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Phosphorus (P) is central to storing and transferring energy and information in living cells, including those of microalgae. Many microalgal species dwelling in low P environments are naturally equipped to take up and store P whenever it becomes available through a complex phenomenon known as “luxury P uptake.” Its research is required for better understanding of the nutrient geochemical cycles in aquatic environments but also for biotechnological applications such as sequestration of nutrients from wastewater and production of algal fertilizers. Here, we report on our recent insights into luxury P uptake and polyphosphate formation originating from physiological, ultrastructural, and transcriptomic evidence. The cultures pre-starved of P and re-fed with inorganic phosphate (Pi) exhibited a bi-phasic kinetics of Pi uptake comprising fast (1–2 h after re-feeding) and slow (1–3 d after re-feeding) phases. The rate of Pi uptake in the fast phase was ca. 10 times higher than in the slow phase with an opposite trend shown for the cell division rate. The transient peak of polyphosphate accumulation was determined 2–4 h after re-feeding and coincided with the period of slow cell division and fast Pi uptake. In this phase, the microalgal cells reached the highest P content (up to 5% of dry cell weight). The P re-feeding also reversed the characteristic changes in cell lipids induced by P starvation, namely increase in the major membrane glycolipid (DGDG/MGDG) ratio and betaine lipids. These changes were reversed upon Pi re-feeding of the starved culture. Electron microscopy revealed the ordered organization of vacuolar polyphosphate indicative of the possible involvement of an enzyme (complex) in their synthesis. A candidate gene encoding a protein similar to the vacuolar transport chaperone (VTC) protein, featuring an expression pattern corresponding to polyphosphate accumulation, was revealed. Implications of the findings for efficient biocapture of phosphorus are discussed.

Summary An obligate methanotrophic bacterium, strain MTS, was isolated from a methane-fed microaerobic denitrifying bioreactor. 16S rRNA and DNA–DNA hybridization analysis revealed that this organism was most closely related to Methylocystis parvus, a Type II methanotroph, belonging to the α-subclass of the Proteobacteria. The metabolism of the bacterium under microaerobic and anaerobic conditions was studied by 13C-NMR. 13C-labelled poly-β-hydroxybutyrate (PHB) formation occurred in cell suspensions incubated with 13C-labelled methane at low (5–10%) oxygen concentration. Under these conditions low levels of succinate, acetate and 2,3-butanediol were formed and excreted into the culture medium. Intracellular PHB degradation was observed in intact cells under anaerobic conditions in the absence of an exogenous carbon source during a long-term incubation of 90 days. Multiple 13C-labelled β-hydroxybutyrate, butyrate, acetate, acetone, isopropanol, 2,3-butanediol and succinate were identified as products in in vivo13C-NMR spectra and in the spectra of culture medium during the dynamic PHB degradation. The isolated obligate methanotroph clearly shows a fermentative metabolism of PHB under anaerobic conditions. The excreted products may serve as substrates for denitrifying bacteria.

This study introduces a stable-isotope metabolic approach employing [U-(13)C]glucose that, as a novelty, allows selective profiling of the human intestinal microbial metabolic products of carbohydrate food components, as well as the measurement of the kinetics of their formation pathways, in a single experiment. A well-established, validated in vitro model of human intestinal fermentation was inoculated with standardized gastrointestinal microbiota from volunteers. After culture stabilization, [U-(13)C]glucose was added as an isotopically labeled metabolic precursor. System lumen and dialysate samples were taken at regular intervals. Metabolite concentrations and isotopic labeling were determined by NMR, GC, and enzymatic methods. The main microbial metabolites were lactate, acetate, butyrate, formate, ethanol, and glycerol. They together accounted for a (13)C recovery rate as high as 91.2%. Using an NMR chemical shift prediction approach, several minor products that showed (13)C incorporation were identified as organic acids, amino acids, and various alcohols. Using computer modeling of the (12)C contents and (13)C labeling kinetics, the metabolic fluxes in the gut microbial pathways for synthesis of lactate, formate, acetate, and butyrate were determined separately for glucose and unlabeled background substrates. This novel approach enables the study of the modulation of human intestinal function by single nutrients, providing a new rational basis for achieving control of the short-chain fatty acids profile by manipulating substrate and microbiota composition in a purposeful manner.

Non-bilayer lipids account for about half of the total lipid content in chloroplast thylakoid membranes. This lends high propensity of the thylakoid lipid mixture to participate in different phases which might be functionally required. It is for instance known that the chloroplast enzyme violaxanthin de-epoxidase (VDE) requires a non-bilayer phase for proper functioning in vitro but direct evidence for the presence of non-bilayer lipid structures in thylakoid membranes under physiological conditions is still missing. In this work, we used phosphatidylglycerol (PG) as an intrinsic bulk lipid label for 31P-NMR studies to monitor lipid phases of thylakoid membranes. We show that in intact thylakoid membranes the characteristic lamellar signal is observed only below 20 degrees C. But at the same time an isotropic phase is present, which becomes even dominant between 14 and 28 degrees C despite the presence of fully functional large membrane sheets that are capable of generating and maintaining a transmembrane electric field. Tris-washed membranes show a similar behavior but the lamellar phase is present up to higher temperatures. Thus, our data show that the location of the phospholipids is not restricted to the bilayer phase and that the lamellar phase co-exists with a non-bilayer isotropic phase.

Phosphite (Phi, H(2)PO(3)(-)), being the active part of several fungicides, has been shown to influence not only the fungal metabolism but also the development of phosphate-deficient plants. However, the mechanism of phosphite effects on plants is still widely unknown. In this paper we analysed uptake, subcellular distribution and metabolic effects of Phi in tobacco BY-2 cells using in vivo(31)P nuclear magnetic resonance ((31)P-NMR) spectroscopy. Based on the kinetic properties of the phosphate transport system of tobacco BY-2 cells, it was demonstrated that phosphite inhibited phosphate uptake in a competitive manner. To directly follow the fate of phosphate and phosphite in cytoplasmic and vacuolar pools of tobacco cells, we took advantage of the pH-sensitive chemical shift of the Phi anion. The NMR studies showed a distinct cytoplasmic accumulation of Phi in Pi-deprived cells, whereas Pi resupply resulted in a rapid efflux of Phi. Pi-preloaded cells shifted Phi directly into vacuoles. These studies allowed for the first time to follow Phi flux processes in an in vivo setting in plants. On the other hand, the external Pi nutrition status and the metabolic state of the cells had a strong influence on the intracellular compartmentalization of xenobiotic Phi.

The genome sequence of Archaeoglobus fulgidus VC16 encodes three CO dehydrogenase genes. Here we explore the capacity of A.?fulgidus to use CO as growth substrate. Archaeoglobus fulgidus VC16 was successfully adapted to growth medium that contained sulfate and CO. In the presence of CO and sulfate the culture OD660 increased to 0.41 and sulfide, carbon dioxide, acetate and formate were formed. Accumulation of formate was transient. Similar results, except that no sulfide was formed, were obtained when sulfate was omitted. Hydrogen was never detected. Under the conditions tested, the observed concentrations of acetate (18?mM) and formate (8.2?mM) were highest in cultures without sulfate. Proton NMR spectroscopy indicated that CO2, and not CO, is the precursor of formate and the methyl group of acetate. Methylviologen-dependent formate dehydrogenase activity (1.4??mol formate oxidized min?1?mg?1) was detected in cell-free extracts and expected to have a role in formate reuptake. It is speculated that formate formation proceeds through hydrolysis of formyl-methanofuran or formyl-tetrahydromethanopterin. This study demonstrates that A.?fulgidus can grow chemolithoautotrophically with CO as acetogen, and is not strictly dependent on the presence of sulfate, thiosulfate or other sulfur compounds as electron acceptor.

16S rRNA-based stable isotope probing (SIP) and nuclear magnetic resonance (NMR) spectroscopy-based metabolic profiling were used to identify bacteria fermenting glucose under conditions simulating the human intestine. The TIM-2 in vitro model of the human intestine was inoculated with a GI tract microbiota resembling that of the small intestine, to which subsequently 4, 20 or 40 mM of [U-(13)C]-glucose were added. RNA was extracted from lumen samples after 0 (control), 1, 2 and 4 h and subjected to density-gradient ultracentrifugation. Phylogenetic analysis of unlabeled 16S rRNA revealed a microbial community dominated by lactic acid bacteria and Clostridium perfringens. Distinct (13)C-incorporation into bacterial RNA was only observed for the 40-mM addition. 16S rRNA fingerprinting showed an activity drop of Lactobacillus fermentum after glucose addition, while Streptococcus bovis and C. perfringens were identified as the most active glucose-fermenters. Accordingly, NMR analysis identified lactate, acetate, butyrate and formate as the principal fermentation products, constituting up to 91% of the (13)C-carbon balance. RNA-SIP combined with metabolic profiling allowed us to detect differential utilization of a general model carbohydrate, indicating that this approach holds great potential to identify bacteria involved in the fermentation of dietary relevant oligo- and polymeric carbohydrates in the human intestine.

Intracellular pH homeostasis in the filamentous fungus Aspergillus niger was measured in real time by 31P NMR during perfusion in the NMR tube of fungal biomass immobilized in Ca2+-alginate beads. The fungus maintained constant cytoplasmic pH (pHcyt) and vacuolar pH (pHvac) values of 7.6 and 6.2, respectively, when the extracellular pH (pHex) was varied between 1.5 and 7.0 in the presence of citrate. Intracellular metabolism did not collapse until a ΔpH over the cytoplasmic membrane of 6.6–6.7 was reached (pHex 0.7–0.8). Maintenance of these large pH differences was possible without increased respiration compared to pHex 5.8. Perfusion in the presence of various hexoses and pentoses (pHex 5.8) revealed that the magnitude of ΔpH values over the cytoplasmic and vacuolar membrane could be linked to the carbon catabolite repressing properties of the carbon source. Also, larger ΔpH values coincided with a higher degree of respiration and increased accumulation of polyphosphate. Addition of protonophore (carbonyl cyanide m-chlorophenylhydrazone, CCCP) to the perfusion buffer led to decreased ATP levels, increased respiration and a partial (1 µm CCCP), transient (2 µm CCCP) or permanent (10 µm CCCP) collapse of the vacuolar membrane ΔpH. Nonlethal levels of the metabolic inhibitor azide (N3–, 0.1 mm) caused a transient decrease in pHcyt that was closely paralleled by a transient vacuolar acidification. Vacuolar H+ influx in response to cytoplasmic acidification, also observed during extreme medium acidification, indicates a role in pH homeostasis for this organelle. Finally, 31P NMR spectra of citric acid producing A. niger mycelium showed that despite a combination of low pHex (1.8) and a high acid-secreting capacity, pHcyt and pHvac values were still well maintained (pH 7.5 and 6.4, respectively).

The hyperthermophilic archaeon Pyrococcus furiosus possesses a modified Embden—Meyerhof pathway, including an unusual ADP-dependent glucokinase (ADP-GLK) and an ADP-dependent phosphofructokinase. In the present study, we report the characterization of a P. furiosus galactokinase (GALK) and its comparison with the P. furiosus ADP-GLK. The pyrococcal genes encoding the ADP-GLK and GALK were functionally expressed in Escherichia coli, and the proteins were subsequently purified to homogeneity. Both enzymes are specific kinases with an optimal activity at approx. 90°C. Biochemical characterization of these enzymes confirmed that the ADP-GLK is unable to use ATP as the phosphoryl group donor, but revealed that GALK is ATP-dependent and has an extremely high affinity for ATP. There is a discussion about whether the unusual features of these two classes of kinases might reflect adaptations to a relatively low intracellular ATP concentration in the hyperthermophilic archaeon P. furiosus.

The pathway of propionate conversion in a syntrophic coculture of Smithella propionica and Methanospirillum hungatei JF1 was investigated by 13 C-NMR spectroscopy. Cocultures produced acetate and butyrate from propionate. [3- 13 C]propionate was converted to [2- 13 C]acetate, with no [1- 13 C]acetate formed. Butyrate from [3- 13 C]propionate was labeled at the C2 and C4 positions in a ratio of about 1:1.5. Double-labeled propionate (2,3- 13 C) yielded not only double-labeled acetate but also single-labeled acetate at the C1 or C2 position. Most butyrate formed from [2,3- 13 C]propionate was also double labeled in either the C1 and C2 atoms or the C3 and C4 atoms in a ratio of about 1:1.5. Smaller amounts of single-labeled butyrate and other combinations were also produced. 1- 13 C-labeled propionate yielded both [1- 13 C]acetate and [2- 13 C]acetate. When 13 C-labeled bicarbonate was present, label was not incorporated into acetate, propionate, or butyrate. In each of the incubations described above, 13 C was never recovered in bicarbonate or methane. These results indicate that S. propionica does not degrade propionate via the methyl-malonyl-coenzyme A (CoA) pathway or any other of the known pathways, such as the acryloyl-CoA pathway or the reductive carboxylation pathway. Our results strongly suggest that propionate is dismutated to acetate and butyrate via a six-carbon intermediate.

Poly-β-hydroxybutyrate (PHB) formation under aerobic conditions via incorporation of [13C-2]acetate as a cosubstrate and its intracellular degradation under anaerobic conditions in a Type II methanotroph was studied by 13C NMR. During PHB synthesis in the presence of labelled acetate, low levels of β-hydroxybutyrate, butyrate, acetone, isopropanol, 2,3-butanediol and succinate were observed. Subsequent anaerobic PHB breakdown showed enhanced levels of these products at the expense of PHB. Fermentative metabolism occurring during anaerobic PHB degradation was confirmed in experiments with fully 13C-enriched cells, which were grown on 13C-labelled methane. β-hydroxybutyrate, butyrate, acetate, acetone, isopropanol, 2,3-butanediol and succinate were detected as multiple 13C-labelled compounds in the culture medium. Our results suggest that intracellular PHB degradation can be used as a reserve energy source by methanotrophs under anoxic conditions. Journal of Industrial Microbiology & Biotechnology (2001) 26, 15–21.

13C NMR was used to study the effect of oxygen on methanol oxidation by a type II methanotrophic bacterium isolated from a bioreactor in which methane was used as electron donor for denitrification. Under high (35–25%) oxygen conditions the first step of methanol oxidation to formaldehyde was much faster than the following conversions to formate and carbon dioxide. Due to this the accumulation of formaldehyde led to a poisoning of the cells. A more balanced conversion of 13C-labelled methanol to carbon dioxide was observed at low (1–5%) oxygen concentrations. In this case, formaldehyde was slowly converted to formate and carbon dioxide. Formaldehyde did not accumulate to inhibitory levels. The oxygen-dependent formation of formaldehyde and formate from methanol is discussed kinetically and thermodynamically. Journal of Industrial Microbiology & Biotechnology (2001) 26, 9–14.

The microbial population from a reactor using methane as electron donor for denitrification under microaerophilic conditions was analyzed. High numbers of aerobic methanotrophic bacteria (3 10(7) cells/ml) and high numbers of acetate-utilizing denitrifying bacteria (2 10(7) cells/ml) were detected, but only very low numbers of methanol-degrading denitrifying bacteria (4 10(4) cells/ml) were counted. Two abundant acetate-degrading denitrifiers were isolated which, based on 16S rRNA analysis, were closely related to Mesorhizobium plurifarium (98.4% sequence similarity) and a Stenotrophomonas sp. (99.1% sequence similarity). A methanol-degrading denitrifying bacterium isolated from the bioreactor morphologically resembled Hyphomicrobium sp. and was moderately related to H. vulgare (93.5% sequence similarity). The initial characterization of the most abundant methanotrophic bacterium indicated that it belongs to class II of the methanotrophs. "In vivo" 13C-NMR with concentrated cell suspensions showed that this methanotroph produced acetate under oxygen limitation. The microbial composition of reactor material together with the NMR experiments suggest that in the reactor methanotrophs excrete acetate, which serves as the direct electron donor for denitrification.

Propionate is an important intermediate in the anaerobic degradation of complex organic matter to methane and carbon dioxide. The metabolism of propionate-forming and propionate-degrading bacteria is reviewed here. Propionate is formed during fermentation of polysaccharides, proteins and fats. The study of the fate of 13C-labelled compounds by nuclear magnetic resonance (NMR) spectroscopy has contributed together with other techniques to the present knowledge of the metabolic routes which lead to propionate formation from these substrates. Since propionate oxidation under methanogenic conditions is thermodynamically difficult, propionate often accumulates when the rates of its formation and degradation are unbalanced. Bacteria which are able to degrade propionate to the methanogenic substrates acetate and hydrogen can only perform this reaction when the methanogens consume acetate and hydrogen efficiently. As a consequence, propionate can only be degraded by obligatory syntrophic consortia of microorganisms. NMR techniques were used to study the degradation of propionate by defined and less defined cultures of these syntrophic consortia. Different types of side-reactions were reported, like the reductive carboxylation to butyrate and the reductive acetylation to higher fatty acids.

Applications of nuclear magnetic resonance (NMR) to study a variety of physiological and biochemical aspects of bacteria with a role in the sulfur cycle are reviewed. Then, a case-study of high resolution13 C-NMR spectroscopy on sludges from bioreactors used for treating sulfate and sulfide rich wastewaters is presented.13 C-NMR was used to study the effect of sulfate and butyrate on propionate conversion by mesophilic anaerobic (methanogenic and sulfate reducing) granular sludge and microaerobic (sulfide oxidizing) flocculant sludge. In the presence of sulfate, propionate was degraded via the randomising pathway in all sludge types investigated. This was evidenced by scrambling of [3-13C]propionate into [2-13C]propionate and the formation of acetate equally labeled in the C1 and C2 position. In the absence of sulfate, [3-13C]propionate scrambled to a lesser extend without being degraded further. Anaerobic sludges converted [2,3-13C]propionate partly into the higher fatty acid 2-methyl[2,3-13C]butyrate during the simultaneous degradation of [2,3-13C]propionate and butyrate. [4,5-13C]valerate was also formed in the methanogenic sludges. Up to 10% of the propionate present was converted via these alternative degradation routes. Labeled butyrate was not detected in the incubations, suggesting that reductive carboxylation of propionate does not occur in the sludges.

An obligate methanotrophic bacterium, strain MTS, was isolated from a methane-fed microaerobic denitrifying bioreactor. 16S rRNA and DNA-DNA hybridization analysis revealed that this organism was most closely related to Methylocystis parvus, a Type II methanotroph, belonging to the α-subclass of the Proteobacteria. The metabolism of the bacterium under microaerobic and anaerobic conditions was studied by (13) C-NMR. (13) C-labelled poly-β-hydroxybutyrate (PHB) formation occurred in cell suspensions incubated with (13) C-labelled methane at low (5-10%) oxygen concentration. Under these conditions low levels of succinate, acetate and 2,3-butanediol were formed and excreted into the culture medium. Intracellular PHB degradation was observed in intact cells under anaerobic conditions in the absence of an exogenous carbon source during a long-term incubation of 90 days. Multiple (13) C-labelled β-hydroxybutyrate, butyrate, acetate, acetone, isopropanol, 2,3-butanediol and succinate were identified as products in in vivo(13) C-NMR spectra and in the spectra of culture medium during the dynamic PHB degradation. The isolated obligate methanotroph clearly shows a fermentative metabolism of PHB under anaerobic conditions. The excreted products may serve as substrates for denitrifying bacteria.

The degradation of volatile fatty acids by flocculant mesophilic (35±2°C) syntrophic sludge grown in an anaerobic hybrid reactor fed with a mixture of propionate, n-butyrate and ethanol (1:1:2 on COD basis) at a volumetric loading rate of 3.7 kg COD per m3 per day was examined. The propionate degradation rate amounted to 1.1 mmol per g volatile suspended solids per day. The same propionate degradation rate was measured in the presence of 10 mM acetate, but it decreased by 30% when 10 mM n-butyrate was added. 13C-Nuclear magnetic resonance spectroscopy demonstrated interconversion of 1% to 10% of both the propionate and butyrate pool, during their simultaneous degradation. Propionate formation from [2-13C]butyrate was not via a direct decarboxylation. 13C-Propionate was converted via at least three different pathways. The first pathway was syntrophic conversion of propionate via the randomising pathway, evidenced by scrambling of [3-13C]propionate into [2-13C]propionate. Secondly, reductive carboxylation occurred, i.e. [3-13C]propionate and [2,3-13C]propionate were partly (2 to 10%) converted into [4-13C]butyrate and [3,4-13C]butyrate, respectively. Reductive carboxylation probably involved a transcarboxylase, as 13C-bicarbonate was not incorporated in the carboxyl group of butyrate. Thirdly, propionate was converted into higher fatty acids: [2,3-13C]propionate was converted into [4,5-13C]valerate and 2-methyl[2,3-13C]butyrate.

The xylose metabolism of Bacteroides xylanolyticus X5-1 was studied by determining specific enzyme activities in cell free extracts, by following 13C-label distribution patterns in growing cultures and by mass balance calculations. Enzyme activities of the pentose phosphate pathway and the Embden-Meyerhof-Parnas pathway were sufficiently high to account for in vivo xylose fermentation to pyruvate via a combination of these two pathways. Pyruvate was mainly oxidized to acetyl-CoA, CO2 and a reduced cofactor (ferredoxin). Part of the pyruvate was converted to acetyl-CoA and formate by means of a pyruvate-formate lyase. Acetyl-CoA was either converted to acetate by a combined action of phosphotransacetylase and acetate kinase or reduced to ethanol by an acetaldehyde dehydrogenase and an ethanol dehydrogenase. The latter two enzymes displayed both a NADH- and a NADPH-linked activity. Cofactor regeneration proceeded via a reduction of intermediates of the metabolism (i.e. acetyl-CoA and acetaldehyde) and via proton reduction. According to the deduced pathway about 2.5 mol ATP are generated per mol of xylose degraded.

A syntrophic propionate-oxidizing bacterium was able to grow in the absence of methanogens by fermentation of fumarate to succinate and CO2. The pathway involved in the conversion of fumarate was investigated. During fermentation of unlabelled fumarate cells incorporated [1-13C]acetate, [2-13C]acetate and [1,2-13C]acetate into C2 and C3 of succinate, but not into C1 and C4. This observation, together with the absence of essential enzymes, showed that the citric acid cycle is not used for the oxidation of fumarate to CO2. All the enzymes of the acetyl-CoA cleavage pathway were detected, indicating that this pathway is involved in fumarate oxidation to CO2. During growth on propionate this acetyl-CoA cleavage pathway may have an anaplerotic function.

Oxidation of succinate to fumarate is an energetically difficult step in the biochemical pathway of propionate oxidation by syntrophic methanogenic cultures. Therefore, the effect of fumarate on propionate oxidation by two different propionate-oxidizing cultures was investigated. When the methanogens in a newly enriched propionate-oxidizing methanogenic culture were inhibited by bromoethanesulfonate, fumarate could act as an apparent terminal electron acceptor in propionate oxidation. 13 C-nuclear magnetic resonance experiments showed that propionate was carboxylated to succinate while fumarate was partly oxidized to acetate and partly reduced to succinate. Fumarate alone was fermented to succinate and CO 2 . Bacteria growing on fumarate were enriched and obtained free of methanogens. Propionate was metabolized by these bacteria when either fumarate or Methanospirillum hungatii was added. In cocultures with Syntrophobacter wolinii , such effects were not observed upon addition of fumarate. Possible slow growth of S. wolinii on fumarate could not be demonstrated because of the presence of a Desulfovibrio strain which grew rapidly on fumarate in both the absence and presence of sulfate.

The cytoplasm of anaerobic ammonium oxidizing (anammox) bacteria consists of three compartments separated by membranes. It has been suggested that a proton motive force may be generated over the membrane of the innermost compartment, the "anammoxosome". 31P nuclear magnetic resonance (NMR) spectroscopy was employed to investigate intracellular pH differences in the anammox bacterium Kuenenia stuttgartiensis. With in vivo NMR, spectra were recorded of active, highly concentrated suspensions of K. stuttgartiensis in a wide-bore NMR tube. At different external pH values, two stable and distinct phosphate peaks were apparent in the recorded spectra. These peaks were equivalent with pH values of 7.3 and 6.3 and suggested the presence of a proton motive force over an intracytoplasmic membrane in K. stuttgartiensis. This study provides for the second time--after discovery of acidocalcisome-like compartments in Agrobacterium tumefaciens--evidence for an intracytoplasmic pH gradient in a chemotrophic prokaryotic cell.

This study introduces a stable-isotope metabolic approach employing [U-(13)C]glucose that, as a novelty, allows selective profiling of the human intestinal microbial metabolic products of carbohydrate food components, as well as the measurement of the kinetics of their formation pathways, in a single experiment. A well-established, validated in vitro model of human intestinal fermentation was inoculated with standardized gastrointestinal microbiota from volunteers. After culture stabilization, [U-(13)C]glucose was added as an isotopically labeled metabolic precursor. System lumen and dialysate samples were taken at regular intervals. Metabolite concentrations and isotopic labeling were determined by NMR, GC, and enzymatic methods. The main microbial metabolites were lactate, acetate, butyrate, formate, ethanol, and glycerol. They together accounted for a (13)C recovery rate as high as 91.2%. Using an NMR chemical shift prediction approach, several minor products that showed (13)C incorporation were identified as organic acids, amino acids, and various alcohols. Using computer modeling of the (12)C contents and (13)C labeling kinetics, the metabolic fluxes in the gut microbial pathways for synthesis of lactate, formate, acetate, and butyrate were determined separately for glucose and unlabeled background substrates. This novel approach enables the study of the modulation of human intestinal function by single nutrients, providing a new rational basis for achieving control of the short-chain fatty acids profile by manipulating substrate and microbiota composition in a purposeful manner.

Phosphite (Phi, H(2)PO(3)(-)), being the active part of several fungicides, has been shown to influence not only the fungal metabolism but also the development of phosphate-deficient plants. However, the mechanism of phosphite effects on plants is still widely unknown. In this paper we analysed uptake, subcellular distribution and metabolic effects of Phi in tobacco BY-2 cells using in vivo(31)P nuclear magnetic resonance ((31)P-NMR) spectroscopy. Based on the kinetic properties of the phosphate transport system of tobacco BY-2 cells, it was demonstrated that phosphite inhibited phosphate uptake in a competitive manner. To directly follow the fate of phosphate and phosphite in cytoplasmic and vacuolar pools of tobacco cells, we took advantage of the pH-sensitive chemical shift of the Phi anion. The NMR studies showed a distinct cytoplasmic accumulation of Phi in Pi-deprived cells, whereas Pi resupply resulted in a rapid efflux of Phi. Pi-preloaded cells shifted Phi directly into vacuoles. These studies allowed for the first time to follow Phi flux processes in an in vivo setting in plants. On the other hand, the external Pi nutrition status and the metabolic state of the cells had a strong influence on the intracellular compartmentalization of xenobiotic Phi.

Malic acid is a potential biomass-derivable “building block” for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production from glucose proceeds via carboxylation of pyruvate, followed by reduction of oxaloacetate to malate. This redox- and ATP-neutral, CO 2 -fixing pathway has a theoretical maximum yield of 2 mol malate (mol glucose) −1 . A previously engineered glucose-tolerant, C 2 -independent pyruvate decarboxylase-negative S. cerevisiae strain was used as the platform to evaluate the impact of individual and combined introduction of three genetic modifications: (i) overexpression of the native pyruvate carboxylase encoded by PYC2 , (ii) high-level expression of an allele of the MDH3 gene, of which the encoded malate dehydrogenase was retargeted to the cytosol by deletion of the C-terminal peroxisomal targeting sequence, and (iii) functional expression of the Schizosaccharomyces pombe malate transporter gene Sp MAE1 . While single or double modifications improved malate production, the highest malate yields and titers were obtained with the simultaneous introduction of all three modifications. In glucose-grown batch cultures, the resulting engineered strain produced malate at titers of up to 59 g liter −1 at a malate yield of 0.42 mol (mol glucose) −1 . Metabolic flux analysis showed that metabolite labeling patterns observed upon nuclear magnetic resonance analyses of cultures grown on 13 C-labeled glucose were consistent with the envisaged nonoxidative, fermentative pathway for malate production. The engineered strains still produced substantial amounts of pyruvate, indicating that the pathway efficiency can be further improved.

The genome sequence of Archaeoglobus fulgidus VC16 encodes three CO dehydrogenase genes. Here we explore the capacity of A. fulgidus to use CO as growth substrate. Archaeoglobus fulgidus VC16 was successfully adapted to growth medium that contained sulfate and CO. In the presence of CO and sulfate the culture OD(660) increased to 0.41 and sulfide, carbon dioxide, acetate and formate were formed. Accumulation of formate was transient. Similar results, except that no sulfide was formed, were obtained when sulfate was omitted. Hydrogen was never detected. Under the conditions tested, the observed concentrations of acetate (18 mM) and formate (8.2 mM) were highest in cultures without sulfate. Proton NMR spectroscopy indicated that CO2, and not CO, is the precursor of formate and the methyl group of acetate. Methylviologen-dependent formate dehydrogenase activity (1.4 micromol formate oxidized min(-1) mg(-1)) was detected in cell-free extracts and expected to have a role in formate reuptake. It is speculated that formate formation proceeds through hydrolysis of formyl-methanofuran or formyl-tetrahydromethanopterin. This study demonstrates that A. fulgidus can grow chemolithoautotrophically with CO as acetogen, and is not strictly dependent on the presence of sulfate, thiosulfate or other sulfur compounds as electron acceptor.

16S rRNA-based stable isotope probing (SIP) and nuclear magnetic resonance (NMR) spectroscopy-based metabolic profiling were used to identify bacteria fermenting glucose under conditions simulating the human intestine. The TIM-2 in vitro model of the human intestine was inoculated with a GI tract microbiota resembling that of the small intestine, to which subsequently 4, 20 or 40 mM of [U-(13)C]-glucose were added. RNA was extracted from lumen samples after 0 (control), 1, 2 and 4 h and subjected to density-gradient ultracentrifugation. Phylogenetic analysis of unlabeled 16S rRNA revealed a microbial community dominated by lactic acid bacteria and Clostridium perfringens. Distinct (13)C-incorporation into bacterial RNA was only observed for the 40-mM addition. 16S rRNA fingerprinting showed an activity drop of Lactobacillus fermentum after glucose addition, while Streptococcus bovis and C. perfringens were identified as the most active glucose-fermenters. Accordingly, NMR analysis identified lactate, acetate, butyrate and formate as the principal fermentation products, constituting up to 91% of the (13)C-carbon balance. RNA-SIP combined with metabolic profiling allowed us to detect differential utilization of a general model carbohydrate, indicating that this approach holds great potential to identify bacteria involved in the fermentation of dietary relevant oligo- and polymeric carbohydrates in the human intestine.

NMR analysis of (13)C-labelling patterns showed that the Embden-Meyerhof (EM) pathway is the main route for glycolysis in the extreme thermophile Caldicellulosiruptor saccharolyticus. Glucose fermentation via the EM pathway to acetate results in a theoretical yield of 4 mol of hydrogen and 2 mol of acetate per mole of glucose. Previously, approximately 70% of the theoretical maximum hydrogen yield has been reached in batch fermentations. In this study, hydrogen and acetate yields have been determined at different dilution rates during continuous cultivation. The yields were dependent on the growth rate. The highest hydrogen yields of 82 to 90% of theoretical maximum (3.3 to 3.6 mol H(2) per mol glucose) were obtained at low growth rates when a relatively larger part of the consumed glucose is used for maintenance. The hydrogen productivity showed the opposite effect. Both the specific and the volumetric hydrogen production rates were highest at the higher growth rates, reaching values of respectively 30 mmol g(-1) h(-1) and 20 mmol l(-1) h(-1). An industrial process for biohydrogen production will require a bioreactor design, which enables an optimal mix of high productivity and high yield.

The pathway of methanol conversion by a thermophilic anaerobic consortium was elucidated by recording the fate of carbon in the presence and absence of bicarbonate and specific inhibitors. Results indicated that about 50% of methanol was directly converted to methane by the methylotrophic methanogens and 50% via the intermediates H2/CO2 and acetate. The deprivation of inorganic carbon species [Σ(HCO3 −+CO2)] in a phosphate-buffered system reduced the rate of methanol conversion. This suggests that bicarbonate is required as an electron (H2) sink and as a co-substrate for the efficient and complete removal of the chemical oxygen demand. Nuclear magnetic resonance spectroscopy was used to investigate the route of methanol conversion to acetate in bicarbonate-sufficient and bicarbonate-depleted environments. The proportions of [1,2-13C]acetate, [1-13C]acetate and [2-13C]acetate were determined. Methanol was preferentially incorporated into the methyl group of acetate, whereas HCO3 − was the preferred source of the carboxyl group. A small amount of the added H13CO3 − was reduced to form the methyl group of acetate and a small amount of the added 13CH3OH was oxidised and found in the carboxyl group of acetate when 13CH3OH was converted. The recovery of [13C]carboxyl groups in acetate from 13CH3OH was enhanced in bicarbonate-deprived medium. The small amount of label incorporated in the carboxyl group of acetate when 13CH3OH was converted in the presence of bromoethanesulfonic acid indicates that methanol can be oxidised to CO2 prior to acetate formation. These results indicate that methanol is converted through a common pathway (acetyl-CoA), being on the one hand reduced to the methyl group of acetate and on the other hand oxidised to CO2, with CO2 being incorporated into the carboxyl group of acetate.

Intracellular pH homeostasis in the filamentous fungus Aspergillus niger was measured in real time by 31P NMR during perfusion in the NMR tube of fungal biomass immobilized in Ca2+-alginate beads. The fungus maintained constant cytoplasmic pH (pH(cyt)) and vacuolar pH (pH(vac)) values of 7.6 and 6.2, respectively, when the extracellular pH (pH(ex)) was varied between 1.5 and 7.0 in the presence of citrate. Intracellular metabolism did not collapse until a Delta pH over the cytoplasmic membrane of 6.6-6.7 was reached (pH(ex) 0.7-0.8). Maintenance of these large pH differences was possible without increased respiration compared to pH(ex) 5.8. Perfusion in the presence of various hexoses and pentoses (pH(ex) 5.8) revealed that the magnitude of Delta pH values over the cytoplasmic and vacuolar membrane could be linked to the carbon catabolite repressing properties of the carbon source. Also, larger Delta pH values coincided with a higher degree of respiration and increased accumulation of polyphosphate. Addition of protonophore (carbonyl cyanide m-chlorophenylhydrazone, CCCP) to the perfusion buffer led to decreased ATP levels, increased respiration and a partial (1 microm CCCP), transient (2 microm CCCP) or permanent (10 microm CCCP) collapse of the vacuolar membrane Delta pH. Nonlethal levels of the metabolic inhibitor azide (N3-, 0.1 mm) caused a transient decrease in pH(cyt) that was closely paralleled by a transient vacuolar acidification. Vacuolar H+ influx in response to cytoplasmic acidification, also observed during extreme medium acidification, indicates a role in pH homeostasis for this organelle. Finally, 31P NMR spectra of citric acid producing A. niger mycelium showed that despite a combination of low pH(ex) (1.8) and a high acid-secreting capacity, pH(cyt) and pH(vac) values were still well maintained (pH 7.5 and 6.4, respectively).

Humic acid-like (HA-like) and core-humic acid-like (core-HA-like) were characterized during the high-rate composting process by CP-MAS 13C NMR, pyrolysis-gas chromatography (GC)/mass spectrometry (MS), and elemental analysis. Results obtained indicated that humification proceeded through a relative concentration of aromatic fractions due to the faster degradation of the O-alkyl and alkyl fractions. Core-HA-like, after purification of the parent material, showed a large reduction of the O-alkyl fraction in terms of HA-like. We concluded that HA-like consisted of refractory organic molecules, such as lignin and biopolymers, which formed a stable structure (core-HA-like) coated with degradable material associated with the core by weak physical association, ether or ester bounds.

The amount of energy that can be conserved via halorespiration by Desulfitobacterium dehalogenans JW/IU-DC1 was determined by comparison of the growth yields of cells grown with 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) and different electron donors. Cultures that were grown with lactate, pyruvate, formate, or hydrogen as an electron donor and Cl-OHPA as an electron acceptor yielded 3.1, 6.6, 1.6, and 1.6 g (dry weight) per mol of reduction equivalents, respectively. Fermentative growth on pyruvate yielded 14 g (dry weight) per mol of pyruvate oxidized. Pyruvate was not fermented stoichiometrically to acetate and lactate, but an excess of acetate was produced. Experiments with 13 C-labeled bicarbonate showed that during pyruvate fermentation, approximately 9% of the acetate was formed from the reduction of CO 2 . Comparison of the growth yields suggests that 1 mol of ATP is produced per mol of acetate produced by substrate-level phosphorylation and that there is no contribution of electron transport phosphorylation when D. dehalogenans grows on lactate plus Cl-OHPA or pyruvate plus Cl-OHPA. Furthermore, the growth yields indicate that approximately 1/3 mol of ATP is conserved per mol of Cl-OHPA reduced in cultures grown in formate plus Cl-OHPA and hydrogen plus Cl-OHPA. Because neither formate nor hydrogen nor Cl-OHPA supports substrate-level phosphorylation, energy must be conserved through the establishment of a proton motive force. Pyruvate ferredoxin oxidoreductase, lactate dehydrogenase, formate dehydrogenase, and hydrogenase were localized by in vitro assays with membrane-impermeable electron acceptors and donors. The orientation of chlorophenol-reductive dehalogenase in the cytoplasmic membrane, however, could not be determined. A model is proposed, which may explain the topology analyses as well as the results obtained in the yield study.

Sugar metabolism and exopolysaccharide (EPS) production was analysed in Lactococcus lactis by in vivo 31 P NMR. Transient production of several sugar phosphates, transient depletion of intracellular phosphate, transient production of ATP and UTP, transient acidification of the medium and alkalinisation of the cytoplasm could be observed in a period of 20 min upon energisation by the addition of glucose. EPS and non-EPS producing variants showed similar NMR spectra, the exception being two pH-dependent resonances observed in the former. They were already observed before addition of glucose and their response to glucose incubation reflected exposure to the medium. They are presumably phosphorylated poly- or oligosaccharides being loosely adhered to cell walls. By freezing and perchloric acid extraction of the cell material, different types of phosphorylated compounds could be recognised in the NMR spectra such as fructose-1-6-diphosphate, nucleotides (like ADP, ATP, UTP and TDP) and several nucleotide sugars. The ongoing work is focused on identifying the unknown peaks and quantifying the differences between wild-type cells and the EPS producing variant.

The main pathway for the fermentation of maltose or cellobiose by the hyperthermophile Pyrococcus furiosus was investigated by in vivo NMR and by enzyme measurements. Addition of [1-13C]glucose to cell suspensions resulted in the formation of C2-labeled acetate and C3- labeled alanine. No label was recovered in CO2 or HCO3-. In the presence of [3-13C]glucose, the label ended up in the C1 atom of alanine and in HCO3- and CO2. These labeling patterns indicate that glucose is converted along an Embden-Meyerhof pathway, and they disagree with the previously proposed nonphosphorylated Entner- Doudoroff pathway (pyroglycolysis). The NMR data were supported by enzyme measurements. Hexokinase (8.7 units/mg), phosphoglucose isomerase (6.8 units/mg), phosphofructokinase (0.81 unit/mg), and aldolase (0.26 unit/mg) were present in cell-free extracts (specific activities at 90 degrees C). Remarkably, the two kinases required ADP as the phosphoryl group donor instead of ATP. No activity was found with pyrophosphate. These are the first descriptions of ADP-dependent (AMP-forming) kinases to date. Since P. furiosus is a phylogenetically ancient organism, these enzymes may represent an ancestral kind of metabolism.

SUMMARY: The dynamic response of cellular carbohydrates to a NaCl shock in Rhizobium leguminosarum biovar trifolii TA-1 (0.25 M-NaCl) and Rhizobium meliloti SU-47 (0.4 M-NaCl) grown in NaCl-free medium was investigated in non-growing cell cultures and in cell suspensions, using in vivo NMR. After transferring cells grown in a NaCl-free medium to a glutamic-acid-free medium containing mannitol and NaCl, both strains immediately responded to the increased osmotic pressure by augmenting the cellular trehalose content of the cell. Without mannitol in the medium trehalose synthesis was slower, but clearly detectable. Its synthesis paralleled the breakdown of the reserve materials glycogen and poly-β-hydroxybutyric acid (PHB). NMR experiments with 25-fold-concentrated cell suspensions using 13C1-mannitol as substrate revealed that 15-20% of the trehalose synthesized was derived from mannitol, but 80-85% was from other sources. Trehalose was mainly formed from the internal pool of glycogen and/or PHB, whether mannitol was present or not, and reached 135 and 280 μg (mg cell protein)-1 in the strains TA-1 and SU-47, respectively. At low osmolarity, intracellular trehalose was metabolized by strains TA-1 and SU-47. Intracellularly accumulated phosphoglycerol-substituted and neutral cyclic (1,2)-β-glucans of SU-47 cells grown in the absence of NaCl were neither degraded nor excreted after exposure to NaCl. Strain TA-1, which only makes neutral cyclic (1,2)-β-glucans, continued to synthesize and excrete cyclic (1,2)-β-glucans after exposure to NaCl. By using in vivo 31P-NMR, a sharp peak at 1.34 p.p.m. was present in cell suspensions of strain SU-47. This peak, representing glycerol-1-phosphate-substituted cyclic glucans, was absent in strain TA-1.

The role of carboxylation reactions in propionate metabolism was studied with in vivo high-resolution NMR in two syntrophic propionate oxidizing cocultures and compared with anaerobic propionate forming bacteria with well-established biochemical properties. The inclusion of [ 13 C]propionate and H 13 CO 3 − gave insight into the process of randomization at the level of propionate in relation to the type of the (de)carboxylating enzyme involved. Propionibacterium but not Veillonella and Desulfobulbus showed a propionate randomization in the absence of substrate. These differences are explained by the type of carboxylation mechanism and the energy state of the cells. Both syntrophic cocultures tested degrade propionate via the succinate pathway involving a transcarboxylase.

High resolution (h.r.) 13C NMR is increasingly applied to studies of carbohydrate metabolism in plant cells and tissues, as an analytical technique allowing non-invasive observation of endogenous intermediates and end products in intact biomass (1). One of the major advantages of h.r. 13C NMR apart from being non-destructive is its high spectral resolution (2), as it shares a large spectral width (about 250 ppm of the applied magnetic field) with relatively narrow linewidths of the individual resonances(10–15 Hz in spectra of cell suspensions). High resolution 13C NMR therefore allows the separate observation of the resonances of all chemically distinct carbon atoms present within mobile, low-molecular weight compounds, as cellular intermediates and endproducts generally are. We have recently exploited some of the advantages offered by h.r. 13C NMR in a study of the long-term utilization of sucrose by embryogenic cell suspensions of Daucus carota (cv Flakkese) growing in the presence of the phytohormone 2,4-D and by somatic embryos derived from these cultures in the absence of 2,4-D (3).

Carotenoid difference spectra were recorded of chromatophores of Rhodopseudomonas sphaeroides energized with low light intensities versus chromatophores under dark conditions. The amplitudes of the peaks and troughs were dependent on the light intensities and the duration of illumination, but the shape of the difference spectra remained constant. Sharp isosbestic points were found at 515, 500, 481, 465, and 452 mm. When potassium-valinomycin-induced diffusion potentials (inside negative) were imposed on the chromatophores “mirror images” of the light-induced difference spectra were recorded with the same isosbestic points and the same positions but different relative amplitudes of the peaks and troughs. The results are explained in terms of changes in the shape of the vibrational splittings of the carotenoid main electronic transition. This could be the result of changes in the fluidity of the environment of the carotenoids upon energization of the membrane. Prolonged periods of illumination with low or high light intensities resulted in irreversible changes of the difference spectra. Short periods of illumination with high light intensities resulted in reversible elevations of the baseline and red shifts of peaks, troughs, and baseline crossings.

The composition of the polyol pools in Aspergillus nidulans mycelium during active growth on sucrose depends strongly on pH. At pH 2.5, only mannitol is present. A comparison between nitrate- and ammonium-grown cultures shows stimulation of the arabitol content with nitrate a former nitrogen source. When starved mycelium is incubated either with natural-abundance or 13C-enriched glucose, label appears rapidly in mannitol and arabitol, regardless of the nitrogen source or the pH used.

The disappearance of 2-13C-acetate and the subsequent incorporation of label into cellular metabolites were followed in denitrifying cells of Thiobacillus versutus by 13C NMR spectroscopy. In cells grown under acetate-limitation, the specific rate of consumption was idependent of the density of the cell suspension. An isotopic steady state was reached within 30 min if sufficient substrate was added to the cell suspension. In cells grown under nitrate-limitation, the consumption of 2-13C-acetate proceeded at a significantly lower rate. The decrease and final disappearance of 2-13C-acetate were accompanied by incorporation of 13C into glutamate, glutamine, and by the release of labeled HCO3−and CO2. The appearance of a broad resonance being the methyl endgroup of poly-3-hydroxybutyrate (PHB) was indicative for PHB mobilization during the incubation. The sequence of label incorporation and the distribution among the various carbon nuclei were consistent with the operation of the tricarboxylic acid cycle.

Natural-abundance high-resolution 13C NMR spectra (linewidth, 10 Hz) of the hyphal fungus Aspergillus nidulans have been obtained after growth on glycolytic or gluconeogenic carbon sources. Various polyols, some tricarboxylic acid-cycle intermediates and amino acids, and some phospholipids and fatty acyl compounds are present. The polyols found are mannitol, arabitol, erythritol, and glycerol. The nature of the carbon source has a pronounced effect on the pool sizes of the various polyols. All are present when the fungus is grown on sucrose or sucrose/acetate under strongly aerobic conditions. When grown on acetate, both arabitol and glycerol levels are low, whereas on glycerol erythritol is also hardly detectable. The effect of oxygen is most outspoken in glycolytically grown cultures. Limited oxygenation leads to low levels of arabitol and glycerol. Strong oxygenation changes the ratio of erythritol to mannitol, favoring the C4 polyol. An increase in oxygen supply leads to (i) stimulation of the fluxes through the pentose phosphate pathway and glycolysis, (ii) an overflow of reduced metabolites both at the pentose phosphate pathway level (erythritol and arabitol) and at the C3 level of the glycolytic pathway (glycerol), and (iii) the usual accumulation of mannitol. Upon starvation, glycerol, the other three polyols, and the tricarboxylic acid-cycle intermediates and their associated amino acids disappear in this order. As in yeast, gluconeogenic substrates lead to the synthesis of trehalose, which also occurs when mycelium is grown on acetate/sucrose under limiting aeration. A transient formation of trehalose has been observed upon incubation of starved mycelium, cultured on different substrates, with [13C]glucose.

In vivo high-resolution 13C-NMR was used to study propionate oxidation in a methanogenic coculture. The addition of 3-13C-labelled propionate clearly showed succinate as an intermediate. The ultimate breakdown product acetate was labelled equally in the C-1 and C-2 positions. In addition, de novo synthesis of [2-13C]proprionate was observed. The 13C-label randomised completely between C-2 and C-3 of proprionate. The results presented give further evidence for the involvement of the methylmalonyl-CoA pathway.

The uptake and utilization of sucrose by embryogenic suspension cultures of carrot (Daucus carota L.) growing in the presence of 2,4-D and by somatic embryos derived from these cultures was monitored using (13)C nuclear magnetic resonance. The exogeneously supplied sucrose was completely hydrolyzed before cell entry; glucose was taken up preferentially when the cells were cultured in the presence of 2,4-D, while glucose and fructose were utilized at similar rates by somatic embryos in the absence of 2,4-D. Both suspension cells and somatic embryos accumulated high intracellular levels predominantly of glucose and sucrose, the latter being resynthesized intracellularly from the constitutive hexoses. Initially, fructose was converted mainly into glucose and sucrose rather than being catabolized directly through glycolysis or the pentose phosphate pathway. Carbohydrate supply that exceeded cellular demand resulted in intracellular accumulation of mono- or disaccharides. The capacity of cultured carrot cells to produce somatic embryos appeared to be positively correlated with high intracellular levels of glucose.

500 MHz 1H NMR spectra were obtained of solutions containing oligomeric and monomeric forms of Chl a/b-P2, the major light-harvesting chlorophyll a/b-protein complex of photosystem II, isolated from thylakoid membranes of barley (Hordeum vulgare). Oligomers showed only a broad unresolved spectrum, but for monomers several downfield-shifted chlorophyll proton resonances were observed, assigned to the alpha and beta methine protons and the formyl proton of Chl-b. Identifying the observed shifts as ring-current shifts, these NMR data can be matched with previously obtained optical data confirming the trimeric arrangement of Chl-b in Chl a/b-P2 protein, with a distance between the chromophore centers of approximately 12 A.