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1.
Several metabolic fluxes were analyzed during gradual transitions from aerobic to oxygen-limited conditions in chemostat cultures of Pseudomonas mendocina growing in synthetic medium at a dilution rate of 0.25 h-1. P. mendocina growth was glucose limited at high oxygen partial pressures (70 and 20% pO2) and exhibited an oxidative type of metabolism characterized by respiratory quotient (RQ) values of 1.0. A similar RQ value was obtained at low pO2 (2%), and detectable levels of acetic, formic, and lactic acids were determined in the extracellular medium. RQs of 0.9 +/- 0.12 were found at 70% pO2 for growth rates ranging from 0.025 to 0.5 h-1. At high pO2, the control coefficients of oxygen on catabolic fluxes were 0.19 and 0.22 for O2 uptake and CO2 production, respectively. At low pO2 (2%), the catabolic and anabolic fluxes were highly controlled by oxygen. P. mendocina showed a mixed-type fermentative metabolism when nitrogen was flushed into chemostat cultures. Ethanol and acetic, lactic, and formic acids were excreted and represented 7.5% of the total carbon recovered. Approximately 50% of the carbon was found as uronic acids in the extracellular medium. Physiological studies were performed under microaerophilic conditions (nitrogen flushing) in continuous cultures for a wide range of growth rates (0.03 to 0.5 h-1). A cell population, able to exhibit a near-maximum theoretical yield of ATP (YmaxATP = 25 g/mol) with a number of ATP molecules formed during the transfer of an electron towards oxygen along the respiration chain (P/O ratio) of 3, appears to have adapted to microaerophilic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

2.
Several metabolic fluxes were analyzed during gradual transitions from aerobic to oxygen-limited conditions in chemostat cultures of Pseudomonas mendocina growing in synthetic medium at a dilution rate of 0.25 h-1. P. mendocina growth was glucose limited at high oxygen partial pressures (70 and 20% pO2) and exhibited an oxidative type of metabolism characterized by respiratory quotient (RQ) values of 1.0. A similar RQ value was obtained at low pO2 (2%), and detectable levels of acetic, formic, and lactic acids were determined in the extracellular medium. RQs of 0.9 +/- 0.12 were found at 70% pO2 for growth rates ranging from 0.025 to 0.5 h-1. At high pO2, the control coefficients of oxygen on catabolic fluxes were 0.19 and 0.22 for O2 uptake and CO2 production, respectively. At low pO2 (2%), the catabolic and anabolic fluxes were highly controlled by oxygen. P. mendocina showed a mixed-type fermentative metabolism when nitrogen was flushed into chemostat cultures. Ethanol and acetic, lactic, and formic acids were excreted and represented 7.5% of the total carbon recovered. Approximately 50% of the carbon was found as uronic acids in the extracellular medium. Physiological studies were performed under microaerophilic conditions (nitrogen flushing) in continuous cultures for a wide range of growth rates (0.03 to 0.5 h-1). A cell population, able to exhibit a near-maximum theoretical yield of ATP (YmaxATP = 25 g/mol) with a number of ATP molecules formed during the transfer of an electron towards oxygen along the respiration chain (P/O ratio) of 3, appears to have adapted to microaerophilic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

3.
Factors affecting growth and nitrogen fixation of Spirillum lipoferum.   总被引:21,自引:11,他引:10       下载免费PDF全文
Spirillum lipoferum grows vigorously on malate, succinate, lactate, or pyruvate, moderately on galactose or acetate, and poorly on glucose or citrate. It reduces 15N2. Acetylene reduction rates decrease rapidly when the pH of the culture rises above 7.8. The organism is highly aerobic and had doubling times as low as 2 h when grown on NH4+. However, S. lipoferum reduces N2 well only under microaerophilic conditions. The optimal pO2 for acetylene reduction by stagnant cultures was 0.006 to 0.02 atm depending upon the cell density; aerated cultures grew well at dissolved O2 concentration corresponding to a pO2 of about 0.008 atm. Shaking S. lipoferum with air temporarily inactivates its nitrogenase; reactivation is inhibited by chloramphenicol. The organism assimilated 20 to 24 mg of N/g of organic acid oxidized during growth. The strains studied can be placed in two groups based upon their morphology and physiological characteristics.  相似文献   

4.
Azotobacter vinelandii was grown diazotrophically at different dissolved oxygen concentrations (in the range of 3 to 216 microM) in sucrose-limited continuous culture. The specific nitrogenase activity, measured on the basis of acetylene reduction in situ, was dependent solely on the growth rate and was largely independent of oxygen and sucrose concentration. FeMo (Av1) and Fe (Av2) nitrogenase proteins were quantified after Western blotting (immunoblotting). When the cultures were grown at a constant dilution rate (D, representing the growth rate, mu) of 0.15.h-1, the cellular levels of both proteins were constant regardless of different dissolved oxygen concentrations. The same was true when the organisms were grown at D values above 0.15.h-1. At a lower growth rate (D = 0.09.h-1), however, and at lower oxygen concentrations cellular levels of both nitrogenase proteins were decreased. This means that catalytic activities of nitrogenase proteins were highest at low oxygen concentrations, but at higher oxygen concentrations they increased with growth rate. Under all conditions tested, however, the Av1:Av2 molar ratio was 1:(1.45 +/- 0.12). Cellular levels of flavodoxin and FeS protein II were largely constant as well. In order to estimate turnover of nitrogenase proteins in the absence of protein synthesis, chloramphenicol was added to cultures adapted to 3 and 216 microM oxygen, respectively. After 2 h of incubation, no significant decrease in the cellular levels of Av1 and Av2 could be observed. This suggests that oxygen has no significant effect on the breakdown of nitrogenase proteins.  相似文献   

5.
柱孢鱼腥藻固氮酶防氧的呼吸保护   总被引:1,自引:0,他引:1  
柱孢鱼腥藻生长在缺氮情况下,发现其固氮活性增加的同时也减少了对氧的敏感性。缺氮生长细胞的乙炔还原活性给氧抑制一半时的氧分压(pO_2)是0.5atm.,而有氮生长细胞的半抑制浓度为0.35atm.。这表明蓝藻有可能通过增加呼吸耗氧而提高了它的固氮酶活性。呼吸作用与固氮酶活性之间存在着密切的关系。无论在有氮、缺氮还是光诱导固氮酶形成的情况下,其固氮活性均随着呼吸速率的变化而变化。本研究结果,支持了柱孢鱼腥藻固氮酶的主要防氧手段是呼吸保护的观点。  相似文献   

6.
Rhizobium trifolii 0403 was treated with 16.6 mM succinate and other nutrients and thereby induced to grow in nitrogen-free medium. The organism grew microaerophilically on either semisolid or liquid medium, fixing atmospheric nitrogen to meet metabolic needs. Nitrogen fixation was measured via 15N incorporation (18% 15N enrichment in 1.5 doublings) and acetylene reduction. Nitrogen-fixing cells had a Km for acetylene of 0.07 atm (ca. 7.09 kPa), required about 3% oxygen for optimum growth in liquid medium, and showed a maximal specific activity of 5 nmol of acetylene reduced per min per mg of protein at 0.04 atm (ca. 4.05 kPa) of acetylene. The doubling time on N-free liquid medium ranged from 1 to 5 days, depending on oxygen tension, with an optimum temperature for growth of about 30°C. Nodulation of white clover by the cultures showing in vitro nitrogenase activity indicates that at least part of the population maintained identity with wild-type strain 0403.  相似文献   

7.
It was shown possible to use feeding of hydrogen peroxide as a method for investigating the impact of dissolved oxygen concentrations on growth and development of microorganisms. The influence of pO2 on the respiration intensity was studied in penicillin- and erythromycin-producing cultures and it was found that dependence of the respiration intensity on pO2 had the form of a curve with saturation, at pO2 equal to zero the value of the culture respiration intensity being different from zero. A mathematical model accounting for the presence in the fermentation broth of microbial agglomerates with the average size depending on the agitation conditions is proposed for describing the relationships.  相似文献   

8.
Azoarcus sp. strain BH72 is an aerobic diazotrophic bacterium that was originally found as an endophyte in Kallar grass. Anticipating that these bacteria are exposed to dissolved O2 concentrations (DOCs) in the nanomolar range during their life cycle, we studied the impact of increasing O2 deprivation on N2 fixation and respiration. Bacteria were grown in batch cultures, where they shifted into conditions of low pO2 upon depletion of O2 by respiration. During incubation, specific rates of respiration (qO2) and efficiencies of carbon source utilization for N2 reduction increased greatly, while the growth rate did not change significantly, a phenomenon that we called "hyperinduction." To evaluate this transition from high- to low-cost N2 fixation in terms of respiratory kinetics and nitrogenase activities at nanomolar DOC, bacteria which had shifted to different gas-phase pO2s in batch cultures were subjected to assays using leghemoglobin as the O2 carrier. As O2 deprivation in batch cultures proceeded, respiratory Km (O2) decreased and Vmax increased. Nitrogenase activity at nanomolar DOC increased to a specific rate of 180 nmol of C2H4 min-1 mg of protein-1 at 32 nM O2. Nitrogenase activity was proportional to respiration but not to DOC in the range of 12 to 86 nM O2. Respiration supported N2 fixation more efficiently at high than at low respiratory rates, the respiratory efficiency increasing from 0.14 to 0.47 mol of C2H4 mol of O2 consumed-1. We conclude that (i) during hyperinduction, strain BH72 used an increasing amount of energy generated by respiration for N2 fixation, and (ii) these bacteria have a high respiratory capacity, enabling them to develop ecological niches at very low pO2, in which they may respire actively and fix nitrogen efficiently at comparatively high rates.  相似文献   

9.
Aerobic respiration and dissimilative iron reduction were studied in pure, batch cultures of Pseudomonas sp. strain 200 ("Pseudomonas ferrireductans"). Specific respiratory inhibitors were used to identify elements of electron transport chains involved in the reduction of molecular oxygen and Fe(III). When cells were grown at a high oxygen concentration, dissimilative iron reduction occurred via an abbreviated electron transport chain. The induction of alternative respiratory pathways resulted from growth at low oxygen tension (less than 0.01 atm [1 atm = 101.29 kPa]). Induced cells were capable of O2 utilization at moderately increased rates; dissimilative iron reduction was accelerated by a factor of 6 to 8. In cells grown at low oxygen tension, dissimilative iron reduction appeared to be uncoupled from oxidative phosphorylation. Models of induced and uninduced electron transport chains, including a mathematical treatment of chemical inhibition within the uninduced, aerobic electron transport system, are presented. In uninduced cells respiring anaerobically, electron transport was limited by ferrireductase activity. This limitation may disappear among induced cells.  相似文献   

10.
J Kuhla  J Oelze 《Journal of bacteriology》1988,170(11):5325-5329
Azotobacter vinelandii was grown diazotrophically in chemostat cultures limited by sucrose, citrate, or acetate. Specific activities of cellular oxygen consumption (qO2) and nitrogenase (acetylene reduction) were measured in situ at different dilution rates (D, representing the specific growth rate mu at steady state). Sucrose-limited cultures exhibited linear relationships between qO2 and D, each of which, however, depended on the dissolved oxygen concentration in the range of 12 to 192 microM O2. From these plots, qO2 required for maintenance processes (mO2) were extrapolated. mO2 values did not increase linearly with increasing dissolved oxygen concentrations. With citrate- or acetate-limited cultures qO2 also depended on D. At 108 microM O2, however, qO2 and mO2 of the latter cultures were significantly lower than those of sucrose-limited cultures. Specific rates of acetylene reduction increased linearly with D, irrespective of the type of limitation and of the dissolved oxygen concentration (J. Kuhla and J. Oelze, Arch. Microbiol. 149:509-514, 1988). The reversible switch-off of nitrogenase activity under oxygen stress also depended on D and was independent of qO2, mO2, or the limiting substrate. Increased switch-off effects resulting from increased stress heights could be compensated for by increasing D. Since D represents not only the supply of the carbon source but also the supply of electrons and energy, the results suggest that the flux of electrons to the nitrogenase complex, rather than qO2, stabilizes nitrogenase activity against oxygen inactivation in aerobically growing A. vinelandii.  相似文献   

11.
The effect of the partial pressure of oxygen (PO2) on the formation of vesicles, which are thought to be the site of N2 fixation in Frankia, was studied in HFPCcI3, an effective isolate from Casuarina cunninghamiana. Unlike other actinorhizal root nodules, vesicles are not produced by the endophyte in Casuarina nodules. However, in culture under aerobic conditions, large, phase-bright vesicles are formed in HFPCcI3 within 20 h following removal of NH+4 from the culture medium and reach peak numbers within 72 to 96 h. In vivo acetylene reduction activity parallels vesicle formation. Optimum rates of acetylene reduction in short-term assays occurred at 20% O2 (0.2 atm (1 atm = 101.325 kPa] in the gas phase. O2 uptake (respiration) determined polarographically showed diffusion-limited kinetics and remained unsaturated by O2 until 300 microM O2. In contrast, respiration in NH+4-grown cells was saturated by O2 between 8 and 10 microM O2. These results indicate the presence of a diffusion barrier associated with the vesicles. Vesicle development was repressed in cells incubated in N-free media sparged with gas mixtures with PO2 between 0.001 and 0.003 atm. Nitrogenase was induced under these conditions, but acetylene reduction was extremely O2 sensitive. The kinetics of O2 uptake as a function of dissolved O2 concentration in avesicular cells were similar to those in NH+4-grown cells indicating the lack of a diffusion barrier. These results demonstrate that vesicle formation and the development of the O2 protection mechanisms of nitrogenase are regulated by ambient PO2 in HFPCcI3.  相似文献   

12.
Carbon dioxide and oxygen concentrations have a profound effect on the lag period of chemoautotrophically grown Hydrogenomonas eutropha. Minimum lag periods and high growth rates were obtained in shaken flask cultures with a prepared gas mixture containing 70% H(2), 20% O(2), and 10% CO(2). However, excessively long lag periods resulted when the same gas mixture was sparged through the culture. The lag period was shortened in sparged cultures by decreasing both the pO(2) and the pCO(2), indicating that gas medium equilibration had not occurred in shaken cultures. The lag period was completely eliminated at certain concentrations of O(2) and CO(2). The optimum pO(2) was 0.05 atm, but the optimum pCO(2) varied according to the pH of the medium and physiological age of the inoculum. At pH 6.4, the pCO(2) required to obtain immediate growth of exponential, postexponential, and stationary phase inocula at equal specific rates was 0.02, 0.05, and 0.16 atm, respectively. With each 0.3-unit increase in the pH of the medium, a 50% decrease in the CO(2) concentration was needed to permit growth to occur at the same rate. The pCO(2) changes required to compensate for the pH changes of the medium had the net effect of maintaining a constant bicarbonate ion concentration. Initial growth of H. eutropha was therefore indirectly related to pCO(2) and directly dependent upon a constant bicarbonate ion concentration.  相似文献   

13.
Electron paramagnetic resonance (EPR) oximetry is being widely used to measure the oxygen consumption of cells, mitochondria, and submitochondrial particles. However, further improvement of this technique, in terms of data analysis, is required to use it as a quantitative tool. Here, we present a new approach for quantitative analysis of cellular respiration using EPR oximetry. The course of oxygen consumption by cells in suspension has been observed to have three distinct zones: pO(2)-independent respiration at higher pO(2) ranges, pO(2)-dependent respiration at low pO(2) ranges, and a static equilibrium with no change in pO(2) at very low pO(2) values. The approach here enables one to comprehensively analyze all of the three zones together-where the progression of O(2) diffusion zones around each cell, their overlap within time, and their potential impact on the measured pO(2) data are considered. The obtained results agree with previously established methods such as high-resolution respirometry measurements. Additionally, it is also demonstrated how the diffusion limitations can depend on cell density and consumption rate. In conclusion, the new approach establishes a more accurate and meaningful model to evaluate the EPR oximetry data on cellular respiration to quantify related parameters using EPR oximetry.  相似文献   

14.
The effects of thiamine deprivation on the growth, respiration, and activity of several enzymes of the phytoflagellate protozoanPolytomella agilis were studied. Vitamin deprivation had no effect on the exponential growth rate; the peak population of cultures grown without thiamine was 50% of the control level. The rates of oxygen consumption in control and thiamine-deprived cultures were not significantly different from each other. The activities of pyruvate dehydrogenase and oxoglutarate dehydrogenase in vitamin-deprived cells were 14% and 30%, respectively, of the control values. In these cells, the succinic dehydrogenase activity was 10% and mitochondrial ATPase activity was twice that of control cells. Vitamin deprivation had no effect on the activities of malate dehydrogenase and isocitrate lyase, but pyruvic carboxylase activity increased fourfold. These results indicate a complex role for thiamine in the regulation of growth, respiration, and metabolism in this organism.  相似文献   

15.
All oxygen levels are detrimental to the nitrogenase activity ofSynechococcus RF-1 cells. In continuous light, cultures maintain a high dissolved oxygen concentration and a continuous but usually low rate of nitrogenase activity.Cultures adapted to a light-dark regimen will reduce acetylene almost exclusively during the dark periods. When switched to continuous light, they continue to exhibit a diurnal rhythm in nitrogenase activity. While in continuous light, each upsurge of nitrogenase activity coincides with a marked drop in the net oxygen production rate; this drop is due largely to a concomitant increase in the dark respiration rate of the culture.The endogenous nitrogenase activity rhythm can be induced in continuous light by periodically lowering the oxygen concentration of the culture by either bubbling nitrogen through it or by treating the culture with 3(3,4-dichlorophenol)-1,1-dimethylurea (DCMU or diuron).  相似文献   

16.
Growth of a Bacterium Under a High-Pressure Oxy-Helium Atmosphere   总被引:3,自引:3,他引:0       下载免费PDF全文
Growth of a barotolerant marine organism, EP-4, in a glutamate medium equilibrated with an oxy-helium atmosphere at 500 atmospheres (atm; total pressure) (20°C) was compared with control cultures incubated at hydrostatic pressures of 1 and 500 atm. Relative to the 1-atm control culture, incubation of EP-4 at 500 atm in the absence of an atmosphere resulted in an approximately fivefold reduction in the growth rate and a significant but time variant reduction in the rate constants for the incorporation of substrate into cell material and respiration. Distinct from the pressurized control and separate from potential effects of dissolution of helium upon decompression of subsamples, exposure of the organism to high-pressure oxy-helium resulted in either a loss of viability of a large fraction of the cells or the arrest of growth for one-third of the experimental period. After these initial effects, however, the culture grew exponentially at a rate which was three times greater than the 500-atm control culture. The rate constant for the incorporation of substrate into cell material was also enhanced twofold in the presence of high-pressure oxy-helium. Dissolved oxygen was well controlled in all of the cultures, minimizing any potential toxic effects of this gas.  相似文献   

17.
Mechanism of nitrogenase switch-off by oxygen.   总被引:5,自引:1,他引:4       下载免费PDF全文
Oxygen caused a reversible inhibition (switch-off) of nitrogenase activity in whole cells of four strains of diazotrophs, the facultative anaerobe Klebsiella pneumoniae and three strains of photosynthetic bacteria (Rhodopseudomonas sphaeroides f. sp. denitrificans and Rhodopseudomonas capsulata strains AD2 and BK5). In K. pneumoniae 50% inhibition of acetylene reduction was attained at an O2 concentration of 0.37 microM. Cyanide (90 microM), which did not affect acetylene reduction but inhibited whole-cell respiration by 60 to 70%, shifted the O2 concentration that caused 50% inhibition of nitrogenase activity to 2.9 microM. A mutant strain of K. pneumoniae, strain AH11, has a respiration rate that is 65 to 75% higher than that of the wild type, but its nitrogenase activity is similar to wild-type activity. Acetylene reduction by whole cells of this mutant was inhibited 50% by 0.20 microM O2. Inhibition by CN- of 40 to 50% of the O2 uptake in the mutant shifted the O2 concentration that caused 50% inhibition of nitrogenase to 1.58 microM. Thus, when the respiration rates were lower, higher oxygen concentrations were required to inhibit nitrogenase. Reversible inhibition of nitrogenase activity in vivo was caused under anaerobic conditions by other electron acceptors. Addition of 2 mM sulfite to cell suspensions of R. capsulata B10 and R. sphaeroides inhibited nitrogenase activity. Nitrite also inhibited acetylene reduction in whole cells of the photodenitrifier R. sphaeroides but not in R. capsulata B10, which is not capable of enzymatic reduction of NO2-. Lower concentrations of NO2- were required to inhibit the activity in NO3- -grown cells, which have higher activities of nitrite reductase.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

18.
Azotobacter vinelandii was grown at constant growth rate in a chemostat with different molar ratios of sucrose to ammonium (C/N) in the influent media. Both compounds were consumed at essentially the same ratios as were present in the influent media. At low (C/N)-ratios, the cultures were ammonium-limited. At increased (C/N)-ratio ammonium-assimilating cultures additionally began to fix dinitrogen. The (C/N)-ratio at which nitrogenase activity became measurable, increased when the ambient oxygen concentration was increased. Immunoblotting revealed the appearance of nitrogenase proteins when the activity became detectable. Nitrogenase activity as determined either by acetylene reduction or by total nitrogen fixation gave constant relative activities of 1:3.8 (mol of N2 fixed per mol of acetylene reduced) under all sets of conditions used in this investigation. In spite of the oxygen dependent variation of the (C/N)-ratio, nitrogenase became active when the ammonium supply was less than about 14 nmol of ammonium per g of protein. This suggests that oxygen was not directly involved in the onset of dinitrogen fixation.  相似文献   

19.
Summary Using a root nodule cuvette and a continuous flow gas exchange system, we simultaneously measured the rates of carbon dioxide evolution, oxygen uptake and acetylene reduction by nodules ofAlnus rubra. This system allowed us to measure the respiration rates of single nodules and to determine the effects of oxygen concentration and temperature on the energy cost of nitrogen fixation. Energy cost was virtually unchanged (2.8–3.5 moles of carbon dioxide or oxygen per mole of ethylene) from 16 to 26°C (pO2=20 kPa) while respiration and nitrogenase activity were highly temperature dependent. At temperatures below 16°C, nitrogenase activity decreased more than did respiration and as a result, energy cost rose sharply. Acetylene reduction ceased below 8°C. Inhibition of nitrogenase activity at low temperatures was rapidly reversed upon return to higher temperatures. At high temperatures (above 30°C) nitrogenase activity declined irreversibly, while respiration and energy cost increased.Energy cost was nearly unchanged at oxygen partial pressures of 5 to 20 kPa (temperature of 20°C). Respiration and nitrogenase activity were strongly correlated with oxygen tension. Below 5 kPa, acetylene reduction and oxygen uptake decreased sharply while production of carbon dioxide increased, indicating fermentation. Fermentation alone was unable to support nitrogenase activity. Acetylene reduction was independent of oxygen concentration from 15 to 30 kPa. Nitrogenase activity decreased and energy cost rose above 30 kPa until nearly complete inactivation of nitrogenase at 70–80 kPa. Activity declined gradually, such that acetylene reduction at a constant oxygen concentration was stable, but showed further inactivation when oxygen concentration was once again increased. Alder nodules appear to consist of a large number of compartments that differ in the degree to which nitrogenase is protected from excess oxygen.Supported by United States Department of Agriculture Grant 78-59-2252-0-1-005-1  相似文献   

20.
The aim of this work was to study how the concentration of oxygen dissolved in the cultural broth influenced the respiration and morphology of the yeast Candida utilis in batch and continuous cultures. Highly effective respiration was registered in cells growing for a certain period of time at low oxygen concentrations limiting the growth; the respiration was characterized by low values of the Michaelis constant kc and the critical concentration of dissolved oxygen Ccr. When passing from the low oxygen concentration to a high one, the character of cellular respiration changed abruptly in the cells whose growth was limited with oxygen for a long time. The morphology of the culture limited with oxygen was characterized by an increase in the percentage of elongated forms in the population. The respiration of the cells cultivated at high oxygen concentrations, when their growth was either non-limited or limited by glucose, was distinguished by high Ccr values and slow respiration rates at small oxygen concentrations while the dependence of the respiration rate on the concentration of oxygen had an about S-shaped character.  相似文献   

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