Chemical composition of the peptidoglycan-free cell walls of methanogenic bacteria

Cell walls were prepared from freeze-dried samples of 7 strains of Methanobacterium by mechanical disintegration of the cells followed by incubation with trypsin. Electron microscopy revealed the presence of sacculi exhibiting the shape of the original cells, on which no surface structure could be detected. Ultrathin sections of the isolated sacculi showed a homogenously electron dense layer of about 10–15 nm in width. The ash content varied between 8 and 18% of dry weight. The sacculi of all the strains contained Lys: Ala: Glu: GlcNAc or GalNAc in a molar ratio of about 1:1.2:2:1. In one strain (M. ruminantium M 1) alanine is replaced by threonine, however. Neutral sugars and-in some strains-additional amounts of the amino sugars were present in variable amounts, and could be removed by formamide extraction or HF treatment without destroying the sacculi. No muramic acid or d-amino acids typical of peptidoglycan were found. Therefore, the sacculi of the methanobacteria consist of a different polymer containing a set of three l-amino acids and one N-acetylated amino sugar. From cells of Methanospirillum hungatii no sacculi, but tube-like sheaths could be isolated, which tend to fracture perpendicularly to the long axis of the sheath along the fibrills seen on the surface. The sheaths consist of protein containing 18 amino acids and small amounts of neutral sugars. They are resistent to the proteinases tested and are not disintegrated by boiling in 2% sodium dodecylsulfate for 30 min.The three Gram-negative strains Black Sea isolate JR-1, Cariaco isolate JR-1 and Methanobacterium mobile do not contain a rigid sacculus, but merely a SDS-sensitive surface layer composed of regularly arranged protein subunits. This evidence indicates that, within the methanogens, different cell wall polymers characteristic of particular groups of organisms may have evolved during evolution, and supports the hypothesis that the evolution of the methanogens was separated from that of the peptidoglycan-containing procaryotic organisms at a very early stage.Non Standard Abbreviations SDS sodium dodecylsulfate - EDTA ethylenediaminetetra acetic acid - DNP dinitrophenyl Dedicated to Prof. Dr. Adolf Butenandt on the occasion of his 75th birthday     

Pathways of energy conservation in methanogenic archaea

Methanogenic archaea are strictly anaerobic organisms that derive their metabolic energy from the conversion of a restricted number of substrates to methane. H₂+CO₂ and formate are converted to CH₄ via the CO₂-reducing pathway, while methanol and methylamines are metabolized by the methylotrophic pathway. A limited number of methanogenic organisms utilize acetate by the aceticlastic pathway. Redox reactions involved in these processes are partly catalyzed by membrane-bound enzyme systems that generate or, in the case of endergonic reactions, use electrochemical ion gradients. The H₂:heterodisulfide oxidoreductase, the F₄₂₀H₂:heterodisulfide oxidoreductase and the CO:heterodisulfide oxidoreductase, are novel systems that generate a proton motive force by redox-potential-driven H⁺ translocation. The methyltetrahydromethanopterin:coenzyme M methyltransferase is a unique, reversible sodium ion pump that couples methyl transfer with the transport of Na⁺ across the cytoplasmic membrane. Formylmethanofuran dehydrogenase is a reversible ion pump that catalyzes formylation and deformylation, of methanofuran. In summary, the pathways are coupled to the generation of an electrochemical sodium ion gradient and an electrochemical proton gradient. Both ion gradients are used directly for ATP synthesis via membrane integral ATP synthases. The function of the above-mentioned systems and their components in the metabolism of methanogens are described in detail.Abbreviations DCCD N,N dicyclohexylcarbodiimide - F ₄₂₀ (N-l-Lactyl--l-glutamyl)-l-glutamic acid phosphodiester of 7,8 didemethyl-8-hydroxy-5-deazariboflavin-5-phosphate - H ₄MPT Tetrahydromethanopterin - HS-CoM 2-Mercaptoethanesulfonate - HS-HTP 7-Mercaptoheptanoyl-O-phospho-l-threonine - MF Methanofuran - Ms Methanosarcina - Mc Methanococcus - Mb Methanobacterium - SF 6847 3,5-Di-tert-butyl-4-hydroxybenzylidene-malononitrile - Electrochemical sodium ion gradient - Electrochemical proton gradient     

One carbon metabolism in methanogenic bacteria

Two strains of Methanosarcina (M. Barkeri strain MS, isolated from sewage sludge, and strain UBS, isolated from lake sediments) were found to have similar cellular properties and to have DNA base compositions of 44 mol percent guanosine plus cytosine. Strain MS was selected for further studies of its one-carbon metabolism. M. barkeri grew autotrophically via H₂ oxidation/CO₂ reduction. The optimum temperature for growth and methanogenesis was 37°C. H₂ oxidation proceeded via an F₄₂₀-dependent NADP⁺-linked hydrogenase. A maximum specific activity of hydrogenase in cell-free extracts, using methyl viologen as electron acceptor, was 6.0 mol min · mg protein at 37°C and the optimum pH (9.0). M. barkeri also fermented methanol andmethylamine as sole energy sources for growth. Cell yields during growth on H₂/CO₂ and on methanol were 6.4 and 7.2 mg cell dry weight per mmol CH₄ formed, respectively. During mixotrophic growth on H₂/CO₂ plus methanol, most methane was derived from methanol rather than from CO₂. Similar activities of hydrogenase were observed in cell-free extracts from H₂/CO₂-grown and methanol-grown cells. Methanol oxidation apparently proceeded via carrierbound intermediates, as no methylotrophy-type of methanol dehydrogenase activity was observed in cell-free extracts. During growth on methanol/CO₂, up to 48% of the cell carbon was derived from methanol indicating that equivalent amounts of cell carbon were derived from CO₂ and from an organic intermediate more reduced than CO₂. Cell-free extracts lacked activity for key cell carbon synthesis enzymes of the Calvin cycle, serine path, or hexulose path.Abbreviations CAPS cycloaminopropane sulfonic acid - CH₃-SCoM methyl coenzyme M - DCPIP 2,6-dichlorophenolindophenol - DEAE diethylaminoethyl - dimethyl POPOP 1,4-bis-2-(4-mothyl-5-phenyloxazolyl)-benzene - DNA deoxyribonucleic acid - dpm dismtegrations per min - DTT dithiothreitol - EDTA ethylenediamine tetraacetic acid - F₄₂₀ factor 420 - G+C guanosine plus cytosine - NAD⁺ nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - PBBW phosphate buffered basal Weimer - PMS phenazine methosulfate - PPO 2,5-diphenyloxazole - rRNA ribosomal ribonucleic acid - RuBP ribulose-1,5-bisphosphate - Tris tris-hydroxymethyl-aminomethane - max maximum specific growth rate     

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     

Isomerization and biodegradation of beta-cypermethrin by Pseudomonas aeruginosa CH7 with biosurfactant production

Pseudomonas aeruginosa CH7, isolated from activated sludge, was able not only to isomerize and degrade beta-cypermethrin but also to utilize it as the sole source of carbon and energy for growth and produce biosurfactant. The strain effectively degraded beta-cypermethrin with inocula biomass of 0.1-0.2 g L⁻¹ at 25-35 °C, pH 6-9, and a final concentration of beta-cypermethrin 25-900 mg L⁻¹. Via response surface methodology analysis, we found the optimal condition was 29.4 °C, pH 7.0, and inocula biomass of 0.15 g L⁻¹; under these conditions, about 90% of the beta-cypermethrin could be degraded within 12 days. Noticeably, biosurfactant was detected in the MSM culture of strain CH7, suggesting that the biosurfactant (rhamnolipid) could potentially enhance the degradation of beta-cypermethrin by promoting the dissolution, adsorption, and absorption of the hydrophobic compounds. Therefore, CH7 may serve as a promising strain in the bioremediation of wastewater and soil polluted by beta-cypermethrin.     

Effect of coculture of anaerobic fungi isolated from ruminants and non-ruminants with methanogenic bacteria on cellulolytic and xylanolytic enzyme activities

Neocallimastix strain N1, an isolate from a ruminant (sheep), was cocultured with three Methanobacterium formicicum strains, Methanosarcina barkeri, and Methanobrevibacter smithii. The coculture with Methanobacterium formicicum strains resulted in the highest production of cellulolytic and xylanolytic enzymes. Subsequently four anaerobic fungi, two Neocallimastix strains (N1 and N2) from a ruminant and two Piromyces species from non-ruminants (E2 and R1), were grown in coculture with Methanobacterium formicicum DSM 3637 on filter paper cellulose and monitored over a 7-day period for substrate utilisation, fermentation products, and secretion of cellulolytic and xylanolytic enzymes. Methanogens caused a shift in fermentation products to more acetate and less ethanol, lactate and succinate. Furthermore the cellulose digestion rate increased by coculture. For cocultures of Neoallimastix strains with Methanobacterium formicicum strains the cellulolytic and xylanolytic enzyme production increased. Avicelase, CMCase and xylanase were almost completely secreted into the medium, while 40–60% of the -glucosidase was found to be cell bound. Coculture had no significant effect on the location of cellulolytic and xylanolytic enzymes.     

Competition between sulfate-reducing and methanogenic bacteria for H2 under resting and growing conditions

The basis for the outcome of competition between sulfidogens and methanogens for H₂ was examined by comparing the kinetic parameters of representatives of each group separately and in co-culture. Michaelis-Menten parameters (V _max and K _m) for four methanogens and five sulfate-reducing bacteria were determined from H₂-depletion data. Further, Monod growth parameters (_max, K _s, Y _H2) for Desulfovibrio sp. G11 and Methanospirillum hungatei JF-1 were similarly estimated. H₂ K _m values for the methanogenic bacteria ranged from 2.5 M (Methanospirillum PM1) to 13 M for Methanosarcina barkeri MS; Methanospirillum hungatei JF-1 and Methanobacterium PM2 had intermediate H₂ K _m estimates of 5 M. Average H₂ K _m estimates for the five sulfidogens was 1.2 M. No consistent difference among the V _max estimates for the above sulfidogens (mean=100 nmol H₂ min^-1 mg^-1 protein) and methanogens (mean=110 nmol H₂ min^-1 mg^-1 protein) was found. A two-term Michaelis-Menten equation accurately predicted the apparent H₂ K _m values and the fate of H₂ by resting co-cultures of sulfate-reducers and methanogens. Half-saturation coefficients (K _s) for H₂-limited growth of Desulfovibrio sp. G11 (2–4 M) and Methanospirillum JF-1 (6–7 M) were comparable to H₂ K _m estimates obtained for these organisms. Maximum specific growth rates for Desulfovibrio sp. G11 (0.05 h^-1) were similar to those of Methanospirillum JF-1 (0.05–0.06 h^-1); whereas G11 had an average yield coefficient 4 x that of JF-1. Calculated _max and V _max/K _m values for the methanogens and sulfidogens studied predict that the latter bacterial group will process more H₂ whether these organisms are in a growing or resting state, when the H₂ concentration is in the first-order region.     

Quantification of ratios of Bacteria and Archaea in methanogenic microbial community by fluorescence in situ hybridization and fluorescence spectrometry

16S rRNA-targeted oligonucleotide probes for Bacteria (Eub338) and Archaea (Arc915) were used for whole-cell, fluorescence in situ hybridization (FISH) to quantify the ratio of these microbial groups in an anaerobic digester. The quantity of specifically bound (hybridized) probe was measured by fluorescence spectrometry and evaluated by analysing the dissociation curve of the hybrids, by the measurement of the binding with a nonsense probe, and by the competitive inhibition of the binding of the labelled probe by the corresponding unlabelled probe. Specific binding of oligonucleotide probes with the biomass of anaerobes was 40–50% of their total binding. The ratio of Arc915 and Eub338 probes hybridized with rRNAs of the cells in anaerobic sludge was 0.50. Measurement of FISH by fluorescence spectrometry appears to be a suitable method for quantification of the microbial community of anaerobes.     

Carbon monoxide-dependent energy metabolism in anaerobic bacteria and archaea

Despite its toxicity for the majority of living matter on our planet, numerous microorganisms, both aerobic and anaerobic, can use carbon monoxide (CO) as a source of carbon and/or energy for growth. The capacity to employ carboxidotrophic energy metabolism anaerobically is found in phylogenetically diverse members of the Bacteria and the Archaea. The oxidation of CO is coupled to numerous respiratory processes, such as desulfurication, hydrogenogenesis, acetogenesis, and methanogenesis. Although as diverse as the organisms capable of it, any CO-dependent energy metabolism known depends on the presence of carbon monoxide dehydrogenase. This review summarizes recent insights into the CO-dependent physiology of anaerobic microorganisms with a focus on methanogenic archaea. Carboxidotrophic growth of Methanosarcina acetivorans, thought to strictly rely on the process of methanogenesis, also involves formation of methylated thiols, formate, and even acetogenesis, and, thus, exemplifies how the beneficial redox properties of CO can be exploited in unexpected ways by anaerobic microorganisms.     

Biomass Enhancement in Maize and Soybean in Response to Glutamate Dehydrogenase Isomerization

The relationship between nutrient composition, crop biomass, and glutamate dehydrogenase (GDH) isoenzyme pattern was investigated in soybean (Glycine max) and maize (Zea mays) by monitoring the nutrient induced isomerization of the enzyme from the seedling stage to the mature crop. GDH was extracted from the leaves of the plants, and the isoenzymes were fractionated by isoelectric focusing followed by native polyacrylamide gel electrophoresis. The isomerization V_max values for soybean GDH, similar to maize GDH increased curvilinearly from 200 – 400 μmol mg⁻¹ min⁻¹ as the inorganic phosphate nutrient applied to the soil decreased from 50 − 0 mM. In soybean, combinations of N and K, P, or S nutrients induced the acidic and neutral isoenzymes, and gave biomass increases 25 – 50 % higher than the control plant. GDH isoenzymes were suppressed in soybean that received nutrients without N, K, or P and accordingly the biomass was about 30 % lower than the control. Treatment of maize with NPK nutrients increased the GDH V_max values from 138.9 at the vegetative to 256.4 μmol mg⁻¹ min⁻¹ at the reproductive phase, and suppressed the basic isoenzymes, but induced both the acidic and neutral isoenzymes thereby inducing seed production (27.0 ± 1.4 g per plant); whereas both the acidic and basic isoenzymes were suppressed in the control maize, and seeds did not develop. Simultaneous induction of the acidic, neutral, and basic isoenzymes of GDH indicated the occurrence of senescence. Therefore in maize and soybean, the induction of the acidic and basic isoenzymes of GDH led to the enhancement of biomass. This revised version was published online in July 2006 with corrections to the Cover Date.     

Involvement of the ntrA gene product in the anaerobic metabolism of Escherichia coli

Summary The ntrA gene product, required for expression of genes involved in nitrogen fixation (nif) and regulation (ntr), was shown to be necessary for the expression of the two enzymes of the anaerobically inducible formate hydrogenlyase (FHL) pathway, formate dehydrogenase (FDH_H) and hydrogenase isoenzyme 3. Consistent with this finding, the gene encoding the selenopolypeptide (fdhF) of FDH_H was shown to have a nif consensus promoter. The levels of six other anaerobically inducible enzymes were examined and found to be ntrA independent. Significantly, these latter six enzymes are dependent upon the fnr gene product for their expression while FDH_H and hydrogenase 3 are fnr independent. These findings indicate that there are at least two classes of anaerobically regulated promoters: one class which is ntrA dependent and fnr independent and a second class which is fnr dependent and ntrA independent.     

Interrelation between the pathways of isoprenoid biosynthesis and carbon source catabolism in anaerobic and facultatively anaerobic bacteria

Data on the interrelation between the pathways of the carbon source catabolism and isoprenoid biosynthesis in anaerobic and facultatively anaerobic bacteria were obtained. Two pathways of isoprenoid biosynthesis (nonmevalonate and mevalonate) were revealed in the representatives of the genus Clostridium. The nonmevalonate pathway of isoprenoid biosynthesis and the glycolytic pathway of substrate oxidation are typical of glucose-grown bacteria, whereas the pentose phosphate cycle operates in xylose-grown bacteria. The mevalonate pathway of isoprenoid biosynthesis was revealed in strain Clostridium thermosaccharolyticum DSM 571 grown in the presence of mevinolin, as well as in a number of lactic acid bacteria. Mevinolin is known to react with the lactate dehydrogenase complex, preventing reduction of pyruvate. The nonmevalonate pathway of isoprenoid biosynthesis was revealed in Bifidobacterium bifidum. The role of different metabolic pathways in isoprenoid biosynthesis is discussed.     

Oxidation of ethanol by methanogenic bacteria

Methanogenium organophilum, a non-autotrophic methanogen able to use primary and secondary alcohols as hydrogen donors, was grown on ethanol. Per mol of methane formed, 2 mol of ethanol were oxidized to acetate. In crude extract, an NADP⁺-dependent alcohol dehydrogenase (ADH) with a pH optimum of about 10.0 catalyzed a rapid (5 mol/min·mg protein; 22°C) oxidation of ethanol to acetaldehyde; after prolonged incubation also acetate was detectable. With NAD⁺ only 2% of the activity was observed. F₄₂₀ was not reduced. The crude extract also contained F₄₂₀: NADP⁺ oxidoreductase (0.45 mol/min·mg protein) that was not active at the pH optimum of ADH. With added acetaldehyde no net reduction of various electron acceptors was measured. However, the acetaldehyde was dismutated to ethanol and acetate by the crude extract. The dismutation was stimulated by NADP⁺. These findings suggested that not only the dehydrogenation of alcohol but also of aldehyde to acid was coupled to NADP⁺ reduction. If the reaction was started with acetaldehyde, formed NADPH probably reduced excess aldehyde immediately to ethanol and in this way gave rise to the observed dismutation. Acetate thiokinase activity (0.11 mol/min·mg) but no acetate kinase or phosphotransacetylase activity was observed. It is concluded that during growth on ethanol further oxidation of acetaldehyde does not occur via acetylCoA and acetyl phosphate and hence is not associated with substrate level phosphorylation. The possibility exists that oxidation of both ethanol and acetaldehyde is catalyzed by ADH. Isolation of a Methanobacterium-like strain with ethanol showed that the ability to use primary alcohols also occurs in genera other than Methanogenium.Non-standard abbreviations ADH alcohol dehydrogenase - Ap₅ALi₃ P¹,P⁵-Di(adenosine-5-)pentaphosphate - DTE dithioerythritol (2,3-dihydroxy-1,4-dithiolbutane) - F₄₂₀ N-(N-l-lactyl--l-glutamyl)-l-glutamic acid phosphodiester of 7,8-dimethyl-8-hydroxy-5-deazariboflavin-5-phosphate - Mg. Methanogenium - OD₅₇₈ optical density at 578 nm - PIPES 1,4-piperazine-diethanesulfonic acid - TRICINE N-(2-hydroxy-1,1-bis[hydroxymethyl]methyl)-glycine - Tris 2-amino-2-hydroxy-methylpropane-1,3-diol - U unit (mol substrate/min)     

Isolation and characterization of mutants of mesophilic methanogenic bacteria resistant to analogues of DNA bases and nucleosides

Spontaneous mutants of mesophilic Methanobacterium, Methanobrevibacter and Methanosarcina species resistant to 6-mercaptopurine, 5-fluorouracil, 8-azaguanine, 6-azauracil or 5-fluorodeoxyuridine were isolated. Low level resistant mutants were unstable but highly resistant strains (resistance factor greater than 10-fold) were stable and showed growth characteristics comparable to the parent. Wild type strains showed linear uptake of hypoxanthine and uracil into cells, but guanine uptake was only detected in Methanosarcina mazei. 6-Mercaptopurine-resistant clones of Methanobacterium and Methanobrevibacter species and 8-azaguanine-resistant clones of Methanosarcina mazei showed reduced uptake of hypoxanthine and guanine respectively, but no evidence for altered permeability of 5-fluoro-and 6-azauracil-resistant strains to uracil was obtained. Double resistant mutants of Methanobacterium sp. strain FR-2 were characterised. Although these generally exhibited reduced specific growth rates, several were selected which showed similar growth to the parent.Abbreviations DSM Deutsche Sammlung von Mikroorganismen, Federal Republic of Germany - MJC minimum inhibitory concentration - cfu colony forming unit - MP 6-mercaptopurine - FU 5-fluorouracil - FDU 5-fluorodeoxyuridine - AG 8-azaguanine - AU 6-azauracil - DA l-deazaadenosine     

Studies on the biosynthesis of coenzyme F420 in methanogenic bacteria

Coenzyme F₄₂₀ is a 8-hydroxy-5-deazaflavin present in methanogenic bacteria. We have investigated whether the pyrimidine ring of the deazaflavin originates from guanine as in flavin biosynthesis, in which the pyrimidine ring of guanine is conserved. For this purpose the incorporation of [2-¹⁴C]guanine and of [8-¹⁴C]guanine into F₄₂₀ by growing cultures of Methanobacterium thermoautotrophicum was studied. Only in the case of [2-¹⁴C]guanine did F₄₂₀ become labeled. The specific radioactivity of the deazaflavin and of guanine isolated from nucleic acids of [2-¹⁴C]guanine grown cells were identical. This finding suggests that the pyrimidine ring of the deazaflavin and of flavins are synthesized by the same pathway.F₄₂₀ did not become labeled when M. thermoautotrophicum was grown in the presence of methyl-[¹⁴C] methionine, [U-¹⁴C]phenylalanine or [U-¹⁴C]tyrosine. This excludes that C-5 of the deazaflavin is derived from the methyl group of methionine and that the benzene ring comes from phenylalanine or tyrosine.     

Elimination of n-butylated hydroxytoluene methylation during fatty acid analysis by gas chromatography

An improved method for fatty acids analysis with optimum recovery of highly polyunsaturated fatty acids methyl esters in biological systems is presented. The method is based on transesterification of phospholipid and triacylglycerols to fatty acid methyl esters using a commercially available reagent, Methyl-Prep II. Without proper precautions, as much as 50% of n-butylated hydroxytoluene (BHT) added to prevent oxidation of polyunsaturated fatty acids, could be methylated during the transesterification step. Methylated BHT elutes close to 14:0 (myristic acid) and no longer functions as an antioxidant, but the modified conditions virtually eliminate the methylation of BHT. Sample extraction and methylation was completed in 30 min at room temperature. A chelator (diethylenetriamine-pentaacetic acid; DTPA) is also added to prevent peroxidation of metal catalyzed free radical chain reactions. The standard deviations of the major fatty acids from multiple human plasma samples prepared on different days were less than 5%. The recovery of arachidonic acid, 20:4, from plasma was improved using the new method, and the recovery for docosahexaenoic acid, 22:6, spiked to human plasma was found to be 99%.     

Lipid Formation by Aqueous Fischer-Tropsch-Type Synthesis over a Temperature Range of 100 to 400 °C

The formation of lipid compounds during anaqueous Fischer-Tropsch-type reaction was studied withsolutions of oxalic acid as the carbon and hydrogensource. The reactions were conducted in stainlesssteel vessels by heating the oxalic acid solution atdiscrete temperatures from 100 to 400 °C, atintervals of 50 °C for two days each. Themaximum lipid yield, especially for oxygenatedcompounds, is in the window of 150–250 °C. At atemperature of 100 °C only a trace amount oflipids was detected. At temperatures above150 °C the lipid components ranged from C₁₂to >C₃₃ and included n-alkanols, n-alkanoic acids, n-alkyl formates, n-alkanals, n-alkanones, n-alkanes, andn-alkenes, all with essentially no carbon numberpreference. The n-alkanes increased inconcentration over the oxygenated compounds attemperatures of 200 °C and above, with a slightreduction in their carbon number ranges due tocracking. It was also noted that the n-alkanoicacids increased while n-alkanols decreased withincreasing temperature above 200 °C. Attemperatures above 300 °C synthesis competeswith cracking and reforming reactions. At 400 °Csignificant cracking was observed and polynucleararomatic hydrocarbons and their alkylated homologswere detected. The results of this work suggest thatthe formation of lipid compounds by aqueous FTTreactions proceeds by insertion of a CO group at theterminal end of a carboxylic acid functionality toform n-oxoalkanoic acids, followed by reductionto n-alkanoic acids, to n-alkanals, thento n-alkanols. The n-alkenes areintermediate homologs for n-alkan-2-ones andn-alkanes. This proposed mechanism for aqueousFTT synthesis differs from the surface-catalyzedstepwise FT process (i.e., gaseous) of polymerization of methylene reported in the literature.     

Identification of replication origins in the genome of the methanogenic archaeon,<Emphasis Type="Italic"> Methanocaldococcus jannaschii</Emphasis>

Methanocaldococcus jannaschii has been notorious as an archaeon in which the replication origins are difficult to identify. Although extensive efforts have been exerted on this issue, the locations of replication origins still remain elusive 7 years after the publication of its complete genome sequence in 1996. Ambiguous results were obtained in identifying the replication origins of M. jannaschii based on all theoretical and experimental approaches. In the genome of M. jannaschii, we found that an ORF (MJ0774), annotated as a hypothetical protein, is a homologue of the Cdc6 protein. The position of the gene is at a global minimum of the x component of the Z curve, i.e., RY disparity curve, which has been used to identify replication origins in other Archaea. In addition, an intergenic region (694,540–695,226 bp) that is between the cdc6 gene and an adjacent ORF shows almost all the characteristics of known replication origins, i.e., it is highly rich in AT composition (80%) and contains multiple copies of repeat elements and AT stretches. Therefore, these lines of evidence strongly suggest that the identified region is a replication origin, which is designated as oriC1. The analysis of the y component of the Z curve, i.e., MK disparity curve, suggests the presence of another replication origin corresponding to one of the peaks in the MK disparity curve at around 1,388 kb of the genome.Communicated by G. Antranikian     

The aerobic and anaerobic microbiology of pustular acne lesions

Specimens from 32 pustular acne lesions that were inoculated on media supportive for the growth of aerobic and anaerobic bacteria showed bacterial growth. Only aerobic or facultative bacteria were recovered in 15 (47%) specimens, only anaerobic bacteria in 11 (34%) specimens, and mixed aerobic and anaerobic bacteria in 6 (18%) specimens. A total of 57 isolates, 31 anaerobes (1.0 per specimen) and 26 aerobes (0.8 per specimen) were recovered. The predominant isolates were Staphylococcus sp. (19 isolates), Peptostreptococcus sp. (15), and Propionibacterium sp. (10). Twelve (37.5%) of the comedones yielded only one organism. This retrospective study highlighted the polymicrobial nature of over two-thirds of culture positive pustular acne lesions and suggests the potential for pathogenic role of aerobic and anaerobic organisms other than P. acnes and Staphylococcus sp. in acne vulgaris.     

Production of hydrogen from marine macro-algae biomass using anaerobic sewage sludge microflora

Hydrogen was produced from various marine macro-algae (seaweeds) through anaerobic fermentation using an undefined bacterial consortium. In this study, anaerobic fermentation from various marine macro-algae for Ulva lactuca, Porphyra tenera, Undaria pinnatifida, and Laminaria japonica was studied. From this analysis Laminaria japorica was determined to be the optimum substrate for hydrogen production. When L. japornica was used as the carbon source for enhanced hydrogen production, the optimum fermentation temperature, substrate concentration, initial pH, and pretreatment condition were determined to be 35°C, 5%, 7.5, and BT120 (Ball mill and thermal treatments at 120°C for 30 min), respectively. In addition, hydrogen production was improved when the sludge was heat-treated at 65°C for 20 min. Under these conditions, about 4,164 mL of hydrogen was produced from 50 g/L of dry algae (L. japonica) for 50 h, with a hydrogen concentration around 34.4%. And the maximum hydrogen production rate and yield were found to be 70 mL/L·h and 28 mL/g dry algae, respectively.