Reductive dehalogenation of bromoform in aqueous solution.

The hybrid semiconducter-macrocycle catalyst TiO2-cobalt phthalocyanine promotes the solar photolysis of aqueous bromoform under anaerobic conditions. The major decomposition products are dibromoethane and HBr. Bromomethane and methane were produced only after prolonged photolysis (30 hr). Acetone, derived from added 2-propanol, was the only observed oxidation product. Preliminary experiments showed that electrolytic reduction of aqueous carbon tetrachloride at a vitamin B12-modified silver electrode produced the expected lower homologues but with surprisingly high yields of methane.

Influence of peat on Fenton oxidation.

A diagnostic probe was used to estimate the activity of Fenton-derived hydroxyl radicals (.OH), reaction kinetics, and oxidation efficiency in batch suspensions comprised of silica sand, crushed goethite (alpha-FeOOH) ore, peat, and H2O2 (0.13 mM). A simple method of kinetic analysis is presented and used to estimate the rate of .OH production (POH) and scavenging term (ks), which were used to establish the influence of organic matter (Pahokee peat) in Fenton systems. POH was greater in the peat-amended systems than in the unamended control, and ks was approximately the same. Any increase in scavenging of .OH that resulted from the addition of peat was insignificant in comparison to radical scavenging by reaction with H2O2. Also, treatment efficiency, defined as the ratio of probe conversion to H2O2 consumption over the same period was greater in the peat-amended system. Results suggest that .OH production is enhanced in the presence of peat by one or more peat-dependent mechanisms. Fe concentration and availability in the peat, reduction of Fe(III) to Fe(II) by the organic matter, and reduction of organic-complexed Fe(III) to Fe(II) are discussed in the context of the Fenton mechanism.

Microcapsules for new animal drugs.

Microencapsulation techniques have been developed and refined for many years. However, in recent years, great strides have been made in controlling the microcapsule size down to the nanometer range when necessary. Progress has also been made in controlling the ratio of wall/capsule and the distribution of microcapsule diameter size in the batching of the capsules. One area of application for microencapsulation is the convenience of dosing large groups of animals once with a sustained-release dosage form rather than with repeated administration since this involves greater effort at animal management. For example, targeted dosages can be achieved by a specialized drug delivery system. Since animal systems are varied and ruminant animals have a digestive system that is different from and more complex than that of other animals, oral administration of a drug substance to be retained through the rumen to the abomasum for subsequent dissolution and/or excretion may be affected by a microencapsulation process. Miller and Gordon of USDA achieved this for control of fecal breeding flies by systemic retention of a microencapsulated pesticide drug into the manure. Perhaps one of the most challenging areas is that of pharmaceuticals. There is now considerable interest in the area of microparticulate drug delivery systems. Examples of how desirable nutritional properties and taste preference are achieved and how these provide improved animal products will be discussed. Additional areas of interest and some possible future systems that have advantages over conventional systems will be discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

Behavior of organic carbon during subsurface wetland treatment in the Sonoran Desert.

We examined the fate of organics during wetland treatment of secondary effluent and groundwater (control) flows in parallel, research-scale, subsurface-flow (SSF) wetland raceways at the Constructed Ecosystem Research Facility (CERF) located in Tucson, Arizona. The CERF facility enabled us to distinguish experimentally among effects on effluent quality due to season-dependent processes of evapotranspiration (ET) and wetlands-derived production of organics. Organics of wastewater and wetlands origin were compared in terms of their contributions to dissolved organic carbon (DOC) in wetland effluent. Elevated temperatures and associated biochemical activities increased DOC levels in wetland effluents during summer. In other words, DOC removal efficiency was negatively correlated to temperature. The contributions of ET and wetland-derived organics to elevation of DOC in wetland effluents during summer were roughly comparable. The elevation of organic carbon concentration during wetland polishing of wastewater effluent will lead to higher levels of disinfection by-products when treated waters are chlorinated prior to reuse. Results of this work are relevant to water managers in arid regions, which may incorporate wetlands into sequential wastewater treatments leading to potable reuse of reclaimed water.

Precision measurement of the weak mixing angle in Møller scattering.

We report on a precision measurement of the parity-violating asymmetry in fixed target electron-electron (Møller) scattering: A(PV) = [-131 +/- 14(stat) +/- 10(syst)] x 10(-9), leading to the determination of the weak mixing angle sin2(thetaW(eff) = 0.2397 +/- 0.0010(stat) +/- 0.0008(syst), evaluated at Q2 = 0.026 GeV2. Combining this result with the measurements of sin2(thetaW(eff) at the Z0 pole, the running of the weak mixing angle is observed with over 6sigma significance. The measurement sets constraints on new physics effects at the TeV scale.

A Test to Determine Microquantities of Milkfat in Cleaning Solutions by Surface Film Displacement 1.

Determining microquantities of soil is a major challenge in evaluating cleaning systems. A highly sensitive test was therefore developed to determine trace quantities of milkfat in cleaning solutions. It involved extraction of cleaning solution with organic solvents and subsequent determination of surface film displacement. The area displaced was directly related to the quantity of milkfat in the test solution with a correlation coefficient of 0.99. Usefulness of the test in evaluating cleaning systems has been shown in other research.

Effects of electron donor and acceptor conditions on reductive dehalogenation of tetrachloromethane by Shewanella putrefaciens 200.

Shewanella putrefaciens 200 is a nonfermentative bacterium that is capable of dehalogenating tetrachloromethane to chloroform and other, unidentified products under anaerobic conditions. Since S. putrefaciens 200 can respire anaerobically by using a variety of terminal electron acceptors, including NO3-, NO2-, and Fe(III), it provides a unique opportunity to study the competitive effects of different electron acceptors on dehalogenation in a single organism. The results of batch studies showed that dehalogenation of CT by S. putrefaciens 200 was inhibited by O2, 10 mM NO3-, and 3 mM NO2-, but not by 15 mM Fe(III), 15 mM fumarate, or 15 mM trimethylamine oxide. Using measured O2, Fe(III), NO2-, and NO3- reduction rates, we developed a speculative model of electron transport to explain inhibition patterns on the basis of (i) the kinetics of electron transfer at branch points in the electron transport chain, and (ii) possible direct inhibition by nitrogen oxides. In additional experiments in which we used 20 mM lactate, 20 mM glucose, 20 mM glycerol, 20 mM pyruvate, or 20 mM formate as the electron donor, dehalogenation rates were independent of the electron donor used. The results of other experiments suggested that sufficient quantities of endogenous substrates were present to support transformation of tetrachloromethane even in the absence of an exogenous electron donor. Our results should be significant for evaluating (i) the bioremediation potential at sites contaminated with both halogenated organic compounds and nitrogen oxides, and (ii) the bioremediation potential of iron-reducing bacteria at contaminated locations containing significant amounts of iron-bearing minerals.

Reductive dissolution of Fe(III) oxides by Pseudomonas sp. 200.

The kinetics and mechanism of reductive dissolution of Fe(III) oxides were examined in pure, batch cultures of Pseudomonassp. 200. Primary factors controlling hematite dissolution kinetics were mineral surface area (or concentration of high-energy surface sites), ligand concentration, and cell number. In the presence of nitrilotriacetic acid (NTA), saturation kinetics were apparent in the relationship governing reductive dissolution of hematite. A kinetic expression was developed in which overall iron-reduction rate is functionally related to the concentrations of both NTA and Fe(III).Addition of NTA resulted in a 20-fold increase in the microbial rate of mineral (reductive) dissolution. Mechanisms in which NTA served as a bridging ligand, shuttling respiratory electrons from the membrane-bound microbial electron transport chain to the metal center of the iron oxide, or accelerated the departure of Fe(II) centers (bound to ligand) from the oxide surface following reduction have been postulated. Experimental results indicated that cell-mineral contact was essential for reductive dissolution of goethite.

Inhibitor studies of dissimilative Fe(III) reduction by Pseudomonas sp. strain 200 ("Pseudomonas ferrireductans")

Aerobic respiration and dissimilative iron reduction were studied in pure, batch cultures of Pseudomonas sp. strain 200 ("Pseudomonas ferrireductans"). Specific respiratory inhibitors were used to identify elements of electron transport chains involved in the reduction of molecular oxygen and Fe(III). When cells were grown at a high oxygen concentration, dissimilative iron reduction occurred via an abbreviated electron transport chain. The induction of alternative respiratory pathways resulted from growth at low oxygen tension (less than 0.01 atm [1 atm = 101.29 kPa]). Induced cells were capable of O2 utilization at moderately increased rates; dissimilative iron reduction was accelerated by a factor of 6 to 8. In cells grown at low oxygen tension, dissimilative iron reduction appeared to be uncoupled from oxidative phosphorylation. Models of induced and uninduced electron transport chains, including a mathematical treatment of chemical inhibition within the uninduced, aerobic electron transport system, are presented. In uninduced cells respiring anaerobically, electron transport was limited by ferrireductase activity. This limitation may disappear among induced cells.

Kinetics and mechanism of dissimilative Fe(III) reduction by Pseudomonas sp. 200.

The kinetics and mechanism of Fe(III) reduction to Fe(II) were studied in pure batch cultures of Pseudomonas sp. 200. The rate of iron reduction has been mechanistically related to aqueous phase iron speciation. In the absence of microbial activity the iron reduction rate was negligible. Initial rates of microbial iron reduction were accelerated more than 20-fold by the addition of equimolar quantities of nitrilotriacetic acid (NTA) to media initially containing 1.86 x 10(-3)M total Fe(III). Numerical techniques were utilized to quantify relationships between the observed rate of Fe(II) production and the calculated (equilibrium) aqueous phase speciation. These results indicate that soluble ferric iron species are not equivalent in terms of their susceptibility to bacterial (dissimilative) iron reduction. The concentration of Fe(NTA)(OH)(2) (2-) correlated strongly with observed iron reduction rates. Ferrous iron species appeared to inhibit the reduction process.

Involvement of cytochromes in the anaerobic biotransformation of tetrachloromethane by Shewanella putrefaciens 200.

Shewanella putrefaciens 200 is an obligate respiratory bacterium that can utilize a variety of terminal electron acceptors, e.g., NO3-, NO2-, Fe(III), and trimethylamine N-oxide, in the absence of O2. The bacterium catalyzed the reductive transformation of tetrachloromethane (CT) under anaerobic conditions. The only identified product was trichloromethane (CF), but CF production was not stoichiometric. No dichloromethane, chloromethane, or methane was produced. A chloride mass balance indicated that fully dechlorinated products were not formed. Studies with [14C]CT suggested that a portion of the transformed CT reacted with biomass to form unidentified soluble and insoluble products. Intermediate production of a trichloromethyl radical can explain observed product distribution without significant CO2 formation. Evidence suggests that respiratory c-type cytochromes are responsible for the dehalogenation ability of S. putrefaciens 200. Previous growth under microaerobic conditions ([O2], < 2.5 microM) results in (i) a 2.6-fold increase in specific heme c content and (ii) a 2.3-fold increase in specific rates of anaerobic CT transformation. Manipulation of heme content by growth on iron-free medium or medium amended with delta-aminolevulinic acid showed that CT transformation rates increase with increases in specific heme c content. Transformation of CT is inhibited by CO. Dehalogenation studies with periplasmic, cytoplasmic, and membrane fractions indicated that only periplasmic and membrane fractions possessed dehalogenation ability. Cytochromes c were the predominant cytochromes present. Membranes were also found to contain smaller amounts of cytochrome b. Observed CT transformation patterns are consistent with a cometabolic description involving fortuitous CT reduction by reduced c-type cytochromes.

Reductive Dechlorination of Trichloroethylene and Tetrachloroethylene under Aerobic Conditions in a Sediment Column.

Biodegradation of trichloroethylene and tetrachloroethylene under aerobic conditions was studied in a sediment column. Cumulative mass balances indicated 87 and 90% removal for trichloroethylene and tetrachloroethylene, respectively. These studies suggest the potential for simultaneous aerobic and anaerobic biotransformation processes under bulk aerobic conditions.

Factors affecting competition between type I and type II methanotrophs in two-organism, continuous-flow reactors.

Competition experiments were performed in a continuous-flow reactor using Methylosinus trichosporium OB3b, a type II methanotroph, and Methylomonas albus BG8, a type I methanotroph. The experiments were designed to establish conditions under which type II methanotrophs, which have significant cometabolic potential, prevail over type I strains. The primary determinants of species selection were dissolved methane, copper, and nitrate concentrations. Dissolved oxygen and methanol concentrations played secondary roles. M. trichosporium OB3b proved dominant under copper and nitratelimited conditions. The ratio of M. trichosporium to M. albus in the reactor increased ten-fold in less than 100 hours following the removal of copper from the reactor feed. Numbers of M. albus declined to levels that were below detection limits (<106/ml) under nitrogen-limited conditions. In the latter experiment, the competitive success of M. trichosporiumdepended on the maintenance of an ambient dissolved oxygen level below about 7.5 × 10(-5) M, or 30% of saturation with air. The ability of M. trichosporium to express soluble methane monooxygenase under copper limitation and nitrogenase under nitrate limitation was very significant. M. albus predominated under methane-limited conditions, especially when low levels of methanol were simultaneously added with methane to the reactor. The results imply that nitrogen limitation can be used to select for type II strains such as M. trichosporium OB3b.

Regulation of Dissimilatory Fe(III) Reduction Activity in Shewanella putrefaciens.

Under anaerobic conditions, Shewanella putrefaciens is capable of respiratory-chain-linked, high-rate dissimilatory iron reduction via both a constitutive and inducible Fe(III)-reducing system. In the presence of low levels of dissolved oxygen, however, iron reduction by this microorganism is extremely slow. Fe(II)-trapping experiments in which Fe(III) and O(2) were presented simultaneously to batch cultures of S. putrefaciens indicated that autoxidation of Fe(II) was not responsible for the absence of Fe(III) reduction. Inhibition of cytochrome oxidase with CN resulted in a high rate of Fe(III) reduction in the presence of dissolved O(2), which suggested that respiratory control mechanisms did not involve inhibition of Fe(III) reductase activities or Fe(III) transport by molecular oxygen. Decreasing the intracellular ATP concentrations by using an uncoupler, 2,4-dinitrophenol, did not increase Fe(III) reduction, indicating that the reduction rate was not controlled by the energy status of the cell. Control of electron transport at branch points could account for the observed pattern of respiration in the presence of the competing electron acceptors Fe(III) and O(2).

Reductive dehalogenation of gas-phase chlorinated solvents using a modified fuel cell.

The reductive dehalogenation of gas-phase chlorinated alkanes (CCl4, CHCl3, and 1,1,1-trichloroethane) and alkenes (perchloroethene (PCE) and trichloroethene (TCE)) was conducted in a modified fuel cell. The fuel-cell performance was a function of cathode material, electric potential, temperature, target compound identity and gas-phase concentration, partial pressure of O2 in the cathode chamber, and cathode condition (time in service). TCE conversion was approximately first order in TCE concentration with half-lives of fractions of a second. Under the same reactor conditions, CCl4 transformation was faster than CHCl3, and TCE reduction was faster than PCE. Rates of both CCl4 and PCE transformation increased substantially with temperature in the range of 30-70 degrees C. At 70 degrees C and a potential (potential of the cathode minus that of the anode) of -0.4 V, single-pass CCl4 conversions were approximately 90%. Mean residence time for gases in the porous cathode was much less than 1 s. The presence of even 5% O2(g) in the influent to the cathode chamber had a deleterious effect on reactor performance. Performance also deteriorated with time in service, perhaps due to the accumulation of HCl on the cathode surface. Conversion efficiency was restored, however, by temporarily eliminating the halogenated target(s) from the influent stream or by briefly reversing fuel-cell polarity.

Applications of a colorimetric plate assay for soluble methane monooxygenase activity.

A straightforward method is described for screening methanotrophic colonies for soluble methane monooxygenase (sMMO) activity on solid media. Such activity results in the development of a colored complex between 1-naphthol, which is formed when sMMO reacts with naphthalene, and o-dianisidine (tetrazotized). Methanotrophic colonies expressing sMMO turned deep purple when exposed successively to naphthalene and o-dianisidine. The method was evaluated within the contexts of two potential applications. The first was for the enumeration of Methylosinus trichosporium OB3b in a methane-amended, unsaturated soil column dedicated to vinyl chloride treatment. The second application was for the isolation and enumeration of sMMO-bearing methanotrophs from sanitary landfill soils. The technique was effective in both applications.

Isolation of copper biochelates from Methylosinus trichosporium OB3b and soluble methane monooxygenase mutants.

Methylosinus trichosporium OB3b produces an extracellular copper-binding ligand (CBL) with high affinity for copper. Wild-type cells and mutants that express soluble methane monooxygenase (sMMO) in the presence and absence of copper (sMMOc) were used to obtain cell exudates that were separated and analyzed by size exclusion high-performance liquid chromatography. A single chromatographic peak, when present, contained most of the aqueous-phase Cu(II) present in the culture medium. In mutant cultures that were unable to acquire copper, extracellular CBL accumulated to high levels both in the presence and in the absence of copper. Conversely, in wild-type cultures containing 5 microM Cu(II), extracellular CBL was maintained at a low, steady level during exponential growth, after which the external ligand was rapidly consumed. When Cu(II) was omitted from the growth medium, the wild-type organism produced the CBL at a rate that was proportional to cell density. After copper was added to this previously Cu-deprived culture, the CBL and copper concentrations in the medium decreased at approximately the same rate. Apparently, the extracellular CBL was produced throughout the period of cell growth, in the presence and absence of Cu(II), by both the mutant and wild-type cultures and was reinternalized or otherwise utilized by the wild-type cultures when it was bound to copper. CBL produced by the mutant strain facilitated copper uptake by wild-type cells, indicating that the extracellular CBLs produced by the mutant and wild-type organisms are functionally indistinguishable. CBL from the wild-type strain did not promote copper uptake by the mutant. The molecular weight of the CBL was estimated to be 500, and its association constant with copper was 1.4 x 10(16) M-1. CBL exhibited a preference for copper, even in the presence of 20-fold higher concentrations of nickel. External complexation may play a role in normal copper acquisition by M. trichosporium OB3b. The sMMOc phenotype is probably related to the mutant's inability to take up CBL-complexed copper, not to a defective CBL structure.

Phenotypic characterization of copper-resistant mutants of Methylosinus trichosporium OB3b.

Cultures of Methylosinus trichosporium OB3b grown in the presence of very low concentrations of copper synthesize a soluble methane monooxygenase (sMMO) that efficiently catalyzes the oxidation of trichloroethylene and other organic pollutants. Recently, we isolated five M. trichosporium OB3b mutants that express sMMO activity when grown in the presence of elevated copper concentrations (P.A. Phelps, S. K. Agarwal, G. E. Speitel, Jr., and G. Georgiou, Appl. Environ. Microbiol. 58:3701-3708, 1992). Here we show that, in contrast to the results for the wild-type cells, the addition of copper to mutant cultures grown on methane and nitrate as the nitrogen source has no noticeable effect on the growth rate and sMMO expression. In vitro experiments indicated that the copper-resistant phenotype does not arise from an increased stability of sMMO to copper deactivation. Furthermore, the mutant cultures exhibit altered speciation of copper in the extracellular fluid and have substantially decreased levels of cell-associated copper. On the basis of these results, we propose that the mutant phenotype arises from defects in copper uptake and metabolism rather than from changes in sMMO expression or enzyme stability.

Observation of parity nonconservation in møller scattering.

We report a measurement of the parity-violating asymmetry in fixed target electron-electron (Møller) scattering: A(PV)=[-175+/-30(stat)+/-20(syst)] x 10(-9). This first direct observation of parity nonconservation in Møller scattering leads to a measurement of the electron's weak charge at low energy Q(e)(W)=-0.053+/-0.011. This is consistent with the standard model expectation at the current level of precision: sin((2)theta(W)(M(Z))((-)MS)=0.2293+/-0.0024(stat)+/-0.0016(syst)+/-0.0006(theory).