Effect of oxygen supply and dilution rate on the production of 2,3-butanediol in continuous bioreactor by Klebsiella pneumoniae

Kinetics of 2,3-butanediol production by Klebsiella pneumoniae (NRRL B199) from glucose have been studied in a continuous bioreactor. The effect of oxygen supply rate and dilution rate on the product output rate and yield of 2,3-butanediol were investigated. For a feed glucose concentration of 100 g l⁻¹, the optimum oxygen transfer rate is between 25.0–35.0 mmol l⁻¹ h⁻¹. Under these conditions, maximum product concentration obtained was 35 g l⁻¹ at a dilution rate of 0.1 h⁻¹ and the maximum product output rate obtained was 4.25 g l⁻¹ h⁻¹. The product yield based on the substrate utilized approached the theoretical value (50%) at low values of oxygen transfer rate but decreased with increasing oxygen transfer rate.     

Rapid assessment of oxygen transfer impact for Corynebacterium glutamicum

Oxygen supply is crucial in industrial application of microbial systems, such as Corynebacterium glutamicum, but oxygen transfer is often neglected in early strain characterizations, typically done under aerobic conditions. In this work, a new procedure for oxygen transfer screening is presented, assessing the impact of maximum oxygen transfer conditions (OTR_max) within microtiter plate-based cultivation for enhanced throughput. Oxygen-dependent growth and productivity were characterized for C. glutamicum ATCC13032 and C. glutamicum DM1933 (lysine producer). Biomass and lysine product yield are affected at OTR_max below 14 mmol L^?1 h^?1 in a standardized batch process, but not by further increase of OTR_max above this threshold value indicating a reasonable tradeoff between power input and oxygen transfer capacity OTR_max. The described oxygen transfer screening allows comparative determination of metabolic robustness against oxygen transfer limitation and serves identification of potential problems or opportunities later created during scale-up.     

Enhancement of oxygen absorption into sodium sulfite solutions

Oxygen absorption enhancement in a sodium sulfite solution was studied in the absence and presence of copper catalyst both for absorption across the liquid surface in a stirred cell and for absorption from individual bubbles rising through a turbulent liquid. The enhancement factor was determined from the ratio of oxygen and argon mass transfer coefficients, measured under identical experimental conditions in the same batch of liquid. It has been found that the oxygen absorption is not chemically enhanced, as long as the mass transfer coefficient, k_L⁰, is high enough, i.e., higher than the value 1.4 × 10^?4 m sec^?1 for the sulfite solution we used. An analysis of our data as well as literature data indicates that the sulfite system is poorly suited for studies of the volumetric mass transfer coefficient of physical absorption (k_L⁰a) in fermentors, inasmuch as oxygen absorption can be chemically enhanced while the degree of enhancement depends on the operating conditions of batch aeration, as well as on the concentration of trace impurities with catalytic effects upon the sulfite solution used.     

Enhancement of oxygen transfer in Hybridoma cell culture by using a perfluorocarbon as an oxygen carrier

Summary In order to increase the oxygen transfer in a bioreactor for Hybridoma cell culture, a perfluorocarbon, Flutec ^R ppll was used in a modified Celligen ^TM system. There was no harmful effect of ppll on the cell growth and on the production of monoclonal antibody.     

Impact of oxygen mass transfer on nitrification reactions in suspended carrier reactor biofilms

Biofilm-internal and external mass transfer resistance was investigated in laboratory-scale nitrifying suspended carrier reactors (SCR), demonstrating the importance of these factors for these increasingly popular reactor systems. Controlled respirometric experiments revealed that oxygen mass transfer resistance regulated the process performance up to a DO concentration of 20 mg L^?1. External mass transfer exerts significant control over the overall reaction rate, thus biofilm models must adequately account for this resistance. Whilst carrier type and characteristics have some influence, biofilm structure seems primarily responsible for differences in mass transfer and nitrification performance. Heterogeneous biofilms grown under high ammonium loadings had much greater area-specific rates than the gel-like biofilms sourced from low loaded systems.Being a mass-transfer controlled process, the overall reaction rate of these SCR systems could be immediately increased by elevating the DO above normal operating levels (up to 20 mg L^?1). Long-term oxygen deficiency in the lower biofilm sections does not negatively affect the biomass activity.     

Correlation of liquid dispersion and oxygen transfer in bubble column

Summary In steady state, attained by continuous aeration after oxygen saturation of water in a bubble column, vertical composition distribution of liquid and gas phases has been determined. It has been assumed that, as a result of absorption at the bottom of the column, desorption in the upper section and vertical dispersion of dissolved oxygen flux, a closed oxygen circulation is created. Determination of the axial dispersion coefficient from hydrodynamic and oxygen transfer data verifies the mathematical model proposed. The results allow conclusions to be drawn about supersaturation and desorption and other phenomena expected in biological systems.Abbreviations C[-] Dimensionless oxygen concentration Unit=0.21 bar oxygen partial pressure or dissolved oxygen level in equilibrium with latter - E[m²/s] Axial dispersion coefficient - F[m²] Horizontal cross-section area - k _L a[s^-1] Overall oxygen transfer coefficient - u; u ₂[m/s; cm/s] Superficial velocity: related to state of bubbles leaving the sparger - x; x _atm[-] Signal registered in the experiment; signal recorded in O₂ saturated water, or water vapor saturated air stream, at temperature identical to the experiment under atmospheric pressure - y[m] Water column height - [s^-1] Dimensionless oxygen flux Indices a asorption - d desorption - g gas - l liquid - k dispersion - m measured value/in the case of hydrodynamically measured E/ Dedicated to Professor Dr. H. J. Rehm on the occasion of his 60th birthday     

Characterization of oxygen uptake and mass transfer in flocculent yeast cell cultures with or without a flocculation additive

Summary The effect of solids concentration and the presence of the charged polymeric additive Magna Floc LT25 on gas-liquid oxygen transfer and oxygen consumption rates have been evaluated for flocculent yeast cells grown in batch fermentations. No significant differences were found for oxygen consumption due to the presence of the additive. Low biomass concentrations have a positive effect on oxygen transfer rate, when the additive was present in the medium. For biomass concentrations above 1 g/L, an increase in biomass concentration leads to a reduction on oxygen transfer rates regardless of the presence of the additive.     

Bioreactor scale-up and oxygen transfer rate in microbial processes: An overview

In aerobic bioprocesses, oxygen is a key substrate; due to its low solubility in broths (aqueous solutions), a continuous supply is needed. The oxygen transfer rate (OTR) must be known, and if possible predicted to achieve an optimum design operation and scale-up of bioreactors. Many studies have been conducted to enhance the efficiency of oxygen transfer. The dissolved oxygen concentration in a suspension of aerobic microorganisms depends on the rate of oxygen transfer from the gas phase to the liquid, on the rate at which oxygen is transported into the cells (where it is consumed), and on the oxygen uptake rate (OUR) by the microorganism for growth, maintenance and production.     

Vertical migration of aggregated aerobic and anaerobic ammonium oxidizers enhances oxygen uptake in a stagnant water layer

Ammonium can be removed as dinitrogen gas by cooperating aerobic and anaerobic ammonium-oxidizing bacteria (AerAOB and AnAOB). The goal of this study was to verify putative mutual benefits for aggregated AerAOB and AnAOB in a stagnant freshwater environment. In an ammonium fed water column, the biological oxygen consumption rate was, on average, 76 kg O₂ ha⁻¹ day⁻¹. As the oxygen transfer rate of an abiotic control column was only 17 kg O₂ ha⁻¹ day⁻¹, biomass activity enhanced the oxygen transfer. Increasing the AnAOB gas production increased the oxygen consumption rate with more than 50% as a result of enhanced vertical movement of the biomass. The coupled decrease in dissolved oxygen concentration increased the diffusional oxygen transfer from the atmosphere in the water. Physically preventing the biomass from rising to the upper water layer instantaneously decreased oxygen and ammonium consumption and even led to the occurrence of some sulfate reduction. Floating of the biomass was further confirmed to be beneficial, as this allowed for the development of a higher AerAOB and AnAOB activity, compared to settled biomass. Overall, the results support mutual benefits for aggregated AerAOB and AnAOB, derived from the biomass uplifting effect of AnAOB gas production.     

Microelectrode measurement of oxygen tensions in collagen-hepatocyte gels

Summary Microelectrodes have been used for the measurement of oxygen tension in various biological systems.(Silver, 1987) Although not previously reported, microelectrodes allow the direct measurement of oxygen tension profiles within collagen gels containing entrapped hepatocytes (collagcn-hepatocyte gels). These oxygen tension profiles, along with hepatocyte oxygen consumption data, allowed the estimation of a diffusion coefficient for oxygen in collagen-hepatocyte gels, D _g = 2.99 × 10^–5 cm²/s.     

The effects of the oxygen transfer coefficient and substrate concentration on the xylose fermentation by Debaryomyces hansenii

Relevant production of xylitol by Debaryomyces hansenii requires semiaerobic conditions since in aerobic conditions the accumulated reduced adenine dinucleotide coenzyme is fully reoxidized leading to the conversion of xylitol into xylulose. For oxygen transfer coefficient values from 0.24 to 1.88 min^-1, in shake flasks experiments, biomass formation increased proportionally to the aeration rate as shown in the oxygen transfer coefficient and xylose concentration isoresponse contours. The metabolic products under study, xylitol and ethanol were mainly growth associated. However, for oxygen transfer coefficient above 0.5 min^-1 higher initial xylose concentration stimulated the rate of production of xylitol. This fact was less evident for ethanol production. The direct relationship between increased biomass and products formation rate, indicated that the experimental domain in respect to the aeration rate was below the threshold level before the decreasing in metabolic production rates reported in literature for xylose-fermenting yeasts. The fact that ethanol was produced, albeit in low levels, throughout the experimental design indicated that the semiaerobic conditions were always attained. Debaryomyces hansenii showed to be an important xylitol producer exhibiting a xylitol/ethanol ratio above four and a carbon conversion of 54% for xylitol.Abbreviations K_La oxygen transfer coefficient - DO(T) dissolved oxygen (tension) - OUR oxygen uptake rate - NAD(H) oxidised (reduced) nicotinamide adenine dinucleotide - NADP(H) oxidised (reduced) nicotinamide adenine dinucleotide phosphate - CRC catabolic reduction charge - C oxygen concentration in the culture medium - C^* oxygen concentration at saturation conditions - Y_i response from experiment i - parameters of the polynomial model - x experimental factor level (coded units) - R² coefficient of multiple determination - t time     

Upregulation of cardioprotective SUR2A by sub-hypoxic drop in oxygen

The effects of hypoxia on gene expression have been vigorously studied, but possible effects of small changes in oxygen tension have never been addressed. SUR2A is an atypical ABC protein serving as a regulatory subunit of sarcolemmal ATP-sensitive K⁺ (K_ATP) channels. Up-regulation of SUR2A is associated with cardioprotection and improved physical endurance. Here, we have found that a 24 h-long exposure to slightly decreased ambient fractional concentration of oxygen (20% oxygen), which is an equivalent to oxygen tension at 350 m above sea level, significantly increased levels of SUR2A in the heart despite that this drop of oxygen did not affect levels of O₂, CO₂ and hematocrit in the blood or myocardial levels of ATP, lactate and NAD/NADH/NAD⁺. Hearts from mice exposed to 20% oxygen were significantly more resistant to ischaemia-reperfusion when compared to control ones. Decrease in fractional oxygen concentration of just 0.9% was associated with phosphorylation of ERK1/2, but not Akt, which was essential for up-regulation of SUR2A. These findings indicate that a small drop in oxygen tension up-regulates SUR2A in the heart by activating ERK signaling pathway. This is the first report to suggest that a minimal change in oxygen tension could have a profound signaling effect.     

The primary structure of D1 near the QB pocket influences oxygen evolution

Photosystem II (PS II) is the site of oxygen evolution. Activation of dark adapted samples by a train of saturating flashes produces oxygen with a yield per flash which oscillates with a periodicity of four. Damping of the oxygen oscillations is accounted for by misses and double hits. The mechanisms hidden behind these parameters are not yet fully understood. The components which participate in charge transfer and storage in PS II are believed to be anchored to the heterodimer formed by the D1 and D2 proteins. The secondary plastoquinone acceptor Q_B binds on D1 in a loop connecting the fourth and fifth helices (the Q_B pocket). Several D1 mutants, mutated in the Q_B binding region, have been studied over the past ten years.In the present report, our results on nine D1 mutants of Synechocystis PCC 6714 and 6803 are analyzed. When oxygen evolution is modified, it can be due to a change in the electron transfer kinetics at the level of the acceptor side of PS II and also in some specific mutants to a long ranging effect on the donor side of PS II. The different properties of the mutants enable us to propose a classification in three categories. Our results can fit in a model in which misses are substantially determined by the fraction of centers which have Q_A ^- before each flash due to the reversibility of the electron transfer reactions. This idea is not new but was more thoroughly studied in a recent paper by Shinkarev and Wraight (1993). However, we will show in the discussion that some doubts remain as to the true origin of misses and double hits.Abbreviations BQ p-benzoquinone - Chl chlorophyll - D1 and D2 proteins of the core of PS II - DCMU 3-(3,4-dichlorophenyl)-1,1 dimethyl urea - OEC oxygen evolving complex - P₆₈₀ chlorophyll center of PS II acting as the primary donor - PS II Photosystem II - Q_A and Q_B primary and secondary quinone electron acceptor - TL thermoluminescence     

Toward an expanded oxygen atom transfer reactivity scale: Computational investigation of the energetics of oxo transfer reaction couples

The computational prediction of gas phase enthalpy (neutral substrates) and aqueous free energy (anion substrates) changes has been evaluated for the oxygen atom transfer reaction X + 1/2O₂ → XO. Several density functionals (SVWN, BP86, B3LYP) at double- and triple-ζ levels were surveyed, along with one composite ab initio method (G3(MP2)). Results are presented for extensive main group test sets for which experimental thermochemistry is available. In addition, several minimal reaction couples of the type [M^IVOL₂]/[M^VIO₂L₂] (M = Mo, W) have been examined. Overall, the results suggest a computational approach to the energetics of oxo transfer is feasible, potentially affording an expanded oxo transfer reactivity scale.     

Effect of surface contaminants on oxygen transfer in bubble column reactors

Gas hold-up (ɛ_g), sauter mean bubble diameter (d₃₂) and oxygen transfer coefficient (k_La) were evaluated at four different alkane concentrations (0.05, 0.1, 0.3 and 0.5 vol.%) in water over the range of superficial gas velocity (u_g) of (1.18–23.52) × 10⁻³ m/s at 25 °C in a laboratory-scale bubble column bioreactor. Immiscible hydrocarbons (n-decane, n-tridecane and n-hexadecane) were utilized in the experiments as impurity. A type of anionic surfactant was also employed in order to investigate the effect of addition of surfactant to organic-aqueous systems on sauter mean bubble diameter, gas hold-up and oxygen transfer coefficient. Influence of addition of alkanes on oxygen transfer coefficient and gas hold-up, was shown to be dependent on the superficial gas velocity. At superficial gas velocity below 0.5 × 10⁻³ m/s, addition of alkane in air–water medium has low influence on oxygen transfer coefficient and also gas hold-up, whereas; at higher gas velocities slight addition of alkane increases oxygen transfer coefficient and also gas hold-up. Increase in concentration of alkane resulted in increase in oxygen transfer coefficient and gas hold-up and roughly decrease in sauter mean bubble diameter, which was attributed to an increase in the coalescence-inhibiting tendency in the presence of surface contaminant molecules. Bubbles tend to become smaller with decreasing surface tension of hydrocarbon, thus, oxygen transfer coefficient increases due to increasing of specific gas–liquid interfacial area (a). Empirical correlations were proposed for evaluating gas hold-up as a function of sauter mean bubble diameter, superficial gas velocity and interfacial surface tension as well as evaluating Sherwood number as a function of Schmidt, Reynolds and Bond numbers.     

Simple system for controlling dissolved oxygen concentration in laboratory fermentors

A simple system for the control of oxygen transfer in laboratory fermentation units has been devised. This system employs pure oxygen to elevate the driving force for oxygen transfer. In addition, the aeration is controlled in a closed loop manner in order to minimize total exit flow of the gas from the fermentor. Fermentation study using Bacillus stearothermophilus grown on n-dodecane at 55°C illustrates excellent carbon recovery as well as the ease of controlling oxygen transfer.     

Experimental evaluation of liquid film resistance in oxygen transport to microbial cells

A membrane probe was used to monitor the dissolved oxygen concentrations in continuous cultures of Candida utilis and Micrococcus roseus growing at low dissolved oxygen concentrations and various agitation levels. For the yeast fermentations, increasing the agitation level within the range of 0.1 to 0.3 w per liter lowered steady-state dissolved oxygen concentrations in the fermentor. The steady-state dissolved oxygen concentration in the fermentor was not influenced by the agitation level within the range of 0.3 to 1.8 w per liter. With M. roseus, no effect of agitation on steady-state dissolved oxygen concentrations in the fermentor was observed within the range of 0.1 to 1.8 w per liter. It was concluded that, under the conditions used, a measurable transfer barrier from the liquid to the yeast cells existed at agitation levels below 0.3 w per liter and that this barrier did not exist at agitation levels above 0.3 w per liter. The transfer barrier from the liquid to the yeast surface could be represented by a stagnant film of liquid 0.6 × 10^-4 cm thick surrounding the cell at an agitation level of 0.10 w per liter. This film represented an oxygen concentration drop of 1.3 × 10^-7 M from the bulk of the medium to the cells under the experimental conditions.     

Intracellular regulation of oxygen consumption in the isolated crayfish stretch receptor neuron

Morphological correlations of the functional regulation of oxygen consumption have been studied on single isolated crayfish mechanoreceptor neurons. An enhancement of oxygen consumption is promoted by the following: (1) redistribution of mitochondria and an increase in cytochrome oxidase (CO) activity in mitochondria near the plasmatic membrane, (2) coordination of mitochondria aggregation rhythms with pO₂ rhythms in the medium external for a cell, (3) a decrease in the area of high CO activity and mitochondria and a shortening of the oxygen diffusion pathway, (4) an increase of the CO activity gradient from the neuron body periphery to its center, (5) a transfer of oxygen with the water flow during neuron body hydration and cytoplasm dilution during the transfer of a portion of the gel into sol, (6) cyclic changes in the ratio of the neuron body and hillock sizes at which there is a transfer of oxygen with the water flow into the neuron body, its mitochondrial uptake in the neuron body, and transfer of the oxygen-free water from the neuron body into the axonal hillock and further into the external medium.     

On-line monitoring of oxygen in Tubespin, a novel, small-scale disposable bioreactor

A novel, optical sensor was fixed in a new type of disposable bioreactor, Tubespin, for the on-line (real-time) monitoring of dissolved oxygen concentrations during cell culture. The cell density, viability and volumetric mass transfer coefficient were also determined to further characterize the bioreactors. The k_La value of the Tubespin at standard conditions was 24.3 h⁻¹, while that of a spinner flask was only 2.7 h⁻¹. The maximum cell density in the Tubespin bioreactor reached 6 × 10⁶ cells mL⁻¹, which was two times higher than the cell density in a spinner flask. Furthermore, the dynamic dissolved oxygen level was maintained above 90% air-saturation in the Tubespin, while the value was only 1.9% in a spinner flask. These results demonstrate the competitive advantage of using the Tubespin system over spinner flasks for process optimization and scale-down studies of oxygen transfer and cell growth.     

Role of oxygen fixation in hydroxyproline biosynthesis by etiolated seedlings

Etiolated maize and soybean seedlings were grown for several days in atmospheres enriched with O¹⁸. Hydroxyproline subsequently isolated from the seedlings by column and thin-layer chromatography was labeled with excess O¹⁸, but proline was not. Control experiments in which seedlings were grown in H₂O¹⁸ and unlabeled atmospheres demonstrated that neither proline nor hydroxyproline was labeled with excess O¹⁸. It was concluded that oxygen fixation is an essential feature of hydroxyproline biosynthesis in these seedlings, and that the hydroxyl oxygen atom in hydroxyproline is derived from molecular oxygen and not from water; similar results have been reported previously for sycamore cell suspensions.