Continuous removal of Cr(VI) from aqueous solution catalysed by palladised biomass of Desulfovibrio vulgaris

Growth-decoupled cells of Desulfovibrio vulgaris NCIMB 8303 can be used to reduce Pd(II) to cell-bound Pd(0) (Bio-Pd(0)), a bioinorganic catalyst capable of reducing hexavalent chromium to less toxic Cr(III), using formate as the electron donor. Magnetic resonance imaging showed that Bio-Pd(0), immobilized in chitosan and agar beads, is distinguishable from the surrounding gel and is evenly dispersed within the immobilization matrix. Agar-immobilized Bio-Pd(0) and 'chemical Pd(0)' were packed into continuous-flow reactors, and challenged with a solution containing 100 microM Cr(VI) (pH 7) at a flow rate of 2.4 ml h(-1). Agar-immobilized chemical Pd(0) columns lost Cr(VI) reducing ability by 160 h, whereas columns containing immobilized Bio-Pd(0) maintained 90% reduction until 680 h, after which reduction efficiency was gradually lost.     

Reduction of Cr(VI) by immobilized cells of Desulfovibrio vulgaris NCIMB 8303 and Microbacterium sp. NCIMB 13776

Hexavalent chromium, a carcinogen and mutagen, can be reduced to Cr(III) by Desulfovibrio vulgaris NCIMB 8303 and Microbacterium sp. NCIMB 13776. This study examined Cr(VI) reduction by immobilized cells of the two strains in a common solution matrix using various entrapment matrices. Chitosan and PVA-borate beads did not retain integrity and supported low or no reduction of Cr(VI) by the cells. A commercial preparation (Lentikats) was stable but also did not support Cr(VI) reduction. K-carrageenan beads were stable in batch suspensions but gel integrity was lost after only 5 h in a flow-through system in the presence of 100 microM Cr(VI). The best immobilization matrices were agar and agarose, where the initial rates of reduction of Cr(VI) (from 500 microM solution) for D. vulgaris NCIMB 8303 and Microbacterium sp. NCIMB 13776 were 127 (agar) and 130 (agarose), and 15 (agar) and 12 (agarose) nmol h(-1) mg dry cell wt(-1), respectively. The higher removal of Cr(VI) by D. vulgaris was also seen in 14-mL packed-bed flow-through columns, where, at a flow rate of 2.4 mL h(-1), the percentage removal of Cr(VI) was approximately 95% and 60% for D. vulgaris and Microbacterium sp., respectively (agar-immobilized cells). The Cr(VI) reducing activities of D. vulgaris and Microbacterium sp. were lost after 159 and 140 h, respectively. Examination of the beads for structural integrity within the columns in situ using magnetic resonance imaging after 24 and 100 h of continuous operation against Cr(VI) (with negligible Cr retained within the columns) showed that agar beads were more stable with time. The most appropriate system for development of a continuous bioprocess is thus the use of D. vulgaris NCIMB 8303 immobilized in an agar gel matrix.     

Reduction of Cr(VI) by "palladized" biomass of Desulfovibrio desulfuricans ATCC 29577

A novel catalytic activity of palladium [Pd(0)]-coated cells of Desulfovibrio desulfuricans ATCC 29577 ["bio-Pd(0)"] is demonstrated. Reduction of 700 microM Cr(VI) occurred within 24 h using formate (25 mM) or hydrogen (1 atm) as the electron donor, under conditions whereby cells lacking bound Pd(0), or palladium metal manufactured via chemical reduction of soluble Pd(II), did not reduce Cr(VI). The biomass-bound Pd(0) also functioned in the continuous removal of 400 microM Cr(VI) from a 1 mM solution under H(2) (flow residence time approximately 5 h), where chemically prepared Pd(0) was ineffective. This demonstrates a new type of active bioinorganic catalysis, whereby the presence of biomass bound to Pd(0) confers a novel catalytic capability not seen with Pd base metal or biomass alone.     

Induction and partial purification of bacteriophages from Desulfovibrio vulgaris (Hildenborough) and Desulfovibrio desulfuricans ATCC 13541.

Bacteriophages were induced from cultures of Desulfovibrio vulgaris NCIMB 8303 and Desulfovibrio desulfuricans ATCC 13541 by UV light. The optimum time of UV exposure was 1 min and the maximum yield of phage was obtained 9-10 h after UV treatment. The two phage preparations were compared by restriction enzyme analysis and Southern blot hybridization. The nucleic acid from both phages was cut by restriction endonucleases specific for double-stranded DNA. The phage DNAs from D. vulgaris and D. desulfuricans showed different restriction enzyme cleavage patterns. No homology was observed between a 25 kb probe from the D. vulgaris phage DNA and the phage DNA from D. desulfuricans. Protein profiles of the phages from both sources were also studied; the D. vulgaris phage contained two major bands corresponding to Mr values of 37 000 and 56 000 while the D. desulfuricans phage contained only one major band, of Mr 38 000.     

Characterization of periplasmic hydrogenase from Desulfovibrio vulgaris Miyazaki K

Periplasmic hydrogenase [hydrogen:ferricytochrome c3 oxidoreductase, EC 1.12.2.1] from Desulfovibrio vulgaris Miyazaki K (MK) was purified to homogeneity. Its chemical and immunological properties were examined and compared with those of other Desulfovibrio hydrogenases. The pure enzyme showed a specific activity of 1,000 mumol H2 evolution min-1 (mg protein)-1. The enzyme had a molecular weight of 50,000 as estimated by gel filtration and consisted of a single polypeptide chain. The absorption spectrum of the enzyme was characteristic of an iron-sulfur protein and the extinction coefficients at 400 and 280 nm were 34 and 104 mM-1. cm-1, respectively. It contained 9.4 mol iron and 6.9 mol of acid-labile sulfide per mol. The amino acid composition of the preparation was very similar to the value reported for D. desulfuricans NRC 49001 hydrogenase. Rabbit antisera were prepared against the enzyme of D. vulgaris MK. Ouchterlony double diffusion and immunotitration tests of crude extracts from several strains of Desulfovibrio revealed that the enzyme from MK cells was immunologically identical with those from D. vulgaris Hildenborough and D. desulfuricans NRC 49001, but different from those from D. vulgaris Miyazaki F (MF) and Miyazaki Y, and D. desulfuricans Essex 6 strains. It is concluded that among Desulfovibrio hydrogenases, those from D. vulgaris MK, D. vulgaris Hildenborough and D. desulfuricans NRC 49001 form one group in terms of both subunit structure and antigenicity.     

Modeling reduction of uranium U(VI) under variable sulfate concentrations by sulfate-reducing bacteria

The kinetics for the reduction of sulfate alone and for concurrent uranium [U(VI)] and sulfate reduction, by mixed and pure cultures of sulfate-reducing bacteria (SRB) at 21 +/- 3 degrees C were studied. The mixed culture contained the SRB Desulfovibrio vulgaris along with a Clostridium sp. determined via 16S ribosomal DNA analysis. The pure culture was Desulfovibrio desulfuricans (ATCC 7757). A zero-order model best fit the data for the reduction of sulfate from 0.1 to 10 mM. A lag time occurred below cell concentrations of 0.1 mg (dry weight) of cells/ml. For the mixed culture, average values for the maximum specific reaction rate, V(max), ranged from 2.4 +/- 0.2 micromol of sulfate/mg (dry weight) of SRB. h(-1)) at 0.25 mM sulfate to 5.0 +/- 1.1 micromol of sulfate/mg (dry weight) of SRB. h(-1) at 10 mM sulfate (average cell concentration, 0.52 mg [dry weight]/ml). For the pure culture, V(max) was 1.6 +/- 0.2 micromol of sulfate/mg (dry weight) of SRB. h(-1) at 1 mM sulfate (0.29 mg [dry weight] of cells/ml). When both electron acceptors were present, sulfate reduction remained zero order for both cultures, while uranium reduction was first order, with rate constants of 0.071 +/- 0.003 mg (dry weight) of cells/ml. min(-1) for the mixed culture and 0.137 +/- 0.016 mg (dry weight) of cells/ml. min(-1) (U(0) = 1 mM) for the D. desulfuricans culture. Both cultures exhibited a faster rate of uranium reduction in the presence of sulfate and no lag time until the onset of U reduction in contrast to U alone. This kinetics information can be used to design an SRB-dominated biotreatment scheme for the removal of U(VI) from an aqueous source.     

Reduction of uranium by Desulfovibrio desulfuricans.

The possibility that sulfate-reducing microorganisms contribute to U(VI) reduction in sedimentary environments was investigated. U(VI) was reduced to U(IV) when washed cells of sulfate-grown Desulfovibrio desulfuricans were suspended in a bicarbonate buffer with lactate or H2 as the electron donor. There was no U(VI) reduction in the absence of an electron donor or when the cells were killed by heat prior to the incubation. The rates of U(VI) reduction were comparable to those in respiratory Fe(III)-reducing microorganisms. Azide or prior exposure of the cells to air did not affect the ability of D. desulfuricans to reduce U(VI). Attempts to grow D. desulfuricans with U(VI) as the electron acceptor were unsuccessful. U(VI) reduction resulted in the extracellular precipitation of the U(IV) mineral uraninite. The presence of sulfate had no effect on the rate of U(VI) reduction. Sulfate and U(VI) were reduced simultaneously. Enzymatic reduction of U(VI) by D. desulfuricans was much faster than nonenzymatic reduction of U(VI) by sulfide, even when cells of D. desulfuricans were added to provide a potential catalytic surface for the nonenzymatic reaction. The results indicate that enzymatic U(VI) reduction by sulfate-reducing microorganisms may be responsible for the accumulation of U(IV) in sulfidogenic environments. Furthermore, since the reduction of U(VI) to U(IV) precipitates uranium from solution, D. desulfuricans might be a useful organism for recovering uranium from contaminated waters and waste streams.     

Reduction of uranium by Desulfovibrio desulfuricans.

The possibility that sulfate-reducing microorganisms contribute to U(VI) reduction in sedimentary environments was investigated. U(VI) was reduced to U(IV) when washed cells of sulfate-grown Desulfovibrio desulfuricans were suspended in a bicarbonate buffer with lactate or H2 as the electron donor. There was no U(VI) reduction in the absence of an electron donor or when the cells were killed by heat prior to the incubation. The rates of U(VI) reduction were comparable to those in respiratory Fe(III)-reducing microorganisms. Azide or prior exposure of the cells to air did not affect the ability of D. desulfuricans to reduce U(VI). Attempts to grow D. desulfuricans with U(VI) as the electron acceptor were unsuccessful. U(VI) reduction resulted in the extracellular precipitation of the U(IV) mineral uraninite. The presence of sulfate had no effect on the rate of U(VI) reduction. Sulfate and U(VI) were reduced simultaneously. Enzymatic reduction of U(VI) by D. desulfuricans was much faster than nonenzymatic reduction of U(VI) by sulfide, even when cells of D. desulfuricans were added to provide a potential catalytic surface for the nonenzymatic reaction. The results indicate that enzymatic U(VI) reduction by sulfate-reducing microorganisms may be responsible for the accumulation of U(IV) in sulfidogenic environments. Furthermore, since the reduction of U(VI) to U(IV) precipitates uranium from solution, D. desulfuricans might be a useful organism for recovering uranium from contaminated waters and waste streams.     

Reduction of Chromate by Desulfovibrio vulgaris and Its c(3) Cytochrome

Washed cell suspensions of Desulfovibrio vulgaris rapidly reduced Cr(VI) to Cr(III) with H(2) as the electron donor. The c(3) cytochrome from this organism functioned as a Cr(VI) reductase. D. vulgaris may have advantages over previously described Cr(VI) reducers for the bioremediation of Cr(VI)-contaminated waters.     

Continuous removal of Cr(VI) from aqueous solution catalysed by palladised biomass of Desulfovibrio vulgaris

Growth-decoupled cells of Desulfovibrio vulgaris NCIMB 8303 can be used to reduce Pd(II) to cell-bound Pd(0) (Bio-Pd⁰), a bioinorganic catalyst capable of reducing hexavalent chromium to less toxic Cr(III), using formate as the electron donor. Magnetic resonance imaging showed that Bio-Pd⁰, immobilized in chitosan and agar beads, is distinguishable from the surrounding gel and is evenly dispersed within the immobilization matrix. Agar-immobilized Bio-Pd⁰ and `chemical Pd⁰' were packed into continuous-flow reactors, and challenged with a solution containing 100 m Cr(VI) (pH 7) at a flow rate of 2.4 ml h^–1. Agar-immobilized chemical Pd⁰ columns lost Cr(VI) reducing ability by 160 h, whereas columns containing immobilized Bio-Pd⁰ maintained 90% reduction until 680 h, after which reduction efficiency was gradually lost.     

Nonaheme cytochrome c, a new physiological electron acceptor for [Ni,Fe] hydrogenase in the sulfate-reducing bacterium Desulfovibrio desulfuricans Essex: primary sequence, molecular parameters, and redox properties

A nonaheme cytochrome c was purified to homogeneity from the soluble and the membrane fractions of the sulfate-reducing bacterium Desulfovibrio desulfuricans Essex. The gene encoding for the protein was cloned and sequenced. The primary structure of the multiheme protein was highly homologous to that of the nonaheme cytochrome c from D. desulfuricans ATCC 27774 and to that of the 16-heme HmcA protein from Desulfovibrio vulgaris Hildenborough. The analysis of the sequence downstream of the gene encoding for the nonaheme cytochrome c from D. desulfuricans Essex revealed an open reading frame encoding for an HmcB homologue. This operon structure indicated the presence of an Hmc complex in D. desulfuricans Essex, with the nonaheme cytochrome c replacing the 16-heme HmcA protein found in D. vulgaris. The molecular and spectroscopic parameters of nonaheme cytochrome c from D. desulfuricans Essex in the oxidized and reduced states were analyzed. Upon reduction, the pI of the protein changed significantly from 8.25 to 5.0 when going from the Fe(III) to the Fe(II) state. Such redox-induced changes in pI have not been reported for cytochromes thus far; most likely they are the result of a conformational rearrangement of the protein structure, which was confirmed by CD spectroscopy. The reactivity of the nonaheme cytochrome c toward [Ni,Fe] hydrogenase was compared with that of the tetraheme cytochrome c(3); both the cytochrome c(3) and the periplasmic [Ni,Fe] hydrogenase originated from D. desulfuricans Essex. The nonaheme protein displayed an affinity and reactivity toward [Ni,Fe] hydrogenase [K(M) = 20.5 +/- 0.9 microM; v(max) = 660 +/- 20 nmol of reduced cytochrome min(-1) (nmol of hydrogenase)(-1)] similar to that of cytochrome c(3) [K(M) = 12.6 +/- 0.7 microM; v(max) = 790 +/- 30 nmol of reduced cytochrome min(-1) (nmol of hydrogenase)(-1)]. This shows that nonaheme cytochrome c is a competent physiological electron acceptor for [Ni,Fe] hydrogenase.     

Reduction of Cr(VI) by Desulfovibrio vulgaris and Microbacterium sp.

Many industrial wastes contain Cr(VI), a carcinogen and mutagen, the toxicity of which can be ameliorated by reduction to Cr(III). Microbacterium sp. NCIMB 13776 andDesulfovibrio vulgaris NCIMB 8303 reduced Cr(VI) to Cr(III) anoxically using 25 mM sodium citrate buffer (pH 7), with 25 mM sodium acetate and 25 mM sodium formate as electron donors at 30 °C, under which conditions the rates of reduction of 500 M sodium chromate were 77 and 6 nmol h^–1 mg dry cell wt for D. vulgaris and Microbacterium sp., respectively, these being increased to 127 and 17 nmol h^–1 mg dry cell wt in the presence of 20 mM MOPS/NaOH buffer.     

Bioreduction and biocrystallization of palladium by Desulfovibrio desulfuricans NCIMB 8307

The reduction of Pd(II) to Pd(0) was accelerated by using the sulfate-reducing bacterium Desulfovibrio desulfuricans NCIMB 8307 at the expense of formate or H(2) as electron donors at pH 2-7. With formate no reduction occurred at pH 2, but with H(2) 50% of the activity was retained at pH 2, with the maximum rate (1.3-1.4 micromol min(-1) mg dry cells(-1)) seen at pH 3-7, which was similar to the rate with formate at neutral pH. Excess nitrate was inhibitory to Pd(II) reduction using formate, but not H(2). Chloride ion was inhibitory as low as 100 mM using formate but with H(2) only ca. 25% inhibition was observed at 500 mM Cl(-) and H(2) was concluded to be the electron donor of choice for the potential remediation of industrial wastes. Deposited Pd was visible on the cells using transmission and scanning electron microscopy and analysis by energy dispersive X-ray microanalysis (EDAX) identified the deposit as Pd, confirmed as Pd(0) by X-ray powder diffraction analysis (XRD). The crystal size of the biodeposited Pd(0) was determined to be only 50% of the size of Pd(0) crystals manufactured chemically from Pd(II) at the expense of H(2) and, unlike the chemically manufactured material, the biocrystal size was independent of the pH. The "biological" Pd(0) functioned as a superior chemical catalyst in a test reaction which liberated hydrogen from hypophosphite. Pd, and also Pt and Rh, could be recovered by resting cell suspensions under H(2) from an industrial processing wastewater, suggesting a possible future application of bioprocessing technology for precious metals.     

Hexavalent chromium reduction in <Emphasis Type="Italic">Desulfovibrio vulgaris</Emphasis> Hildenborough causes transitory inhibition of sulfate reduction and cell growth

Desulfovibrio vulgaris Hildenborough is a well-studied sulfate reducer that can reduce heavy metals and radionuclides [e.g., Cr(VI) and U(VI)]. Cultures grown in a defined medium had a lag period of approximately 30 h when exposed to 0.05 mM Cr(VI). Substrate analyses revealed that although Cr(VI) was reduced within the first 5 h, growth was not observed for an additional 20 h. The growth lag could be explained by a decline in cell viability; however, during this time small amounts of lactate were still utilized without sulfate reduction or acetate formation. Approximately 40 h after Cr exposure (0.05 mM), sulfate reduction occurred concurrently with the accumulation of acetate. Similar amounts of hydrogen were produced by Cr-exposed cells compared to control cells, and lactate was not converted to glycogen during non-growth conditions. D. vulgaris cells treated with a reducing agent and then exposed to Cr(VI) still experienced a growth lag, but the addition of ascorbate at the time of Cr(VI) addition prevented the lag period. In addition, cells grown on pyruvate displayed more tolerance to Cr(VI) compared to lactate-grown cells. These results indicated that D. vulgaris utilized lactate during Cr(VI) exposure without the reduction of sulfate or production of acetate, and that ascorbate and pyruvate could protect D. vulgaris cells from Cr(VI)/Cr(III) toxicity. J.D. Wall and M.W. Fields are both affiliated to the Virtual Institute of Microbial Stress and Survival (). M.E. Clark and S.B. Thieman contributed equally to this work.     

Reduction of Hexavalent Uranium from Organic Complexes by Sulfate- and Iron-Reducing Bacteria

The influence of organic-hexavalent-uranium [U(VI)] complexation on U(VI) reduction by a sulfate-reducing bacterium (Desulfovibrio desulfuricans) and an iron-reducing bacterium (Shewanella alga) was evaluated. Four aliphatic ligands (acetate, malonate, oxalate, and citrate) and an aromatic ligand (tiron [4,5-dihydroxy-1,3-benzene disulfonic acid]) were used to study complexed-uranium bioavailability. The trends in uranium reduction varied with the nature and the amount of U(VI)-organic complex formed and the type of bacteria present. D. desulfuricans rapidly reduced uranium from a monodentate aliphatic (acetate) complex. However, reduction from multidentate aliphatic complexes (malonate, oxalate, and citrate) was slower. A decrease in the amount of organic-U(VI) complex in solution significantly increased the rate of reduction. S. alga reduced uranium more rapidly from multidentate aliphatic complexes than from monodentate aliphatic complexes. The rate of reduction decreased with a decrease in the amount of multidentate complexes present. Uranium from an aromatic (tiron) complex was readily available for reduction by D. desulfuricans, while an insignificant level of U(VI) from the tiron complex was reduced by S. alga. These results indicate that selection of bacteria for rapid uranium reduction will depend on the organic composition of waste streams.     

Microbial reduction of technetium by Escherichia coli and Desulfovibrio desulfuricans: enhancement via the use of high-activity strains and effect of process parameters

Escherichia coli and Desulfovibrio desulfuricans reduce Tc(VII) (TcO(4)(-)) with formate or hydrogen as electron donors. The reaction is catalyzed by the hydrogenase component of the formate hydrogenlyase complex (FHL) of E. coli and is associated with a periplasmic hydrogenase activity in D. desulfuricans. Tc(VII) reduction in E. coli by H(2) and formate was either inhibited or repressed by 10 mM nitrate. By contrast, Tc(VII) reduction catalyzed by D. desulfuricans was less sensitive to nitrate when formate was the electron donor, and unaffected by 10 mM or 100 mM nitrate when H(2) was the electron donor. The optimum pH for Tc(VII) reduction by both organisms was 5.5 and the optimum temperature was 40 degrees C and 20 degrees C for E. coli and D. desulfuricans, respectively. Both strains had an apparent K(m) for Tc(VII) of 0.5 mM, but Tc(VII) was removed from a solution of 300 nM TcO(4)(-) within 30 h by D. desulfuricans at the expense of H(2). The greater bioprocess potential of D. desulfuricans was shown also by the K(s) for formate (>25 mM and 0.5 mM for E. coli and D. desulfuricans, respectively), attributable to the more accessible, periplasmic localization of the enzyme in the latter. The relative rates of Tc(VII) reduction for E. coli and D. desulfuricans (with H(2)) were 12.5 and 800 micromol Tc(VII) reduced/g biomass/h, but the use of an E. coli HycA mutant (which upregulates FHL activities by approx. 50%) had a similarly enhancing effect on the rate of Tc reduction. The more rapid reduction of Tc(VII) by D. desulfuricans compared with the E. coli strains was also shown using cells immobilized in a hollow-fiber reactor, in which the flow residence times sustaining steady-state removal of 80% of the radionuclide were 24.3 h for the wild-type E. coli, 4.25 h for the upregulated mutant, and 1.5 h for D. desulfuricans.     

Factors affecting microbial sulfate reduction by Desulfovibrio desulfuricans in continuous culture: limiting nutrients and sulfide concentration

The effects of sulfate and nitrogen concentrations of the rate and stoichiometry of microbial sulfate reduction were investigated for Desulfovibrio desulfuricans grown on lactate and sulfate in a chemostat at pH 7.0. Maximum specific growth rates (mu(max)), half-saturation coefficients (K(sul)), and cell yield (Y(c/Lac)) of 0.344 +/- 0.007 and 0.352 +/- 0.003 h (-1), 1.8 +/- 0.3 and 1.0 +/- 0.2 mg/L, and 0.020 +/- 0.003 and 0.017 +/- 0.003 g cell/g lactate, respectively, were obtained under sulfate-limiting conditions at 35 degrees C and 43 degrees C. Maintenance energy requirements for D. desulfuricans were significant under sulfate-limiting conditions. The extent of extracellular polymeric substance (EPS) produced was related to the carbon: nitrogen ratio in the medium. EPS production rate increased with decreased nitrogen loading rate. Nitrogen starvation also resulted in decreased cell size of D. desulfuricans. The limiting C : N ratio (w/w) for D. desulfuricans was in the range of 45 : 1 to 120 : 1. Effects of sulfide on microbial sulfate reduction, cell size, and biomass production were also ivestigated at pH 7.0. Fifty percent inhibition of lactate utilization occurred at a total sulfide concentration of approximately 500 mg/L. The cell size of D. desulfuricans decreased with increasing total sulfide concentration. Sulfide inhibition of D. desulfuricans was observed to be a reversible process. (c) 1992 John Wiley & Sons, Inc.     

Uranium reduction by Desulfovibrio desulfuricans strain G20 and a cytochrome c3 mutant

Previous in vitro experiments with Desulfovibrio vulgaris strain Hildenborough demonstrated that extracts containing hydrogenase and cytochrome c3 could reduce uranium(VI) to uranium(IV) with hydrogen as the electron donor. To test the involvement of these proteins in vivo, a cytochrome c3 mutant of D. desulfuricans strain G20 was assayed and found to be able to reduce U(VI) with lactate or pyruvate as the electron donor at rates about one-half of those of the wild type. With electrons from hydrogen, the rate was more severely impaired. Cytochrome c3 appears to be a part of the in vivo electron pathway to U(VI), but additional pathways from organic donors can apparently bypass this protein.     

Towards an integrated system for bio-energy: hydrogen production by <Emphasis Type="Italic">Escherichia coli</Emphasis> and use of palladium-coated waste cells for electricity generation in a fuel cell

Escherichia coli strains MC4100 (parent) and a mutant strain derived from this (IC007) were evaluated for their ability to produce H₂ and organic acids (OAs) via fermentation. Following growth, each strain was coated with Pd(0) via bioreduction of Pd(II). Dried, sintered Pd-biomaterials (‘Bio-Pd’) were tested as anodes in a proton exchange membrane (PEM) fuel cell for their ability to generate electricity from H₂. Both strains produced hydrogen and OAs but ‘palladised’ cells of strain IC007 (Bio-Pd_IC007) produced ~threefold more power as compared to Bio-Pd_MC4100 (56 and 18 mW respectively). The power output used, for comparison, commercial Pd(0) powder and Bio-Pd made from Desulfovibrio desulfuricans, was ~100 mW. The implications of these findings for an integrated energy generating process are discussed.     

Periplasmic oxygen reduction by Desulfovibrio species

Washed cells of Desulfovibrio vulgaris strain Marburg (DSM 2119) reduced oxygen to water with H(2) as electron donor at a mean rate of 253 nmol O(2) min(-1) (mg protein)(-1). After separating the periplasm from the cells, more than 60% of the cytochrome c activity and 90% of the oxygen-reducing activity were found in the periplasmic fraction. Oxygen reduction and the reduction of cytochrome c with H(2) were inhibited by CuCl(2). After further separation of the periplasm by ultrafiltration (exclusion sizes 30, 50, and 100 kDa), oxygen reduction with H(2) occurred with the retentates only. Ascorbate plus tetramethyl-p-phenylenediamine (TMPD), however, were also oxidized by the filtrates. The stoichiometry of 1 mol O(2) reduced per 2 mol ascorbate oxidized indicated the formation of water. Our experiments present evidence that in D. vulgaris periplasmic hydrogenase and cytochrome c play a major role in oxygen reduction. Preliminary studies with other Desulfovibrio species indicated a similar function of periplasmic c-type cytochromes in D. desulfuricans CSN and D. termitidis KH1.