Restricted oxygen supply and excretion of metabolites

Summary The effect of restricted oxygen supply on the excretion of metabolites was studied in Pseudomonas acidovorans (DSM 39), P. delafieldii (DSM 64) and a mutant strain of Paracoccus denitrificans unable to accumulate poly-3-hydroxybutanoic acid. Different metabolites were produced at distinct submaximum respiration rates by these strains. These metabolites were, in order of decreasing respiration rates; 2-oxoglutarate, 2-oxo-3-methylbutanoate, cisaconitate, 3-hydroxybutanoate, succinate, hydrogen gas, formate, acetate, butanoate, acetoin, meso- and D,L-2,3-butanediol, and ethanol. Poly-3-hydroxy-butanoic acid (PHB) accumulated intracellularly at almost the same respiration rates at which the excretion of 3-hydroxybutanoate occurred.The production of ethanol, 2,3-butanediol, butanoate, formate, and hydrogen gas indicate the function of enzymes such as ethanol and butanediol dehydrogenases, pyruvate formate lyase, formate hydrogen lyase, and butanoyl-CoA dehydrogenase. These enzymes are not expected to be present in strict aerobes at different degrees of restricted oxygen supply.Excreted metabolites are indicators of the degree to which the oxygen demand of cells is met. On the other hand, a fermentation process designed for the production of a distinct metabolite can be controlled by maintaining the appropriate oxygen supply.     

Effect of ozone on respiration of adult Sitophilus oryzae (L.), Tribolium castaneum (Herbst) and Rhyzopertha dominica (F.)

The effect of ozone on the respiration of three species of adult stored-product Coleoptera was tested in an air-tight flask. Sitophilus oryzae (L.), Rhyzopertha dominica (F.) and Tribolium castaneum (Herbst) adults were exposed to atmosphere containing 0.1, 0.2 or 0.4 μg/ml initial ozone at 23–25 °C and 50% r.h. Carbon dioxide (CO₂) production reflected the respiration rates of insects and was determined with a gas chromatograph (GC). The experiments showed that the effects of ozone on respiration had two distinct phases. Phase 1 involved a lower respiration rate of the adult stored-product Coleoptera under ozone atmosphere and reflected the need for insects to reduce ozone toxicity. After 1 h, CO₂ production of S. oryzae was 3.19, 2.63, 2.27 and 1.99 μl/mg for the ozone concentration of 0, 0.1, 0.2 and 0.4 μg/ml, respectively. The results also showed that there were decreases in the rate of respiration in R. dominica and T. castaneum with an increase in ozone concentration. During phase 2, respiration of S. oryzae, R. dominica, and T. castaneum adults treated with ozone increased as the ozone degraded to oxygen. After 7 h, the effect of ozone on CO₂ production, relative to the control, changed from a decrease to an increase. The findings in relation to control strategies were discussed.     

Oxygen transfer rate,respiration and yields in batch and chemostat cultures ofKlebsiella aerogenes

The effect was studied of oxygen supply on the changes in total and specific rate of oxygen consumption by the cells, oxygen transfer rate, saturation concentrations of dissolved oxygen and the yields of batch and continuous cultivations. Experiments were done on the microorganismKlebsiella aerogenes CCM 2318 growing on synthetic glucose medium. Continuous cultivations were carried out at dilution rates of 0.96 and 0.178 h⁻¹. The rate of oxygen transfer was determined by the sulphite method and the coefficient K_La was assessed using the dynamic method with a correction for changes in the saturations of dissolved oxygen. A lowered oxygen supply in batch cultivations caused deformations in the course of cell respiration. Comparison of results of batch and continuous cultivations showed that the highest yields Y_x/s and Y_x/o are attained at low dilution rates without oxygen limitation. Batch cultivations, on the other hand, exhibit the lowest yields and the highest cell respiration levels. In both types of cultivations, a respiration peak was ascertained under the conditions of growth limitation by oxygen.     

Respiration activity of suspension cell culture of <Emphasis Type="Italic">Polyscias filicifolia</Emphasis> bailey, <Emphasis Type="Italic">Stephania glabra</Emphasis> (Roxb.) miers,and <Emphasis Type="Italic">Dioscorea deltoidea</Emphasis> wall

Peculiarities of respiration of cells cultures producing biologically active compounds (isoprenoids and alkaloids) were investigated in order to optimize productivity of culture growth and biosynthesis. It had been revealed that studied cells cultures of Dioscorea deltoidea Wall (producer of furistanol glycosides), Stephania glabra (Roxb.) Miers (producer of stepharin alkaloid) and Polyscias filicifolia Bailey (complex of biologically active agents) differ both in joint respiration activity and in ratio between cytochrome and cyanide-resistant respiration, while changes of rate of total oxygen consumption and activity of alternative oxidase during growth were found to be individual for every investigated culture. Maximum rate of oxygen consumption for cells of D. deltoidea and S. glabra was marked in the period preceding active synthesis of secondary metabolites (lag phase for D. deltoidea and exponential phase for S. glabra). The revealed trends can be used for further monitoring and regulation of growth and biosynthesis of secondary metabolites in producing cell cultures during deep cultivation.     

A comparison of net and gross rates of oxygen production as a function of light intensity in some natural plankton populations and in a Synechococcus culture

In vitrorates of gross and net oxygen production were measuredas a function of light intensity in some plankton communitiescollected from Bedford Basin, Nova Scotia, and in a monoclonalculture of Synechococcus. The rate of gross oxygen productionwas measured by a technique in which the stable oxygen isotope,¹⁸O, serves as a photosynthetic tracer Net oxygen productionwas measured by automated Winkler technique. The rate of communityrespiration in the light was then determined by the differencebetween gross and net rates of oxygen production. In the naturalpopulations examined, neither gross nor net oxygen productionrates were significantly inhibited at the highest light intensitymeasured (500–800 µE m^–2 s^–1) In a samplein which the dark respiration rate was small relative to themaximal rate of production [P_max;sensu Platt et al (1980) JMar. Res., 38, 687–701] the rates of ‘light’respiration were 3 times greater. In two other communities,with high rates of dark respiration relative to P_maxthe ratesof ‘light’ respiration were closer to rates of darkrespiration. In the Synechococcus clone, both gross and netoxygen production rates were inhibited at high light intensities.Rates of ‘light’ respiration were found to varyas a function of light intensity. The greatest rates of respirationwere measured in samples incubated at light intensities thatwere just saturating (100 µE m^–2 s^–1). Therates of ¹⁴C production were also measured as a function oflight intensity The photosynthetic quotients, based on ¹⁴C productionrates and gross oxygen production rates, average 1 9     

Simultaneous Measurement of Dissolved Oxygen and Oxidation-Reduction Potentials in the Aerobic Culture

The most dominant factor influencing the oxidation-reduction potentials (E) in the cultured system was oxygen tension. H was an useful index to express the degree of oxygen supply in place of dissolved oxygen (P_L) under a limited oxygen supply. The conversion of microbial products caused by the change in oxygen supply was clearly analyzed by the use of E value. Bacillus subtilis excreted lactic acid at the E value ?220 mV, 2,3-butyleneglycol at ?195 mV and acetoin at ?160mV as the main product. E also gave the significant information concerning the changes in cell’s respiration. Cyanide at the concentration of 10^?5m, azide at 10^?3m and 2,4-dinitrophenol (DNP) at 10^?2m inhibited cell respiration causing the decrease in E and the increase in P_L, and DNP at 0.4×10^?3m promoted oxygen uptake of the cells causing the decrease in both E and P_L.     

Variability of photosynthesis-irradiance curves and ecosystem respiration in a small river

• 1 We investigated photosynthesis‐irradiance relationships (P‐I curves; P = oxygen production rate due to photosynthesis, I = light irradiance rate at the water surface) and ecosystem respiration in a 9 km long reach of a river that is characterised by light conditions favouring primary production, high ambient nutrient concentrations, a high re‐aeration rate, and frequent spates. We addressed the question of how disturbances (spates) and season influence photosynthesis and ecosystem respiration.
• 2 We used an oxygen mass‐balance model of the river to identify ecosystem respiration rates and the two parameters of a hyperbolic P‐I function (P_max = maximum oxygen production rate due to photosynthesis, α = the initial slope of the P‐I function). The model was fitted to dissolved oxygen concentrations quasi‐continuously recorded at the end of the reach. We estimated parameters for 137 three‐day periods (during the years 1992–97) and subsequently explored the potential influence of season and disturbances (spates) on P_max, α and ecosystem respiration using stepwise regression analysis.
• 3 Photosynthesis‐irradiance relationships and ecosystem respiration were subject to distinct seasonal variation. Only a minor portion of the variability of P‐I curves could be attributed to disturbance (spates), while ecosystem respiration did not correlate with disturbance related parameters. Regular seasonal variation in photosynthesis and ecosystem respiration apparently prevailed due to the absence of severe disturbances (a lack of significant bedload transport during high flow).

     

Feeding strategy for the production of the new antibiotic myxovirescin A from Myxococcus virescens (Myxobacterales)

Summary Myxovirescin A is a new antibiotic from Myxococcus virescens. Conditions of growth on peptone media result in the antibiotic being secreted during the transition to the stationary phase. When growth is exponential, no detectable production occurs.In an attempt to improve production of the antibiotic, peptone was fed to the peptone-limited culture at differing feed rates. Product formation was found to be dependent on the peptone supply, and the product concentration could be improved from 0.04 to 2 mg/l myxovirescin A.Article No. 13 on antibiotics from gliding bacteria Article No. 12: Thierbach G, Reichenbach H (1983). The effect of the new antibiotic myxothiazol on the respiration of Paracoccus denitrificans. Arch Microbiol 134: 104–107     

Conversions of Microbial Products in Relation to Oxygen Supply in Inosine Fermentation

On the basis of the ratio (cell’s respiration rate/maximum oxygen demand of cells, r_{a b}/K_rM) as a new criterion of oxygen supply, symptoms of oxygen deficiency was described in inosine fermentation. Conversions of the products were observed to occur in relation to the extent of oxygen deficiency. When oxygen demand of the cells was satisfied (r_{a b}/K_rM = 1.0), the cells accumulated exclusively inosine. Under limited oxygen supply at the value of r_{a b}/K_rM 0.5~0.9, on the other hand, inosine formation was inhibited and acetoin was the predominant product. When oxygen supply was limited more strictly at the value of r_{a b}/K_rM smaller than 0.3, the cells excreted 2,3-butyleneglycol as the main product.     

Citric acid production from glucose. I. Growth and excretion kinetics in a stirred fermentor

The growth and citric acid production kinetics of Saccharomycopsis lipolytica on glucose are investigated in an aerated stirred fermentor. Cellular growth first proceeds exponentially until exhaustion of ammonia in the fermentation medium. Cells then continue to grow at a reduced rate with a concomitant decrease in intracellular nitrogen content. Citric and isocitric acid production starts at the end of the growth phase. During about 80 hr excretion proceeds at a constant rate of 0.7 g/liter/hr for citric acid and 0.1 g/liter/hr for isocitric acid. The final citric and isocitric acid concentrations are 95 and 10g/liter, respectively. During acid excretion cellular respiration accounts for 60 and 35% of consumed oxygen and glucose. Both acid and CO₂ production rates follow a Michaelis–Menten-type dependence on oxygen concentration with Michaelis–Menten constants of 0.9 and 0.15 mg/liter for acid and CO₂ productions, respectively.     

Molar growth yields,respiration and cytochrome profiles of Beneckea natriegens when grown under carbon limitation in a chemostat

The effect of growth rate on the physiology of Beneckea natriegens was studied in chemostat culture. The molar growth yields (Y) from glucose and oxygen, the specific rates of oxygen (q _{O 2}) and glucose (q _glc) consumption and the specific rate of CO₂ production (q _{CO 2}) were linearly dependent on the growth rate over the dilution rate 0.17 h^-1 to 0.60 h^-1. Further increase in the dilution rate resulted in a decrease in growth yield and respiration rate and these changes were coincident with increases in the specific rate of glucose utilisation and of acetate production. The affinity of Beneckea natriegens for glucose was similar when measured either directly in chemostat culture or in a closed oxygen electrode system using harvested bacteria. The total content of cytochromes decreased with increasing growth rate. However, the quantity of CO-binding cytochromes remained independent of growth rate and correlated with the potential respiration rate.     

Partitioning of river metabolism identifies phytoplankton as a major contributor in the regulated Murray River (Australia)

1. River metabolism was measured over an annual cycle at three sites distributed along a 1000 km length of the lowland Murray River, Australia. 2. Whole system metabolism was measured using water column changes in dissolved oxygen concentrations while planktonic and benthic metabolism were partitioned using light‐dark bottles and benthic chambers. 3. Annual gross primary production (GPP) ranged from 775 to 1126 g O₂ m^?2 year^?1 which in comparison with rivers of similar physical characteristics is moderately productive. 4. Community respiration (CR) ranged from 872 to 1284 g O₂ m^?2 year^?1 so that annual net ecosystem production (NEP) was near zero, suggesting photosynthesis and respiration were balanced and that allochthonous organic carbon played a minor role in fuelling metabolism. 5. Planktonic rates of gross photosynthesis and respiration were similar to those of the total channel, indicating that plankton were responsible for much of the observed metabolism. 6. Respiration rates correlated with phytoplankton standing crop (estimated as the sum of GPP plus the chlorophyll concentration in carbon units), yielding a specific respiration rate of ?1.1 g O₂ g C^?1 day^?1. The respiration rate was equivalent to 19% of the maximum rate of phytoplankton photosynthesis, which is typical of diatoms. 7. The daily GPP per unit phytoplankton biomass correlated with the mean irradiance of the water column giving a constant carbon specific photon fixation rate of 0.35 gO₂ g Chl a^?1 day^?1 per μmole photons m^?2 s^?1 (ca. 0.08 per mole photons m^?2 on a carbon basis) indicating that light availability determined daily primary production. 8. Annual phytoplankton net production (NP) estimates at two sites indicated 25 and 36 g C m^?2 year^?1 were available to support riverine food webs, equivalent to 6% and 11% of annual GPP. 9. Metabolised organic carbon was predominantly derived from phytoplankton and was fully utilised, suggesting that food‐web production was restricted by the energy supply.     

Metabolite profiles of the biocontrol yeast<Emphasis Type="Italic"> Pichia anomala</Emphasis> J121 grown under oxygen limitation

The biocontrol yeast Pichia anomala J121 prevents mould growth during the storage of moist grain under low oxygen/high carbon dioxide conditions. Growth and metabolite formation of P. anomala was analyzed under two conditions of oxygen limitation: (a) initial aerobic conditions with restricted oxygen access during the growth period and (b) initial microaerobic conditions followed by anaerobiosis. Major intra- and extracellular metabolites were analyzed by high-resolution magic-angle spinning (HR-MAS) NMR and HPLC, respectively. HR-MAS NMR allows the analysis of major soluble compounds inside intact cells, without the need for an extraction step. Biomass production was higher in treatment (a), whereas the specific ethanol production rate during growth on glucose was similar in both treatments. This implies that oxygen availability affected the respiration and not the fermentation of the yeast. Following glucose depletion, ethanol was oxidized to acetate in treatment (a), but continued to be produced in (b). Arabitol accumulated in the culture substrate of both treatments, whereas glycerol only accumulated in treatment (b). Trehalose, arabitol, and glycerol accumulated inside the cells in both treatments. The levels of these metabolites were generally significantly higher in treatment (b) than in (a), indicating their importance for P. anomala during severe oxygen limitation/anaerobic conditions.     

Respiration and oxygen transport in soybean nodules

Summary The respiration rate of individual soybean (Glycine max Merr.) nodules was measured as a function of pO₂ and temperature. At 23°, as the pO₂ was increased from 0.1 to 0.9 atm, there was a linear increase in respiration rate. At 13°, similar results were obtained, except that there was an abrupt saturation of respiration at approximately 0.5 atm pO₂. When measurements were made on the same nodule, the rate of increase in respiration with pO₂ was the same at 13° and 23°. Additional results were that 5% CO in the gas phase had no effect on respiration, except for a small decrease in the pO₂ at which respiration became saturated. Also, nodules still attached to the soybean root displayed the same respiratory behavior as detached nodules. A model for oxygen transport in the nodule is presented which explains these results quantitatively. The essence of the model is that the respiration rate of the central tissue of the nodule is almost entirely determined by the rate of oxygen diffusion to the respiratory enzymes. Evidence is given that the nodule cortex is the site of almost all of the resistance to oxygen diffusion within the nodule.     

Effect of oxygen supply on growth ofKlebsiella aerogenes in a multistage tower fermentor

The effect of the rate of oxygen supply on biomass growth, consumption of carbon source formation of metabolic by-products, biomass yeilds referred to C-source and oxygen, respiration rate and the respiratory quotient was studied in a multistage tower fermentor with an interstage backflow, i.e. with a continuous reinoculation of the preceding stages. Experiments were done with Klebsiella aerogenes CCM 2318 in a synthetic glucose medium with constant glucose concentration in the feed, at pH 7.0. temperature 30 degrees C, and dilution rates 0.6 and 0.178 h-1 (referred to one stage). Different behavior of the culture was found at different dilution rates both with oxygen and under oxygen limitation. As compared with the chemostat system, the regime with an interstage backflow exhibited differences in respiration rate and CO2 formation; this attests to a considerably different physiological state of the cells.     

Physiological aspects of surface-immobilized Catharanthus roseus cells

Summary Growth and alkaloid production of surface-immobilized C. roseus cells were studied in a 2-1 bioreactor. Media designed to maximize cell growth or alkaloid production were employed. Nitrate and carbohydrate consumption rates as well as growth rates and biomass yields of immobilized cultures were equal or somewhat lower than for cell suspension cultures. Respiration rate (O₂ consumption and CO₂ production rates) of immobilized C. roseus cell cultures was obtained by on-line analysis of inlet and outlet gas composition using a mass spectrometer. Respiration rate increased during the growth phase and decreased once the nitrogen or the carbon source was depleted from the medium. The respiration rate of immobilized C. roseus cells resembled rates reported in the literature for suspension cultures. Offprint requests to: Denis Rho     

Respiration of Senecio Shoots: Inhibition during Photosynthesis, Resistance to Cyanide and Relation to Growth and Maintenance

Respiration and dry matter producation were measured in shoots of senecia aquaticus Hill, which is flood tolerant and in shoots of S. jacobaea L., which is flood- sensitive. Both species were grown in culture solutions of high and of low oxygen concentration Growth of food of S. jacobaca was unaffected by a low oxygen supply bur growth of S. jacobaca was severly hampered by a low oxygen concentration in the root medium. Kinetic data about the rate of apparent photosynthesis at low oxygen conetration and different carbon dioxide concentrations indicated that at light saturation respiration was strongly repressed during photosynthesis. Shoot growth respiration, i.e. the amount of carbon dioxide produced for synthesis of shoot dry, matter appeared to be absent on S. jacobaea and to be very low (13.mg CO₂/g dry shoots) in S. aquaticus. In comparison with values prepiration rate was 2.8. 2.0. 1.5 and 1.3mg CO₂/h.g dry shoots in aerobically and anaerobically growth S. jacobaea and in aerobically and anaerobically growth S. aquabaea respectively. These values were also low in comprision with values previously found for roots of the same species. Shoot dark respiration on S. aquaticus was inbihitedd by a com bination on CN and salicylhydroxamic acid (SHAM), but not by application on one of these inhibitors alone. It was therefore concluded that an alternative oxidative pathway was present but not active in shoots of S. aquaticus. In the absence of inhibited of the cylochorome pathway. The low value of growth respiration and maintenance respiration rate in the shoots as compared with those in the roots of the investigated Sencio species are discussed in relation to the activity of the alternative oxidative pathway and to the possibilbity of a direct supply of ATP by photosynthesis intead of respiratory meta bolism.     

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures was examined, using on-line off-gas analyses to measure the oxygen and carbon dioxide consumption rates continuously. A cell suspension from continuous cultures at steady state was used as the inoculum. It was observed that a dynamic phase occurred in the initial phase of the experiment. In this phase the bacterial ferrous iron oxidation and growth were uncoupled. After about 16 h the bacteria were adapted and achieved a pseudo-steady state, in which the specific growth rate and oxygen consumption rate were coupled and their relationship was described by the Pirt equation. In pseudo-steady state, the growth and oxidation kinetics were accurately described by the rate equation for competitive product inhibition. Bacterial substrate consumption is regarded as the primary process, which is described by the equation for competitive product inhibition. Subsequently the kinetic equation for the specific growth rate, μ, is derived by applying the Pirt equation for bacterial substrate consumption and growth. The maximum specific growth rate, μ _max, measured in the batch culture agrees with the dilution rate at which washout occurs in continuous cultures. The maximum oxygen consumption rate, q _O2,max, of the cell suspension in the batch culture was determined by respiration measurements in a biological oxygen monitor at excess ferrous iron, and showed changes of up to 20% during the course of the experiment. The kinetic constants determined in the batch culture slightly differ from those in continuous cultures, such that, at equal ferric to ferrous iron concentration ratios, biomass-specific rates are up to 1.3 times higher in continuous cultures. Received: 8 February 1999 / Accepted: 17 February 1999     

Are altitudinal limits of equatorial stream insects reflected in their respiratory performance?

1. We measured respiration of the larvae of aquatic insects from streams in the Ecuadorian Andes in relation to oxygen saturation at 5, 8, 11, 14 and 17 °C. Polycentropus (Polycentropodidae), Lachlania (Oligoneuriidae), Anchytarsus (Ptilodactylidae) and Anacroneuria (Perlidae) represented genera absent from the highest altitudes, reaching 2720, 2930, 3120, 3450 m a.s.l., respectively, while Claudioperla (Gripopterygidae) and Anomalocosmoecus (Limnephilidae) occurred only above 2900 m a.s.l. Our purpose was to determine whether natural altitudinal limits were reflected in physiological critical points on respiration versus oxygen curves and by the effect of temperature on the ability to oxy‐regulate. 2. For all six genera, respiration was affected by oxygen saturation and temperature. Respiration (mg O₂ g⁻¹ AFDM h⁻¹) at 70% oxygen saturation (Michaelis–Menten fitted) varied from 2.6 to 7.6 between genera at 17 °C, and from 1.3 to 2.5 at 5 °C. Q₁₀ values for this temperature interval ranged 1.5–2.9 (mean 2.3). The two “high‐altitude” genera had higher respiration rates at low temperature and oxygen saturation, and their respiration rate saturated at lower temperatures, than three of the four “low‐altitude” genera. 3. The oxy‐regulatory capacity (critical points and initial decrease in respiration versus oxygen regressions) varied among genera and was affected by temperature. Lachlania, Claudioperla and Anomalocosmoecus had a higher ability to oxy‐regulate at low than at high temperatures, Anacroneuria was not clearly affected by temperature, while Polycentropus and Anchytarsus had a greater oxy‐regulatory capacity at high than at low temperature. These results indicate that the ability to oxy‐regulate is related to the temperature (altitude) at which species naturally occur. 4. Upper altitudinal limits of the six genera were not reflected in their respiratory performance, because all genera had critical minima of temperature and oxygen saturation much lower than those occurring at the limits of their natural distribution. So, the altitudinal limit could not be attributed to absolute short‐term physiological tolerance of low temperature and oxygen concentration. 5. Multiple regressions (based on respiration experiments and previously obtained relationships between water temperature, oxygen saturation and altitude) were used to predict how respiration rates should vary with altitude. At the upper limit of the four “low‐altitude” genera, respiration rates were 50–68% of those predicted at the centre of the range. With an arbitrary increase of 400 m above the actual limit, the effect of temperature would be a 13% decrease, and that of oxygen a 2% decrease, in respiration rate of Polycentropus, Lachlania and Anacroneuria, while respiration in Anchytarsus would be reduced by 5% by both factors. 6. It seems that, while the immediate decrease in respiration with increased altitude is caused mainly by a decrease in temperature, the long‐term survival of a species at given altitudes might be more affected by oxygen saturation. Further quantitative and long‐term studies on survival and recruitment in populations and communities are needed to determine the importance of temperature and oxygen for altitudinal limits of aquatic insects.