Acetate photometabolism in Ectothiorhodospira shaposhnikovii]

The cells of Ectothiorhodospira were cultivated under autotrophic conditions and assimilated 14C-acetate in the presence of bicarbonate. The label was incorporated rapidly into phosphoglyceric acid (PGA) and phosphorous esters (PE) of sugars, into compounds of the tricarboxylic acid cycle (TAC), aspartate, glutamate. After some time the content of the label became the highest in glutamate and decreased in other compounds. The same products were formed upon the assimilation of acetate by the cells cultivated in its presence. However, the amount of PGA and PE of sugars, especially those formed in the presence of sulphides, was less, and their curve had a positive slope. Fluoroacetate inhibited the incorporation of 14C from acetate in the cells and caused an increase of labeled citrate. Iodacetate inhibited almost completely the fixation of CO2 by the cells in the presence of sulphide; the fixation of carbon dioxide constituted about 60 percent of the control if both sulphide and acetate were present in the medium. Therefore, the assimilation of acetate by Ect. shaposhnikovii can be accomplished via the glyoxylate shunt and TAC, and also as a result of action of pyruvate synthase and the production of C4- and C5-organic acids with the participation of CO2. The pathways of acetate metabolism depend on the growth conditions and on the presence of sulphide in the medium.     

Propionate metabolism of Ectothiorhodospira shaposhnikovii during growth in the dark

Assimilation of propionate by Ectothiorhodospira shaposhnikovii growing under the aerobic conditions in the dark, like in the light involves carboxylation of propionyl-CoA with the participation of biotin-dependent carboxylase. The succinate being formed is transformed in the reactions of the citric acid cycle and the glyoxylate shunt. This is corroborated by the determination of the enzyme activity manifested by cell extracts, by the composition and kinetics of labeled products, as well as by the action of the inhibitors of the citric acid cycle (fluoroacetate, malonate) and avidin on the assimilation of 14C-propionate and 14CO2 by the cells.     

Carbon dioxide fixation in the metabolism of propylene and propylene oxide by Xanthobacter strain Py2.

Evidence for a requirement for CO2 in the productive metabolism of aliphatic alkenes and epoxides by the propylene-oxidizing bacterium Xanthobacter strain Py2 is presented. In the absence of CO2, whole-cell suspensions of propylene-grown cells catalyzed the isomerization of propylene oxide (epoxypropane) to acetone. In the presence of CO2, no acetone was produced. Acetone was not metabolized by suspensions of propylene-grown cells, in either the absence or presence of CO2. The degradation of propylene and propylene oxide by propylene-grown cells supported the fixation of 14CO2 into cell material, and the time course of 14C fixation correlated with the time course of propylene and propylene oxide degradation. The degradation of glucose and propionaldehyde by propylene-grown or glucose-grown cells did not support significant 14CO2 fixation. With propylene oxide as the substrate, the concentration dependence of 14CO2 fixation exhibited saturation kinetics, and at saturation, 0.9 mol of CO2 was fixed per mol of propylene oxide consumed. Cultures grown with propylene in a nitrogen-deficient medium supplemented with NaH13CO3 specifically incorporated 13C label into the C-1 (major labeled position) and C-3 (minor labeled position) carbon atoms of the endogenous storage compound poly-beta-hydroxybutyrate. No specific label incorporation was observed when cells were cultured with glucose or n-propanol as a carbon source. The depletion of CO2 from cultures grown with propylene, but not glucose or n-propanol, inhibited bacterial growth. We propose that propylene oxide metabolism in Xanthobacter strain Py2 proceeds by terminal carboxylation of an isomerization intermediate, which, in the absence of CO2, is released as acetone.     

Growth of Seliberia carboxydohydrogena carboxy bacteria with an altered composition of the gas mixture

The growth and gas exchange of Seliberia carboxydohydrogena Z-1062 were studied in the regime of turbidostat when the conditions of gaseous nutrition were changed: a decrease in hydrogen concentration and an increase in carbon monoxide concentration, growth on two carbon sources (CO+CO2) and on two energy sources (H2+CO). The inhibition of the bacterial growth by CO was expressed in a decrease of the specific growth rate and in the reduced effectiveness of using a gaseous substrate. When the concentration of carbon monoxide was elevated from 0 to 40% and that of hydrogen was reduced from 80 to 40%, the specific growth rate of the cells was decreased from 0.4 to 0.04 h-1; here, the economic coefficient in terms of hydrogen fell from 3.6 to 0.62 g/g. The CO-oxidizing system of the bacterium was shown to be resistant. The rate of CO oxidation by the culture was from 0.6 to 0.8 L/h per 1 g of the synthesized biomass at the following concentration of gases in the medium (%); H2, 80-40; CO2, 5; O2, 15; CO, 10-40. The rate of CO oxidation by the culture rose when hydrogen concentration was decreased and CO concentration was increased.     

Reductive Pentose Cycle and Formate Assimilation in Rhodopseudomonas palustris

Rhodopseudomonas palustris assimilated formate autotrophically as carbon dioxide and hydrogen arising from the activity of the formic hydrogenlyase system. Kinetic analyses of cell suspensions pulse-labeled with (14)C-formate or (14)C-bicarbonate showed similar distributions of incorporated radioactivity. In both cases phosphate esters were the first assimilation products. Ribulose diphosphate carboxylase, phosphoribose isomerase, and phosphoribulokinase, characteristic enzymes of the reductive pentose cycle, were present in extracts of cells grown on formate.     

The omega-hydroxylation of arachidonic acid by human polymorphonuclear leukocytes

Incubations of [1-14C]arachidonic acid with unstimulated human polymorphonuclear leukocytes resulted in the formation of four new metabolites in a previously described reverse-phase HPLC system. Three of these metabolites were largely suppressed in a CO/O2 (80/20, by vol.) atmosphere indicating a cytochrome-P450-dependent monooxygenase reaction. In agreement with this assumption is their NADPH/O2-dependent formation in the microsomal fraction. One metabolite was identified by gas chromatography/mass spectrometry analysis as omega-hydroxy-arachidonic acid and the two others were secondary products identified as omega-carboxy-arachidonic acid and 5,20-dihydroxy-E,Z,Z,Z-6,8,11,14-eicosatetraenoic acid. Since the affinity for arachidonate of the omega-monooxygenase was quite low and the presence of LTB4 suppressed the omega-hydroxylation of arachidonate, we conclude that the known LTB4 omega-monooxygenase is responsible for the formation of omega-hydroxy-arachidonate. It is unlikely, however, that significant concentrations of these metabolites are formed by activated polymorphonuclear leukocytes in vivo. The fourth metabolite remains tightly associated with the leukocytes but has not been further characterized.     

Method for measuring substrate preferences by individual members of microbial consortia proposed for bioaugmentation

In this study we used the assimilation of isotope labeled CO(2) to measure the substrate preferences by two different bioaugmentation mixtures proposed for bioremediation of diesel oil contamination. All active microorganisms assimilate CO(2) in various carboxylation processes involved in growth. The CO(2) assimilation by the two mixtures was measured upon addition of glucose, diesel oil or specific compounds present in diesel oil (naphthalene, toluene, hexadecane, and octane). It was shown that within short term incubations with diesel oil (<5 h), one bioaugmentation mixture was superior to the other regarding the assimilation of CO(2). This observation was confirmed in a labor-intensive long term microcosm study (60 days). The applied method open various possibilities for fast pre-testing of substrate-preferences by microbial-bioaugmentation mixtures without microcosm experiments, onsite tests, and complicated chemical analysis. This study also demonstrates the possibility to obtain further information on the substrate preferences at a single cell level of phylogenetically defined microbial subgroups in bioaugmentation mixtures, based on combined analyses of microautoradiography and fluorescence in situ hybridization.     

Hydrothermal upgrading of biomass: effect of K2CO3 concentration and biomass/water ratio on products distribution

Catalytic hydrothermal treatment of wood biomass was performed at 280 degrees C for 15 min in the presence of K2CO3 with different concentrations and biomass/water ratio (thermal). Oil products were extracted from both liquid and solid portion by different solvents and analyzed them individually. The biomass to water ratio has an important effect on product distribution and composition of oil products. Oil 1 (ether extract) with K2CO3 contained mainly phenolic compounds. Benzenediol derivatives were observed with 0.94 M K2CO3 concentration and they were not formed at lower concentrations (0.235 and 0.47 M). The decrease of solid residue was achieved to 4% with 0.94 M K2CO3 at 280 degrees C for 15 min. The volatility distribution of hydrocarbons (ether extract) were characterized by using C-NP gram. The distribution of oxygenated hydrocarbons changed depending upon the biomass to water ratio and concentration of K2CO3 solution.     

Heterotrophic Carbon Metabolism by Beggiatoa alba

The assimilation and metabolism of CO(2) and acetate by Beggiatoa alba strain B18LD was investigated. Although B. alba was shown to require CO(2) for growth, the addition of excess CO(2) (as NaHCO(3)) to the medium in a closed system did not stimulate growth. Approximately 24 to 31% of the methyl-labeled acetate and 38 to 46% of the carboxyl-labeled acetate were oxidized to (14)CO(2) by B. alba. The apparent V(max) values for combined assimilation and oxidation of [2-(14)C]acetate by B. alba were 126 to 202 nmol min(-1) mg of protein(-1) under differing growth conditions. The V(max) values for CO(2) assimilation by heterotrophic and mixotrophic cells were 106 and 131 pmol min(-1) mg of protein(-1), respectively. The low V(max) values for CO(2) assimilation, coupled with the high V(max) values for acetate oxidation, suggested that the required CO(2) was endogenously produced from acetate. Moreover, exogenously supplied acetate was required by B. alba for the fixation of CO(2). From 61 to 73% of the [(14)C]acetate assimilated by washed trichomes was incorporated into lipid. Fifty-five percent of the assimilated [2-(14)C]acetate was incorporated into poly-beta-hydroxybutyric acid. This was consistent with chemical data showing that 56% of the heterotrophic cell dry weight was poly-beta-hydroxybutyric acid. Succinate and CO(2) were incorporated into cell wall material, proteins, lipids, nucleic acids, and amino and organic acids, but not into poly-beta-hydroxybutyric acid. Glutamate and succinate were the major stable products after short-term [1-(14)C]acetate assimilation. Glutamate and aspartate were the first stable (14)CO(2) fixation products, whereas glutamate, a phosphorylated compound, succinate, and aspartate were the major stable (14)CO(2) fixation products over a 30-min period. The CO(2) fixation enzymes isocitrate dehydrogenase (nicotinamide adenine dinucleotide phosphate; reversed) and malate dehydrogenase (nicotinamide adenine dinucleotide phosphate; decarboxylating) were found in cell-free extracts of both mixotrophically grown and heterotrophically grown cells. The data indicate that the typical autotrophic CO(2) fixation mechanisms are absent from B. alba B18LD and that the CO(2) and acetate metabolism pathways are probably linked.     

Biotransformation products and mineralization potential for hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in abiotic versus biological degradation pathways with anthraquinone-2,6-disulfonate (AQDS) and <Emphasis Type="Italic">Geobacter metallireducens</Emphasis>

This study investigated extracellular electron shuttle-mediated RDX biodegradation and the distribution of ring cleavage metabolites generated by biological degradation (cells) versus the products formed by abiotic degradation (reduced electron shuttles), and when the two pathways were acting simultaneously. All pathways were influenced by pH. Buffered suspensions (pH 6.8/7.9/9.2) were performed with cell-free anthrahydroquinone-2,6-disulfonate as the sole electron donor, cells (Geobacter metallireducens) + acetate, or cells/acetate + anthraquinone-2,6-disulfonate as an electron shuttle. The metabolites identified included methylenedinitramine, formaldehyde, nitrous oxide, nitrite, ammonium and carbon dioxide. As pH increased, the rates of RDX reduction by AH(2)QDS also increased. Cells alone reduced RDX faster at the lower pH values. However, at all pH the rates of the electron shuttle-mediated pathways were consistently the fastest, and the proportion of carbon present as formaldehyde, which is a precursor to mineralization, was highest in the presence of electron shuttles. Formaldehyde accounted for 45/51/54% of the carbon in electron shuttle amended cell suspensions as opposed to 13/42/45% of carbon without shuttles at the pH 6.8/7.9/9.2, respectively. Approximately 7-20% of RDX was mineralized to CO(2) in the presence of cells at all pH tested; AQDS increased the extent of (14)CO(2) produced. Nitrous oxide and nitrite were end products in the strictly abiotic pathway, but nitrite was depleted in the presence of cells to form ammonium. Understanding the different products formed in the abiotic versus biological pathways and the influence of pH is critical to developing mixed biotic-abiotic remediation strategies for RDX.     

Fermentation of Fructose and Synthesis of Acetate from Carbon Dioxide by Clostridium formicoaceticum

Clostridium formicoaceticum ferments fructose labeled with (14)C in carbon 1, 4, 5, or 6 via the Embden Meyerhof pathway. In fermentations of fructose in the presence of (14)CO(2), acetate is formed labeled equally in both carbons. Extracts convert the methyl groups of 5-methyltetrahydrofolate and methyl-B(12) to the methyl group of acetate in the presence of pyruvate. Formate dehydrogenase, 10-formyltetrahydrofolate synthetase, 5,10-methenyltetrahydrofolate cyclohydrolase, 5,10-methylenetetrahydrofolate dehydrogenase, and 5,10-methylenetetrahydrofolate reductase are present in extracts of C. formicoaceticum. These enzymes are needed for the conversion of CO(2) to 5-methyltetrahydrofolate. It is proposed that acetate is totally synthesized from CO(2) via the reactions catalyzed by the enzymes listed above and that 5-methyltetra-hydrofolate and a methylcorrinoid are intermediates in this synthesis.     

Fermentativer Abbau von 2-14C-Mannose durch Leuconostoc mesenteroides

Mannose-2-14C has been fermented by Leuconostoc mesenteroides, CO2 ethanol and D-lactic acid were formed in a molar ratio of 1:1:1. A small amount of acetic acid was found as by-product. It could easily be isolated from the main products of the fermentation and it did not disturb further degradation procedures. The methyl-C-atom of ethanol, which was derived from C-2 of the mannose, had nearly the same specific radioactivity as mannose-2-14C. All other C-atoms of the degradation products were only very slightly labeled. Their content of radioactivity was in any case lower than 3% of the specific radioactivity of the degraded mannose. This procedure is applicable for the degradation of 14C-labeled mannose.     

On methylamine assimilation in a bacterium

Summary Kinetic analyses of ¹⁴C-methylamine and ¹⁴C-bicarbonate assimilation in a gram-negative diplococcus suggests that methylamine assimilation occurs via a glycine-serine hydroxymethylation sequence rather than autotrophically by a Calvin cycle.Dedicated to Prof. C. B. van Niel on the occasion of his 70th birthday. These and related experiments were first reported in June, 1962, in a seminar in the General Microbiology course in Pacific Grove. The stimulating and pleasant atmosphere provided by Prof. Van Niel during this course and the subsequent visit in his laboratories is remembered with pleasure.     

Asparagine Biosynthesis by Cotton Roots: Carbon Dioxide Fixation and Cyanide Incorporation 1

Asparagine is the dominant amino acid in cotton root tips (Acala SJ-1). Two biosynthetic pathways may be operative. First, asparagine is an ultimate product of nonphotosynthetic CO(2) fixation. Whereas short term (14)CO(2) labeling experiments indicate that malate is the predominant product, asparagine appears exponentially and does not appear to be in an active metabolic pool. Other products labeled with (14)CO(2) are citrate, aspartate, and glutamate. No neutral components are labeled. Secondly, asparagine is synthesized via a pathway starting with cyanide. Major amino acid products labeled with (14)CN(-) are beta-cyanoalanine and asparagine. Similarly to CO(2) fixation, asparagine synthesized from cyanide is not in an active metabolic pool. Other products labeled include anion and neutral components. The exact nature of the latter is not known.     

Evidence for the presence of phosphoriboisomerase and ribulose-1,5-diphosphate carboxylase in extracts of Desulfovibrio vulgaris.

Cell extracts of Desulfovibrio vulgaris were found to incorporate 14CO2 into acid-stable products when ribose-5-phosphate or ribulose-1,5-diphosphate was used as a substrate. This CO2 fixation required adenosine triphosphate and produced 3-phosphoglyceric acid as one of the products. The assimilation of CO2 by pentose phosphates was unrelated to the pyruvate-CO2 exchange reaction. The pyruvate-CO2 exchange did not require adenosine triphosphate, did not produce phosphorylated compounds, and, unlike the pentose phosphate system, required an acidic protein fraction for activity.     

Absence of microbial mineralization of lignin in anaerobic enrichment cultures.

The existence of anaerobic biodegradation of lignin was examined in mixed microflora. Egyptian soil samples, in which rapid mineralization of organic matter takes place in the presence of an important anaerobic microflora, were used to obtain the anaerobic enrichment cultures for this study. Specifically, 14CO2 or [14C]lignin wood was used to investigate the release of labeled gaseous or soluble degradation products of lignin in microbial cultures. No conversion of 14C-labeled lignin to 14CO2 or 14CH4 was observed after 6 months of incubation at 30 degrees C in anaerobic conditions with or without NO3-. A small increase in soluble radioactivity was observed in certain cultures, but it could not be related to the release of catabolic products during the anaerobic biodegradation of lignin.     

Regulation of hexose phosphate metabolism in Acetobacter xylinum

The metabolism of glucose and fructose was studied in resting succinate-grown cells of Acetobacter xylinum. From fructose only cellulose and CO(2) were formed by the cells, whereas from glucose, gluconate was formed much more rapidly than these two products. The molar ratio of sugar converted into cellulose to sugar converted into CO(2) was significantly greater than unity for both hexoses. The pattern of label retention in the cellulose formed by the cells from specifically (14)C-labelled glucose, fructose or gluconate corresponded to that of hexose phosphate in a pentose cycle. On the other hand, the isotopic configuration of cellulose arising from variously singly (14)C-labelled pyruvate did not agree with the operation of a pentose cycle on gluconeogenic hexose phosphate. Readily oxidizable tricarboxylic acid-cycle intermediates such as acetate, pyruvate or succinate promoted cellulose synthesis from fructose and gluconate although retarding their oxidation to CO(2). The incorporation into cellulose of C-1 of fructose was greatly increased in the presence of these non-sugar substrates, although its oxidation to CO(2) was greatly diminished. It is suggested that the flow of hexose phosphate carbon towards cellulose or through the pentose cycle in A. xylinum is regulated by an energy-linked control mechanism.     

The role of biotin and sodium in the decarboxylation of oxaloacetate by the membrane-bound oxaloacetate decarboxylase from Klebsiella aerogenes

The biotin-containing oxaloacetate decarboxylase from Klebsiella aerogenes catalyzed the Na+-dependent decarboxylation of oxaloacetate to pyruvate and bicarbonate (or CO2) but not the reversal of this reaction, not even in the presence of an oxaloacetate trapping system. The enzyme catalyzed an avidin-sensitive isotopic exchange between [1-14C]pyruvate and oxaloacetate, which indicated the intermediate formation of a carboxybiotin enzyme. Sodium ions were not required for this partial reaction, but promoted the second partial reaction, the decarboxylation of the carboxybiotin enzyme, thus accounting for the Na+ requirement of the overall reaction. Therefore, the 14CO2-enzyme which was formed upon incubation of the decarboxylase with [4-15C]oxaloacetate, could only be isolated if Na+ ions were excluded. Preincubation of the decarboxylase with avidin also prevented its labelling with 14CO2. The isolated 14CO2-labelled oxaloacetate decarboxylase revealed the following properties. It was slowly decarboxylated at neutral pH and rapidly upon acidification. The 14CO2 residues of the 14CO2-enzyme could be transferred to pyruvate yielding [4-14C]oxaloacetate. In the presence of Na+ this 14CO2 transfer was repressed by the simultaneous decarboxylation of the 14CO2-enzyme. However, Na+ alone was insufficient as a cofactor for the decarboxylation of the isolated 14CO2-enzyme, since this required pyruvate in addition to Na+. It is therefore concluded that the decarboxylation of oxaloacetate proceeds over a CO2-enzyme--pyruvate complex and that free CO2-enzyme is an abortive reaction intermediate. The activation energy of the enzymic decarboxylation of oxaloacetate changed with temperature and was about 113 kJ below 11 degrees C, 60 kJ between 11 degrees C and 31 degrees C and 36 kJ between 31--45 degrees C.     

Phosphoenolpyruvate Carboxylation and Aspartate Synthesis in Acetobacter suboxydans

Dialyzed extracts of Acetobacter suboxydans ATCC 621 catalyze (14)CO(2) assimilation in the presence of phosphoenolpyruvate and a divalent cation. The formation of (14)C-oxalacetate was demonstrated and found not to be dependent upon the presence of orthophosphate or diphosphonucleotides. Oxalacetate synthesis was stimulated by orthophosphate and inhibited by aspartate. All attempts to demonstrate a reversible carboxylation mechanism have failed. (14)C-aspartate was synthesized when phosphoenolpyruvate, H(14)Co(3) (-), pyridoxal phosphate, and glutamate were added to dialyzed extracts. Chromatographic and spectrophotometric analyses and chemical degradation further demonstrate the presence of a reversible aspartate aminotransferase. The function of oxalacetate synthesis in a bacterium that reportedly lacks an operative tricarboxylic acid cycle is discussed.     

Absence of microbial mineralization of lignin in anaerobic enrichment cultures

The existence of anaerobic biodegradation of lignin was examined in mixed microflora. Egyptian soil samples, in which rapid mineralization of organic matter takes place in the presence of an important anaerobic microflora, were used to obtain the anaerobic enrichment cultures for this study. Specifically, 14CO2 or [14C]lignin wood was used to investigate the release of labeled gaseous or soluble degradation products of lignin in microbial cultures. No conversion of 14C-labeled lignin to 14CO2 or 14CH4 was observed after 6 months of incubation at 30 degrees C in anaerobic conditions with or without NO3-. A small increase in soluble radioactivity was observed in certain cultures, but it could not be related to the release of catabolic products during the anaerobic biodegradation of lignin.