Relationship between Methanogenic Cofactor Content and Maximum Specific Methanogenic Activity of Anaerobic Granular Sludges

In this study we investigated whether a relationship exists between the methanogenic activity and the content of specific methanogenic cofactors of granular sludges cultured on different combinations of volatile fatty acids in upflow anaerobic sludge blanket or fluidized-bed reactors. Significant correlations were measured in both cases between the contents of coenzyme F₄₂₀−2 or methanopterin and the maximum specific methanogenic activities on propionate, butyrate, and hydrogen, but not acetate. For both sludges the content of sarcinapterin appeared to be correlated with methanogenic activities on propionate, butyrate, and acetate, but not hydrogen. Similar correlations were measured with regard to the total content of coenzyme F₄₂₀−4 and F₄₂₀−5 in sludges from fluidized-bed reactors. The results indicate that the contents of specific methanogenic cofactors measured in anaerobic granular sludges can be used to estimate the hydrogenotrophic or acetotrophic methanogenic potential of these sludges.     

Fluorimetric monitoring of methanogenesis in anaerobic digestors

A new parameter, Q_CH4(F₄₂₀), is proposed to determine the potential methanogenic activity in the mixed microbial communities of anaerobic digestors. It is based on the particular fluorimetric properties of F420, a coenzyme common to and specific for methanogenic bacteria.     

On-line monitoring of the methanogenic fermentation by measurement of culture fluorescence

Summary Real-time on-line fluorescence measurements of the coenzymes NAD(P)H and F₄₂₀ were evaluated as indicators of stability in a glucose-fed anaerobic methanogenic digester. A probe designed forin situ fluorimetric measurement of NAD(P)H provided an assessment of activity of the total microbial community, while the response of a fluorescence probe designed to measure coenzyme F₄₂₀ correlated well with methanogenic activity. The two fluorescence-monitoring probes responded directly to fermentation imbalance during periods of substrate overloading and corresponded to traditional offline measurements, suggesting that the probes may be suitable candidates for inclusion in an on-line process control system for anaerobic digestion.Florida Agricultural Experimental Station, Journal Series no. R-00326     

Fed-batch cultivation of methanobacterium formicicum and its fluorometric monitoring

Fed-batch cultivation of Methanobacterium formicicum Z-281 was performed under substrate limiting conditions. Specific rate of culture growth amounted to 0.041 h^?1, cell yield related to substrate was 0.6 mg proteine/mM formate. Significant increase of coenzyme F₄₂₀ content in cells depending on time of cultivation was observed. Increase of the fluorescence intensity of the medium was also observed with the accumulation of biomass. These parameters were suggested for tracking culture growth. The possibility of application of fluorescent factors for methanogenic population control in the anaerobic digester is discussed.     

Anaerobic treatment of protein-containing waste waters: correlation between coenzyme F420 and methane production

Summary Anaerobic treatment of gelatine-containing model waste water and baker's yeast manufacturing effluent was investigated in upflow anaerobic sludge blanket (UASB) reactors. During start up a correlation between coenzyme F ₄₂₀ content and methane production in the reactor was observed. By monitoring coenzyme F ₄₂₀ concentrations a certain prediction of methanogenic activities was possible.     

Relationship of Intracellular Coenzyme F420 Content to Growth and Metabolic Activity of Methanobacterium bryantii and Methanosarcina barkeri

The use of F₄₂₀ as a parameter for growth or metabolic activity of methanogenic bacteria was investigated. Two representative species of methanogens were grown in batch culture: Methanobacterium bryantii (strain M.o.H.G.) on H₂ and CO₂, and Methanosarcina barkeri (strain Fusaro) on methanol or acetate. The total intracellular content of coenzyme F₄₂₀ was followed by high-resolution fluorescence spectroscopy. F₄₂₀ concentration in M. bryantii ranged from 1.84 to 3.65 μmol · g of protein⁻¹; and in M. barkeri grown with methanol it ranged from 0.84 to 1.54 μmol · g⁻¹ depending on growth conditions. The content of F₄₂₀ in M. barkeri was influenced by a factor of 2 depending on the composition of the medium (minimal or complex) and by a factor of 3 to 4 depending on whether methanol or acetate was used as the carbon source. A comparison of F₄₂₀ content with protein, cell dry weight, optical density, and specific methane production rate showed that the intracellular content of F₄₂₀ approximately followed the increase in biomass in both strains. In contrast, no correlation was found between specific methane production rate and intracellular F₄₂₀ content. However, qCH₄(F₄₂₀), calculated by dividing the methane production rate by the coenzyme F₄₂₀ concentration, almost paralleled qCH₄(protein). These results suggest that F₄₂₀ may be used as a specific parameter for estimating the biomass, but not the metabolic activity, of methanogens; hence qCH₄(F₄₂₀) determined in mixed populations with complex carbon substrates must be considered as measure of the actual methanogenic activity and not as a measure of potential activity.     

Quantitation of coenzyme F420 in methanogenic sludge by the use of reversed-phase high-performance liquid chromatography and a fluorescence detector

Summary With the use of acetone extraction, reversed-phase High-Performance Liquid Chromatography and fluorimetric monitoring, the quantity of coenzyme F₄₂₀ in mixed liquors and rumen contents can be measured. A synthetic analog of coenzyme F₄₂₀ is used as an internal standard to compensate for differences in fluorimetric monitoring. The method allows the detection of one picomol of coenzyme F₄₂₀ and the differentiation between different forms of the coenzyme known to be present in various methanogenic bacteria.     

Separation and quantification of cofactors from methanogenic bacteria by high-performance liquid chromatography: optimum and routine analyses

Four assays were developed, employing high-performance liquid chromatography, which gave optimal detection and separation of derivatives of 7-methylpterin, coenzyme F₄₂₀, factor F₄₃₀ or vitamin B₁₂. In addition an assay was developed in which thirteen of these cofactors can be separated and quantified simultaneously and which can be used in routine analysis of methanogenic populations in anaerobic digesters. The application of the different assays is demonstrated by analyses of extracts of pure cultures of Methanobacterium thermoautotrophicum and Methanosarcina barkeri and of sludge from a methanogenic fluidized bed reactor. Mixtures of authentic methanogenic cofactors were used in reference analyses and a relative peak area method was employed to identity the various cofactors in the extracts.     

Monitoring of specific methanogenic activity of granular sludge by confocal laser scanning microscopy during start-up of thermophilic upflow anaerobic sludge blanket reactor

Confocal, laser-scanning microscopy was applied to acquire coenzyme F₄₂₀-based autofluorescence images of middle sections of sludge granules during start-up of a thermophilic reactor that were seeded with mesophilically-grown microorganisms of granular sludge. Digital images were analyzed to calculate weighted averages of autofluorescence. The values were related (r ²=0.97) to specific methanogenic activities of granular sludge as the granules developed to steady state.     

Function of H2-forming methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum in coenzyme F420 reduction with H2

Summary In most methanogenic archaea, two hydrogenase systems that can catalyze the reduction of coenzyme F₄₂₀ (F₄₂₀) with H₂ are present: (1) the F₄₂₀-reducing hydrogenase, which is a nickel iron-sulfur flavoprotein composed of three different subunits, and (2) the N ⁵, N¹⁰-methylenetetrahydromethanopterin dehydrogenase system, which is composed of H₂-forming methylenetetrahydromethanopterin dehydrogenase and F₄₂₀-dependent methylenetetrahydromethanopterin dehydrogenase, both metal-free proteins without an apparent prosthetic group. We report here that in nickel-limited chemostat cultures of Methanobacterium thermoautotrophicum, the specific activity of the F₄₂₀-reducing Ni/Fe-hydrogenase was essentially zero, whereas that of the H₂-forming methylenetetrahydromethanopterin dehydrogenase was six times higher, and that of the F₄₂₀-dependent methylenetetrahydromethanopterin dehydrogenase was four times higher than in cells grown under non-nickel-limited conditions. This evidence supports the hypothesis that when M. thermoautotrophicum grows under conditions of nickel limitation, the reduction of F₄₂₀ with H₂ is catalyzed by the metal-free methylenetetrahydromethanopterin dehydrogenase system. Received: 9 September 1997 / Accepted: 30 October 1997     

A method for estimating phosphate in the presence of phytate and its application to the determination of phytase

Quantification of coenzymes and related compounds from methanogens was performed in extracts obtained from whole cells with aqueous ethanol at 80°C. By means of high-performance liquid chromatography the following compounds could be detected and quantified in extracts from Methanobacterium thermoautotrophicum: coenzyme MF₄₃₀, the prosthetic group of methylcoenzyme M reductase, F₅₆₀, an oxidation product of this compound, coenzyme F₄₂₀, F₃₄₂, methanopterin, and carboxytetrahydromethanopterin, previously known as YFC. Coenzyme MF₄₃₀, coenzyme F₄₂₀, and methanopterin could be determined in extracts from Methanosarcina barkeri. Structural differences were noticed between the coenzymes from the methanogenic bacteria studied.     

In vivo measurement of F420 fluorescence in cultures of Methanobacterium thermoautotrophicum

Growth of Methanobacterium thermoautotrophicum could be followed by measuring the intensity of fluorescence directly in the culture vessel, avoiding conventional time-consuming extraction procedures of fluorescent coenzymes. The influence of light scattering by the bacteria was investigated. It could be shown, that light scattering had only little effect on the measurement of coenzyme F₄₂₀ fluorescence. However, culture fluorescence did not correlate to methanogenic activity, due to superposition of bacterial fluorescence by fluorescence from cell-free coenzyme which accumulates in the culture medium. By use of time-resolved laser spectroscopy, different fluorescence lifetimes were obtained for intracellular (1.0 ns) and extracellular (2.5 ns) components, respectively. A combination of this technique with photobleaching measurements for direct determination of F₄₂₀ content of bacteria in a culture is proposed.     

Metabolic characteristics of an aerobe isolated from a methylotrophic methanogenic enrichment culture

An anaerobic methylotrophic methanogenic enrichment culture, with sustained metabolic characteristics, including that of methanation for over a decade, was the choice of the present study on interspecies interactions. Growth and methanation by the enrichment were suppressed in the presence of antibiotics, and no methanogen grown on methanol could be isolated using stringent techniques. The present study confirmed syntrophic metabolic interactions in this enrichment with the isolation of a strain ofPseudomonas sp. The organism had characteristic metabolic versatility in metabolizing a variety of substrates including alcohols, aliphatic acids, amino acids, and sugars. Anaerobic growth was favoured with nitrate in the growth medium. Cells grown anaerobically with methanol, revealed maximal nitrate reductase activity. Constitutive oxidative activity of the membrane system emerged from the high-specific oxygen uptake and nitrate reductase activities of the aerobically and anerobically grown cells respectively. Cells grown anaerobically on various alcohols effectively oxidized methanol in the presence of flavins, cofactor FAD and the methanogenic cofactor F₄₂₀, suggesting a constitutive alcohol oxidizing capacity. In cells grown anaerobically on methanol, the rate of methanol oxidation with F₄₂₀ was three times that of FAD. Efficient utilization of alcohols in the presence of F₄₂₀ is a novel feature of the present study. The results suggest that utilization of methanol by the mixed culture would involve metabolic interactions between thePseudomonas sp. and the methanogen(s). Methylotrophic, methanogenic partnership involving an aerobe is a novel feature hitherto unreported among anaerobic syntrophic associations and is of ecological significance.     

Methanogenic potential activity of mixed liquors: Fluorimetric monitoring

Summary Fluorimetric monitoring of potential methanogenic activity (coenzyme F₄₂₀) of bacterial community from methane digesters is until now resstricted to a few particular cases because severe interference frequnetly occurs, or the concentration is too low when digesting highly diluted wastes. A method of purification and/or concentration, based on purification of the cellular fraction of the sample by centrifugation and washing, is proposed. In biomethanation of spent liquor from citric acid fermentation, a typical case where interference largely obscures the F₄₂₀ fluorescence signal, precision is shown to be better than ± 5% (confidence interval [t.05 (3)]) for F₄₂₀ concentration down to 200 nM, and the detection limit is below 10 nM.     

Interconversion of F430 derivatives of methanogenic bacteria

F₄₃₀ is the prosthetic group of the methylcoenzyme M reductase of methanogenic bacteria. The compound isolated from Methanosarcina barkeri appears to be identical to the one obtained from the only distinctly related Methanobacterium thermoautotrophicum. F₄₃₀ is thermolabile and in the presence of acetonitrile or C10 _in4 ^sup- two epimerization products are obtained upon heating; in the absence of these compounds F₄₃₀ is oxidized to 12, 13-didehydro-F₄₃₀. The latter is stereoselectively reduced under H₂ atmosphere to F₄₃₀ by cell-free extracts of M. barkeri or M. thermoautotrophicum. H₂ may be replaced by the reduced methanogenic electron carrier coenzyme F₄₂₀.Abbreviations CH₃S-CoM methylcoenzyme M, 2-methylthioethanesulfonic acid - HS-CoM coenzyme M, 2-mercaptoethanesulfonic acid - F₄₃₀ Ni(II) tetrahydro-(12, 13)-corphin with a uroporphinoid (III) ligand skeleton - 13-epi-F₄₃₀ and 12,13-di-epi-F₄₃₀ the 12, 13- and 12, 13-derivatives of F₄₃₀ - 12, 13-didehydro-F₄₃₀ F₄₃₀ oxidized at C-12 and C-13 - coenzyme F₄₂₀ 7,8-didemethyl-8-hydroxy-5-deazaflavin derivative - coenzyme F₄₂₀H₂ reduced coenzyme F₄₂₀ - MV⁺ methylviologen semiquinone - HPLC high-performance liquid chromatography     

Comparison of coenzyme F420 from Methanobacterium bryantii with 7- and 8-hydroxy-10-methyl-5-deazaisoalloxazine

Syntheses of 7-hydroxy-10-methyl-5-deazaisoalloxazine (7-HMDI) and 8-hydroxy-10-methyl-5-deazaisoalloxazine (8-HMDI) are described. The physicochemical and biological properties of 8-HMDI, in contrast to those of 7-HMDI, are very analogous to those of F₄₂₀, a coenzyme found in methanogenic bacteria.     

Immunogold localization of coenzyme F420-reducing formate dehydrogenase and coenzyme F420-reducing hydrogenase in Methanobacterium formicicum

The ultrastructural locations of the coenzyme F₄₂₀-reducing formate dehydrogenase and coenzyme F₄₂₀-reducing hydrogenase of Methanobacterium formicicum were determined using immunogold labeling of thin-sectioned, Lowicryl-embedded cells. Both enzymes were located predominantly at the cell membrane. Whole cells displayed minimal F₄₂₀-dependent formate dehydrogenase activity or F₄₂₀-dependent hydrogenase activity, and little activity was released upon osmotic shock treatment, suggesting that these enzymes are not soluble periplasmic proteins. Analysis of the deduced amino acid sequences of the formate dehydrogenase subunits revealed no hydrophobic regions that could qualify as putative membrane-spanning domains.Abbreviation PBST Phosphate-buffered saline containing 0.1% (v/v) Triton X-100     

Quantification of coenzyme F420 analogues from methanogenic bacteria by HPLC and fluorimetry

Summary A quantitative assay for analogues of coenzyme F₄₂₀ is presented. The assay combines separation of the coenzymes by binary reversed-phase high performance liquid chromatography with detection by fluorescence, yielding high specificity and sensitivity. Quantification is by calibration with a coenzyme F₄₂₀ standard or by employing coenzyme F₄₂₀ fragments as internal standards.     

Tat–Dependent Translocation of an F420–Binding Protein of Mycobacterium tuberculosis

F₄₂₀ is a unique cofactor present in a restricted range of microorganisms, including mycobacteria. It has been proposed that F₄₂₀ has an important role in the oxidoreductive reactions of Mycobacterium tuberculosis, possibly associated with anaerobic survival and persistence. The protein encoded by Rv0132c has a predicted N–terminal signal sequence and is annotated as an F₄₂₀–dependent glucose-6-phosphate dehydrogenase. Here we show that Rv0132c protein does not have the annotated activity. It does, however, co–purify with F₄₂₀ during expression experiments in M. smegmatis. We also show that the Rv0132c–F₄₂₀ complex is a substrate for the Tat pathway, which mediates translocation of the complex across the cytoplasmic membrane, where Rv0132c is anchored to the cell envelope. This is the first report of any F₄₂₀–binding protein being a substrate for the Tat pathway and of the presence of F₄₂₀ outside of the cytosol in any F₄₂₀–producing microorganism. The Rv0132c protein and its Tat export sequence are essentially invariant in the Mycobacterium tuberculosis complex. Taken together, these results show that current understanding of F₄₂₀ biology in mycobacteria should be expanded to include activities occurring in the extra-cytoplasmic cell envelope.     

High-yield production of coenzyme F420 in Escherichia coli by fluorescence-based screening of multi-dimensional gene expression space

Coenzyme F₄₂₀ is involved in bioprocesses such as biosynthesis of antibiotics by streptomycetes, prodrug activation in Mycobacterium tuberculosis, and methanogenesis in archaea. F₄₂₀-dependent enzymes also attract interest as biocatalysts in organic chemistry. However, as only low F₄₂₀ levels are produced in microorganisms, F₄₂₀ availability is a serious bottleneck for research and application. Recent advances in our understanding of the F₄₂₀ biosynthesis enabled heterologous overproduction of F₄₂₀ in Escherichia coli, but the yields remained moderate. To address this issue, we rationally designed a synthetic operon for F₄₂₀ biosynthesis in E. coli. However, it still led to the production of low amounts of F₄₂₀ and undesired side-products. In order to strongly improve yield and purity, a screening approach was chosen to interrogate the gene expression-space of a combinatorial library based on diversified promotors and ribosome binding sites. The whole pathway was encoded by a two-operon construct. The first module (“core”) addressed parts of the riboflavin biosynthesis pathway and F_O synthase for the conversion of GTP to the stable F₄₂₀ intermediate F_O. The enzymes of the second module (“decoration”) were chosen to turn F_O into F₄₂₀. The final construct included variations of T7 promoter strengths and ribosome binding site activity to vary the expression ratio for the eight genes involved in the pathway. Fluorescence-activated cell sorting was used to isolate clones of this library displaying strong F₄₂₀-derived fluorescence. This approach yielded the highest titer of coenzyme F₄₂₀ produced in the widely used organism E. coli so far. Production in standard LB medium offers a highly effective and simple production process that will facilitate basic research into unexplored F₄₂₀-dependent bioprocesses as well as applications of F₄₂₀-dependent enzymes in biocatalysis.