A novel approach to controlling dissolved oxygen levels in laboratory experiments

Hypoxia is a widespread environmental stressor that affects marine, estuarine and freshwater systems worldwide. Investigating the effects of hypoxia on aquatic animals in the natural environment is difficult and expensive. Laboratory experiments provide an alternative that allows manipulation of environmental variables and simulation of altered conditions that are expected in the future. However, controlling dissolved oxygen (DO) levels precisely in laboratory test situations is challenging and generally costly and time intensive. In this paper, we describe a novel chamber design that is capable of maintaining low DO levels precisely for a period of at least 4 days with minimal re-adjustment and nitrogen gas requirement. The system is simple, inexpensive and easy to use, and offers the additional benefit of being portable. The utility of this design is demonstrated with an experiment to test the effects of hypoxia and high salinity on hatch rates and yolk-sac larvae survival in the estuarine fish black bream Acanthopagrus butcheri (Munro, 1949). The results show that early life-stage black bream are sensitive to hypoxia and increased salinity, indicating that deterioration in estuarine environmental conditions may have negative effects on spawning success and recruitment to fish populations. This new laboratory technique will be useful for testing future scenarios for the estuaries of southern Australia as predicted by climate change models. Other potential applications and modifications for further development of this new technique are discussed.     

A device for simultaneously controlling multiple treatment levels of dissolved oxygen in laboratory experiments

Hypoxia is common in freshwater, estuarine, and shallow or enclosed marine systems. Research on the sub-lethal effects of hypoxia on feeding and growth includes both field and laboratory studies. Extended regulation of dissolved oxygen (DO) is difficult to precisely control in the laboratory. Here we describe a device that is capable of simultaneously monitoring and controlling DO at multiple treatment levels, in replicate experimental tanks, for indefinite periods of time. Each treatment level may be static or fluctuate on a diel cycle. The device consists of a series of independent aquarium systems, each of which is monitored and adjusted by a computer program. A data file and chart of DO levels in each treatment level is recorded. A computer program that repeatedly measures and adjusts DO controls the device. Because this apparatus is software-controlled, the user has the flexibility of altering treatment parameters at any time without interrupting experiments.     

Feedforward-feedback control of dissolved oxygen concentration in a predenitrification system

As the largest single energy-consuming component in most biological wastewater treatment systems, aeration control is of great interest from the point of view of saving energy and improving wastewater treatment plant efficiency. In this paper, three different strategies, including conventional constant dissolved oxygen (DO) set-point control, cascade DO set-point control, and feedforward-feedback DO set-point control were evaluated using the denitrification layout of the IWA simulation benchmark. Simulation studies showed that the feedforward-feedback DO set-point control strategy was better than the other control strategies at meeting the effluent standards and reducing operational costs. The control strategy works primarily by feedforward control based on an ammonium sensor located at the head of the aerobic process. It has an important advantage over effluent measurements in that there is no (or only a very short) time delay for information; feedforward control was combined with slow feedback control to compensate for model approximations. The feedforward-feedback DO control was implemented in a lab-scale wastewater treatment plant for a period of 60 days. Compared to operation with constant DO concentration, the required airflow could be reduced by up to 8–15% by employing the feedforward-feedback DO-control strategy, and the effluent ammonia concentration could be reduced by up to 15–25%. This control strategy can be expected to be accepted by the operating personnel in wastewater treatment plants.     

The study of neural network-based controller for controlling dissolved oxygen concentration in a sequencing batch reactor

The design and development of the neural network (NN)-based controller performance for the activated sludge process in sequencing batch reactor (SBR) is presented in this paper. Here we give a comparative study of various neural network (NN)-based controllers such as the direct inverse control, internal model control (IMC) and hybrid NN control strategies to maintain the dissolved oxygen (DO) level of an activated sludge system by manipulating the air flow rate. The NN inverse model-based controller with the model-based scheme represents the controller, which relies solely upon the simple NN inverse model. In the IMC, both the forward and inverse models are used directly as elements within the feedback loop. The hybrid NN control consists of a basic NN controller in parallel with a proportional integral (PI) controller. Various simulation tests involving multiple set-point changes, disturbances rejection and noise effects were performed to review the performances of these various controllers. From the results it can be seen that hybrid controller gives the best results in tracking set-point changes under disturbances and noise effects.     

Experimental simulation of dissolved oxygen fluctuations in large fermentors: effect on Streptomyces clavuligerus

An experimental Monte Carlo method was used to study the effect of fluctuations in oxygen concentration on the synthesis of antibiotics by Streptomyces clavuligerus. Air was supplied to the culture in a 2-L fermentor in random cycles following the lognormal distribution in order to model the circulation within large production-scale vessels. Each cycle consisted of air supply for 5 s followed by no aeration for the balance of the cycle time which ranged from 8 to 44 s, with a mean time of 20 s. Comparable experiments were also conducted with constant period cycling of air and with continuous supply of air. The yields of cephamycin C and its precursor, penicillin N, were suppressed by the Monte Carlo simulation of circulation in a large tank, as compared to constant period cycling. The concentration of dissolved oxygen remained at a low, ca. 5% of saturation, for 5-10 h longer during the Monte Carlo experiment than during the periodic aeration experiment. The biosynthetic enzymes, which are sensitive to oxygen levels, were likely affected not only by the mean time of cycling but also by the distribution of the cycles.     

Propagation of Bacillus popilliae in laboratory fermentors

The insect pathogen Bacillus popilliae Dutky causes a fatal milky disease of Japanese beetle larvae. Spores of the bacterium offer a biological means of controlling this insect. While satisfactory sporulation in vitro has not yet been accomplished, conditions have been developed for the proliferation of vegetative cells in shaken flasks and aerated fermentors. Vegetative cultures are maintained by frequent transfer or by lyophilization. Media based on yeast extract are used routinely, but corn steep liquor and casein hydrolyzates afford comparable yields of 5 × 10⁸ cells/ml. in 16–24 hr. Nutritional requirements have been established for growth in a synthetic medium. Oxygen availability affects the pathway of carbohydrate catabolism and is necessary for optimal growth. In rapidly growing cultures, a short period of maximum viability is characteristically followed by rapid death of the cells. When inoculum size and transfer time are suitably manipulated, viable cell yields reach 1–2 × 10⁹/ml. Alternative methods of propagation, including the addition of particulate carbon, and procedures designed either to neutralize acids or to remove metabolic products by dialysis, do not markedly enhance the yield of cells per volume of medium, although viability may be prolonged.     

Scaling oxygen mass transfer in agitated fermentors

There are many scaling formulas that predict the oxygen mass transfer coefficient as k(L).a = constant.(Hp/V)(alpha)Vs(beta) Exponents alpha and beta frequently are scale dependent themselves. A general formula has been derived from the work of Calderbank,(1) Miller,(2) and Tilton,(3) resulting in k(L).a = C(1) phi + C(2) log (Pm/V) phi where phi equals the gas-holdup fraction and Pm/V equals the effective mechanical power input per unit of volume. This formula is consistent with the formula of Westerterp(4) modified by Miller.(2) Gas holdup can be predicted in several ways. Gas-sparged isothermal expansion power input, used for predicting phi, demonstrates that scaling can be done by using either superficial air velocity or volume per volume per minute for aeration.The importance of mixing in replenishing oxygen at the boundary layers of microorganisms will be assessed and compared with the k(L).a as the oxygen transfer ratelimiting step.     

Adaptive control of dissolved oxygen concentration in a bioreactor

A new adaptive DO (dissolved oxygen) concentration control algorithm considering DO electrode dynamics with response time delay has been developed. A system model with two time-varying parameters was used to relate the DO concentration with two control variables: air flow rate and agitation speed. Parameters of this model were estimated on-line using a regularized constant trace recursive least-squares method. An extended Kalman filter was used to remove the effect of noises from the DO concentration measurements and thus to improve control performance. A discrete one-step ahead control scheme was adopted to determine control actions based on the parameter estimation results. Experimental results showed that the new adaptive DO concentration control algorithm performed better than other algorithms tested, a PID controller and adaptive algorithms without the DO electrode dynamics.     

Effect of dissolved oxygen concentration on sludge settleability

This laboratory study presents a detailed evaluation of the effects of dissolved oxygen concentration and accumulation of storage polymers on sludge settleability in activated sludge systems with an aerobic selector. The oxygen and substrate availability regime were simulated in laboratory sequencing batch reactor systems. The experiments showed that low dissolved oxygen concentration (1.1 mg O₂ l^–1) had a strong negative effect on sludge settleability, leading to the proliferation of filamentous bacteria (Thiothrix spp., Type 021N and Type 1851). This negative effect was stronger at high chemical oxygen demand loading rate. This indicates that a compartmentalised (plug flow) aerobic contact tank, designed at short hydraulic residence time to guarantee a strong substrate gradient, with low dissolved oxygen concentration, might be worse for sludge settleability than an "overdesigned" completely mixed contact tank. Contrary to the general hypothesis, the maximum specific acetate uptake rate, poly--hydroxybutyrate production rate, and resistance to short starvation periods are similar in both poor- and well-settling sludge. The results of this study support our previous hypothesis on the importance of substrate gradients for the development of filamentous structures in biological flocs, from soluble organic substrate gradients to dissolved oxygen gradients in sludge flocs.     

An algorithm for adaptive control of dissolved oxygen concentration in batch culture

An effective automatic control algorithm for set-point control of dissolved oxygen concentration in batch culture has been developed. Adaptation of PI controller to the variable state of batch culture is based on analytically obtained functional relations between the controller parameters and the state variables: oxygen uptake rate, stirring speed, and saturation value of dissolved oxygen concentration, which are measured or estimated on-line. Results of experimental investigation of the adaptive control system are presented.     

Influence of dissolved oxygen concentration on the oxygen kinetics of Gluconobacter oxydans

Summary As shown in earlier studies in production scale bioreactors oxygen limited zones occur. Microorganisms in these reactors are therefore subjected to concentrations of oxygen varying with time. To simulate these conditions, the effect of low oxygen concentrations upon product formation and kinetics of oxygen of Gluconobacter oxydans are studied at laboratory scale.Under these oxygen limited conditions comparable kinetic parameters for oxygen are observed as under normally aerated conditions.So, a saturation constant for oxygen K _{O 2}=6.9 mol/l is observed, which is equivalent to a DOT value of about 3% of air saturation.For optimization purposes of production scale conditions, gassing with oxygen enriched air or with pure oxygen is one of the possibilities.To study the effect of high oxygen concentrations upon kinetics and product formation, the organisms are also cultivated under these extreme conditions. Although at oxygen concentrations larger then 60% saturation with pure oxygen, still growth was observed, the growth rate and also the product formation rate were strongly diminished.From these experiments it can be concluded that gassing with pure oxygen to achieve higher oxygen transfer rates at production scale will be restricted.     

Compact automated displacement gas metering system for measurement of low gas rates from laboratory fermentors

An automated metering system was developed for measuring biogas production from laboratory scale biogas digestors. The gas metering system is based on the principle of liquid displacement with a 100-mL reversible cycle and registration. The gas meter is made entirely of plastic and rubber materials resistant to the corrosive components of biogas (e.g., H(2)S) and requires a 12 to 15 VDC power supply.     

On the methodology of dissolved oxygen saturation concentration measurements

Summary An experimental method for the determination of the concentration of dissolved oxygen saturation in real fermentation media is described. It is based on a joint analysis of gas and liquid phases, applying a mass spectrometer for gas analyses and an oxygen electrode for the liquid phase measurements. This method enables the experimental measurement of oxygen solubilities in real fermentation broth within the bioreactor during the process and its application seems to be of general validity.     

Maximizing mouse embryonic stem cell production in a stirred tank reactor by controlling dissolved oxygen concentration and continuous perfusion operation

One of the key challenges in stem cell bioprocessing is the large-scale cultivation of stem cells in order to meet the demanding meaningful cell numbers needed for biomedical applications, especially for clinical settings. Mouse embryonic stem cells [1], used as a model system herein, were cultivated on microcarriers in a fully controlled stirred tank reactor (STR) [2]. The impact of varying the concentration of dissolved oxygen (at 5%, 10%, 20% and 30% DO) and operating under a continuous perfusion mode on cell growth and pluripotency maintenance was investigated. In addition, in order to further optimize the feeding strategy of the STR operating under continuous perfusion toward maximal cell production, the influence of different medium residences times (12 h, 24 h, 32 h, 48 h and 96 h) was evaluated. Overall, the maximal cell concentration of 7.9–9.2 × 10⁶ cells/mL were attained after 11 days, with no passaging required, under a DO of 10–20% in the continuous perfused bioreactor with cell retention and medium residences times of 32–48 h. Importantly, mESC expanded under these conditions, retained the expression of pluripotency markers (Oct4, Nanog and Ssea-1), as well as their differentiation potential into cells of the three embryonic germ layers.The STR-based cultivation platform optimized herein represents a major contribution toward the development of large-volume production systems of differentiated cell derivatives for a wide range of biomedical applications.     

Monitoring and controlling the dissolved oxygen (DO) concentration within the high aspect ratio vessel (HARV)

A probe-type oxygen sensor was developed utilizing a radioluminescent (RL)-based light source and a ruthenium-based sensing chemistry for monitoring the dissolved oxygen (DO) concentration in a modified version of the NASA-designed high aspect ratio vessel (HARV), a batch rotating wall vessel. This sensor provided the means to monitor the DO concentration in the HARV without influencing the flow pattern, thereby retaining the low shear HARV environment conducive to the formation of 3-dimensional cell aggregates. This sensor lost significant signal as a result of exposure to the first three autoclave cycles, but only minimal change in signal was observed following exposure to subsequent autoclave cycles. A new calibration model requiring only one fitted parameter was developed that accurately fit data over the entire range from 0% to 100% oxygen saturation. The ability for DO concentration control within the vessel was demonstrated by using this sensor to monitor the DO concentration inside the HARV.     

Mixing and oxygen transfer in conventional stirred fermentors

A set of experiments has been performed in an industrial 112 m(3) fermentor in order to get a complete map of oxygen concentration and temperature distribution in the system. Five fermentations of non-Newtonian broths of two different strains, in various operating conditions, were examined. A simple model has been developed which takes into account both the mixing and the mass-transfer properties of the fermentor, and a dimensionless parameter has been identified which is sufficient to characterize the oxygen axial distribution in the reactor in any operating condition.     

Optimal dissolved oxygen concentration for the production of chitinases by Serratia marcescens

Chitinase production by Serratia marcescens 990E was maximal (34 U/ml) for a dissolved oxygen concentration ranging from 20 to 50% saturation. Chitin addition (6 g/l) at the end of fermentation reactivated the growth but did not bring about further chitinase accumulation. Four chitinases with different isoelectric points were separated.