Control of the production of individual fusicoccins at different dissolved oxygen concentrations

The regulatory effect of different concentrations of dissolved oxygen on the production of fusicoccins by the fungus Fusicoccum amygdali Del. was studied. The maximum output of total fusicoccins was obtained by using a profiled dissolved oxygen tension (DOT) regime, in which the DOT was maintained at 15–20% during the biomass growth phase and at 5–8% during the fusicoccins production phase. In comparison with the profiled regime, the maintenance of DOT at 15–20% during the whole fermentation shortened the fusicoccins production phase. The fermentation performance at a low DOT (5–8%) inhibited both the accumulation of biomass and the production of fusicoccins. At high DOT (40–50%), an accelerated accumulation of the biomass with an expressed autolysis of mycelia took place, and the production of fusicoccins was lowered. The qualitative composition of individual fusicoccins varied substantially at different DOTs. Fusicoccins, A, C, D, J, H, 16-O-demethyl-J, detretpentenylfusicoccin and some minor fusicoccin metabolites were found in the fermentation broth using the method of liquid secondary ion mass spectrometry. It was established that the profiled DOT regime (15–20% to 5–8%) provided both the maximum concentration of fusicoccins and an enhanced accumulation of the main metabolite – fusicoccin A (FC A). The performance of the fermentation at a DOT of 15–20% decreased the content of FC A by 2–6% in comparison with the profiled DOT regime, and increased the content of fusicoccin C to 14–20% of the total fusicoccins. Fermentation at DOT of 5–8% was characterized by the highest content of the precursors of FC A, the less oxidized fusicoccins H and J, the contents of which were in range 7–12% and 16–17% of total fusicoccins, respectively.     

不同溶氧对谷氨酸棒杆菌代谢的影响

【目的】以谷氨酸棒杆菌为研究对象,分别控制在0、30%、50%3种溶氧水平下进行发酵,分析不同溶氧水平下代谢的变化。【方法】通过检测发酵代谢物中有机酸、氨基酸的含量,以及测定代谢途径中关键酶活性及其编码基因的表达情况来考察不同溶氧水平下物质代谢发生的变化。通过检测胞内还原力和ATP的含量来分析不同溶氧水平对能量代谢产生的影响。【结果】谷氨酸棒杆菌代谢支路受溶氧的影响而发生改变,氨基酸、有机酸的产量也随之改变。特别是在低溶氧(0)情况下,细胞内氧化磷酸化减弱,导致维持生命活动所必需的ATP供应减少,因此细胞通过增强底物水平磷酸化来产生ATP以满足生命活动的需求。在此情况下,胞内NADH得到较多积累,TCA循环代谢流量减小,而转向糖酵解、乙醛酸循环等,并且这个过程伴随多种杂酸包括乳酸、缬氨酸、亮氨酸等的产生,必将影响目的产物的产量。【结论】研究结果对于进一步采取措施优化溶氧的控制策略,提高目的产物的产量具有指导意义。     

Testing a method for measuring dissolved oxygen concentrations in microhabitats in fresh water

It was previously known that polyethylene film is permeable to oxygen, and that water-filled polyethylene bags can be used for determination of dissolved oxygen in fresh-water habitats: analysis of oxygen concentrations in the bag water indicates the concentrations in the habitat where the bag has been resting for some time. Field experiments show that the bag method (with minor modifications) is reasonably accurate for ecological studies (a deviation of 0–0.20 ml O₂/l as compared with samples collected with a Ruttner water sampler).The bag method is simple in use. Its main advantage is its usefulness in many microhabitats in fresh-water where other methods may fail. Oxygen deficit in a dense helophyte stand is demonstrated using the bag method.     

Oxygen consumption kinetics of Nitrosomonas europaea and Nitrobacter hamburgensis grown in mixed continuous cultures at different oxygen concentrations

Chemolithotrophic ammonium- and nitrite-oxidizing bacteria are dependent on the presence of oxygen for the production of nitrite and nitrate, respectively. In oxygen-limited environments, they have to compete with each other as well as with other organotrophic bacteria for the available oxygen. The outcome of the competition will be determined by their specific affinities for oxygen as well as by their population sizes. The effect of mixotrophic growth by the nitrite-oxidizing Nitrobacter hamburgensis on the competition for limiting amounts of oxygen was studied in mixed continuous culture experiments with the ammonium-oxidizing Nitrosomonas europaea at different levels of oxygen concentrations.The specific affinity for oxygen of N. europaea was in general higher than of N. hamburgensis. In transient state experiments, when oxic conditions were switched to anoxic, N. hamburgensis was washed out and nitrite accumulated. However, grown at low oxygen concentration, the specific affinity for oxygen of N. hamburgensis increased and became as great as that of N. europaea. Due to its larger population size, the nitrite-oxidizing bacterium became the better competitor for oxygen and ammonium accumulated in the fermentor. It is suggested that continuously oxygen-limited environments present a suitable ecological niche for the nitrite-oxidizing N. hamburgensis.     

Long-term effect of dissolved oxygen on partial nitrification performance and microbial community structure

In this study, the performance of partial nitrification via nitrite and microbial community structure were investigated and compared in two sequencing batch reactors (SBR) with different dissolved oxygen (DO) levels. Both reactors achieved stable partial nitrification with nitrite accumulation ratio of above 95% by using real-time aeration duration control. Compared with high DO (above 3 mg/l on average) SBR, simultaneous nitrification and denitrification (SND) via nitrite was carried out in low DO (0.4–0.8 mg/l) SBR. The average efficiencies of SND in high DO and low DO reactor were 7.7% and 44.9%, and the specific SND rates were 0.20 and 0.83 mg N/(mg MLSS h), respectively. Low DO did not produce sludge with poorer settling properties but attained lower turbidities of the effluent than high DO. Fluorescence in situ hybridization (FISH) analysis in both the reactors showed that ammonia-oxidizing bacteria (AOB) were the dominant nitrifying bacteria and nitrite-oxidizing bacteria (NOB) did not be recovered in spite of exposing nitrifying sludge to high DO. The morphology of the sludge from both two reactors according to scanning electron microscope indicated that small rod-shaped and spherical clusters were dominant, although filamentous bacteria and few long rod-shaped coexisted in the low DO reactor. By selecting properly DO level and adopting process control method is not only of benefit to the achievement of novel biological nitrogen removal technology, but also favorable to sludge population optimization.     

Superoxide dismutase and catalase in Azotobacter vinelandii grown in continuous culture at different dissolved oxygen concentrations

Superoxide dismutase and catalase activities were studied in Azotobacter vinelandii grown diazotrophically at different ambient oxygen concentrations in continuous culture. Activities were expressed either as specific activity or activity per cell. Specific superoxide dismutase activity increased by a factor of 1.6 with increasing oxygen concentration from about 1% to 90% air saturation of the growth medium whereas specific catalase activity increased only slightly, if at all. Since cell volumes increased in parallel to increases in the oxygen concentration cellular superoxide dismutase activities increased by a factor of 4.3 while cellular catalase activities increased by a factor of 3.3. Under all conditions only the Fe-containing form of superoxide dismutase was detected. The possible function of these enzymes in the protection nitrogenase from oxygen damage is discussed.Abbreviation SOD superoxide dismutase     

Nitrification rates in the lower river Rhine as a monitor for ecological recovery

The rate of in situ nitrification was tested as an indicator of the toxicological quality of the river Rhine. Concentration changes of ammonium ions over 85 to 133 km long reaches of three river branches downstream of the densely populated Ruhr-area (F.R.G.) were calculated from a data base for the period 1972 to 1986. Concentrations of ammonium in the river exceeded values of 1 mg N/l in winter. Because of the very high input of ammonium, bacterial nitrification dominated over other nitrogen processes. Relative rates of nitrification in the three river branches were proportional to the water temperature for the individual years. Nitrification rates in the river increased by a factor of ca. 4 during the period of 1972 to 1986. Toxic substances, whose concentrations decreased in the same period of time, were proposed as inhibitors of in situ nitrification rather than e.g. a low oxygen saturation of the water. The improvement of the conditions in the river, indicated by the in situ rate of nitrification, was also documented by data on macrofauna and fish populations.     

Levels and activities of nitrogenase proteins in Azotobacter vinelandii grown at different dissolved oxygen concentrations.

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.     

Comparison of oxygen consumption in freshwater mussels (Unionidae) from different habitats during declining dissolved oxygen concentration

The rate of oxygen consumption (OC) of 9 species of freshwater mussels was measured under declining dissolved oxygen (DO) concentrations. The effects of temperature for some species also was investigated. The pattern of the OC vs. DO curve for each species was used in a hyperbolic model to compare abilities to regulate OC under low oxygen conditions. At 24.5 °C, Pyganodon grandis (from lakes), Amblema plicata and Quadrula pustulosa (from mud or sand in large rivers), Elliptio complanata (from pool areas in rivers), and Elliptio fisheriana and Elliptio lanceolata (from bank margins of rivers) were better able to maintain OC under low DO than were Villosa iris and Villosa constricta (which inhabit riffles) and Pleurobema cordatum (found in rivers with moderate flow). Villosa iris was especially sensitive to low oxygen conditions. The ability to maintain normal OC at low DO was improved considerably at 16.5 °C for V. iris, P. grandis and E. complanata. It is concluded that oxygen regulation ability appears to be related to the degree of hypoxia a species normally experiences in its habitat type, and it is enhanced at low temperature. The measurement of OC vs. DO may be a useful technique for estimating DO water quality criteria for endangered species because it is noninvasive.     

Long-term continuous monitoring of dissolved oxygen in cell culture medium for perfused bioreactors using optical oxygen sensors

For long-term growth of mammalian cells in perfused bioreactors, it is essential to monitor the concentration of dissolved oxygen (DO) present in the culture medium to ascertain the health of the cells. An optical oxygen sensor based on dynamic fluorescent quenching was developed for long-term continuous measurement of DO for NASA-designed rotating perfused bioreactors. Tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) chloride is employed as the fluorescent dye indicator. A pulsed, blue LED was chosen as the excitation light source. The sensor can be sterilized using an autoclave. The sensors were tested in a perfused rotating bioreactor supporting a BHK-21 (baby hamster kidney) cell culture over one 28-day, one 43-day, and one 180-day cell runs. The sensors were initially calibrated in sterile phosphate-buffered saline (PBS) against a blood-gas analyzer (BGA), and then used continuously during the entire cell culture without recalibration. In the 180-day cell run, two oxygen sensors were employed; one interfaced at the outlet of the bioreactor and the other at the inlet of the bioreactor. The DO concentrations determined by both sensors were compared with those sampled and measured regularly with the BGA reference. The sensor outputs were found to correlate well with the BGA data throughout the experiment using a single calibration, where the DO of the culture medium varied between 25 and 60 mm Hg at the bioreactor outlet and 80-116 mm Hg at the bioreactor inlet. During all 180 days of culture, the precision and the bias were +/-5.1 mm Hg and -3.8 mm Hg at the bioreactor outlet, and +/- 19 mm Hg and -18 mm Hg at inlet. The sensor dynamic range is between 0 and 200 mm Hg and the response time is less than 1 minute. The resolution of the sensor is 0.1 mm Hg at 50 mm Hg, and 0.25 mm Hg at 130 mm Hg.     

Nitrification and utilization of ammonium and nitrate during oil bioremediation at different soil water potentials

Bioremediation of petroleum spills requires aerobic soil conditions and readily available N, which may be susceptible to leaching. Our objectives were to determine the influence of soil water potential on nitrification in the presence of crude oil, the toxicity of oil to NHj‐oxidizing bacteria, and the preferences of microorganisms for NH⁺ ₄ or NO^? ₃. A Weswood clay loam was amended with crude oil to contain 0, 5, and 10% by soil dry weight, and N was added to achieve C:N ratios of 90:1 and 120:1. Soil water potentials were maintained at ‐0.02, ‐0.1, and ‐1.0 kJ/kg or allowed to fluctuate between ‐0.02 and ‐3 kJ/kg. Concentrations of NH⁺ ₄ and NO₃ ^?were measured during an incubation period of 40 d. Nitrification in soil not amended with oil was rapid at water potentials of ‐0.02 and ‐0.1 kJ/kg but inactive at a water potential of ‐1.0 kJ/kg. Oil reduced nitrification rates and populations of NH⁺ ₄‐oxidizing bacteria. Little NO^? ₃ accumulated when the C:N ratio was 120:1, but when the C:N ratio was 90:1, up to 150 μg of NO₃‐N/g of soil accumulated at a soil water potential of ‐0.02 kJ/kg. Soil water potential in the range used did not greatly influence the extent of oil bioremediation but significantly influenced nitrification. Ammonium was preferentially used over NO^? ₃ by microorganisms during oil bioremediation. Nitrate accumulation from urea applied to stimulate oil bioremediation was low when N application matched requirements for oil bioremediation, and nitrification was restricted by controlling soil water content.     

Linking microbial community structure to membrane biofouling associated with varying dissolved oxygen concentrations

In order to elucidate how dissolved oxygen (DO) concentration influenced the generation of extracellular polymeric substance (EPS) and soluble microbial products (SMP) in mixed liquor and biocake, 16S rDNA fingerprinting analyses were performed to investigate the variation of the microbial community in an aerobic membrane bioreactor (MBR). The function of microbial community structure was proved to be ultimately responsible for biofouling. Obvious microbial community succession from the subphylum of Betaproteobacteria to Deltaproteobacteria was observed in biocake. High concentration of EPS in biocake under the low DO concentration (0.5 mg L⁻¹) caused severe biofouling. The correlation coefficient of membrane fouling rate with EPS content in biocake (0.9941-0.9964) was much higher than that in mixed liquor (0.6689-0.8004).     

Temporal and spatial variations of dissolved oxygen concentrations in some Dutch polder ditches

This study deals with the temporal and spatial variations of dissolved oxygen concentration, temperature and occasionally pH in three polder ditches in the Netherlands. A routine programme including different environmental parameters served to characterize the ditch environment. Conclusions concerning water quality criteria are presented briefly.     

Nitrification in coniferous forest soils

Net nitrification rates tend to be low or negligible in the forest floor of many coniferous forests of North-East Scotland. The most likely process controls are substrate availability, pH, allelopathy, water potential, nutrient status and temperature. These are discussed in relation to field and laboratory studies of net and potential rates of nitrification.Fungi make up by far the largest part of the nitrifier community in the coniferous forest floor. Very little is known about the distribution and activity of autotrophs in these systems, although it is certain that in vitro evidence suggesting autotrophs cannot nitrify at pH levels characteristic of coniferous forest soils is unrealistic.Because of the metabolic diversity of nitrifying fungi, a variety of organic and inorganic nitrification pathways may exist in coniferous forests. The possible involvement of free radicles in fungal nitrification in coniferous forest soils is also suggested.A complete understanding of nitrification in coniferous forest soils can only result from field characterisation of N flux such as through the use of ¹⁵N. This must be combined with ecophysiological characterisation of the organisms involved in order that the complexity of nitrification in coniferous forest soils can be resolved.     

Method for determining oxygen consumption rates of static cultures from microplate measurements of pericellular dissolved oxygen concentration

We describe a simple protocol for determining the oxygen consumption of cells in static culture. The protocol is based on a noninvasive oxygen-sensing microplate and a simple mathematical model derived from Fick's Law. The applicability of the model is confirmed by showing the correlation of computed oxygen consumption rate (OCR) values to actual cell densities ascertained by direct cell counting and/or MTT for HL60 and U937 cells cultured in suspension. Correlation between computed OCR and these other indications of cell number was quite good, as long as the cultures were not diffusion-limited for oxygen. The impact of the geometric factors of media depth and well size were confirmed to be consistent with the model. Based on this demonstrated correlation, we also developed a simple, completely noninvasive algorithm for ascertaining the per-cell oxygen utilization rate (OUR), which is the ratio of OCR to cell number, and a fundamental cell characteristic. This is accomplished by correlating the known seed densities to extrapolated determinations of OCR at time zero. Such determinations were performed for numerous cell types, in varying well sizes. Resulting OUR values are consistent with literature values acquired by far more painstaking methods, and ranged from <0.01 fmol.min(-1).cell(-1) for bacteria to 0.1-10 fmol.min(-1).cell(-1) for immortalized mammalian and insect cell lines to >10 fmol.min(-1).cell(-1) for primary hepatocytes. This protocol for determining OCR and OUR is extremely simple and broadly applicable and can afford rapid, informative, and noninvasive insight into the state of the culture.     

Tolerance of parr of Arctic charr, Salvelinus alpinus, to reduced dissolved oxygen concentrations

One year old pan Item Windermere (north west England) tolerated lower incipient lethal levels of oxygen [18 20 mg 1 ¹: 15 17% air saturation value (ASV)] at lower (5, 10°C) than at higher (15, 20°C) an elimation temperatures (2·2 2·4 mg 1 ¹ 22 25% ASV). Values were not significantly different for two races of charr in the lake and are amongst the lowest recorded for salmenid species.     

Morphometric adaptations of sea bass gills to different dissolved oxygen partial pressures

The total respiratory surface area (RSA) of sea bass Dicentrarchus labrax (initial mass 153 ± 27 g), reared in a brackish water recirculation system at 24° C for 3 months under three different oxygen partial pressures of 60, 90 and 140% saturation (87.0, 130.0 and 203.1 torr. respectively), was 351.98 (± 8.90), 264.86 (± 48.20) and 212.90 (± 57.22) mm² g⁻¹ body mass. RSA was negatively correlated with oxygen availability in the water. In the same experiment, no significant differences in the total length of filaments or frequency of lamellae were observed, although the total length of filaments was shorter in fish cultured under hyperoxia.     

氧对膜生物反应器短程硝化的影响

为了研究膜生物反应器的短程硝化性能以及氧对短程硝化的影响,通过对比耗氧率和供氧率,提出了膜生物反应器短程硝化的控制优化建议。在膜生物反应器硝化过程中,DO小于1 mg/L开始出现亚硝氮积累;DO降到0.5 mg/L,出水氨氮浓度与亚硝氮浓度之比接近1∶1;DO调控在0.5-1 mg/L范围内,有利于前置硝化反应器与后续厌氧氨氧化反应器衔接。膜生物反应器中污泥浓度可达20 g/L,耗氧能力可达19.86 mg O2/(L·s),但最大供氧能力仅为0.369 mg O2/(L·s),供氧成为反应器运行的制约瓶颈,"低DO高流量"曝气是继续提高短程硝化效能的控制策略。     

The effect of oscillating dissolved oxygen concentrations on the metabolism of a Spodoptera frugiperda IPLB-Sf21-AE clonal isolate

The effect of oscillating dissolved oxygen (DO) concentration on the metabolism of a clonal isolate of the Spodoptera frugiperda IPLB-Sf21-AE insect cell line was investigated. Specifically, the effect on cell growth, re- combinant protein synthesis, glucose and glutamine consumption, and lactate accumulation was determined. Prior to conducting the oscillating DO experiments, it was found that the DO concentration could be reduced to 15% air saturation without adversely affecting the growth rate. Under these conditions, glucose and glutamine became depleted as the maximum cell density was reached. The introduction of DO oscillations, that is, cycles consisting of 30 min at 15% DO followed by 30 min of anoxia, significantly altered cell metabolism, including inhibition of cell growth and recombinant protein synthesis. The effect of DO oscillations on glucose consumption was dependent on the experimental conditions. Glucose exhaustion occurred when the DO oscillations contained either an "apparent" anoxia period (nitrogen sparging discontinued upon reaching 0% DO) without pH control or a "true" anoxia period (nitrogen sparging continued throughout anoxia period) with pH control. Glucose consumption was significantly decreased, however, when the cells were exposed to a "true" anoxia period without pH control, that is, low pH inhibited glucose utilization. Glutamine uptake was not significantly affected by DO oscillations. Lactate only accumulated in the oscillating DO runs, a finding consistent with previous results demonstrating that significant lactate accumulation only occurs under DO-limited conditions. (c) 1995 John Wiley & Sons, Inc.     

High density continuous production of murine pluripotent cells in an acoustic perfused bioreactor at different oxygen concentrations

Strategies for the production of pluripotent stem cells (PSCs) rely on serially dissociated adherent or aggregate‐based culture, consequently limiting robust scale‐up of cell production, on‐line control and optimization of culture conditions. We recently developed a method that enables continuous (non‐serially dissociated) suspension culture‐mediated reprogramming to pluripotency. Herein, we use this method to demonstrate the scalable production of PSCs and early derivatives using acoustic filter technology to enable continuous oxygen‐controlled perfusion culture. Cell densities of greater than 1 × 10⁷ cells/mL were achieved after 7 days of expansion at a specific growth rate (µ) of 0.61 ± 0.1 day^?1 with a perfusion rate (D) of 5.0 day^?1. A twofold increase in maximum cell density (to greater than 2.5 × 10⁷ cells/mL) was achieved when the medium dissolved oxygen was reduced (5% DO). Cell densities and viabilities >80% were maintained for extended production periods during which maintenance of pluripotency was confirmed by stable expression of pluripotency factors (SSEA‐1 and Nanog), as well as the capacity to differentiate into all three germ layers. This work establishes a versatile biotechnological platform for the production of pluripotent cells and derivatives in an integrated, scalable and intensified stirred suspension culture. Biotechnol. Bioeng. 2013; 110: 648–655. © 2012 Wiley Periodicals, Inc.