE, suggesting unique metabolic capabilities. The average growth yield was (5.2 ± 1.5) × 10⁸ copies of the 16S rRNA gene/μmol of Cl⁻ (number of samples, 10), with VC being used as the electron acceptor and hydrogen as the electron donor. The maximum VC utilization rate () was determined to be 7.8 × 10⁻¹⁰ μmol of Cl⁻ (copy⁻¹ day⁻¹), indicating a maximum growth rate of 0.4 day⁻¹. These average growth yield and values agree well with values found previously for dechlorinating cultures. Decay coefficients were determined with growth (0.05 day⁻¹) and no-growth (0.09 day⁻¹) conditions. An important limitation of this cPCR assay was its inability to discriminate between active and inactive cells. This is an essential consideration for kinetic studies.     

Protistan Predation Affects Trichloroethene Biodegradation in a Bedrock Aquifer

Despite extensive research on the bottom-up force of resource availability (e.g., electron donors and acceptors), slow biodegradation rates and stalling at cis-dichloroethene (cDCE) and vinyl chloride continue to be observed in aquifers contaminated with trichloroethene (TCE). The objective of this research was to gauge the impact of the top-down force of protistan predation on TCE biodegradation in laboratory microcosms. When indigenous bacteria from an electron donor-limited TCE-contaminated bedrock aquifer were present, the indigenous protists inhibited reductive dechlorination altogether. The presence of protists during organic carbon-amended conditions caused the bacteria to elongate (length:width, ≥10:1), but reductive dechlorination was still inhibited. When a commercially available dechlorinating bacterial culture and an organic carbon amendment were added in he presence of protists, the elongated bacteria predominated and reductive dechlorination stalled at cDCE. When protists were removed under organic carbon-amended conditions, reductive dechlorination stalled at cDCE, whereas in the presence organic carbon and bacterial amendments, the total chlorinated ethene concentration decreased, indicating TCE was converted to ethene and/or CO₂. The data suggested that indigenous protists grazed dechlorinators to extremely low levels, inhibiting dechlorination altogether. Hence, in situ bioremediation/bioaugmentation may not be successful in mineralizing TCE unless the top-down force of protistan predation is inhibited.The bacterially mediated sequential dechlorination of trichloroethene (TCE) to cis-dichloroethene (cDCE), vinyl chloride (VC), ethene, and CO₂ by dehalorespiration is often proposed as the most cost-effective in situ treatment to remediate chlorinated solvent-contaminated aquifers (35, 42). TCE mineralization to CO₂ requires specific electron donors (i.e., acetate and H₂) typically produced from readily fermentable organic carbon, the presence of specific bacterial species, and sulfate-reducing or methanogenic conditions (1, 4, 8, 15, 22, 25, 33, 35, 46). When the rate of mineralization is slow or stalled at one of the progeny (cDCE and VC), the problem is usually attributed to the bottom-up force of resource availability (e.g., the absence of a necessary condition such as suitable electron donors or bacterial species) (1, 4, 10, 22, 26, 43, 46). For example, whereas many bacterial species are capable of degrading TCE to cDCE and VC by dehalorespiration (33), only Dehalococcoides ethenogenes is known to convert VC to ethene (25). Hence, if an indigenous population of D. ethenogenes is not present in situ, the system will likely stall at cDCE or VC even if sufficient electron donor is added. Stalling is problematic because VC is more toxic than TCE (18). In this case, bioaugmentation with D. ethenogenes may trigger complete mineralization.An established link exists for the top-down force of predator-prey relationships between protists and bacteria in a range of surface water systems (13, 19-21, 29). Our previous work with groundwater protists in a wastewater contaminated sandy aquifer demonstrated that size selective predation by protists affects biodegradation of the organic carbon. Our subsequent work in a TCE-contaminated bedrock aquifer at the Bedrock Bioremediation Center (BBC) research site (Portsmouth, NH) suggested that bottom-up resource availability could not totally explain stalls at cDCE. This led us to hypothesize that the top-down force of selective predation by protists on dehalorespiring bacteria inhibited the required compositional shift in the bacterial community to one dominated by D. ethenogenes, thus preventing the conversion of cDCE and VC to ethene and CO₂ (2, 15). This, coupled with bacterial studies by Travis and Rosenberg (41), Lewis (32) and Snyder et al. (40), led us to postulate that protistan predation could have a negative impact on TCE biodegradation. Continuously stirred reactors were used to examine how the presence of protists influenced the rate of bacterially mediated reductive dechlorination. Experiments were conducted using ambient (≤0.8 mM as C) and amended (10 mM as C) organic carbon concentrations with protists present and absent. TCE biodegradation was also assessed when the indigenous community was amended with a commercially available bacterial culture containing D. ethenogenes (34).(A portion of this research was originally submitted to the University of New Hampshire, Durham, by J. Cunningham as an M.S. thesis [12].)     

Effects of bioaugmentation on enhanced reductive dechlorination of 1,1,1-trichloroethane in groundwater: a comparison of three sites

Microcosm studies investigated the effects of bioaugmentation with a mixed Dehalococcoides (Dhc)/Dehalobacter (Dhb) culture on biological enhanced reductive dechlorination for treatment of 1,1,1-trichloroethane (TCA) and chloroethenes in groundwater at three Danish sites. Microcosms were amended with lactate as electron donor and monitored over 600 days. Experimental variables included bioaugmentation, TCA concentration, and presence/absence of chloroethenes. Bioaugmented microcosms received a mixture of the Dhc culture KB-1 and Dhb culture ACT-3. To investigate effects of substrate concentration, microcosms were amended with various concentrations of chloroethanes (TCA or monochloroethane [CA]) and/or chloroethenes (tetrachloroethene [PCE], trichloroethene [TCE], or 1,1-dichloroethene [1,1-DCE]). Results showed that combined electron donor addition and bioaugmentation stimulated dechlorination of TCA and 1,1-dichloroethane (1,1-DCA) to CA, and dechlorination of PCE, TCE, 1,1-DCE and cDCE to ethane. Dechlorination of CA was not observed. Bioaugmentation improved the rate and extent of TCA and 1,1-DCA dechlorination at two sites, but did not accelerate dechlorination at a third site where geochemical conditions were reducing and Dhc and Dhb were indigenous. TCA at initial concentrations of 5 mg/L inhibited (i.e., slowed the rate of) TCA dechlorination, TCE dechlorination, donor fermentation, and methanogenesis. 1 mg/L TCA did not inhibit dechlorination of TCA, TCE or cDCE. Moreover, complete dechlorination of PCE to ethene was observed in the presence of 3.2 mg/L TCA. In contrast to some prior reports, these studies indicate that low part-per million levels of TCA (<3 mg/L) in aquifer systems do not inhibit dechlorination of PCE or TCE to ethene. In addition, the results show that co-bioaugmentation with Dhc and Dhb cultures can be an effective strategy for accelerating treatment of chloroethane/chloroethene mixtures in groundwater, with the exception that all currently known Dhc and Dhb cultures cannot treat CA.     

Interaction of sulfate and glutathione transport in cultured tobacco cells

Photoheterotrophic and heterotrophic suspension cultures of tobacco (Nicotiana tabacum L.) were grown with 1 mM glutathione (reduced; GSH) as sole source of sulfur. Addition of sulfate to both cultures did not alter the rate of exponential growth, but affected the removal of GSH and sulfate in different ways. In photoheterotrophic suspensions, addition of sulfate caused a decline in the net uptake of GSH, whereas sulfate was taken up by the green cells immediately. In heterotrophic suspensions, however, addition of sulfate did not affect the net uptake of GSH and sulfate was only taken up by the cells after the GSH supply in the medium had been exhausted. Apparently, GSH uptake in photoheterotrophic cells is inhibited by sulfate, whereas sulfate uptake is inhibited by GSH in heterotrophic cells. The differences in the effect of GSH on sulfate uptake in photoheterotrophic and heterotrophic tobacco suspensions cannot be attributed to differences in the kinetic properties of sulfate carriers. In short-time transport experiments, both cultures took up sulfate almost entirely by an active-transport system as shown by experiments with metabolic inhibitors; sulfate transport of both cultures obeyed monophasic Michaelis-Menten kinetics with similar app. K_m (photoheterotrophic cells: 16.0±2.0 M; heterotrophic cells: 11.8±1.8 M) and V_max (photoheterotrophic cells: 323±50 nmol·min^-1·g^-1 dry weight; heterotrophic cells: 233±3 nmol·min^-1·g^-1 dry weight). Temperature- and pH-dependence of sulfate transport showed almost identical patterns. However, the cultures exhibited remarkable differences in the inhibition of sulfur influx by GSH in short-time transport experiments. Whereas 1 mM GSH inhibited sulfate transport into heterotrophic tobacco cells completely, sulfate transport into photoheterotrophic cells proceeded at more than two-thirds of its maximum velocity at this GSH concentration. The mode of action of GSH on sulfate transport in chloroplast-free tobacco cell does not appear to be direct: a 14-h exposure to 1 mM GSH was found to be necessary to completely block sulfate transport; a 4-h time of exposure did not affect this process. Consequently, glutathione does not seem to be a product of sulfur metabolism acting on sulfate-carrier entities by negative feedback control. When transferred to the whole plant, the observed differences in sulfate and glutathione influx into green and chloroplast-free cells may be interpreted as a regulatory device to prevent the uptake of excess sulfate by plants.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - DNP dinitrophenol - DW dry weight - FW fresh weight - GSH reduced glutathione     

Role of sodium ions for sulfate transport and energy metabolism in Desulfovibrio salexigens

The sodium ion gradient and the membrane potential were found to be the driving forces of sulfate accumulation in the marine sulfate reducer Desulfovibrio salexigens. The protonmotive force of –158 mV, determined by means of radiolabelled membrane-permeant probes, consisted of a membrane potential of –140 mV and a pH gradient (inside alkaline) of 0.3 at neutral pH_out. The sodium ion gradient, as measured with silicone oil centrifugation and atomic absorption spectroscopy, was eightfold ([Na⁺]_out/[Na⁺]_in) at an external Na⁺ concentration of 320 mM. The resulting sodium ionmotive force was –194 mV and enabled D. salexigens to accumulate sulfate 20000-fold at low external sulfate concentrations (<0.1 M). Under these conditions high sulfate accumulation occurred electrogenically in symport with three sodium ions (assuming equilibrium with the sodium ion-motive force). With increasing external sulfate concentrations sulfate accumulation decreased sharply, and a second, low-accumulating system symported sulfate electroneutrally with two sodium ions. The sodium-ion gradient was built up by electrogenic Na⁺/H⁺ antiport. This was demonstrated by (i) measuring proton translocation upon sodium ion pulses, (ii) studying uptake of sodium salts in the presence or absence of the electrical membrane potential, and (iii) the inhibitory effect of the Na⁺/H⁺ antiport inhibitor propylbenzilylcholin-mustard HCl (PrBCM). With resting cells ATP synthesis was found after proton pulses (changing the pH by three units), but neither after pulses of 500 mM sodium ions, nor in the presence of the uncoupler tetrachorosalicylanilide (TCS). It is concluded that the energy metabolism of the marine strain D. salexigens is based primarily on the protonmotive force and a protontranslocating ATPase.Abbreviations MOPS morpholinopropanesulfonic acid - TCS tetrachlorosalicylanilide - PrBCM propylbenzilylcholin-mustard HCl - Tris tris(hydroxymethyl)aminomethane - TPP⁺ bromide tetraphenylphosphonium bromide     

Monitoring trichloroethylene mineralization by Pseudomonas cepacia G4 PR1

To analyze the extent of mineralization of trichloroethylene (TCE) without disturbing an actively growing biofilm, a minimal growth medium was formulated that reduces the concentration of chloride ions to the extent that the chloride ions generated from TCE mineralization may be detected with a chloride-ion-specific electrode. By substituting chloride salts with phosphates and nitrates, a chloride-free minimal medium was produced that yields a specific growth rate for Pseudomonas cepacia G4 PR1 which was 93% of that in chloride-ion-containing minimal medium. Furthermore, TCE degradation by resting cell suspensions was similar in both media (85% of 75 M TCE degraded in 6 h), and complete mineralization of TCE was slightly superior in the chloride-free minimal medium (77% compared to 60% of 75 M TCE mineralized in 6 h). In addition, indole-containing, minimal-medium agar plates were developed to indicate the presence of the TCE-degrading enzyme toluene ortho-monooxygenase (fire-engine-red colonies) as well as to distinguish this enzyme from other TCE-degrading enzymes (toluene dioxygenase and toluene para-monooxygenase).     

Effect of Nitrogen Source on Growth and Trichloroethylene Degradation by Methane-Oxidizing Bacteria

The effect of nitrogen source on methane-oxidizing bacteria with respect to cellular growth and trichloroethylene (TCE) degradation ability were examined. One mixed chemostat culture and two pure type II methane-oxidizing strains, Methylosinus trichosporium OB3b and strain CAC-2, which was isolated from the chemostat culture, were used in this study. All cultures were able to grow with each of three different nitrogen sources: ammonia, nitrate, and molecular nitrogen. Both M. trichosporium OB3b and strain CAC-2 showed slightly lower net cellular growth rates and cell yields but exhibited higher methane uptake rates, levels of poly-β-hydroxybutyrate (PHB) production, and naphthalene oxidation rates when grown under nitrogen-fixing conditions. The TCE-degrading ability of each culture was measured in terms of initial TCE oxidation rates and TCE transformation capacities (mass of TCE degraded/biomass inactivated), measured both with and without external energy sources. Higher initial TCE oxidation rates and TCE transformation capacities were observed in nitrogen-fixing mixed, M. trichosporium OB3b, and CAC-2 cultures than in nitrate- or ammonia-supplied cells. TCE transformation capacities were found to correlate with cellular PHB content in all three cultures. The results of this study suggest that the nitrogen-fixing capabilities of methane-oxidizing bacteria can be used to select for high-activity TCE degraders for the enhancement of bioremediation in fixed-nitrogen-limited environments.

Optimal bioremediation conditions within contaminated aquifers are often found to be limited by the availability of nutrients, including nitrogen. Consequently, microorganisms that are capable of degrading contaminants as well as fixing molecular nitrogen as their sole nitrogen source could have a growth advantage in fixed-nitrogen-deficient environments that would be favorable for promoting in situ bioremediation.Trichloroethylene (TCE) is a major groundwater contaminant of concern in the United States due to its suspected carcinogenity and persistence in subsurface environments (31). However, a number of laboratory (1, 4, 13, 16, 18, 19, 22, 23, 26–28, 34) and field studies (3, 15, 24, 25) have shown that TCE can be cometabolically transformed into nontoxic end products (CO₂ and Cl⁻) by methane-oxidizing bacteria at the expense of reducing energy in the form of NADH. Many studies have also reported that some methane-oxidizing cultures (type II) are able to utilize different sources of nitrogen (N) for cellular growth (32, 33), including molecular nitrogen at reduced oxygen partial pressures (11, 12, 20, 33). The types of methanotrophs that are capable of nitrogen fixation also produce a type of oxygenase (i.e., soluble methane monooxygenase [sMMO]) which exhibits high activity with respect to the oxidation of TCE.Poly-β-hydroxybutyrate (PHB) is an internal reducing-energy storage polymer that can be used as an alternative reducing-energy source by a number of methane-oxidizing cultures under starvation conditions (9). Recently, a number of studies observed a correlation between TCE transformation capacities (T_c; mass of TCE transformed per mass of cells inactivated) and microbial PHB content (7, 16, 17), suggesting that PHB might be used as an alternative NADH source for TCE oxidation by methane-oxidizing bacteria in the absence of growth substrate. It has also been shown that the synthesis of PHB is stimulated in cells grown under nutrient-limited conditions, including nitrogen-fixing conditions (2, 9, 10, 21). As a result of the characteristics of methane-oxidizing microorganisms described above, it may be possible to select for nitrogen-fixing methane oxidizers in fixed-nitrogen-limited subsurface environments such that the burden of nutrient addition to the subsurface for the sustained growth of these contaminant degraders is diminished while contaminant degradation is enhanced during in situ bioremediation.A recent study conducted by us (7) explored the feasibility of using the nitrogen-fixing capabilities of methane oxidizers for the enhancement of bioremediation. Our results suggested that nitrogen-fixing mixed cultures were able to degrade TCE as effectively as nitrate-supplied cultures. Further, higher T_c and higher cellular PHB contents were observed in nitrogen-fixing cultures. Of particular interest were observations of lower TCE product toxicity, measured in terms of methane uptake rates following TCE exposure, for nitrogen-fixing cultures than for nitrate- or ammonia-supplied cultures. Since that study was conducted with mixed cultures, it was difficult to elucidate the reasons for the enhanced degradation performance of the nitrogen-fixing methane oxidizers. An understanding of the effects of nitrogen source on cell growth and TCE degradation ability will be particularly beneficial for designing, operating, and implementing in situ- or ex situ-engineered bioremediation systems. This study evaluates nitrogen source effects on methane-oxidizing bacteria, using two pure strains and one mixed chemostat culture. Nitrogen source effects are examined with regard to cellular growth, specific methane uptake rates, specific naphthalene oxidation rates, and TCE degradation ability.     

Kinetic analysis of the inhibition of sulfate transport in human red blood cells by isothiocyanates

Summary A kinetic analysis of anion self-exchange in human red blood cells, in the presence of an irreversible inhibitor, is presented and applied to the study of the inactivation of sulfate transport by three isothiocyanates: 3-isothiocyano-1,5-naphthalenedisulfonic acid, disodium salt (INDS), 1-isothiocyano-4-naphthalene sulfonic acid, sodium salt, monohydrate (INS), and 1-isothiocyano-4-benzenesulfonic acid, sodium salt, monohydrate (IBS). The time dependence of the inhibition of sulfate transport by the isothiocyanates used could be described by a single exponential and could be shown to contain a reversible and an irreversible component. In each case a portion of sulfate efflux was found to be resistant to inactivation. The residual portion of the sulfate efflux varied with inhibition: 4% for INS, 16% for INDS, and 34% for IBS. INS showed the largest reversible inhibitory effect (12% of the flux remaining at 0.2mm inhibitor concentration), while INDS showed the weakest effect (92% of the flux remaining at 0.3mm inhibitor concentration). IBS had the highest rate of inactivation while INDS had the lowest. The kinetic analysis further suggests that all three isothiocyanates bind reversibly to an inhibitory site on the membrane before they bind covalently, and therefore irreversibly, to the same site on the membrane. The equilibrium constant for the dissociation of the reversibly-bound complex,K _i, and the rate of irreversible inactivation after all membrane sites are reversibly bound,k _max, have been computed for all three inhibitors: INDS (K _i=420m,k _max=5.04 hr^–1), INS (K _i=148 m,k _max=6.48 hr^–1), and IBS (K _i=208 m,k _max=8.11 hr^–1).     

Micropropagation of Raphanus sativus L. var. longipinnatus (Japanese radish) cv. Gungjung

Shoot cultures were established from seedling shoot tips of Raphanus sativus var. longipinnatus Bailey cv. Gungjung, (Japanese radish) cultured on a Murashige-Skoog medium supplemented with ca. 4.5–135 M kinetin or N⁶-benzyladenine. The latter cytokinin supported overall better growth, and 22.2 M was adopted for maintenance of established cultures. The nitrate: ammonium levels in the medium proved optimal for growth and shoot proliferation and both these parameters were significantly increased by addition of adenine sulfate or sodium phosphate. Rooting of excised shoots was achieved on auxin containing medium. Indole-3-butyric acid (ca. 5 or 10 M) also enhanced shoot growth. Plants were easily established in soil, appeared morphologically normal, and flowered.     

Transient Expression of β-Glucuronidase in Embryo Axes of Cotton by Agrobacterium and Particle Bombardment Methods

Transient expression of -glucuronidase (GUS) in zygotic embryo axes of two cotton (Gossypium hirsutum L.) cultivars NHH-44 and DCH-32 was induced by Agrobacterium mediated transformation or by particle bombardment. For Agrobacterium transformation, disarmed A. tumefaciens strain GV 2260/p35SGUSINT was used. In cv. NHH-44, the maximum frequency of transient expression (14.28 %) was achieved on spotting Agrobacterium paste on the apical regions of the split embryo axes. The method resulted in a transformed callus line, which showed strong GUS activity. Integration of NPTII gene was confirmed by Southern analysis. Transgene expression by particle bombardment was achieved with p35SGUSINT and pIBGUS plasmids independently. The maximum frequency of GUS expression in 29.16 % explants was observed in cultivar NHH-44 with gold microcarriers (1.1 µm) when bombarded once with rupture disc of 7586 kPa at target cell distance of 6 cm. A transformed callus line was obtained when explants were bombarded with p35SGUSINT and cultured on Murashige and Skoog's medium supplemented with B₅ vitamins, 0.1 mg dm^–3 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea, 0.01 mg dm^–3 -naphthaleneacetic acid, 3 % glucose + 50 mg dm^–3 kanamycin. High GUS activity was observed in callus tissue as well as in somatic embryo like structures achieved in liquid shake cultures.     

Growth and energy production in Bacteroides amylophilus

The molar growth yield (Y _m) of Bacteroides amylophilus strain WP91 on maltose was 68±2 g/mol when determined from batch cultures at the peaks of maximal growth. Continued incubation led to considerable cell lysis. When calculated from batch cultures in exponential phase (specific growth rate, =0.57 h^-1) Y _m was 101 g/mol. The maximum value of Y _m in maltose-limited chemostat cultures at the maximum dilution rate (D) attainable (D==0.39 h^-1) was about 79 g/mol. Ammonia-Fmited chemostat cultures metabolized maltose with a much reduced efficiency and this was associated with a difference in morphology and chemical composition of the cells. The theoretical maximum molar growth yields (Y _m ^max ) were 55 and 114 g/mol for ammonia- and maltose-limited growth respectively. However, if account was taken of extracellular nitrogen-containing material in ammonia-limited cultures, Y _m ^max became 60. The maintenance coefficient (m _s), estimated from the lines relating the specific rate of maltose consumption (q _m) and D (where m _s=q _m at D=0), was 7.4±0.6×10^-4 mol maltose/g x h for both nutrient limitations. A difference in maintenance energy demand, independent of growth-rate, could not account, therefore, for the observed differences in Y _m between ammonia- and maltose-limited growth.     

Lysosomal sulfate efflux following glycosaminoglycan degradation: measurements in enzyme-supplemented Maroteaux-Lamy syndrome fibroblasts and isolated lysosomes

Studies using lysosomal membrane vesicles have suggested that efflux of the sulfate that results from lysosomal glycosaminoglycan degradation is carrier-mediated. In this study, glycosaminoglycan degradation and sulfate efflux were examined using cultured skin fibroblasts and lysosomes deficient in the lysosomal enzymeN-acetylgalactosamine-4-sulfatase. Such fibroblasts store dermatan sulfate lysosomally, which could be labelled biosynthetically with Na ₂ ³⁵ SO₄. The addition of recombinantN-acetylgalactosamine-4-sulfatase to the media of³⁵S labelled fibroblasts degraded up to 82% of the stored dermatan [³⁵S] sulfate over a subsequent 96 h chase and released inorganic [³⁵S] sulfate into the medium. In the presence of 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS), sulfate was reused to a minor extent in newly synthesized proteoglycan. Isolated granules from recombinant enzyme supplemented fibroblasts degraded stored dermatan [³⁵S]sulfate to sulfate which was rapidly released into the medium at a rate that was reduced by the extra-lysosomal presence of the lysosomal sulfate transport inhibitors SITS, Na₂SO₄ and Na₂MoO₄. SITS also inhibited dermatan sulfate turnover, although it had no effect on the action of purified recombinant enzymein vitro. These data imply that sulfate clearance occurred concomitantly with dermatan sulfate turnover in the lysosome even at high substrate loading, and that lysosome-derived sulfate, while available, is reutilized minimally in synthetic pathways.Abbreviations SITS 4-acetamido-4-isothiocyanatostilbene-2,-2-disulfonic acid - GAG glycosaminoglycan - 4S N-acetylgalactosamine-4-sulfatase - r4S recombinant humanN-acetylgalactosamine-4-sulfatase - PBS phosphate buffered saline - BME basal modified Eagle's medium - FBS fetal bovine serum - GalNAc4S-GlcA-GalitolNAc4S -(N-acetyl-d-galactosamine-4-sulfate)-(1–4)--d-glucuronic acid)-(1–3)-N-acetyl-d-[1-³H]galactosaminitol-4-sulfate - DS dermatan sulfate - MPS mucopolysaccharidosis