Effect of pH on Anaerobic Mild Steel Corrosion by Methanogenic Bacteria

Methanogens can use H₂ produced by cathodic depolarization-mediated oxidation of elemental iron to produce methane. Thermodynamic consideration of the cathodic depolarization mechanism predicts more oxidation of Fe⁰ at lower pH. Methanogenic responses to pH by Methanococcus deltae, Methanococcus thermolithotrophicus, and Methanosarcina barkeri were examined. When grown on H₂-CO₂, these bacteria had pH optima from 6.2 to 7.0, but when all H₂ was supplied from Fe⁰, methanogenic pH optima were lower, 5.4 to 6.5. Corrosion was monitored with and without cultures and at various pHs; more corrosion occurred when cultures were present, biologically induced corrosion was greatest at the pH optima for methanogenesis from Fe⁰, and corrosion without cultures increased with a drop in pH.     

Methanococcus thermolithotrophicus,a novel thermophilic lithotrophic methanogen

An autotrophic thermophilic motile coccoid methanogen was isolated from geothermally heated sea sediments close to Naples, Italy. Growth occurs on H₂/CO₂ and on formate between 30 and 70°C with an optimum at 65°C. The optimal doubling time is only 55 min. The NaCl-concentration ranges from 1.3% to 8.3% with an optimum around 4%. By its G+C-content of 31.3 mol%, its subunit envelope, and by DNA-RNA hybridization the new isolate is clearly defined to be a member of the genusMethanococcus. We name itMethanococcus thermolithotrophicus.Abbreviations G+C Guanine + Cytosine - SDS Sodium dodecylsulfate (Sodium lauryl sulfate)     

Energetics of the growth of Methanococcus thermolithotrophicus

Methanococcus thermolithotrophicus was grown in a mineral salts medium at 65° C in a fermenter gassed with H₂ and CO₂, which were the sole carbon and energy sources. Evolution of growth parameters during batch culture experiments showed the existence of an uncoupling phenomenon. The growth was then studied using a continuous technique and steady states for various gas flow rates were obtained. Y _{CH 4}and the maintenance coefficient varied with the gas input. The maximum Y _{CH 4} determined for Methanococcus thermolithotrophicus was 3.33 g·mol^-1 CH₄. An excess of energy and carbon sources induced uncoupling of growth.     

Thermococcus alcaliphilus sp. nov., a new hyperthermophilic archaeum growing on polysulfide at alkaline pH

A novel coccoid-shaped, hyperthermophilic, heterotrophic member of the archaea was isolated from a shallow marine hydrothermal system at Vulcano Island, Italy. The isolate grew between 56 and 90° C with an optimum around 85° C. The pH range for growth was 6.5 to 10.5, with an optimum around 9.0. Polysulfide and elemental sulfur were reduced to H₂S. Sulfur stimulated the growth rate. The isolate fermented yeast extract, peptone, meat extract, tryptone, and casein. Isovalerate, isobutyrate, propionate, acetate, CO₂, NH₃, and H₂S (in the presence of S°) were detected as end products. Growth was not inhibited by H₂. Based on DNA-DNA hybridization and 16S rRNA partial sequences, the new isolate represents a new species of Thermococcus, which we named Thermococcus alcaliphilus. The type strain is isolate AEDII12 (DSM 10322) Received: 7 July 1995 / Accepted: 25 August 1995     

Purification,properties and primary structure of H2-formingN 5,N10-methylenetetrahydromethanopterin dehydrogenase fromMethanococcus thermolithotrophicus

H₂-FormingN ⁵,N¹⁰-methylenetetrahydromethanopterin dehydrogenase (Hmd) is a novel type of hydrogenase found in methanogenic Achaea that contains neither nickel nor iron-sulfur clusters. The enzyme has previously been characterized fromMethanobacterium thermoautotrophicum and fromMethanopyrus kandleri. We report here on the purification and properties of the enzyme fromMethanococcus thermolithotrophicus. Thehmd gene was cloned and sequenced. The results indicate that the enzyme fromMc. thermolithotrophicus is functionally and structurally closely related to the H₂-forming methylene tetrahydromethanopterin dehydrogenase fromMb. thermoautotrophicum andMp. kandleri. From amino acid sequence comparisons of the three enzymes, a phylogenetic tree was deduced that shows branching orders similar to those derived from sequence comparisons of the 16S rRNA of the orders Methanococcales, Methanobacteriales, and Methanopyrales.Abbreviations H ₂ Forming dehydrogenase orHmd - H₂-FormingN ⁵,N¹⁰ methylene tetrahydromethanopterin dehydrogenase - H ₄MPT Tetrahydromethanopterin - CH ₂=H₄MPT N⁵,N¹⁰ Methylene tetrahydromethanopterin - CHH ₄MPT⁺ N⁵,N¹⁰ Methenyltetrahydromethanopterin - MALDI-TOF-MS Matrix-assisted laser desorption     

Growth of Methanococcus thermolithotrophicus in batch and continuous culture on H2 and CO2: influence of agitation

Summary A thermophilic methanogenic bacterium, Methanococcus thermolithotrophicus, was grown on H₂ and CO₂ in both batch and continuous culture, in a fermentor equipped with either a straight blade impeller or a Rushton impeller. Production was continued until 470 l CH₄·l^-1 per day was obtained with a biomass of 3.5 g dry wt. l^-1 under batch conditions.     

Thermococcus acidaminovorans sp. nov., a new hyperthermophilic alkalophilic archaeon growing on amino acids

From a shallow marine hydrothermal system at Vulcano (Italy), a new hyperthermophilic member of the Archaea was isolated. The cells are coccoid – shaped and possess up to five flagella. They grow between 56° and 93°C (optimum 85°C) and pH 5.0–9.5 (optimum 9.0). The organism is strictly anaerobic and grows heterotrophically on defined amino acids and complex organic substrates such as casamino acids, yeast extract, peptone, meat extract, tryptone, and casein. Polysulfide and elemental sulfur are reduced to H₂S. In the absence of polysulfide or elemental sulfur, the isolate grows at a significantly reduced rate. Growth is not influenced by the presence of H₂. DNA–DNA hybridization and 16S rRNA partial sequences indicated that the new isolate belongs to the genus Thermococcus, and represents a new species, Thermococcus acidaminovorans. The type strain is isolate AEDII10 (DSM 11906). Received: September 24, 1997 / Accepted: January 1, 1998     

The effect of pH on hydrogen production with Citrobacter intermedius

Summary Citrobacter intermedius was grown in a 14-liter fermenter under batch anaerobic conditions at the following controlled pH values: 5, 5.75, 6.0, 6.5, 7.0, 7.5, and 8.0. The growth medium was a glucose mineral salts medium with 0.1% ammonium sulfate as the source of sulfur. The optimum pH for H₂ production was 5.75 and 6.0 which gave a yield of 1.1 moles H₂/mole glucose. The optimum H₂-productivity was 144 moles H₂ per hour at pH 6.0.     

Xylanolytic anaerobic thermophiles from Icelandic hot-springs

Anaerobic enrichment cultures inoculated with neutral and alkaline (pH 7.0–9.0) sediment and biomat samples from hot-springs in Hveragerdi and Fluir, Iceland, were screened for growth on beech xylan from pH 8.0 to 10.0 at 68° C: no growth occured in cultures above pH 8.4. Five anaerobic xylanolytic bacteria were isolated from enrichment cultures at pH 8.4; all five microbes were Gram-positive rods with terminal spores, and produced CO₂, H₂, acetate, lactate and ethanol from xylan and xylose. One of the isolates, strain A2, grew from 50 to 75° C, with optimum growth near 68° C, and from pH 5.2 to 9.0 with an optimum between 6.8 and 7.4. Taxonomically, strain A2 was most similar to Clostridium thermohydrosulfuricum. At pH 7.0, the supernatant xylanases of strain A2 had a temperature range from 50 to 78° C with an optimum between 68 and 78° C. At 68° C, xylanase activity occurred from pH 4.9 to 9.1, with an optimum from pH 5.0 to 6.6. At pH 7.0 and 68° C, the K _m of the supernatant xylanases was 2.75 g xylan/l and the V _max was 2.65 × 10^–6 kat/l culture supernatant. When grown on xylose, xylanase production was as high as when grown on xylan. Correspondence to: B. K. Ahring     

Kinetic parameters for hydrogen evolution by the NAD-linked hydrogenase of Alcaligenes eutrophus

The hydrogen-evolving reaction of the purified soluble NAD-linked hydrogenase of Alcaligenes eutrophus was used to determine kinetic parameters of the enzyme. The H₂-evolving activity with methyl viologen as electron mediator was 20-fold as compared to that with NADH. In the assay with dithionite-reduced methyl viologen (K _m 0.7 mM) the hydrogenase was most active at a redox potential of –560 mV and exhibited a pH optimum of 7.0. The K _m for protons, the second substrate for H₂ evolution, was 6.2 nM. With electrochemically reduced methyl viologen the pH optimum was shifted to pH 6.0. Double-reciprocal plots of reaction rates versus proton concentrations intercepted at the ordinate for different methyl viologen concentrations. At different pH values such an intercept was also observed with the dye as the varied substrate. The kinetic data are diagnostic for an ordered bisubstrate mechanism where both substrates are bound before the product H₂ is released. Hydrogenase coupled to thylakoid membranes resulted in a constant H₂ evolution rate over 6 h. The system appeared to be limited by the capacity of the thylakoid membranes.     

Spectral similarities and kinetic differences of two tomato plant peroxidase isoenzymes

The kinetic and spectral properties of peroxidases A and B from the dwarf tomato plant were compared. The absolute absorption spectra were essentially the same for peroxidases A and B and their derivatives. Peroxidases A and B had different pH optima with guaiacol as the hydrogen donor but essentially the same optimum when pyrogallol was the substrate. The substrate concentrations required for optimum activity were different not only for the different substrates but also for each isoenzyme. When pyrogallol was used as the substrate, peroxidases A and B were 80% active when assayed under conditions optimal for the other isoenzyme. When guaiacol was used as the substrate, peroxidase A was completely inactive when assayed under conditions optimal for peroxidase B. In this case the pH was not optimum and the H₂O₂ concentration was inhibitory. Similarly, peroxidase B retained only 9% of its peroxidase activity toward guaiacol when assayed under conditions optimum for peroxidase A. In this case the pH was not optimum and the H₂O₂ was limiting. A possible role for peroxidase isoenzymes is discussed.     

Bioreduction of platinum salts into nanoparticles: a mechanistic perspective

A mechanism for the bioreduction of H₂PtCl₆ and PtCl₂ into platinum nanoparticles by a hydrogenase enzyme from Fusarium oxysporum is proposed. Octahedral H₂PtCl₆ is too large to fit into the active region of the enzyme and, under conditions optimum for nanoparticle formation (pH 9, 65°C), undergoes a two-electron reduction to PtCl₂ on the molecular surface of the enzyme. This smaller molecule is transported through hydrophobic channels within the enzyme to the active region where, under conditions optimal for hydrogenase activity (pH 7.5, 38°C) it undergoes a second two-electron reduction to Pt(0). H₂PtCl₆ was unreactive at pH 7.5, 38°C; PtCl₂ was unreactive at pH 9, 65°C.     

<Emphasis Type="Italic">Desulfurispirillum alkaliphilum</Emphasis> gen. nov. sp. nov., a novel obligately anaerobic sulfur- and dissimilatory nitrate-reducing bacterium from a full-scale sulfide-removing bioreactor

Strain SR 1^T was isolated under anaerobic conditions using elemental sulfur as electron acceptor and acetate as carbon and energy source from the Thiopaq bioreactor in Eerbeek (The Netherlands), which is removing H₂S from biogas by oxidation to elemental sulfur under oxygen-limiting and moderately haloalkaline conditions. The bacterium is obligately anaerobic, using elemental sulfur, nitrate and fumarate as electron acceptors. Elemental sulfur is reduced to sulfide through intermediate polysulfide, while nitrate is dissimilatory reduced to ammonium. Furthermore, in the presence of nitrate, strain SR 1^T was able to oxidize limited amounts of sulfide to elemental sulfur during anaerobic growth with acetate. The new isolate is mesophilic and belongs to moderate haloalkaliphiles, with a pH range for growth (on acetate and nitrate) from 7.5 to 10.25 (optimum 9.0), and a salt range from 0.1 to 2.5 M Na⁺ (optimum 0.4 M). According to phylogenetic analysis, SR 1^T is a member of a deep bacterial lineage, distantly related to Chrysiogenes arsenatis (Macy et al. 1996). On the basis of the phenotypic and genetic data, the novel isolate is placed into a new genus and species, Desulfurispirillum alkaliphilum (type strain SR^T = DSM 18275 = UNIQEM U250). Nucleotide sequence accession number: the GenBank/EMBL accession number of the 16S rRNA gene sequence of strain SR 1^T is DQ666683.     

Methanogenesis in McLean Bog,an Acidic Peat Bog in Upstate New York: Stimulation by H2/CO2 in the Presence of Rifampicin,or by Low Concentrations of Acetate

Acidic peat bog soils produce CH₄ and although molecular biological studies have demonstrated the presence of diverse methano-genic populations in them, few studies have sustained methanogenesis by adding the CH₄ precursors H₂/CO₂ or acetate, and few indigenous methanogens have been cultured. McLean Bog is a small (ca. 70 m across), acidic (pH 3.4–4.3) Sphagnum -dominated bog in upstate New York. Although addition of H₂/CO₂ or 10 mM acetate stimulated methanogenesis in soils from a nearby circumneutral-pH fen, neither of these substrates led to sustained methanogenesis in McLean Bog soil slurries. After a brief period of stimulation by H₂/CO₂, methanogenesis in McLean Bog soil declined, which could be attributed to buildup of large amounts of acetic acid produced from the H₂/CO₂ by acetogens. Addition of the antibiotic rifampicin inhibited acetogenesis (carried out by Bacteria) and allowed methanogenesis (carried out by Archaea) to continue. Using rifampicin, we were able to study effects of temperature, pH, and salts on methanogenesis from H₂/CO₂ in McLean Bog soil samples. The enriched H₂/CO₂-utilizing methanogens showed an optimum for activity near pH 5, and a temperature optimum near 35°C. Methanogenesis was not stimulated by addition of 10 mM acetate, but it was stimulated by 1 mM acetate, and multiple additions were consumed at increasing rates and nearly stoichiometrically converted to CH₄. In conclusion, we have found that both hydrogentrophic and aceticlastic methanogens are present in McLean Bog soils, and that methanogenic activity can be stimulated using H₂/CO₂ in the presence of rifampicin, or using low concentrations of acetate.     

Effect of carbon dioxide and hydrostatic pressure on the pH of culture media and the growth of methanogens at elevated temperature

Summary High pressure/high temperature investigations on thermophilic methanogens require specific precautions to provide well-defined pH conditions in their culture media. Applying CO₂ as carbon source, sufficient buffering capacity of the culture medium is of crucial importance in investigations involving elevated pressures. In order to separate pressure effects on the growth and reproduction of thermophilic methanogens from pressure-induced protonation/deprotonation and increased solubility of gaseous components, direct pH measurements in common culture media in the absence and in the presence of CO₂ were performed at elevated temperature (65° C), and at pressures up to 100 MPa. Neutral phosphate buffer at high pressure shows a significant downward shift of its pH which is strongly enhanced in the presence of CO₂. In minimal media containing acetate, carbonate, formate and phosphate in 100 mM concentrations, 120 mM HEPES is found to provide optimum pH stability: near neutrality the pH change upon CO₂ saturation in the absence and in the presence of HEPES amounts to pH=2.10 and 0.41, respectively; the corresponding pressure dependences are pH/100 MPa=-0.26 and -0.07. As taken from these results, the apparent pressure dependence of the optimum growth ofMethanococcus thermolithotrophicus at 65° C in minimal medium reflects the pH shift below the cutoff point of growth (pH 5.5), rather than pressure-induced growth inhibition. At constant pH, elevated pressure up to 400 MPa is found to increase the rate and yield of growth; at the same time, alterations in the phenotype of the bacterium are observed.     

Desulfurella multipotens sp. nov., a new sulfur-respiring thermophilic eubacterium from Raoul Island (Kermadec archipelago,New Zealand)

A new moderately thermophilic sulfur-reducing eubacterium was isolated from bottom deposits of Green Lake (Raoul Island, Kermadec archipelago, New Zealand). Cells are short rods, 1.5–1.8 by 0.5–0.7 m, single or in pairs, motile with one polar flagellum, gram-negative with S-layer of subunit structure. Growth occurred between 42 and 77°C with the optimum at 58–60°C and at pH from 6.0 to 7.2 with the optimum at 6.4–6.8. The bacterium was obligately anaerobic and obligately sulfur-respiring, and capable of lithoautotrophic growth on a mineral medium with S° and H₂/CO₂ gas phase. In addition to molecular hydrogen, a wide range of substrates can be utilized as energy source in the presence of elemental sulfur: pyruvate, acetate, butyrate, pentadecanate, palmitate, stearate. Products are CO₂ and H₂S. The G+C content of DNA is 33.5 mol%. DNA-DNA homology with the type species of the genus Desulfurella — Desulfurella acetivorans — is 69±2%. A new species, Desulfurella multipotens sp. nov., with the type strain RH-8 is described.     

A proton-deuterium exchange study of three types ofDesulfovibrio hydrogenases

Summary Hydrogenases are among the main enzymes involved in bacterial anaerobic corrosion of metals. The study of their mode of action is important for a full comprehension of this phenomenon. The three types ofDesulfovibrio hydrogenases [(Fe), (NiFe), (NiFeSe)] present different patterns in the pH dependence of their activity. The periplasmic enzyme fromDesulfovibrio salexigens and the cytoplasmic enzyme fromDesulfovibrio baculatus both have pH optima at 7.5 for H₂ uptake and 4.0 for H₂ evolution and H⁺–D₂ exchange reaction (measured by membrane-inlet mass-spectrometry). The H₂ to HD ratio at pH above 5.0 is higher than 1.0. The periplasmic hydrogenase fromD. gigas presents the same pH optimum (8.0) for the H⁺–D₂ exchange as for H₂ consumption. In contrast, the enzyme fromD. vulgaris has the highest activity in H₂ production and in the exchange at pH 5.0. Both hydrogenases have a H₂-to-HD ratio below 1.0.     

Hydrogenase 3 but not hydrogenase 4 is major in hydrogen gas production by Escherichia coli formate hydrogenlyase at acidic pH and in the presence of external formate

Fermenting Escherichia coli is able to produce formate and molecular hydrogen (H₂) when grown on glucose. H₂ formation is possessed by two hydrogenases, 3 (Hyd-3) and 4 (Hyd-4), those, in conjunction with formate dehydrogenase H (Fdh-H), constitute distinct membrane-associated formate hydrogenylases. At slightly alkaline pH (pH 7.5), the production of H₂ was found to be dependent on Hyd-4 and the F₀F₁-adenosine triphosphate (ATPase), whereas external formate increased the activity of Hyd-3. In this study with cells grown without and with external formate H₂ production dependent on pH was investigated. In both types of cells, H₂ production was increased after lowering of pH. At acidic pH (pH 5.5), this production became insensitive either to N,N′-dicyclohexylcarbodiimide or to osmotic shock and it became largely dependent on Fdh-H and Hyd-3 but not Hyd-4 and the F₀F₁-ATPase. The results indicate that Hyd-3 has a major role in H₂ production at acidic pH independently on the F₀F₁-ATPase.     

<Emphasis Type="Italic">Desulfovibrio legallis</Emphasis> sp. nov.: A Moderately Halophilic,Sulfate-Reducing Bacterium Isolated from a Wastewater Digestor in Tunisia

A new moderately halophilic sulfate-reducing bacterium (strain H₁^T) was enriched and isolated from a wastewater digestor in Tunisia. Cells were curved, motile rods (2–3 x 0.5 μm). Strain H₁^T grew at temperatures between 22 and 43°C (optimum 35°C), and at pH between 5.0 and 9.2 (optimum 7.3–7.5). Strain H₁^T required salt for growth (1–45 g of NaCl/l), with an optimum at 20–30 g/l. Sulfate, sulfite, thiosulfate, and elemental sulfur were used as terminal electron acceptors but not nitrate and nitrite. Strain H₁^T utilized lactate, pyruvate, succinate, fumarate, ethanol, and hydrogen (in the presence of acetate and CO₂) as electron donors in the presence of sulfate as electron acceptor. The main end-products from lactate oxidation were acetate with H₂ and CO₂. The G + C content of the genomic DNA was 55%. The predominant fatty acids of strain H₁^T were C_15:0 iso (38.8%), C_16:0 (19%), and C_14:0 iso 3OH (12.2%), and menaquinone MK-6 was the major respiratory quinone. Phylogenetic analysis of the small-subunit (SSU) ribosomal RNA (rRNA) gene sequence indicated that strain H₁^T was affiliated to the genus Desulfovibrio. On the basis of SSU rRNA gene sequence comparisons and physiological characteristics, strain H₁^T is proposed to be assigned to a novel species of sulfate reducers of the genus Desulfovibrio, Desulfovibrio legallis sp. nov. (= DSM 19129^T = CCUG 54389^T).     

Metabolic properties and kinetics of methanogenic granules

Two types of mesophilic methanogenic granules (R- and F-granules) were developed on different synthetic feeds containing acetate, propionate and butyrate as major carbon sources and their metabolic properties were characterized. The metabolic activities of granules on acetate, formate and H₂-CO₂ were related to the feed composition used for their development. These granules performed a reversible reaction between H₂ production from formate and formate synthesis from H₂ plus bicarbonate. Both types of granules exhibited high activity on normal and branched volatile fatty acids with three to five carbons and low activity on ethanol and glucose. The granules performed a reversible isomerization between isobutyrate and butyrate during butyrate or isobutyrate degradation. Valerate and 2-methylbutyrate were produced and consumed during propionate-butyrate degradation. The respective apparent K _m (mm) for various substrates in disrupted R- and F-granules was: acetate, 0.43 and 0.41; propionate, 0.056 and 0.038; butyrate, 0.15 and 0.19; isobutyrate, 0.12 and 0.19; valerate, 0.15 and 0.098. Both granules had an optimum temperature range from 40 to 50° C for H₂-CO₂ and formate utilization and 40° C for acetate, propionate and butyrate utilization and a similar optimum pH. Correspondence to: J. G. Zeikus