Purification and characterization of membrane-bound hydrogenase from Methanosarcina barkeri MS

Hydrogenase was solubilized from the membrane of acetate-grown Methanosarcina barkeri MS and purification was carried out under aerobic conditions. The enzyme was reactivated under reducing conditions in the presence of H₂. The enzyme showed a maximal activity of 120±40 mol H₂ oxidized · min^–1 · min^–1 with methyl viologen as an electron acceptor, a maximal hydrogen production rate of 45±4 mol H₂ · min^–1 · mg^–1 with methyl viologen as electron donor, and an apparent K _m for hydrogen oxidation of 5.6±1.7 M. The molecular weight estimated by gel filtration was 98,000. SDS-PAGE showed the enzyme to consist of two polypeptides of 57,000 and 35,000 present in a 1:1 ratio. The native protein contained 8±2 mol Fe, 8±2 mol S^2–, and 0.5 mol Ni/mol enzyme. Cytochrome b was reduced by hydrogen in a solubilized membrane preparation. The hydrogenase did not couple with autologous F₄₂₀ or ferredoxin, nor with FAD, FMN, or NAD(P)⁺. The physiological function of the membrane-bound hydrogenase in hydrogen consumption is discussed.Abbreviation CoM-S-S-HTP the heterodisulfide of 7-mercaptoheptanoylthrconine phosphate and coenzyme M (mercaptoethanesulfonic acid)     

The kinetics of reactions around the cytochrome bf complex studied in an isolated system

The kinetics of oxidation and reduction of P700, plastocyanin, cytochrome f and cytochrome b-563 were studied in a reconstituted system consisting of Photosystem I particles, cytochrome bf complex and plastocyanin, all derived from pea leaf chloroplasts. Decyl plastoquinol was the reductant of the bf complex. Turnovers of the system were initiated by laser flashes. The reaction between oxidised P700 and plastocyanin was non-homogeneous in that a second-order rate coefficient of c. 5×10^–7 M^–1 s^–1 applied to 80% of the P700⁺ and c. 0.7×10⁷ M^–1 s^–1 to the remainder. In the presence of bf complex, but without quinol, the electron transfer between cytochrome f and oxidised plastocyanin could be described by a second-order rate coefficient of c. 4×10⁷ M^–1 s^–1 (forward), and c. 1.6×10⁷ M^–1 s^–1 (reverse). The equilibrium coefficient was thus 2.5. Unexpectedly, there was little reduction of cytochrome f ⁺ or plastocyanin⁺ by electrons from the Rieske centre. With added quinol, reduction of cytochrome b-563 occurred. Concomitantly, electrons appeared in the oxidised species. It was inferred that either the Rieske centre was not involved in the high-potential chain of electron transfer events, or that, only in the presence of quinol, electrons were quickly passed from the Rieske centre to cytochrome f ⁺. Additionally, the presence of quinol altered the equilibrium coefficient for the cyt f/PC interaction from 2.5 to c. 5. The reaction between quinol and the bf complex was describable by a second-order rate coefficient of about 3×10⁶ M^–1 s^–1. The pattern of the redox reactions around the bf complex could be simulated in detail with a Q-cycle model as previously found for chloroplasts.Abbreviations AQS anthraquinone sulphonate - cyt cytochrome - cyt b-563(H) high-potential cyt b-563 - cyt b-563(L) low potential cyt b-563 - FeS(R) the Rieske protein of the cyt bf complex, containing an Fe₂S₂ centre - PC plastocyanin - PS photosystem - P700 reaction centre in PS I     

Reaction center and UQH2:cytc 2 oxidoreductase act as independent enzymes inRps. sphaeroides

Turnover of the ubiquinol oxidizing site of the UQH₂:cyt c₂ oxidoreductase (b/c ₁ complex) ofRps. sphaeroides can be assayed by measuring the rate of reduction of cytb ₅₆₁ in the presence of antimycin (AA). Oxidation of ubiquinol is a second-order process, with a value ofk ₂ of about 3 × 10⁵ M^–1. The reaction shows saturation at high quinol concentrations, with an apparentK _m of about 6–8 mM (with respect to the concentration of quinol in the membrane). When the quinone pool is oxidized before illumination, reduction of the complex shows a substantial lag (about 1 ms) after a flash, indicating that the quinol produced as a result of the photochemical reactions is not immediately available to the complex. We have suggested that the lag may be due to several factors, including the leaving time of the quinol from the reaction center, the diffusion time to the complex, and the time for the head group to cross the membrane. We have suggested aminimal value for the diffusion coefficient of ubiquinone in the membrane (assuming that the lag is due entirely to diffusion) of about 10^–9 cm^–2 sec^–1. The lag is reduced to about 100 µsec when the pool is significantly reduced, showing that quinol from the pool is more rapidly available to the complex than that from the reaction center. With the pool oxidized, similar kinetics are seen when the reduction of cytb ₅₆₁ occurs through the AA-sensitive site (with reactions at the quinol oxidizing site blocked by myxothiazol). These results show that there is no preferential reaction pathway for transfer of reducing equivalents from reaction center tob/c ₁ complex. Oxidation of cytb ₅₆₁ through the AA-sensitive site can be assayed from the slow phase of the carotenoid electrochromic change, and by comparison with the kinetics of cytb ₅₆₁. As long as the quinone pool is significantly oxidized, the reaction is not rate-determining for the electrogenic process. On reduction of the pool below 1 quinone per complex, a slowing of the electrogenic process occurs, which could reflect a dependence on the concentration of quinone. If the process is second-order, the rate constant must be about 2–5 times greater than that for quinol oxidation, since the effect on rate is relatively small compared with the effect seen at the quinol oxidizing site when the quinol concentration is changed over theE _h range where the first few quinols are produced on reductive titration. When the quinone pool is extracted (experiments in collaboration with G. Venturoli and B. A. Melandri), the slowing of the electrochromic change on reduction of the pool is not enhanced; we assume that this is due to the fact that a minimum of one quinone per active complex is produced by turnover of the quinol oxidizing site. Two lines of research lead us to revise our previous estimate for the minimal value of the quinone diffusion coefficient. These relate to the relation between the diffusion coefficient and the rate constants for processes involving the quinones: (a) The estimated rate constant for reaction of quinone at the AA-site approaches the calculated diffusion limited rate constant, implying an improbably efficient reaction. (b) From a preliminary set of experiments, the activation energy determined by measuring the variation of the rate constant for quinol oxidation with temperature, is about 8 kcal mol^–1. Although we do not know the contribution of entropic terms to the pre-exponential factor, the result is consistent with a considerably larger value for the diffusion coefficient than that previously suggested.     

The kinetics of P515 in relation to the lipid composition of the thylakoid membrane

Flash-induced P515 absorbance changes have been studied in dark-adapted chloroplasts isolated from spinach plants grown under two different light intensities. The slow component (reaction 2), normally present in the P515 response of chloroplasts isolated from plants grown at an intensity of 60 W · m^–2, was largely reduced in chloroplasts isolated from plants grown at an intensity of 6 W · m^–2. This reduction of the slow component in the P515 response appeared to be coincident with an alteration in the lipid composition of the thylakoid membrane. Mainly the ratio monogalactosyldiacylglycerol to digalactosyldiacylglycerol appeared to be altered. In thylakoids from plants grown at 6 W · m^–2, the ratio was approximately 35% lower than that of plants grown at 60 W · m^–2. The amount of both cytochromeb ₅₆₃ and cytochromef was largely reduced in chloroplasts isolated from plants grown at low light intensity. These results may indicate a possible correlation between structural organization of the thylakoid membrane and the kinetics of the flash-induced P515 response.     

Molecular mechanisms of nitrogen dioxide induced epithelial injury in the lung

The lung can be exposed to a variety of reactive nitrogen intermediates through the inhalation of environmental oxidants and those produced during inflammation. Reactive nitrogen species (RNS) include, nitrogen dioxide (·NO₂) and peroxynitrite (ONOO^–). Classically known as a major component of both indoor and outdoor air pollution, ·NO₂ is a toxic free radical gas. ·NO₂ can also be formed during inflammation by the decomposition of ONOO^– or through peroxidase-catalyzed reactions. Due to their reactive nature, RNS may play an important role in disease pathology. Depending on the dose and the duration of administration, ·NO₂ has been documented to cause pulmonary injury in both animal and human studies. Injury to the lung epithelial cells following exposure to ·NO₂ is characterized by airway denudation followed by compensatory proliferation. The persistent injury and repair process may contribute to airway remodeling, including the development of fibrosis. To better understand the signaling pathways involved in epithelial cell death by ·NO₂ or other RNS, we routinely expose cells in culture to continuous gas-phase ·NO₂. Studies using the ·NO₂ exposure system revealed that lung epithelial cell death occurs in a density dependent manner. In wound healing experiments, ·NO₂ induced cell death is limited to cells localized in the leading edge of the wound. Importantly, ·NO₂-induced death does not appear to be dependent on oxidative stress per se. Potential cell signaling mechanisms will be discussed, which include the mitogen activated protein kinase, c-Jun N-terminal Kinase and the Fas/Fas ligand pathways. During periods of epithelial loss and regeneration that occur in diseases such as asthma or during lung development, epithelial cells in the lung may be uniquely susceptible to death. Understanding the molecular mechanisms of epithelial cell death associated with the exposure to ·NO₂ will be important in designing therapeutics aimed at protecting the lung from persistent injury and repair.     

Na/H exchange in cultured epithelial cells from fish gills

Using primary cultures of gill pavement cells from freshwater rainbow trout, a method is described for achieving confluent monolayers of the cells on glass coverslips. A continuous record of intracellular pH was obtained by loading the cells with the pH-sensitive flourescent dye 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and mounting the coverslips in the flowthrough cuvette of a spectrofluorimeter. Experiments were performed in HEPES-buffered media nominally free of HCO₃. Resting intracellular pH (7.43 at extracellular pH=7.70) was insensitive to the removal of Cl^– or the application of 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (0.1 mmol·l^–1), but fell by about 0.3 units when Na⁺ was removed or in the presence of amiloride (0.2 mmol·l^–1). Exposure to elevated ammonia (ammonia prepulse; 30 mmol·l^–1 as NH₄Cl for 6–9 min) produced an increase in intracellular pH (to about 8.1) followed by a slow decay, and washout of the pulse caused intracellular pH to fall to about 6.5. Intracellular non-HCO ₃ ^– buffer capacity was about 13.4 slykes. Rapid recovery of intracellular pH from intracellular acidosis induced by ammonia prepulse was inhibited more than 80% in Na⁺-free conditions or in the presence of amiloride (0.2 mmol·l^–1). Neither bafilomycin A₁ (3 mol·l^–1) nor Cl removal altered the intracellular pH recovery rate. The K _m for Na⁺ of the intracellular pH recovery mechanism was 8.3 mmol·l^–1, and the rate constant at V _max was 0.008·s^–1 (equivalent to 5.60 mmol H⁺·l^–1 cell water·min^–1), which was achieved at external Na⁺ levels from 25 to 140 mmol·l^–1. We conclude that intracellular pH in cultured gill pavement cells in HEPES-buffered, HCO ₃ ^– -free media, both at rest and during acidosis, is regulated by a Na⁺/H⁺ antiport and not by anion-dependent mechanisms or a vacuolar H⁺-ATPase.Abbreviations BCECF 2,7-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein - BCECF/AM 2,7-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein, acetoxymethylester - Cholin-Cl choline chloride - DMSO dimethyl sulfoxide - EDTA ethylene diamine tetra-acetic acid - FBS foetal bovine serum - H ⁺ -ATPase Proton-dependent adenosine triphosphatase - HEPES N-[2-hydroxyethyl]piperazine-N[2-ethanesulfonic acid] - pH _i intracellular pH - pH _e extracellular pH - PBS phosphate-buffered saline - SITS 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid     

Reaction of nitric oxide with the oxidized di-heme and heme–copper oxygen-reducing centers of terminal oxidases: Different reaction pathways and end-products

Inhibition of terminal oxidases by nitric oxide (NO) has been extensively investigated as it plays a role in regulation of cellular respiration and pathophysiology. Cytochrome bd is a tri-heme (b₅₅₈, b₅₉₅, d) bacterial oxidase containing no copper that couples electron transfer from quinol to O₂ (to produce H₂O) with generation of a transmembrane protonmotive force. In this work, we investigated by stopped-flow absorption spectroscopy the reaction of NO with Escherichia coli cytochrome bd in the fully oxidized (O) state. We show that under anaerobic conditions, the O state of the enzyme binds NO at heme d with second-order rate constant k_on = 1.5 ± 0.2 × 10² M⁻¹ s⁻¹, yielding a nitrosyl adduct (d³⁺–NO or d²⁺–NO⁺) with characteristic optical features (an absorption increase at 639 nm and a red shift of the Soret band). The reaction mechanism is remarkably different from that of O cytochrome c oxidase in which the heme–copper binuclear center reacts with NO approximately three orders of magnitude faster, forming nitrite. The data allow us to conclude that in the reaction of NO with terminal oxidases in the O state, Cu_B is indispensable for rapid oxidation of NO into nitrite.     

Lateral hydraulic conductivity of early metaxylem vessels in Zea mays L. roots

The hydraulic conductivity of the lateral walls of early metaxylem vessels (Lp_x in m · s^–1 · MPa^–1) was measured in young, excised roots of maize using a root pressure probe. Values for this parameter were determined by comparing the root hydraulic conductivities before and after steam-ringing a short zone on each root. Killing of living tissue virtually canceled its hydraulic resistance. There were no suberin lamellae present in the endodermis of the roots used. The value of Lp_x ranged between 3 · 10^–7 and 35 · 10^–7 m · s^–1 · MPa^–1 and was larger than the hydraulic conductivity of the untreated root (Lp_r = 0.7 · 10^–7 to 4.0 · 10^–7 m · s^–1 · MPa^–1) by factor of 3 to 13. Assuming that all flow through the vessel walls was through the pit membranes, which occupied 14% of the total wall area, an upper limit of the hydraulic conductivity of this structure could be given(Lp_pm=21 · 10^–7 to 250 · 10^–7 m · s^–1 · MPa^–1). The specific hydraulic conductivity (Lp_cw) of the wall material of the pit membranes (again an upper limit) ranged from 0.3 · 10^–12 to 3.8 · 10^–12 m² · s^–1 · MPa^–1 and was lower than estimates given in the literature for plant cell walls. From the data, we conclude that the majority of the radial resistance to water movement in the root is contributed by living tissue. However, although the lateral walls of the vessels do not limit the rate of water flow in the intact system, they constitute 8–31% of the total resistance, a value which should not be ignored in a detailed analysis of water flow through roots.Abbreviatations and Symbols k_wr (T ₁ ^2/W ) rate constant (half-time) of water exchange across root (s^–1 or s, respectively) - Lp_cw specific hydraulic conductivity of wall material (m² · s^–1 · MPa^–1) - Lp_pm hydraulic conductivity of pit membranes (m · s ^–1 · MPa^–1) - Lp_r hydraulic conductivity of root (m · s^–1 · MPa^–1) - Lp_x lateralhydraulic conductivity of walls of root xylem (m · s ^–1 · MPa^–1) This research was supported by a grant from the Bilateral Exchange Program funded jointly by the Natural Sciences and Engineering Research Council of Canada and the Deutsche Forschungsgemeinschaft to C.A.P., and by a grant from the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 137, to E.S. The expert technical help of Mr. Burkhard Stumpf and the work of Ms. Martina Murrmann and Ms. Hilde Zimmermann in digitizing chart-recorder strips is gratefully acknowledged.     

Isolation of the respiratory burst oxidase: The role of a flavoprotein component

The article reviews the enzymatic and electron transfer properties of a low-potential FAD-dependent flavoprotein that is a component of the NADPH-dependent O ₂ ^– · -generating respiratory burst oxidase of phagocytes. Current methods available for isolation of the respiratory burst oxidase and the flavo-protein component of the complex are also reviewed. These studies and data obtained from affinity-labeling of respiratory burst oxidase components, suggest that the flavoprotein has a molecular weight of 65–67 kD. The prevailing evidence suggests that the flavoprotein functions as a dehydrogenase/electron transferase and can directly catalyse NADPH-dependent O ₂ ^– · formation when isolated. However, in neutrophil plasma membranes, the prevailing evidence suggests that the flavoprotein functions primarily to transfer electrons from NADPH to cytochromeb _–245 and that this latter redox component is the catalytic side of O ₂ ^– · formation. A working model for the arrangement of the flavorprotein and cytochromeb _–245 components of the respiratory burst oxidase in neutrophil membranes is proposed.     

Stomatal responses to carbon dioxide of isolated epidermis from a C3 plant,the Argenteum mutant of Pisum sativum L., and a crassulacean-acid-metabolism plant Kalanchoë daigremontiana Hamet et Perr

The response of stomata in isolated epidermis to the concentration of CO₂ in the gaseous phase was examined in a C₃ species, the Argenteum mutant of Pisum sativum, and a crassulacean-acid-metabolism (CAM) species, Kalanchoë daigremontiana. Epidermis from leaves of both species was incubated on buffer solutions in the presence of air containing various volume fractions of CO₂ (0 to 10000·10^–6). In both species and in the light and in darkness, the effect of CO₂ was to inhibit stomatal opening, the maximum inhibition of opening occurring in the range 0 to 360·10^–6. The inhibition of opening per unit change in concentration was greatest between volume fractions of 0 and 240·10^–6. There was little further closure above the volume fraction of 360·10^–6, i.e. approximately ambient concentration of CO₂. Thus, although leaves of CAM species may experience much higher internal concentrations of CO₂ in the light than those of C₃ plants, this does not affect the sensitivity of their stomata to CO₂ concentration or the range over which they respond. Stomatal responses to CO₂ were similar in both the light and the dark, indicating that effects of CO₂ on stomata occur via mechanisms which are independent of light. The responses of stomata to CO₂ in the gaseous phase took place without the treatments changing the pH of the buffered solutions. Thus it is unlikely that CO₂ elicited stomatal movement by changing either the pH or the HCO ₃ ^– /CO ₃ ^2- equilibria. It is suggested that the concentration of dissolved unhydrated CO₂ may be the effector of stomatal movement and that its activity is related to its reactivity with amines.     

Cycling of inorganic and organic sulfur in peat from Big Run Bog,West Virginia

Total S concentration in the top 35 cm of Big Run Bog peat averaged 9.7 mol·g — wet mass^–1 (123 mol·g dry mass^–1). Of that total, an average of 80.8% was carbon bonded S, 10.4% was ester sulfate S, 4.5% was FeS₂­S, 2.7% was FeS­S, 1.2% was elemental S, and 0.4% was SO₄ ^2–­S. In peat collected in March 1986, injected with³⁵S­SO₄ ^2– and incubated at 4 °C, mean rates of dissimilatory sulfate reduction (formation of H₂S + S⁰ + FeS + FeS₂), carbon bonded S formation, and ester sulfate S formation averaged 3.22, 0.53, and 0.36 nmol·g wet mass^–1·h^–1, respectively. Measured rates of sulfide oxidation were comparable to rates of sulfate reduction. Although dissolved SO₄ ^2– concentrations in Big Run Bog interstitial water (< 200 µM) are low enough to theoretically limit sulfate reducing bacteria, rates of sulfate reduction integrated throughout the top 30–35 cm of peat of 9 and 34 mmol·m^–2·d^–1 (at 4 °C are greater than or comparable to rates in coastal marine sediments. We suggest that sulfate reduction was supported by a rapid turnover of the dissolved SO₄ ^2– pool (average turnover time of 1.1 days). Although over 90% of the total S in Big Run Bog peat was organic S, cycling of S was dominated by fluxes through the inorganic S pools.     

Long-term kinetics of the light-dependent regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in plants with and without 2-carboxyarabinitol 1-phosphate

The light-dependent modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity was studied in two species: Phaseolus vulgaris L., which has high levels of the inhibitor of Rubisco activity, carboxyarabinitol 1-phosphate (CA1P), in the dark, and Chenopodium album L., which has little CA1P. In both species, the ratio of initial to fully-activated Rubisco activity declined by 40–50% within 60 min of a reduction in light from high a photosynthetic photon flux density (PPFD; >700 mol · m^–2 · s^–1) to a low PPFD (65 ± 15 mol · m^–2 · s^–1) or to darkness, indicating that decarbamylation of Rubisco is substantially involved in the initial regulatory response of Rubisco to a reduction in PPFD, even in species with potentially extensive CA1P inhibition. Total Rubisco activity was unaffected by PPFD in C. album, and prolonged exposure (2–6 h) to low light or darkness was accompanied by a slow decline in the activity ratio of this species. This indicates that the carbamylation state of Rubisco from C. album gradually declines for hours after the large initial drop in the first 60 min following light reduction. In P. vulgaris, the total activity of Rubisco declined by 10–30% within 1 h after a reduction in PPFD to below 100 mol · m^–2 · s^–1, indicating CA1P-binding contributes significantly to the reduction of Rubisco capacity during this period, but to a lesser extent than decarbamylation. With continued exposure of P. vulgaris leaves to very low PPFDs (< 30 mol · m^–2 · s^–1), the total activity of Rubisco declined steadily so that after 6–6.5 h of exposure to very low light or darkness, it was only 10–20% of the high-light value. These results indicate that while decarbamylation is more prominent in the initial regulatory response of Rubisco to a reduction in PPFD in P. vulgaris, binding of CA1P increases over time and after a few hours dominates the regulation of Rubisco activity in darkness and at very low PPFDs.Abbreviations CA1P 2-carboxyarabinitol 1-phosphate - CABP 2-carboxyarabinitol 1,5-bisphosphate - k_cat substrate-saturated turnover rate of fully carbamylated enzyme - PPFD photosynthetically active photon flux density (400–700 nm) - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate     

Nitrogen fixation in subarctic streams influenced by beaver (Castor canadensis)

Nitrogen fixation was measured in four subarctic streams substantially modified by beaver (Castor canadensis) in Quebec. Acetylene-ethylene (C₂H₂ C₂H₄) reduction techniques were used during the 1982 ice-free period (May–October) to estimate nitrogen fixation by microorganisms colonizing wood and sediment. Mean seasonal fixation rates were low and patchy, ranging from zero to 2.3 × 10^–3 µmol C₂H₄ · cm^–2 · h^–1 for wood, and from zero to 7.0 × 10^–3 µmol C₂H₄ · g AFDM^–1 · h^–1 for sediment; 77% of all wood and 63% of all sediment measurements showed no C₂H₂ reduction. Nonparametric statistical tests were unable to show a significant difference (p > 0.05) in C₂H₂ reduction rates between or within sites for wood species or by sediment depth.Nitrogen contributed by microorganisms colonizing wood in riffles of beaver influenced watersheds was small (e.g., 0.207 g N · m^–2 · y^–1) but greater than that for wood in beaver ponds (e.g., 0.008 g N · m^–2 · y^–1) or for streams without beaver (e.g., 0.003 g N · m^–2 · y^–1). Although mass specific nitrogen fixation rates did not change significantly as beaver transform riffles into ponds, the nitrogen fixed by organisms colonizing sediment in pond areas (e.g., 5.1 g N · m^–2 · y^–1) was greater than that in riffles (e.g., 0.42 g N · m^–2 · y^–1). The annual nitrogen contribution is proportional to the amount of sediment available for microbial colonization. We estimate that total nitrogen accumulation in sediment, per unit area, is enhanced 9 to 44 fold by beaver damming a section of stream.     

The effect of carbon dioxide on the growth kinetics of fructose-limited chemostat cultures of Acetobacterium woodii DSM 1030

The growth of the anaerobic acetogenic bacterium Acetobacterium woodii DSM 1030 was investigated in fructose-limited chemostat cultures. A defined medium was developed which contained fructose, mineral salts, cysteine · HCl and Ca pantothenate (1 mg · 1^–1) supplied in a vitamin supplement. Growth at high dilution rates was dependent on the presence of CO₂ in the gas phase. The ^max was found to be 0.16 h^–1 and the fructose maintenance requirement was 0.1 to 0.13 mmol fructose · (g dry wt)^–1 · h^–1. A growth yield of 61 g dry wt · (mol fructose)^–1, corrected for the cell maintenance requirement and for incorporation of fructose carbon into cell biomass, was determined from the fructose consumption. A corresponding growth yield of 69 g dry wt · (mol fructose)^–1 was calculated from the acetate production assuming that fructose fermentation was homoacetogenic. A Y_ATP of 12.2 to 13.8 g dry wt · (mol ATP)^–1 was calculated from these growth yields using a value of 5 mol ATP · (mol fructose)^–1 as an estimate of the amount of ATP synthesised from fructose fermentation. The addition of yeast extract (0.5 g · 1^–1) to the medium did not influence the ^max or cell yield. After prolonged growth under fructose-limited conditions the requirement of the culture for CO₂ in the gas phase was reduced.Abbreviations YE yeast extract - IC inorganic carbon - D fermenter dilution rate : h^–1 - M_X maintenance requirement for X: mmol X · (g dry wt)^–1 · h^–1 - X may be fructose (Fruct), fructose consumed in energy metabolism (Fruct [E]), acetate (Ac) - ATP CO₂, NH _inf4 ^sup+ or Pi - qX specific rate of utilisation or consumption of X: mmol X · (g dry wt)^–1 · h^–1 - V fermenter volume: litre - rC · Cell, fermenter cell carbon production: mmol C · h^–1 - Y_X yield of cells on X: g dry wt · (mol X)^–1 - Y _infx ^supmax the yield corrected for cell maintenance: g dry wt · (mol X)^–1 - S_ATP stoichiometry of ATP synthesis from fructose: mol ATP · (mol frucose)^–1 - x cell concentration: g dry wt · 1^–1 - specific growth rate : h^–1 - ^max maximum specific growth rate: h^–1     

Effects of prolonged cycle ergometer exercise on maximal muscle power and oxygen uptake in humans

The mechanical power (W_tot, W·kg^–1) developed during ten revolutions of all-out periods of cycle ergometer exercise (4–9 s) was measured every 5–6 min in six subjects from rest or from a baseline of constant aerobic exercise [50%–80% of maximal oxygen uptake (VO_2max)] of 20–40 min duration. The oxygen uptake [VO₂ (W·kg^–1, 1 ml O₂ = 20.9 J)] and venous blood lactate concentration ([la]_b, mM) were also measured every 15 s and 2 min, respectively. During the first all-out period, W_tot decreased linearly with the intensity of the priming exercise (W_tot = 11.9–0.25·VO₂). After the first all-out period (i greater than 5–6 min), and if the exercise intensity was less than 60% VO_2max, W_tot, VO₂ and [la]_b remained constant until the end of the exercise. For exercise intensities greater than 60% VO_2max, VO₂ and [la]_b showed continuous upward drifts and W_tot continued decreasing. Under these conditions, the rate of decrease of W_tot was linearly related to the rate of increase of V [(d W_tot/dt) (W·kg^–1·s^–1) = 5.0·10^–5 –0.20·(d VO₂/dt) (W·kg^–1·s^–1)] and this was linearly related to the rate of increase of [la]_b [(d VO₂/dt) (W·kg^–1·s^–1) = 2.310^–4 + 5.910^–5·(d [la]_b/dt) (mM·s^–1)]. These findings would suggest that the decrease of W_tot during the first all-out period was due to the decay of phosphocreatine concentration in the exercising muscles occurring at the onset of exercise and the slow drifts of VO₂ (upwards) and of W_tot (downwards) during intense exercise at constant W_tot could be attributed to the continuous accumulation of lactate in the blood (and in the working muscles).     

Ion fluxes inAcetabularia: Vesicular shuttle

Summary Ion flux relations in the unicellular marine algaAcetabularia have been investigated by uptake and washout kinetics of radioactive tracers (²²Na⁺,⁴²K⁺,³⁶Cl^– and⁸⁶Rb⁺) in normal cells and in cell segments with altered compartmentation (depleted of vacuole or of cytoplasm). Some flux experiments were supplemented by simultaneous electrophysiological recordings. The main results and conclusions about the steady-state relations are: the plasmalemma is the dominating barrier for translocation of K⁺ with influx and efflux of about 100 nmol·m^–2·sec^–1×K⁺ passes three- to sevenfold more easily than Rb⁺ does. Under normal conditions, Cl^– (the substrate of the electrogenic pump, which dominates the electrical properties of the plasmalemma in the resting state) shows two efflux components of about 17 and 2 mol·m^–2·sec^–1, and a cytoplasmic as well as vacuolar [Cl^–] of about 420mm ([Cl^–] _o =529mm). At 4°C, when the pump is inhibited, both influx and efflux, as well as the cellular [Cl^–], are significantly reduced. Na⁺ ([Na⁺] _i : about 70mm, [Na⁺] _o : 461mm), which is of minor electrophysiological relevance compared to K⁺, exhibits rapid and virtually temperature-insensitive (electroneutral) exchange (two components with about 2 and 0.2 mol·m^–2·sec^–1 for influx and efflux). Some results with Na⁺ and Cl^– are inconsistent with conventional (noncyclic) compartmentation models: (i) equilibration of the vacuole (with the external medium) can be faster than equilibration of the cytoplasm, (ii) absurd concentration values result when calculated by conventional compartmental analysis, and (iii) large amounts of ions can be released from the cell without changes in the electrical potential of the cytoplasm. These observations can be explained by the particular compartmentation of normalAcetabularia cells (as known by electron micrographs) with about 1 part cytoplasm, 5 parts central vacuole, and 5 parts vacuolar vesicles. These vesicles communicate directly with the central vacuole, with the cytoplasm and with the external medium.     

Nitrite uptake by Chlamydomonas reinhardtii cells immobilized in calcium alginate

When an initial cell loading of about 30–40 µg chlorophyll (Chl)·g^–1 gel and alginate suspension of 3% (w/v) were used for immobilization of Chlamydomonas reinhardtii, the resulting cell beads showed optimum nitrite uptake rate, at 30° C and pH 7.5, of 14 µmol NO _inf2 ^sup– ·mg^–1 Chl·h^–1, the photosynthetic and respiratory activities being about 120 µmol O₂ produced·mg^–1 Chl·h^–1, and 40 µmol O₂ consumed ·mg^–1 Chl·h^–1, respectively. The nitrite uptake activity required CO₂ in the culture and persisted after 8 days of cells immobilization, or in the presence of 0.2 mm ammonium in the medium. Our data indicate that alginate-entrapped C, reinhardtii cells may provide a stable and functional system for removing nitrogenous contaminants from waste-waters.Correspondence to: C. Vílchez     

Process for biological oxidation and control of dissolved iron in bioleach liquors

Iron has a central role in bioleaching and biooxidation processes. Fe²⁺ produced in the dissolution of sulfidic minerals is re-oxidized to Fe³⁺ mostly by biological action in acid bioleaching processes. To control the concentration of iron in solution, it is important to precipitate the excess as part of the process circuit. In this study, a bioprocess was developed based on a fluidized-bed reactor (FBR) for Fe²⁺ oxidation coupled with a gravity settler for precipitative removal of ferric iron. Biological iron oxidation and partial removal of iron by precipitation from a barren heap leaching solution was optimized in relation to the performance and retention time (τ_FBR) of the FBR. The biofilm in the FBR was dominated by Leptospirillum ferriphilum and “Ferromicrobium acidiphilum.” The FBR was operated at pH 2.0 ± 0.2 and at 37 °C. The feed was a barren leach solution following metal recovery, with all iron in the ferrous form. 98–99% of the Fe²⁺ in the barren heap leaching solution was oxidized in the FBR at loading rates below 10 g Fe²⁺/L h (τ_FBR of 1 h). The optimal performance with the oxidation rate of 8.2 g Fe²⁺/L h was achieved at τ_FBR of 1 h. Below the τ_FBR of 1 h the oxygen mass transfer from air to liquid limited the iron oxidation rate. The precipitation of ferric iron ranged from 5% to 40%. The concurrent Fe²⁺ oxidation and partial precipitative iron removal was maximized at τ_FBR of 1.5 h, with Fe²⁺ oxidation rate of 5.1 g Fe²⁺/L h and Fe³⁺ precipitation rate of 25 mg Fe³⁺/L h, which corresponded to 37% iron removal. The precipitates had good settling properties as indicated by the sludge volume indices of 3–15 mL/g but this step needs additional characterization of the properties of the solids and optimization to maximize the precipitation and to manage sludge disposal.     

Secretion of HCO 3 − /OH− in cortical distal tubule of the rat

Secretion of bicarbonate has been described for distal nephron epithelium and attributed to apical Cl^–/HCO ₃ ^– exchange in beta-intercalated cells. We investigated the presence of this mechanism in cortical distal tubules by perfusing these segments with acid (pH 6) 10 mm phosphate Ringer. The kinetics of luminal alkalinization was studied in stationary microperfusion experiments by double-barreled pH (ion-exchange resin)/1 m KCl reference microelectrodes. Luminal alkalinization may be due to influx (into the lumen) of HCO ₃ ^– or OH^–, or efflux of H⁺. The magnitude of the Cl^–/ HCO ₃ ^– exchange component was measured by perfusing the lumen with solutions with or without chloride, which was substituted by gluconate. This component was not different from zero in control and alkalotic (chronic plus acute) Wistar rats. Homozygous Brattleboro rats (BRB), genetically devoid of antidiuretic hormone, were used since this hormone has been shown to stimulate H⁺ secretion, which could mask bicarbonate secretion. In these rats, no evidence for Cl^–/HCO ₃ ^– exchange was found in control BRB and in early distal segments of alkalotic animals, but in late distal tubule a significant component of 0.14±0.033 nmol/cm² · sec was observed, which, however, is small when compared to the reabsorptive flow found in control Wistar rats, of 0.95±0.10 nmol/cm² · sec. In addition, 5×10^–4 m SITS had no effect on distal bicarbonate reabsorption in controls as well as on secretion in alkalotic Wistar and Brattleboro rats, which is compatible with the absence of effect of this drug on the apical Cl^–/HCO ₃ ^– exchange in other tissues. It is concluded that most distal alkalinization is not Cl^– dependent, and that Cl^–/HCO ₃ ^– exchange may be found in cortical distal tubule, but its magnitude is, even in alkalosis, markedly smaller than the reabsorptive flux, which predominates in the rats studied in this paper, keeping luminal pH lower than that of blood.     

Biodegradation of coal slurry transport wastewater organics

Summary Activated sludge was successful in reducing the levels of dissolved organic carbon (DOC) in coal slurry wastewaters. DOC removal by the activated sludge ranged from 61% to 97% with a large percentage (21–41%) of this DOC being completely metabolized to CO₂. Second order kinetic constants (k ₂) developed for DOC removal ranged from 1.39·10^–4 to 2.30·10^–1 liter·day^–1·(mg of sludge)^–1, providing evidence that biological treatment was an effective mechanism for reducing the pollution potential of the slurry wastewaters. After treatment with activated sludge a residual DOC remained in the wastewater and data from ultrafiltration studies indicated that this residual carbon was of MW>1000. The activated sludge preferentially removed the lower (MW<1000) molecular weight compounds and the higher molecular weight DOC was more resistant to biological attack. However, extended acclimation (greater than 1 month) enabled the activated sludge to remove the higher molecular weight DOC from the slurry wastewaters.