Acetate Threshold Concentrations Suggest Varying Energy Requirements during Anaerobic Respiration by Anaeromyxobacter dehalogenans

Acetate threshold concentrations were determined under chlororespiring and Fe(III)-reducing conditions for Anaeromyxobacter dehalogenans strain 2CP-C. The acetate threshold concentrations measured were 69 ± 4, 19 ± 8, and <1 nM for chlororespiration, amorphous Fe(III) reduction, and Fe(III) citrate reduction, respectively. Residual ΔG values of −75.4 kJ/mol of electrons for chlororespiration and −41.5 kJ/mol of electrons for amorphous Fe(III) reduction were calculated at the acetate threshold concentration. By comparing threshold concentrations for different metabolisms in a single organism, this study provides insight into the metabolic use of energy under different growth conditions.     

Geobacter lovleyi sp. nov. strain SZ, a novel metal-reducing and tetrachloroethene-dechlorinating bacterium

A bacterial isolate, designated strain SZ, was obtained from noncontaminated creek sediment microcosms based on its ability to derive energy from acetate oxidation coupled to tetrachloroethene (PCE)-to-cis-1,2-dichloroethene (cis-DCE) dechlorination (i.e., chlororespiration). Hydrogen and pyruvate served as alternate electron donors for strain SZ, and the range of electron acceptors included (reduced products are given in brackets) PCE and trichloroethene [cis-DCE], nitrate [ammonium], fumarate [succinate], Fe(III) [Fe(II)], malate [succinate], Mn(IV) [Mn(II)], U(VI) [U(IV)], and elemental sulfur [sulfide]. PCE and soluble Fe(III) (as ferric citrate) were reduced at rates of 56.5 and 164 nmol min(-1) mg of protein(-1), respectively, with acetate as the electron donor. Alternate electron acceptors, such as U(VI) and nitrate, did not inhibit PCE dechlorination and were consumed concomitantly. With PCE, Fe(III) (as ferric citrate), and nitrate as electron acceptors, H(2) was consumed to threshold concentrations of 0.08 +/- 0.03 nM, 0.16 +/- 0.07 nM, and 0.5 +/- 0.06 nM, respectively, and acetate was consumed to 3.0 +/- 2.1 nM, 1.2 +/- 0.5 nM, and 3.6 +/- 0.25 nM, respectively. Apparently, electron acceptor-specific acetate consumption threshold concentrations exist, suggesting that similar to the hydrogen threshold model, the measurement of acetate threshold concentrations offers an additional diagnostic tool to delineate terminal electron-accepting processes in anaerobic subsurface environments. Genetic and phenotypic analyses classify strain SZ as the type strain of the new species, Geobacter lovleyi sp. nov., with Geobacter (formerly Trichlorobacter) thiogenes as the closest relative. Furthermore, the analysis of 16S rRNA gene sequences recovered from PCE-dechlorinating consortia and chloroethene-contaminated subsurface environments suggests that Geobacter lovleyi belongs to a distinct, dechlorinating clade within the metal-reducing Geobacter group. Substrate versatility, consumption of electron donors to low threshold concentrations, and simultaneous reduction of electron acceptors suggest that strain SZ-type organisms have desirable characteristics for bioremediation applications.     

Characterization of Fe(III) reduction by chlororespiring Anaeromyxobacter dehalogenans

Anaeromyxobacter dehalogenans strain 2CP-C has been shown to grow by coupling the oxidation of acetate to the reduction of ortho-substituted halophenols, oxygen, nitrate, nitrite, or fumarate. In this study, strain 2CP-C was also found to grow by coupling Fe(III) reduction to the oxidation of acetate, making it one of the few isolates capable of growth by both metal reduction and chlororespiration. Doubling times for growth of 9.2 and 10.2 h were determined for Fe(III) and 2-chlorophenol reduction, respectively. These were determined by using the rate of [(14)C]acetate uptake into biomass. Fe(III) compounds used by strain 2CP-C include ferric citrate, ferric pyrophosphate, and amorphous ferric oxyhydroxide. The addition of the humic acid analog anthraquinone 2,6-disulfonate (AQDS) increased the reduction rate of amorphous ferric iron oxide, suggesting AQDS was used as an electron shuttle by strain 2CP-C. The addition of chloramphenicol to fumarate-grown cells did not inhibit Fe(III) reduction, indicating that the latter activity is constitutive. In contrast, the addition of chloramphenicol inhibited dechlorination activity, indicating that chlororespiration is inducible. The presence of insoluble Fe(III) oxyhydroxide did not significantly affect dechlorination, whereas the presence of soluble ferric pyrophosphate inhibited dechlorination. With its ability to respire chlorinated organic compounds and metals such as Fe(III), strain 2CP-C is a promising model organism for the study of the interaction of these potentially competing processes in contaminated environments.     

Characterization of Fe(III) Reduction by Chlororespiring Anaeromxyobacter dehalogenans

Anaeromyxobacter dehalogenans strain 2CP-C has been shown to grow by coupling the oxidation of acetate to the reduction of ortho-substituted halophenols, oxygen, nitrate, nitrite, or fumarate. In this study, strain 2CP-C was also found to grow by coupling Fe(III) reduction to the oxidation of acetate, making it one of the few isolates capable of growth by both metal reduction and chlororespiration. Doubling times for growth of 9.2 and 10.2 h were determined for Fe(III) and 2-chlorophenol reduction, respectively. These were determined by using the rate of [¹⁴C]acetate uptake into biomass. Fe(III) compounds used by strain 2CP-C include ferric citrate, ferric pyrophosphate, and amorphous ferric oxyhydroxide. The addition of the humic acid analog anthraquinone 2,6-disulfonate (AQDS) increased the reduction rate of amorphous ferric iron oxide, suggesting AQDS was used as an electron shuttle by strain 2CP-C. The addition of chloramphenicol to fumarate-grown cells did not inhibit Fe(III) reduction, indicating that the latter activity is constitutive. In contrast, the addition of chloramphenicol inhibited dechlorination activity, indicating that chlororespiration is inducible. The presence of insoluble Fe(III) oxyhydroxide did not significantly affect dechlorination, whereas the presence of soluble ferric pyrophosphate inhibited dechlorination. With its ability to respire chlorinated organic compounds and metals such as Fe(III), strain 2CP-C is a promising model organism for the study of the interaction of these potentially competing processes in contaminated environments.     

Low and high acetate amendments are equally as effective at promoting complete dechlorination of trichloroethylene (TCE)

Experiments with trichloroethylene-contaminated aquifer material demonstrated that TCE, cis-DCE, and VC were completely degraded with concurrent Fe(III) or Fe(III) and sulfate reduction when acetate was amended at stoichiometric concentration; competing TEAPs did not inhibit ethene production. Adding 10× more acetate did not increase the rate or extent of TCE reduction, but only increased methane production. Enrichment cultures demonstrated that ~90 μM TCE or ~22 μM VC was degraded primarily to ethene within 20 days with concurrent Fe(III) or Fe(III) + sulfate reduction. The dechlorination rates were comparable between the low and high acetate concentrations (0.36 vs 0.34 day^?1, respectively), with a slightly slower rate in the 10× acetate amended incubations. Methane accumulated to 13.5 (±0.5) μmol/tube in the TCE-degrading incubations with 10× acetate, and only 1.4 (±0.1) μmol/tube with low acetate concentration. Methane accumulated to 16 (±1.5) μmol/tube in VC-degrading enrichment with 10× acetate and 2 (±0.1) μmol/tube with stoichiometric acetate. The estimated fraction of electrons distributed to methanogenesis increased substantially when excessive acetate was added. Quantitative PCR analysis indicated that 10× acetate did not enhance Dehalococcoides biomass but rather increased the methanogen abundance by nearly one order of magnitude compared to that with stoichiometric acetate. The data suggest that adding low levels of substrate may be equally if not more effective as high concentrations, without producing excessive methane. This has implications for field remediation efforts, in that adding excess electron donor may not benefit the reactions of interest, which in turn will increase treatment costs without direct benefit to the stakeholders.     

Availability of Ferric Iron for Microbial Reduction in Bottom Sediments of the Freshwater Tidal Potomac River

The distribution of Fe(III), its availability for microbial reduction, and factors controlling Fe(III) availability were investigated in sediments from a freshwater site in the Potomac River Estuary. Fe(III) reduction in sediments incubated under anaerobic conditions and depth profiles of oxalate-extractable Fe(III) indicated that Fe(III) reduction was limited to depths of 4 cm or less, with the most intense Fe(III) reduction in the top 1 cm. In incubations of the upper 4 cm of the sediments, Fe(III) reduction was as important as methane production as a pathway for anaerobic electron flow because of the high rates of Fe(III) reduction in the 0- to 0.5-cm interval. Most of the oxalate-extractable Fe(III) in the sediments was not reduced and persisted to a depth of at least 20 cm. The incomplete reduction was not the result of a lack of suitable electron donors. The oxalate-extractable Fe(III) that was preserved in the sediments was considered to be in a form other than amorphous Fe(III) oxyhydroxide, since synthetic amorphous Fe(III) oxyhydroxide, amorphous Fe(III) oxyhydroxide adsorbed onto clay, and amorphous Fe(III) oxyhydroxide saturated with adsorbed phosphate or fulvic acids were all readily reduced. Fe₃O₄ and the mixed Fe(III)-Fe(II) compound(s) that were produced during the reduction of amorphous Fe(III) oxyhydroxide in an enrichment culture were oxalate extractable but were not reduced, suggesting that mixed Fe(III)-Fe(II) compounds might account for the persistence of oxalate-extractable Fe(III) in the sediments. The availability of microbially reducible Fe(III) in surficial sediments demonstrates that microbial Fe(III) reduction can be important to organic matter decomposition and iron geochemistry. However, the overall extent of microbial Fe(III) reduction is governed by the inability of microorganisms to reduce most of the Fe(III) in the sediment.     

Breakdown kinetics of the tri-chromium(III) oxo acetate cluster ([Cr(3)O(OAc)(6)]+) with some ligands of biological interest

Kinetics for the breakdown of the trinuclear chromium acetate cluster, [Cr(3)O(OAc)(6)](+), with a series of monoprotic and diprotic ligands in weakly acidic aqueous media (pH approximately 4 or approximately 5) have been investigated spectrophotometrically at 40-60 degrees C. The results point to an ion-pair equilibrium as the first step followed by associative interchange mechanism forming the mononuclear product of the reaction. Pseudo-first-order rates were determined from absorbance data and associated activation parameters were calculated using the Eyring equation. Enthalpy and entropy terms of the reactions (e.g., histidine, DeltaH(double dagger) = 75 +/- 15 kJ mol(-1), DeltaS(double dagger) = -130 +/- 25 J K(-1) mol(-1); lactic acid, DeltaH(double dagger) = 66 +/- 13 kJ mol(-1), DeltaS(double dagger) = -155 +/- 30 J K(-1) mol(-1); glycine, DeltaH(double dagger) = 31 +/- 6 kJ mol(-1), DeltaS(double dagger) = -225 +/- 45 J K(-1) mol(-1)) are consistent with an associative interchange (I(a)) mechanism, and produce a linear isokinetic plot (slope = 50 degrees C). Rates and activation parameters are comparable to those of substitution reactions of the chromium(III) hexaaqua cation. Other ligands studied included malonic acid and the amino acid, aspartic acid. Observed rates are faster than water exchange rates, but typically slower than anion substitution rates, and indicate that trinuclear chromium(III) clusters are expected to be kinetically stable in neutral to slightly acidic conditions.     

Free radical-induced redox chemistry of platinum-containing anti-cancer drugs

Arrhenius parameters for the reactions of oxidizing hydroxyl radicals and reducing hydrated electrons with cisplatin, transplatin and carboplatin in aqueous solution have been determined using pulsed electron radiolysis and absorption spectroscopy techniques. Under physiological pH and chloride concentration conditions, hydroxyl radical reaction rate constants of (9.99 +/- 0.20) x 10(9), (8.38 +/- 0.55) x 10(9), and (6.03 +/- 0.08) x 10(9) M(-1) s(-1) at 24.0, 20.7 and 24.0 degrees C, respectively, with corresponding activation energies of 12.79 +/- 0.57, 13.88 +/- 1.14, and 14.35 +/- 0.56 kJ mol(-1) for these three reactions, were determined. These oxidations of cisplatin and transplatin to form a Pt(III) transient are significantly faster than reported previously at room temperature. The lower rate constant for carboplatin is consistent with hydroxyl radical reaction partitioning between reaction at the platinum center and the cyclobutanedicarboxylate ligand. The equivalent reductive hydrated electron reaction rate constants measured were (1.99 +/- 0.04) x 10(10) (24.0 degrees C), (1.77 +/- 0.08) x 10(10) (22.0 degrees C), and (8.92 +/- 0.06) x 10(9) M(-1) s(-1) (24.0 degrees C), with corresponding activation energies of 15.75 +/- 1.00, 19.74 +/- 1.82, and 19.99 +/- 0.34 kJ mol(-1). Again, the values determined for cisplatin and transplatin are faster than reported; however, all three values are consistent with direct reduction of the platinum center to form a Pt(I) moiety.     

Effect of nutrient limitation on product formation during continuous fermentation of xylose with <Emphasis Type="Italic">Thermoanaerobacter ethanolicus</Emphasis> JW200 Fe(7)

Thermoanaerobacter ethanolicus JW200 Fe(7) was grown in continuous culture, using xylose as the primary carbon source, with progressively lower concentrations of supplementary yeast extract. This enabled the comparison of metabolic flux to fermentation end-products under carbon-limited and carbon-sufficient (yeast extract-limited) conditions and the determination of process data under fully mass-balanced conditions. Under carbon-limitation, the specific ethanol-formation rate was described by q (p)=40.34 micro +3.74, the specific rate of substrate utilisation for maintenance was 0.31+/-0.02 g x g(-1) x h(-1) and the maximum cell yield on xylose, corrected for maintenance requirements, was 0.15+/-0.04 g x g(-1). Based on the product profiles, these corresponded to a maintenance coefficient of m(ATP)=4.1+/-0.5 mmol x g(-1) x h(-1) and a maximum cell yield of = 14.7+/-0.8 x g x mol(-1). Limitation by a component in yeast extract resulted in incomplete xylose utilisation, increased catabolic flux rates (primarily resulting in increased lactate production, due to limitations in the flux through the phosphoroclastic reaction), a reduction in cell yield = 10.0+/-1.0 g x mol(-1) and an increase in maintenance energy requirements of m(ATP)=7.95+/-0.7 mmol x g(-1). The latter was also reflected in a shift from ethanol to acetate production at lower growth rates. An analysis of ethanol and acetate tolerance indicated that any high-intensity process employing this strain would require a bioreactor design which incorporated continuous ethanol stripping.     

Ferric iron reduction-linked growth yields of Shewanella putrefaciens MR-1

The anaerobic reduction of ferric citrate by Shewanella putrefaciens MR-1 cells was inhibited markedly by p -chloromercuriphenylsulphonate, moderately by potassium cyanide, and to a small extent by 2-heptyl-4-hydroxyquinolone- N -oxide. Iron reduction was accompanied by increases in total cellular protein, with values of 0.33-7.54 g cell protein produced per mol Fe(III) reduced. The growth yields were dependent upon the growth conditions of the inoculum and the initial concentration of Fe(III) citrate in the medium. Specifically, maximum growth yields were obtained when the inoculum was pregrown anaerobically and when the initial Fe(III) citrate concentrations were 5–10 mmol l^-1. Lower growth yields were obtained with initial Fe(III) citrate concentrations of 20–30 mmol l^-1, suggesting that cell growth was partially inhibited by higher concentrations of Fe(III) or Fe(II). Maximal growth yields were also observed early (6–24 h), after which continued increases in cell protein were minimal.     

Competitive Mechanisms for Inhibition of Sulfate Reduction and Methane Production in the Zone of Ferric Iron Reduction in Sediments

Mechanisms for inhibition of sulfate reduction and methane production in the zone of Fe(III) reduction in sediments were investigated. Addition of amorphic iron(III) oxyhydroxide to sediments in which sulfate reduction was the predominant terminal electron-accepting process inhibited sulfate reduction 86 to 100%. The decrease in electron flow to sulfate reduction was accompanied by a corresponding increase in electron flow to Fe(III) reduction. In a similar manner, Fe(III) additions also inhibited methane production in sulfate-depleted sediments. The inhibition of sulfate reduction and methane production was the result of substrate limitation, because the sediments retained the potential for sulfate reduction and methane production in the presence of excess hydrogen and acetate. Sediments in which Fe(III) reduction was the predominant terminal electron-accepting process had much lower concentrations of hydrogen and acetate than sediments in which sulfate reduction or methane production was the predominant terminal process. The low concentrations of hydrogen and acetate in the Fe(III)-reducing sediments were the result of metabolism by Fe(III)-reducing organisms of hydrogen and acetate at concentrations lower than sulfate reducers or methanogens could metabolize them. The results indicate that when Fe(III) is in a form that Fe(III)-reducing organisms can readily reduce, Fe(III)-reducing organisms can inhibit sulfate reduction and methane production by outcompeting sulfate reducers and methanogens for electron donors.     

Biological reduction of nitric oxide in aqueous Fe(II)EDTA solutions

The reduction of nitric oxide (NO) in aqueous solutions of Fe(II)EDTA is one of the core processes in BioDeNOx, an integrated physicochemical and biological technique for NO(x)() removal from industrial flue gases. NO reduction in aqueous solutions of Fe(II)EDTA (20-25 mM, pH 7.2 +/- 0.2) was investigated in batch experiments at 55 degrees C. Reduction of NO to N(2) was found to be biologically catalyzed with nitrous oxide (N(2)O) as an intermediate. Various sludges from full-scale denitrifying and anaerobic reactors were capable to catalyze NO reduction under thermophilic conditions. The NO reduction rate was not affected by the presence of ethanol or acetate. EDTA-chelated Fe(II) was found to be a suitable electron donor for the biological reduction of nitric oxide to N(2), with the concomitant formation of Fe(III)EDTA. In the presence of ethanol, EDTA-chelated Fe(III) was reduced to Fe(II)EDTA. This study strongly indicates that redox cycling of FeEDTA plays an important role in the biological denitrification process within the BioDeNOx concept.     

A Hydrogen-Oxidizing, Fe(III)-Reducing Microorganism from the Great Bay Estuary, New Hampshire

A dissimilatory Fe(III)- and Mn(IV)-reducing bacterium was isolated from bottom sediments of the Great Bay estuary, New Hampshire. The isolate was a facultatively anaerobic gram-negative rod which did not appear to fit into any previously described genus. It was temporarily designated strain BrY. BrY grew anaerobically in a defined medium with hydrogen or lactate as the electron donor and Fe(III) as the electron acceptor. BrY required citrate, fumarate, or malate as a carbon source for growth on H₂ and Fe(III). With Fe(III) as the sole electron acceptor, BrY metabolized hydrogen to a minimum threshold at least 60-fold lower than the threshold reported for pure cultures of sulfate reducers. This finding supports the hypothesis that when Fe(III) is available, Fe(III) reducers can outcompete sulfate reducers for electron donors. Lactate was incompletely oxidized to acetate and carbon dioxide with Fe(III) as the electron acceptor. Lactate oxidation was also coupled to the reduction of Mn(IV), U(VI), fumarate, thiosulfate, or trimethylamine n-oxide under anaerobic conditions. BrY provides a model for how enzymatic metal reduction by respiratory metal-reducing microorganisms has the potential to contribute to the mobilization of iron and trace metals and to the immobilization of uranium in sediments of Great Bay Estuary.     

Energy sources for chemolithotrophs in an arsenic- and iron-rich shallow-sea hydrothermal system

The hydrothermally influenced sediments of Tutum Bay, Ambitle Island, Papua New Guinea, are ideal for investigating the chemolithotrophic activities of micro-organisms involved in arsenic cycling because hydrothermal vents there expel fluids with arsenite (As(III)) concentrations as high as 950 μg L(-1) . These hot (99 °C), slightly acidic (pH ~6), chemically reduced, shallow-sea vent fluids mix with colder, oxidized seawater to create steep gradients in temperature, pH, and concentrations of As, N, Fe, and S redox species. Near the vents, iron oxyhydroxides precipitate with up to 6.2 wt% arsenate (As(V)). Here, chemical analyses of sediment porewaters from 10 sites along a 300-m transect were combined with standard Gibbs energies to evaluate the energy yields (-ΔG(r)) from 19 potential chemolithotrophic metabolisms, including As(V) reduction, As(III) oxidation, Fe(III) reduction, and Fe(II) oxidation reactions. The 19 reactions yielded 2-94 kJ mol(-1) e(-) , with aerobic oxidation of sulphide and arsenite the two most exergonic reactions. Although anaerobic As(V) reduction and Fe(III) reduction were among the least exergonic reactions investigated, they are still potential net metabolisms. Gibbs energies of the arsenic redox reactions generally correlate linearly with pH, increasing with increasing pH for As(III) oxidation and decreasing with increasing pH for As(V) reduction. The calculated exergonic energy yields suggest that micro-organisms could exploit diverse energy sources in Tutum Bay, and examples of micro-organisms known to use these chemolithotrophic metabolic strategies are discussed. Energy modeling of redox reactions can help target sampling sites for future microbial collection and cultivation studies.     

Performance characteristics of a two-stage dark fermentative system producing hydrogen and methane continuously

The performance of a mesophilic two-stage system generating hydrogen and methane continuously from sucrose (10-30 g/L) was investigated. A hydrogen-generating CSTR followed by an upflow anaerobic filter were both inoculated with anaerobically digested sewage sludge, and ORP, pH, gas output, %H(2), %CH(4) and %CO(2) monitored. pH was controlled with NaOH, KOH or Ca(OH)(2). Using NaOH as alkali with 10 g/L sucrose, yields of 1.62 +/- 0.2 mol H(2)/mol hexose added and 323 mL CH(4)/gCOD added to the hydrogen and methane reactors respectively were achieved. The overall chemical oxygen demand (COD) reduction was 92.6% with 0.90 +/- 0.1 g/L sodium and 316 +/- 40 mg/L residual acetate in the methane reactor. Operation at 20 g/L sucrose and NaOH as alkali led to impaired volatile fatty acid (VFA) degradation in the methane reactor with 2.23 +/- 0.2 g/L sodium, 1,885 mg/L residual acetate, a hydrogen yield of 1.47 +/- 0.1 mol/mol hexose added, a methane yield of 294 mL/gCOD added and an overall COD reduction of 83%. Using Ca(OH)(2) as alkali with 20 g/L sucrose gave a hydrogen yield of 1.29 +/- 0.3 mol/mol hexose added, a methane yield of 337 mL/gCOD added and improved the overall COD reduction to 91% with residual acetate concentrations of 522 +/- 87 mg/L. Operation at 30 g/L sucrose with Ca(OH)(2) gave poorer overall COD reduction (68%), a hydrogen yield of 1.47 +/- 0.2 mol/mol hexose added, a methane yield of 138 mL/gCOD added and residual acetate 7,343 +/- 715 mg/L. It was shown that sodium toxicity and overloading are important issues for successful anaerobic digestion of effluent from biohydrogen reactors in high rate systems.     

Reduction of Fe(III) oxide by methanogens in the presence and absence of extracellular quinones

Five methanogens (Methanosarcina barkeri MS, Methanosphaera cuniculi 1R7, Methanobacterium palustre F, Methanococcus voltaei A3 and Methanolobus vulcani PL-12/M) were investigated for their ability to reduce Fe(III) oxide and the soluble quinone anthraquinone-2,6-disulphonate (AQDS). Two species (M. barkeri and M. voltaei) reduced significant amounts of Fe(III) oxide using hydrogen as the electron donor, and 0.1 mM AQDS greatly accelerated Fe(III) reduction by these organisms. Although Fe(III) appeared to inhibit growth and methanogenesis of some strains, hydrogen partial pressures under donor-limited conditions were much lower (<0.5 Pa) in the presence of Fe(III) than in normal media (1-10 Pa) for all species except for M. vulcani. These results demonstrate that electrons were transferred to Fe(III) by hydrogen-utilizing methanogens even when growth and methanogenesis were inhibited. All species except the obligate methylotroph M. vulcani were able to reduce AQDS when their growth substrates were present as electron donors, and rates were highest when organisms used hydrogen as the electron donor. Purified soil humic acids could also be reduced by the AQDS-reducing methanogens. The ability of methanogens to interact with extracellular quinones, humic acids and Fe(III) oxides raises the possibility that this functional group of organ-isms contributes to Fe(III) and humic acid reduction under certain conditions in the environment and provides an alternative explanation for the inhibition of methanogenesis in some Fe(III)-containing ecosystems.     

Cooperativity and intermediates in the equilibrium reactions of Fe(II,III) with ethanethiolate in N-methylformamide solution

The reaction of FeCl(2) or FeCl(3) with sodium ethanethiolate (SEt) in N-methylformamide (NMF) has been reevaluated to rectify a previous Fe(II) oxidation artifact. On titrating Fe(II) with EtS(-) concentrations up to 12 mol Eq, new features in the UV/vis spectrum (epsilon(344)=(3.1+/-0.2)x10(3) M(-1) cm(-1); epsilon(486)=(4.5+/-0.1)x10(2) M(-1) cm(-1)) indicated that the first observable step was the formation of a single complex different from the known tetrahedral tetrathiolate, [Fe(SEt)(4)](2-) . As the EtS(-) concentration increased past 12.5 mol Eq the UV/vis spectrum gradually transformed to that of [Fe(SEt)(4)](2-) (lambda(max)=314 nm). A Hill-formalism fit to the titration data of the initially formed complex indicated cooperative ligation by three ethanethiolate ions, with K(coop)=(1.7+/-0.1)x10(3) M(-3) and Hill "n"=2.4+/-0.1 (r=0.997). The 3:1 EtS(-)-Fe(II) complex is proposed to be [Fe(2)(SEt)(6)](2-). Titration of Fe(III) with EtS(-) showed direct cooperative formation of [Fe(SEt)(4)](-) [epsilon(340)=(3.4+/-0.5)x10(3) M(-1) cm(-1)] with a Hill-formalism K(coop)=(4.3+/-0.1)x10(2) M(-4) and a Hill coefficient "n"=3.7+/-0.2 (r=0.996). Further ligation past [Fe(SEt)(4)](-) was observed at EtS(-) concentrations above 35 mol Eq. The Fe(III) Hill constants are at variance with our previous report. However, the UV/vis spectrum of Fe(III) in NMF solution was found to change systematically over time, consistent with a slow progressive deprotonation of [Fe(nmf)](3+). The observed time-to-time differences in the equilibrium chemistry of Fe(III) with ethanethiolate in NMF thus reflect variation in the microscopic solution composition of FeCl(3) in alkaline NMF solvent. These results are related to the chemistry of nitrogenase FeMo cofactor in alkaline NMF solution.     

The enthalpy change for the reduction of nicotinamide–adenine dinucleotide

The heat of the reaction NAD(+)+propan-2-ol=NADH+acetone+H(+) was determined to be 42.5+/-0.6kJ/mol (10.17+/-0.15kcal/mol) from equilibrium measurements at 9-42 degrees C catalysed by yeast alcohol dehydrogenase. With the aid of thermochemical data for acetone and propan-2-ol the values of DeltaH=-29.2kJ/mol (-6.99kcal/mol) and DeltaG(0)=22.1kJ/mol (5.28kcal/mol) are derived for the reduction of NAD (NAD(+)+H(2)=NADH+H(+)). These values are consistent with analogous but less accurate data for the ethanol-acetaldehyde reaction. Thermodynamic data for the reduction of NAD and NADP are summarized.     

Influence of Biogenic Fe(II) on Bacterial Crystalline Fe(III) Oxide Reduction

Bacterial crystalline Fe(III) oxide reduction has the potential to significantly influence the biogeochemistry of anaerobic sedimentary environments where crystalline Fe(III) oxides are abundant relative to poorly crystalline (amorphous) phases. A review of published data on solid-phase Fe(III) abundance and speciation indicates that crystalline Fe(III) oxides are frequently 2- to S 10-fold more abundant than amorphous Fe(III) oxides in shallow subsurface sediments not yet subjected to microbial Fe(III) oxide reduction activity. Incubation experiments with coastal plain aquifer sediments demonstrated that crystalline Fe(III) oxide reduction can contribute substantially to Fe(II) production in the presence of added electron donors and nutrients. Controls on crystalline Fe(III) oxide reduction are therefore an important consideration in relation to the biogeochemical impacts of bacterial Fe(III) oxide reduction in subsurface environments. In this paper, the influence of biogenic Fe(II) on bacterial reduction of crystalline Fe(III) oxides is reviewed and analyzed in light of new experiments conducted with the acetate-oxidizing, Fe(III)-reducing bacterium (FeRB) Geobacter metallireducens . Previous experiments with Shewanella algae strain BrY indicated that adsorption and/or surface precipitation of Fe(II) on Fe(III) oxide and FeRB cell surfaces is primarily responsible for cessation of goethite ( f -FeOOH) reduction activity after only a relatively small fraction (generally < 10%) of the oxide is reduced. Similar conclusions are drawn from analogous studies with G. metallireducens . Although accumulation of aqueous Fe(II) has the potential to impose thermodynamic constraints on the extent of crystalline Fe(III) oxide reduction, our data on bacterial goethite reduction suggest that this phenomenon cannot universally explain the low microbial reducibility of this mineral. Experiments examining the influence of exogenous Fe(II) (20 mM FeCl 2 ) on soluble Fe(III)-citrate reduction by G. metallireducens and S. algae showed that high concentrations of Fe(II) did not inhibit Fe(III)-citrate reduction by freshly grown cells, which indicates that surface-bound Fe(II) does not inhibit Fe(III) reduction through a classical end-product enzyme inhibition mechanism. However, prolonged exposure of G. metallireducens and S. algae cells to high concentrations of soluble Fe(II) did cause inhibition of soluble Fe(III) reduction. These findings, together with recent documentation of the formation of Fe(II) surface precipitates on FeRB in Fe(III)-citrate medium, provide further evidence for the impact of Fe(II) sorption by FeRB on enzymatic Fe(III) reduction. Two different, but not mutually exclusive, mechanisms whereby accumulation of Fe(II) coatings on Fe(III) oxide and FeRB surfaces may lead to inhibition of enzymatic Fe(III) oxide reduction activity (in the absence of soluble electron shuttles and/or Fe(III) chelators) are identified and discussed in relation to recent experimental work and theoretical considerations.     

Growth of Geobacter sulfurreducens under nutrient-limiting conditions in continuous culture

A system for growing Geobacter sulfurreducens under anaerobic conditions in chemostats was developed in order to study the physiology of this organism under conditions that might more closely approximate those found in the subsurface than batch cultures. Geobacter sulfurreducens could be cultured under acetate-limiting conditions with fumarate or Fe(III)-citrate as the electron acceptor at growth rates between 0.04 and 0.09 h(-1). The molar growth yield was threefold higher with fumarate as the electron acceptor than with Fe(III), despite the lower mid-point potential of the fumarate/succinate redox couple. When growth was limited by availability of fumarate, high steady-state concentrations were detected, suggesting that fumarate is unlikely to be an important electron acceptor in sedimentary environments. The half-saturation constant, Ks, for acetate in Fe(III)-grown cultures (10 microM) suggested that the growth of Geobacter species is likely to be acetate limited in most subsurface sediments, but that when millimolar quantities of acetate are added to the subsurface in order to promote the growth of Geobacter for bioremediation applications, this should be enough to overcome any acetate limitations. When the availability of electron acceptors, rather than acetate, limited growth, G. sulfurreducens was less efficient in incorporating acetate into biomass but had higher respiration rates, a desirable physiological characteristic when adding acetate to stimulate the activity of Geobacter species during in situ uranium bioremediation. These results demonstrate that the ability to study the growth of G. sulfurreducens under steady-state conditions can provide insights into its physiological characteristics that have relevance for its activity in a diversity of sedimentary environments.