Production of acetate by mutant strains of Clostridium thermoaceticum

Summary Mutant strains were derived from Clostridium thermoaceticum ATCC 39 289 by treatment with chemical mutagenic agents and selective enrichment procedures. Some mutant strains exhibited growth when cultured in media containing 20 mabetm (1.75 g l^–1) pyruvate of high-magnesium lime (dolime) above pH 6.0. One strain (G-20) grew and produced acetate when 80 mabetm (7 gl^–1) pyruvate or 50 mabetm (2.3 g l^–1) formate at pH 5.6 was the sole energy source. In a fed-batch process controlled at pH 6.2, this mutant produced 52.5 g l^–1 acetate (equivalent to 72.5 g l^–1 Na acetate) and 67 g l^–1 calcium-magnesium acetate (CMA) in 140 h when dolime was the neutralizing agent, with 93% substrate utilization. This mutant strain holds promise for CMA production due to its better tolerance of dolime and its ability to synthesize high levels of acetic acid. Offprint requests to: S. R. Parekh     

Preliminary compositional nutrient diagnosis norms for cowpea (Vigna unguiculata (L.) Walp.) grown on desert calcareous soil

This study calculated the compositional nutrient diagnosis (CND) norms of cowpea (Vigna unguiculata (L.) Walp.), as well as identified significant nutrient interactions of this crop growing in an irrigated calcareous desert soil. Three genotypes were distributed in rows in a 2-ha field. The soil showed high heterogeneity in its chemical properties. For statistical analysis, 86 foliar composite samples from healthy plants were used. Preliminary CND norms were developed using a cumulative variance ratio function and the ² distribution function. Means and standard deviations of row-centered log ratios V_X of five nutrients (N, P, K, Ca, and Mg) and a filling value R, which included all nutrients not chemically analyzed. Preliminary CND norms are: V_N^*=0.174±0.095, V_P^*=–2.172±0.234, V_K^*=–0.007±0.267, V_Ca^*=–0.022±0.146, V_Mg^*=–1.710±0.132, and V_R5^*=3.728±0.084. These CND norms are associated with dry bean yields higher than 1.88 t ha^–1, and are associated with the following foliar concentrations: 26.2 g N kg^–1, 2.5 g P kg^–1, 22.9 g K kg^–1, 21.6 g Ca kg^–1, and 4 g Mg kg^–1. Cowpea plants growing in desert calcareous soils took up lower amounts of N, P, and K than those considered as optimum in a previous report. Six interactions were strongly indicated for cowpea through principal component analyses: positive for Ca–Mg, and negative for N–Ca, N–Mg, Ca–P, Mg–P, and K–P. Furthermore, two interactions were identified using simple correlations, negative N–P and positive K–Ca.     

Protein synthesis in halophytes: The influence of potassium,sodium and magnesium in vitro

The amino acid (³⁵S-methionine) incorporating activity of an in vitro wheat germ translation system was found to be maximal in 80 to 125 mol m^–3 K with 2 to 4 mol m^–3 Mg both as the acetate. Substitution of Na for K, or chloride for acetate at concentrations above 80 mol m^–3 inhibited incorporation. When the K acetate concentration was raised to 200 mol m^–3, no incorporation of radioactive methionine occurred.Translation by polysomes extracted from leaf tissue of S. maritima, supplemented with postribosomal supernatant from wheat germ, showed activity which was optimal in the presence of 225 mol m^–3 K acetate and 8 mol m^–3 Mg acetate. However, the translation system was not directly comparable with the wheat germ system, as studies with an initiation inhibitor, aurintricarboxylic acid, suggested that the S. maritima system was essentially elongation-dependent, while initiation occurred in the wheat germ system.Elongation-dependent polysomal preparations were extracted from leaves of the glycophytes Pisum sativum, Triticum aestivum, Oryza sativa and Hordeum vulgare, and from the halophytes Atriplex isatidea and Inula crithmoides. Translation by polysomes from the salt-tolerant plants was optimal at higher K and Mg concentrations, than by polysomes from the glycophytes. Furthermore, NaCl was better able partially to substitute for the role of K in polysomal preparations from halophytes than glycophytes.     

High concentrations of acetate with a mutant strain ofC. thermoaceticum

Summary Fed-batch fermentation of glucose by a mutant strain ofC.thermoaceticum resulted in acetate concentrations of 83–100 gL^–1. Excess nutrients were required to maintain cell viability, especially when high cell concentrations were used. The strain was tolerant to high levels of Na, Ca and Mg. Product yield was 0.74–0.80 g acetate/g glucose, and the productivity was 0.60–0.85 gL^–1h^–1.     

Examining water temperature proxies in<Emphasis Type="Italic"> Porites</Emphasis> corals from the Great Barrier Reef: a cross-shelf comparison

Cores from colonies of the coral species Porites sp. were collected from inshore, mid-shelf, and outer reef localities (central Great Barrier Reef) to test the robustness of the major elemental sea surface temperature (SST) proxies (B/Ca, Mg/Ca, Sr/Ca, U/Ca) to the influence of inshore processes. Time series analyses of Sr/Ca, U/Ca, B/Ca, and Mg/Ca are compared to sea surface temperature (SST) in order to provide calibrations for these elements. This study shows that there are significant variations between the corals with respect to some of the proxies. In some cases, variations of ~6 °C are observed for a single U/Ca value. This magnitude of variation is also seen in the Mg/Ca proxy and, to a smaller extent, in the B/Ca–SST relationship. In two of the corals, both Mg/Ca and U/Ca do not follow a seasonal signal. The Mg/Ca and U/Ca ratios for two inshore corals are significantly different than the offshore corals (lower and higher, respectively). The other two proxies (B/Ca and Sr/Ca) do not display any inshore vs. offshore variations except for one inshore site that did not have a clear seasonal signal for either of these proxies. The Sr/Ca–SST relationship is the most robust, with a temperature variation of ~2 °C for a single Sr/Ca value, which is within error for this technique.     

Charybdotoxin blocks with high affinity the Ca-activated K+ channel of Hb A and Hb S red cells: Individual differences in the number of channels

Summary We have investigated the effect of a purified preparation of Charybdotoxin (CTX) on the Ca-activated K⁺ (Ca–K) channel of human red cells (RBC). Cytosolic Ca²⁺ was increased either by ATP depletion or by the Ca ionophore A23187 and incubation in Na⁺ media containing CaCl₂. The Ca–K efflux activated by metabolic depletion was partially (77%) inhibited from 15.8±2.4 mmol/liter cell · hr, to 3.7±1.0 mmol/liter cell · hr by 6nm CTX (n=3). The kinetic of Ca–K efflux was studied by increasing cell ionized Ca²⁺ using A23187 (60 mol/liter cell), and buffering with EGTA or citrate; initial rates of net K⁺ efflux (90 mmol/liter cell K⁺) into Na⁺ medium containing glucose, ouabain, bumetanide at pH 7.4 were measured. Ca–K efflux increased in a sigmoidal fashion (n of Hill 1.8) when Ca²⁺ was raised, with aK _m of 0.37 m and saturating between 2 and 10 m Ca²⁺. Ca–K efflux was partially blocked (71±7.8%, mean ±sd,n=17) by CTX with high affinity (IC₅₀0.8nm), a finding suggesting that is a high affinity ligand of Ca–K channels. CTX also blocked 72% of the Ca-activated K⁺ efflux into 75mm K⁺ medium, which counteracted membrane hyperpolarization, cell acidification and cell shrinkage produced by opening of the K⁺ channel in Na⁺ media. CTX did not block Valinomycin-activated K⁺ efflux into Na⁺ or K⁺ medium and therefore it does not inhibit K⁺ movement coupled to anion conductive permeability.TheV _max, but not theK _m–Ca of Ca–K efflux showed large individual differences varying between 4.8 and 15.8 mmol/liter cell · min (FU). In red cells with Hb A,V _max was 9.36±3.0 FU (mean ±sd,n=17). TheV _max of the CTX-sensitive, Ca–K efflux was 6.27±2.5 FU (range 3.4 to 16.4 FU) in Hb A red cells and it was not significantly different in Hb S (6.75±3.2 FU,n=8). Since there is larger fraction of reticulocytes in Hb S red cells, this finding indicates that cell age might not be an important determinant of theV _max of Ca–K⁺ efflux.Estimation of the number of CTX-sensitive Ca-activated K⁺ channels per cell indicate that there are 1 to 3 channels/per cell either in Hb A or Hb S red cells. The CTX-insensitive K⁺ efflux (2.7±0.9 FU) may reflect the activity of a different channel, nonspecific changes in permeability or coupling to an anion conductive pathway.     

Catalytic and biocatalytic oxidation of glucose to gluconic acid in a modified three-phase reactor

A comparative study of catalytic and biocatalytic glucose oxidation was carried out. Gluconobacter oxydans NBIMCC 1043 strain was used for biocatalytic glucose conversion. In the case of cell recycle coupled with cross-flow microfiltration the productivity and biomass concentration reached 40% and 3 g l^–1 respectively, in comparison to those of batch fermentation (21% and 2.3 g l^–1, respectively).     

Calcium-calcium and calcium-strontium exchange across the membrane of human red cell ghosts

Summary Stationary and nonstationary state⁴⁵Ca fluxes as well as Sr–Ca exchange movements were studied in energy-depleted human erythrocyte ghosts at different intra-and extracellular Ca concentrations. Influx and efflux followed the kinetics of a closed two-compartment system. The influx and efflux rate constants (k _in andk _out, respectively, fractions of total extra- or intracellular⁴⁵Ca that move in one direction per unit time) were similar in magnitude. They decreased with increasing Ca concentration on the cisside and increased with increasing Ca concentration on the trans-side of the membrane. Hence, the fluxes in both directions were characterized by saturation kinetics and appeared to be partially caused by an exchange diffusion mechanism. In the presence of a moderate inward (up to 8mm) or outward (up to 2mm) Ca concentration gradient, k_in andk _out did not vary in the course of an experiment and did not differ significantly from rates which were measured under stationary state conditions. Extracellular Sr induced an outward transport of intracellular Ca against the concentration gradient (counter-transport). The resulting inward Ca concentration gradient (maximal inside-to-outside concentration ratio as 1 to 3) persisted since extra- and intracellular Sr did not equilibrate. Analogous results were obtained studying⁴⁵Ca–⁴⁰Ca countertransport. In net flow experiments Ca–Sr exchange proved to occur on a one-for-one basis. Ca–Sr exchange was additive to the noncoupled Ca and Sr net downhill movements. The experimental results suggest that a specific ATP-independent Ca transfer system exists in the erythrocyte membrane which acts symmetrically on the two sides of the membrane and is restricted to a tightly coupled one-for-one exchange diffusion.     

A novel EDTA-degrading Pseudomonas sp.

A bacterial strain LPM-410 capable of utilizing ethylenediaminetetraacetate (EDTA) as the sole source of energy, carbon, and nitrogen was isolated from sewage sludge and identified as a Pseudomonas sp. on the basis of its phenotypic characteristics. Suspensions of exponential-phase cells degraded EDTA, Mg–, Ca–, Ba–, and Mn–EDTA at constant specific rates ranging from 0.363 to 0.525 mmol EDTA/(g cells h). The more stable chelate, Zn–EDTA, was degraded at a lower rate (0.195 ± 0.030 mmol EDTA/(g cells h)), and here was no degradation of Co–, Cu–, Pb–, and Fe(III)–EDTA.     

Adsorption and exchange of Ca,Mg and K-ions on the root cell walls of clover and rye-grass

Summary The ion exchange properties of clover and rye-grass root cell walls were studied quantitatively. Three sets of experiments were performed: adsorption of Ca, Mg and K ions versus pH, adsorption versus cation concentration and exchange isotherms Ca–Mg and Mg–K. A model has been developed. It allows the satisfactory prediction of results (except for pH curves) with the adjustment of a minimum of parameters. The total charge (RT), on its own, accounts for the difference between the ion exchange properties of the clover and rye grass cell walls. The selectivities observed on root material are very different from those observed on soil exchange complexes. The decreasing affinities of cell walls for cations follow the sequence: Ca>MgK for cell walls. These differences of selectivity are much larger than those usually observed for soil exchange complexes.     

Mammalian chromatin substructure studies with the calcium–magnesium endonuclease and two-dimensional polyacrylamide-gel electrophoresis

The basic regularity of chromatin substructure that has been reported in rat liver chromatin (Hewish & Burgoyne, 1973b) was also detected in mouse chromatin. The regular series of DNA fragments produced by the action of Ca–Mg endonuclease on rat chromatin were studied further. The smallest single-stranded class has a molecular weight of approx. 45000–63000 and the smallest double-stranded class has a molecular weight of approx. 120000–150000. Studies of the substructure of the DNA fragments produced by the Ca–Mg endonuclease have shown that the regular series of double-stranded fragments have regular series of single-stranded fragments within them. It was concluded that the regular series of double-stranded fragments was probably a consequence of the regular series of single-stranded fragments. Digestion time-courses are presented for mouse and rat nuclear DNA.     

Acetate production from lactate and glucose fermentation by Gluconobacter oxydans

Summary The production of acetate from the fermentation of lactate by Gluconobacter oxydans was studied. Batch experiments showed that glucose was the preferred substrate compared to lactate. A fed-batch culture was fed with a mixture of glucose and lactate followed by periodic addition of lactate. The maximum productivity of acetate was 0.16 g/l h but this value decreased during the fedbatch culture due to growth inhibition by acetate.     

<Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> NAD-dependent, <Emphasis Type="SmallCaps">D</Emphasis>-fructose reducing,polyol dehydrogenases activity: screening,medium optimisation and application for enzymatic polyol production

Gluconobacter oxydans LMG 1489 was selected as the best strain for NAD(P)-dependent polyol dehydrogenase production. The highest enzyme activities were obtained when this strain was cultivated on a medium consisting of 30 g glycerol l^–1, 7.2 g peptone l–1 and 1.8 g yeast extract l^–1. Two D-fructose reducing, NAD-dependent intracellular enzymes were present in the G. oxydans cell-free extract: sorbitol dehydrogenase, and mannitol dehydrogenase. Substrate reduction occurred optimally at a low pH (pH 6), while the optimum for substrate oxidation was situated at alkaline pHs (pH 9.5–10.5). The mannitol dehydrogenase was more thermostable than the sorbitol dehydrogenase. The cell-free extract could be used to produce D-mannitol and D-sorbitol enzymatically from D-fructose. Efficient coenzyme regeneration was accomplished by formate dehydrogenase-mediated oxidation of formate into CO₂.     

Kinetic analysis of ethanol production by an acetate-resistant strain of recombinant Zymomonas mobilis

Zymomonas mobilis ZM4/Ac^R (pZB5), a mutant recombinant strain with increased acetate resistance, has been isolated following electroporation of Z. mobilis ZM4/Ac^R. This mutant strain showed enhanced kinetic characteristics in the presence of 12 g sodium acetate l^–1 at pH 5 in batch culture on 40 g glucose, 40 g xylose l^–1 medium when compared to ZM4 (pZB5). In continuous culture, there was evidence of increased maintenance energy requirements/uncoupling of metabolism for ZM4/Ac^R (pZB5) in the presence of sodium acetate; a result confirmed by analysis of the effect of acetate on other strains of Z. mobilis. Nomenclature m Cell maintenance energy coefficient (g g^–1 h^–1)Maximum overall specific growth rate (1 h^–1)Maximum specific ethanol production rate (g g^–1 h^–1)Maximum specific total sugar utilization rate (g g^–1 h^–1)Biomass yield per mole of ATP (g mole^–1 Ethanol yield on total sugars (g g^–1)Biomass yield on total sugars (g g^–1)True biomass yield on total sugars (g g^–1)     

Bacterial Degradation of EDTA

Degradation of EDTA (ethylenediaminetetraacetic acid) or metal–EDTA complexes by cell suspensions of the bacterial strain DSM 9103 was studied. The activity of EDTA degradation was the highest in the phase of active cell growth and decreased considerably in the stationary phase, after substrate depletion in the medium. Exponential-phase cells were incubated in HEPES buffer (pH 7.0) with 1 mM of uncomplexed EDTA or EDTA complexes with Mg²⁺, Ca²⁺, Mn²⁺, Pb²⁺, Co²⁺, Cd²⁺, Zn²⁺, Cu²⁺, or Fe³⁺. The metal–EDTA complexes (Me–EDTA) studied could be divided into three groups according to their degradability. EDTA complexes with stability constants K below 10¹⁶ (log K < 16), such as Mg–EDTA, Ca–EDTA, and Mn–EDTA, as well as uncomplexed EDTA, were degraded by the cell suspensions at a constant rate to completion within 5–10 h of incubation. Me–EDTA complexes with log K above 16 (Zn–EDTA, Co–EDTA, Pb–EDTA, and Cu–EDTA) were not completely degraded during a 24-h incubation, which was possibly due to the toxic effect of the metal ions released. No degradation of Cd–EDTA or Fe(III)–EDTA by cell suspensions of strain DSM 9103 was observed under the conditions studied.     

Kinetic studies of acetate fermentation by Methanosarcina sp. MSTA-1

Summary The effect of three parameters (initial acetate concentration, temperature and pH) on the acetoclastic reaction was studied with the thermophilic methanogenic bacterium Methanosarcina sp. MSTA-1. The optimum temperature for growth ranged around 55° C, and optimum pH was 6.5–7.5, giving a minimum generation time of 12.6–13.9 h (µ_max = 0.050–0.055 h^–1) and a maximum value of the specific acetate consumption rate (q _infs ^supps ) of 14–20 mmol/g cells per hour. Contrary to the methane yield, the growth yield was found to be dependent on culture conditions, especially on incubation temperature. Methanosarcina sp. MSTA-1 showed a low affinity for acetate substrate. Growth at 55° C and at constant pH 7 resulted in a K _m value and a threshold acetate concentration of 10.7 mM and 0.7 mM, respectively. Offprint requests to: R. Moletta     

Direct analysis of nitrocatechol-type glucuronides in urine by capillary electrophoresis–electrospray ionisation mass spectrometry and tandem mass spectrometry

Direct, quantitative capillary electrophoresis–electrospray ionisation mass spectrometric (CE–ESI-MS) and tandem mass spectrometric (CE–ESI-MS–MS) methods are described for the quantitation of 3-O-glucuronides of E- and Z-entacapone isomers (EEG and EZG) and tolcapone (TG) in urine. 3-O-Glucuronide of nitecapone was used as internal standard. Good separation of glucuronides was achieved with 20 mM ammonium acetate as separation solution at pH 6.84. Stacking was used to increase the sensitivity of the method by introducing samples in 5 mM ammonium acetate. CE–ESI-MS and CE–ESI-MS–MS methods are linear with correlation coefficients better than 0.9983 and 0.9982, and repeatable with relative standard deviations below 9 and 14%, respectively. The limit of detection (LOD) in CE–ESI-MS at signal-to-noise ratio 3 is 100 ng/ml for EEG and EZG and 250 ng/ml for TG. The CE–ESI-MS–MS method was the more sensitive; LOD was 7 ng/ml for all compounds, without any concentration of the sample.     

Synthetic analogues of the histidine–chlorophyll complex: a NMR study to mimic structural features of the photosynthetic reaction center and the light-harvesting complex

Mg(II)–porphyrin–ligand and (bacterio)chlorophyl–ligand coordination interactions have been studied by solution and solid-state MAS NMR spectroscopy. ¹H, ¹³C and ¹⁵N coordination shifts due to ring currents, electronic perturbations and structural effects are resolved for imidazole (Im) and 1-methylimidazole (1-MeIm) coordinated axially to Mg(II)-OEP and (B)Chl a. As a consequence of a single axial coordination of Im or 1-MeIm to the Mg(II) ion, 0.9–5.2 ppm ¹H, 0.2–5.5 ppm ¹³C and 2.1–27.2 ppm ¹⁵N coordination shifts were measured for selectively labeled [1,3-¹⁵N]-Im, [1,3-¹⁵N,2-¹³C]-Im and [1,3-¹⁵N,1,2-¹³C]-1-MeIm. The coordination shifts depend on the distance of the nuclei to the porphyrin plane and the perturbation of the electronic structure. The signal intensities in the ¹H NMR spectrum reveal a five-coordinated complex, and the isotropic chemical shift analysis shows a close analogy with the electronic structure of the BChl a–histidine in natural light harvesting 2 complexes. The line broadening of the ligand responses support the complementary IR data and provide evidence for a dynamic coordination bond in the complex.Abbreviations (B)Chl a (bacterio)chlorophyll a - HMBC heteronuclear multiple bond correlation - Im imidazole - LH light-harvesting - 1-MeIm 1-methylimidazole - Mg(II)-Por Mg(II)-porphyrin macrocycle - OEP 2,3,7,8,12,13,17,18-octaethylporphyrin     

Biomass production by alders on four abandoned agricultural soils in Québec

The production of aboveground tissue of three alder species (Alnus crispa (Ait.) Pursh,A. rugosa (Du Roi) Spreng. andA. glutinosa (L) Gaertn.) on four sites ranged from 0.4 t ha^–1 yr^–1 to 4.0 t ha^–1 yr^–1 after four growing seasons. Large differences were observed among the four sites studied and among species. Soil nutrient levels affected the biomass production and foliar symptoms of P and Mg deficiency occurred withA. crispa andA. rugosa. Because of their poor aboveground biomass production (0.4–1.4 t ha^–1 yr^–1),A. crispa andA. rugosa should be used mainly as nurse trees. For its higher potential for biomass production (up to 4.0 t ha^–1 yr^–1), and its apparent higher ability to use P and Mg on deficient sites,A. glutinosa should be used preferably toA. crispa andA. rugosa for the production of biomass.