Biohydrogen production from carbon monoxide and water byRhodopseudomonas palustris P4

A reactor-scale hydrogen (H₂) productionvia the water-gas shift reaction of carbon monoxide (CO) and water was studied using the purple nonsulfur bacterium,Rhodopseudomonas palustris P4. The experiment was conducted in a two-step process: an aerobic/chemoheterotrophic cell growth step and a subsequent anaerobic H₂ production step. Important parameters investigated included the agitation speed, inlet CO concentration and gas retention time. P4 showed a stable H₂ production capability with a maximum activity of 41 mmol H₂ g cell⁻¹h⁻¹ during the continuous reactor operation of 400 h. The maximal volumetric H₂ production rate was estimated to be 41 mmol H₂ L¹h⁻¹, which was about nine-fold and fifteen-fold higher than the rates reported for the photosynthetic bacteriaRhodospirillum rubrum andRubrivivax gelatinosus, respectively. This is mainly attributed to the ability of P4 to grow to a high cell density with a high specific H₂ production activity. This study indicates that P4 has an outstanding potential for a continuous H₂ productionvia the water-gas shift reaction once a proper bioreactor system that provides a high rate of gas-liquid mass transfer is developed.     

Synthesis of acetyl coenzyme A by carbon monoxide dehydrogenase complex from acetate-grown Methanosarcina thermophila.

The carbon monoxide dehydrogenase (CODH) complex from Methanosarcina thermophila catalyzed the synthesis of acetyl coenzyme A (acetyl-CoA) from CH3I, CO, and coenzyme A (CoA) at a rate of 65 nmol/min/mg at 55 degrees C. The reaction ended after 5 min with the synthesis of 52 nmol of acetyl-CoA per nmol of CODH complex. The optimum temperature for acetyl-CoA synthesis in the assay was between 55 and 60 degrees C; the rate of synthesis at 55 degrees C was not significantly different between pHs 5.5 and 8.0. The rate of acetyl-CoA synthesis was independent of CoA concentrations between 20 microM and 1 mM; however, activity was inhibited 50% with 5 mM CoA. Methylcobalamin did not substitute for CH3I in acetyl-CoA synthesis; no acetyl-CoA or propionyl coenzyme A was detected when sodium acetate or CH3CH2I replaced CH3I in the assay mixture. CO could be replaced with CO2 and titanium(III) citrate. When CO2 and 14CO were present in the assay, the specific activity of the acetyl-CoA synthesized was 87% of the specific activity of 14CO, indicating that CO was preferentially incorporated into acetyl-CoA without prior oxidation to free CO2. Greater than 100 microM potassium cyanide was required to significantly inhibit acetyl-CoA synthesis, and 500 microM was required for 50% inhibition; in contrast, oxidation of CO by the CODH complex was inhibited 50% by approximately 10 microM potassium cyanide.     

Structuring of bacterioplankton communities by specific dissolved organic carbon compounds

The main role of microorganisms in the cycling of the bulk dissolved organic carbon pool in the ocean is well established. Nevertheless, it remains unclear if particular bacteria preferentially utilize specific carbon compounds and whether such compounds have the potential to shape bacterial community composition. Enrichment experiments in the Mediterranean Sea, Baltic Sea and the North Sea (Skagerrak) showed that different low-molecular-weight organic compounds, with a proven importance for the growth of marine bacteria (e.g. amino acids, glucose, dimethylsulphoniopropionate, acetate or pyruvate), in most cases differentially stimulated bacterial growth. Denaturing gradient gel electrophoresis 'fingerprints' and 16S rRNA gene sequencing revealed that some bacterial phylotypes that became abundant were highly specific to enrichment with specific carbon compounds (e.g. Acinetobacter sp. B1-A3 with acetate or Psychromonas sp. B3-U1 with glucose). In contrast, other phylotypes increased in relative abundance in response to enrichment with several, or all, of the investigated carbon compounds (e.g. Neptuniibacter sp. M2-A4 with acetate, pyruvate and dimethylsulphoniopropionate, and Thalassobacter sp. M3-A3 with pyruvate and amino acids). Furthermore, different carbon compounds triggered the development of unique combinations of dominant phylotypes in several of the experiments. These results suggest that bacteria differ substantially in their abilities to utilize specific carbon compounds, with some bacteria being specialists and others having a more generalist strategy. Thus, changes in the supply or composition of the dissolved organic carbon pool can act as selective forces structuring bacterioplankton communities.     

Synthesis of acetyl coenzyme A from carbon monoxide, methyltetrahydrofolate, and coenzyme A by enzymes from Clostridium thermoaceticum

Two purified fractions from Clostridium thermoaceticum are shown to catalyze the following reaction: CO + CH3THF + CoA ATP leads to CH3COCoA + THF. The methyltetrahydrofolate (CH3THF) gives rise to the methyl group of the acetyl-coenzyme A (CoA) and the carbon monoxide (CO) and CoA to its carboxyl thio ester group. The role of ATP is unknown. One of the protein fractions (F2) is a methyltransferase, whereas the other fraction (F3) contains CO dehydrogenase and a methyl acceptor which is postulated to be a corrinoid enzyme. The methyltransferase catalyzes the transfer of the methyl group to the methyl acceptor, and the CO is converted to a formyl derivative by the CO dehydrogenase. By a mechanism that is as yet unknown, the formyl derivative in combination with CoA and the methyl of the methyl acceptor are converted to acetyl-CoA. It is also shown that fraction F3 catalyzes the reversible exchange of 14C from [1-14C]acetyl-CoA into 14CO and that ATP is required, but not the methyltransferase. It is proposed that these reactions are part of the mechanism which enables certain autotrophic bacteria to grow on CO. It is postulated that CH3THF is synthesized from CO and tetrahydrofolate which then, as described above, is converted to acetyl-CoA. The acetyl-CoA then serves as a precursor in other anabolic reactions. A similar autotropic pathway may occur in bacteria which grow on carbon dioxide and hydrogen.     

Electrosynthesis of organic compounds from carbon dioxide is catalyzed by a diversity of acetogenic microorganisms

Microbial electrosynthesis, a process in which microorganisms use electrons derived from electrodes to reduce carbon dioxide to multicarbon, extracellular organic compounds, is a potential strategy for capturing electrical energy in carbon-carbon bonds of readily stored and easily distributed products, such as transportation fuels. To date, only one organism, the acetogen Sporomusa ovata, has been shown to be capable of electrosynthesis. The purpose of this study was to determine if a wider range of microorganisms is capable of this process. Several other acetogenic bacteria, including two other Sporomusa species, Clostridium ljungdahlii, Clostridium aceticum, and Moorella thermoacetica, consumed current with the production of organic acids. In general acetate was the primary product, but 2-oxobutyrate and formate also were formed, with 2-oxobutyrate being the predominant identified product of electrosynthesis by C. aceticum. S. sphaeroides, C. ljungdahlii, and M. thermoacetica had high (>80%) efficiencies of electrons consumed and recovered in identified products. The acetogen Acetobacterium woodii was unable to consume current. These results expand the known range of microorganisms capable of electrosynthesis, providing multiple options for the further optimization of this process.     

Kinetics of carbon monoxide and oxygen binding to hemoglobin in human red blood cell suspensions studied by laser flash photolysis

The reaction kinetics of the binding of CO and O₂ to hemoglobin (Hb) in human red blood cell (RBC) suspensions have been examined using a 300 ns dye laser to photodissociate HbCO or HbO₂. Fast (halftime1?0 μs) and slow (5?ms) processes were seen after photolysis. The results indicate that neither the rate constants nor the activation energies for the binding of CO to the fast reacting form of Hb in the RBC are significantly different from that measured in solution in spite of the different environments. Rate constants determined for O₂ binding in RBC were intermediate between rates observed for reaction with fast and slow reacting forms of Hb and probably consist of contributions from each. The slow recombination of CO and O₂ probably has contributions both from reaction with slow reacting forms of Hb and from ligand that had diffused away from the RBC after photolysis.     

Assessment of biological activity and fate of organic compounds in a reactor for the measurement of biodegradable organic carbon in water

A new, rapid method for the determination of biodegradable dissolved organic carbon (BDOC), especially suited to water industry needs, was recently proposed by the authors. This dynamic method measured the BDOC of circulating water continuously pumped over a biofilm attached to a special support (sinterized porous glass) that fills a system of two glass columns. The BDOC value corresponds to the difference in dissolved organic carbon (DOC) between inflow and outflow water samples. The analytical results are not significantly different from those of other bioassays that use indigenous bacteria, and the total duration of the analysis is less than 3 h. However, a problem common to all the BDOC methods based on attached bacteria is the extent to which the decrease in DOC during the BDOC analysis is due to true biodegradation or to adsorption of organic matter to the reactor. In the present study, a reasonable support is provided for the hypothesis that this decrease, at least in the dynamic method, is predominantly due to microbiological activity. After comparing the support (sinterized porous glass) with a good physical adsorbent (granular activated carbon), the influence of temperature, residual chlorine and sodium azide on the reactor performance was tested, and a sensitivity only attributable to biological activity was observed. Another set of experiments were performed to assess the fate and specific elimination of different organic substances, explicable assuming that biodegradation processes were involved.     

2,4,6-trinitrotoluene reduction by carbon monoxide dehydrogenase from Clostridium thermoaceticum

Purified CO dehydrogenase (CODH) from Clostridium thermoaceticum catalyzed the transformation of 2,4,6-trinitrotoluene (TNT). The intermediates and reduced products of TNT transformation were separated and appear to be identical to the compounds formed by C. acetobutylicum, namely, 2-hydroxylamino-4,6-dinitrotoluene (2HA46DNT), 4-hydroxylamino-2,6-dinitrotoluene (4HA26DNT), 2, 4-dihydroxylamino-6-nitrotoluene (24DHANT), and the Bamberger rearrangement product of 2,4-dihydroxylamino-6-nitrotoluene. In the presence of saturating CO, CODH catalyzed the conversion of TNT to two monohydroxylamino derivatives (2HA46DNT and 4HA26DNT), with 4HA26DNT as the dominant isomer. These derivatives were then converted to 24DHANT, which slowly converted to the Bamberger rearrangement product. Apparent K(m) and k(cat) values of TNT reduction were 165 +/- 43 microM for TNT and 400 +/- 94 s(-1), respectively. Cyanide, an inhibitor for the CO/CO(2) oxidation/reduction activity of CODH, inhibited the TNT degradation activity of CODH.     

Microbial production of ethanol from carbon monoxide

Production of ethanol from fermentation of CO has received much attention in the last few years with several companies proposing to use CO fermentation in their ethanol production processes. The genomes of two CO fermenters, Clostridium ljungdahlii and Clostridium carboxidivorans, have recently been sequenced. The genetic information obtained from this sequencing is aiding molecular biologists who are enhancing ethanol and butanol production by genetic manipulation. Several studies have optimized media for CO fermentation, which has resulted in enhanced ethanol production. Also, new reactor designs involving the use of hollow fiber membranes have reduced mass transfer barriers that have hampered previous CO fermentation efforts.     

Fermentative production of ethanol from carbon monoxide

'Too much Carbon Monoxide for me to bear…' are the opening lyrics of the CAKE song Carbon Monoxide (from their 2004 album Pressure Chief), and while this may be the case for most living organisms, several species of bacteria both thrive on this otherwise toxic gas, and metabolize it for the production of fuels and chemicals. Indeed CO fermentation offers the opportunity to sustainably produce fuels and chemicals without impacting the availability of food resources or even farm land. Mounting commercial interest in the potential of this process has in turn triggered greater scrutiny of the molecular and genetic basis for CO metabolism, as well as the challenges associated with the implementation and operation of gas fermentation at scale.     

Production of carbon monoxide from aromatic amino acids by Morganella morganii

Morganella morganii produced CO when cultured in a medium containing casamino acids or peptone as the sole carbon source. Although the production of CO was distinctly enhanced by the addition of hemin to the medium, the amounts of CO produced in the absence of hemin were nearly proportional to the amounts of peptone added to the culture media. Examination of 20 amino acids for their ability to produce CO by resting cells revealed that phenylalanine, tyrosine, histidine and tryptophan were the sources of CO. Oxygen and hemin were necessary for CO production from the amino acids except tryptophan which produced CO in the absence of hemin. When cells were incubated for 4 h at 30° C in the mixture containing 40 mol tyrosine and 1 mol hemin, about 15 mol CO was produced; the activity of CO production was about 1.2 mol CO/h · mg cell nitrogen. Phenylpyruvic acid, p-hydroxyphenylpyruvic acid and imidazolepyruvic acid also produced CO in the presence of hemin, while indolepyruvic acid produced CO regardless of the presence or absence of hemin. The production of CO by the 2-oxo acids proceeded spontaneously and did not require the presence of M. morganii cells.     

UV photolysis for accelerated quinoline biodegradation and mineralization

Sequentially and intimately coupled photolysis with biodegradation were evaluated for their ability to accelerate quinoline-removal and quinoline-mineralization kinetics. UV photolysis sequentially coupled to biodegradation significantly improved biomass-growth kinetics, which could be represented well by the Aiba self-inhibition model: UV photolysis increased the maximum specific growth rate (μ _max) by 15 %, and the inhibition constant (K _SI) doubled. An internal loop photo-biodegradation reactor (ILPBR) was used to realize intimately coupled photolysis with biodegradation. The ILPBR was operated with batch experiments following three protocols: photolysis alone (P), biodegradation alone (B), and intimately coupled photolysis and biodegradation (P&B). For P&B, the maximum quinoline removal rate (r _max) increased by 9 %, K _SI increased by 17 %, and the half-maximum-rate concentration (K _S) decreased by 55 %, compared to B; the composite result was a doubling of the quinoline-biodegradation rate for most of the concentration range tested. The degree of mineralization was increased by both forms of photolysis coupled to biodegradation, and the impact was greater for intimate coupling (18 % increase) than sequential coupling (5 %). The benefits of UV photolysis were greater with intimate coupling than with sequential coupling due to parallel transformation by biodegradation and photolysis.     

Infrared studies of carbon monoxide binding to carbon monoxide dehydrogenase/acetyl-CoA synthase from Moorella thermoacetica

Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) is a bifunctional enzyme that catalyzes the reversible reduction of carbon dioxide into carbon monoxide and the coupled synthesis of acetyl-CoA from the carbon monoxide produced. Exposure of CODH/ACS from Moorella thermoacetica to carbon monoxide gives rise to several infrared bands in the 2100-1900 cm(-1) spectral region that are attributed to the formation of metal-coordinated carbon monoxide species. Infrared bands attributable to M-CO are not detected in the as-isolated enzyme, suggesting that the enzyme does not contain intrinsic metal-coordinated CO ligands. A band detected at 1996 cm(-1) in the CO-flushed enzyme is assigned as arising from CO binding to a metal center in cluster A of the ACS subunit. The frequency of this band is most consistent with it arising from a terminally coordinated Ni(I) carbonyl. Multiple infrared bands at 2078, 2044, 1970, 1959, and 1901 cm(-1) are attributed to CO binding at cluster C of the CODH subunit. All infrared bands attributed to metal carbonyls decay in a time-dependent fashion as CO(2) appears in the solution. These observations are consistent with the enzyme-catalyzed oxidation of carbon monoxide until it is completely depleted from solution during the course of the experiments.     

Formation of carbon monoxide from CO2 and H2 by Methanobacterium thermoautotrophicum

Cell suspensions of Methanobacterium thermoautotrophicum were found to reduce CO2 with H2 to CO at a maximal rate of 100 nmol X min-1 X mg protein-1. Half-maximal rates were obtained at a H2 and a CO2 concentration in the gas phase of 10% and 30%, respectively. The CO concentration in the gas phase surpassed the equilibrium concentration by a factor of more than 15 which indicates that CO2 reduction with H2 to CO was energy-driven. This was substantiated by the observation that the cells only formed CO when they also generated methane and that CO formation was completely inhibited by uncouplers. CO formation by cell suspensions and by growing cells was inhibited by cyanide. Neither methane formation nor the electrochemical proton potential were affected by this inhibitor. Cyanide was shown to inactivate specifically the carbon monoxide dehydrogenase present in M. thermoautotrophicum. It is therefore concluded that reduction of CO2 to CO is catalyzed by this enzyme. CO production by growing cells was 5-10-times slower than by resting cells. This is explained by effective CO assimilation in growing cells; when CO assimilation was inhibited by propyl iodide the rate of CO production immediately increased more than tenfold.     

Synthesis of organic compounds in ultrasonic field]

On the basis of our experimental data, it is suggested that powerful ultrasonic vibrations acted as inductors and activators of primary chemical reactions and that these reactions occurred not only in the atmosphere, but in the hydrosphere as well. Synthesis of various biologically important substances was monitored in aqueous solutions under the influence of ultrasonic treatment. Water and gases of the reduction atmosphere play a significant role in these reactions.     

The dissociation of carbon monoxide from hemoglobin intermediate

To investigate the mechanism of allosteric switching in human hemoglobin, we have studied the dissociation of the ligand (CO) from several intermediate ligation states by a stopped-flow kinetic technique that utilizes competitive binding of CO by microperoxidase. The hemoglobin species investigated include Hb(CO)4, the diliganded symmetrical species (alpha beta-CO)2 and (alpha-CO beta)2, and the di- and monoliganded asymmetrical species (alpha-CO beta-CO)(alpha beta), (alpha-CO beta)(alpha beta-CO), (alpha beta-CO) (alpha beta), and (alpha-CO beta)(alpha beta). They were obtained by rapid reduction with dithionite of the corresponding valence intermediates that in turn were obtained by chromatography or by hybridization. The nature and concentration of the intermediates were determined by isoelectric focusing at -25 degrees C. The study was performed at varying hemoglobin concentrations (0.1, 0.02, and 0.001 mM [heme]), pH (6.0, 7.0, 8.0), with and without inositol hexaphosphate. The results indicate that: (a) hemoglobin concentration in the 0.1-0.02 mM range does not significantly affect the kinetic rates; (b) the alpha chains dissociate CO faster than the beta chains; (c) the symmetrical diliganded intermediates show cooperativity with respect to ligand dissociation that disappears in the presence of inositol hexaphosphate; (d) the monoliganded intermediates dissociate CO faster than the diliganded intermediates; (e) the asymmetrical diliganded intermediates are functionally different from the symmetrical species.