Automated determination of critical oxygen concentration for routinely active fish

Synopsis We describe an automated respirometer and control system that determines critical (metabolism-limiting) oxygen concentration for routinely active fish. A microcomputer monitors fish metabolic rate as oxygen concentration in the closed respirometer declines; the critical oxygen concentration is signaled by metabolic-rate change, which is resolved via statistically based rules that consider both magnitude and consistency of rate deviations. After the critical oxygen concentration is found, data are written to disk, the respirometer is reoxygenated and another trial is initiated. This sequence can be repeated indefinitely without human intervention, allowing replicate estimates from a single fish. Ideally, metabolic rate should be calculated frequently during a trial, to minimize exposure of the fish to sub-critical oxygen concentrations. However, precision of measurement is limited by ‘noise’ related to length of time interval over which changes in oxygen concentration are determined, respirometer chamber volume, and fish respiration rate. Short time intervals lead to excessive noise, whereas long time intervals result in insufficient numbers of rate measurements. In a respirometer chamber of calculable optimum volume, measurements made by averaging oxygen readings taken very rapidly over two to three minute intervals provide a good compromise. We present data from experiments with bluegill,Lepomis macrochirus, to illustrate the method and show that critical oxygen concentrations identified by the system are consistent with estimates made by humans viewing graphs of the same experiments     

A lab-built respirometer for plant and animal cell culture

A very simple off-line respirometer was developed to measure oxygen consumption rates of low respiring and shear-sensitive cell suspensions. The respirometer is composed of a 10 mL glass syringe in which the plunger was substituted with a polarographic dissolved oxygen probe. Mechanical agitation is provided by means of a magnetic stirring bar inside the measuring chamber and a stir plate placed below the respirometer. Abiotic oxygen fluxes occurring in the measurement chamber such as oxygen diffusion and probe oxygen consumption were investigated. The apparent oxygen uptake rate was then corrected for abiotic oxygen fluxes, leading to accurate measurements of respiration rates ranging from 0.5 to 25.0 mM x h(-1). Additionally, the effect of the stirring bar shape and of the test length on the integrity of plant (Eschschzoltzia californica) and animal (NS0) cells was evaluated. Animal cells showed a higher resistance to mechanical stirring inside the respirometer compared to plant cells (0% of broken cells and 78.1% respectively for a polygonal stirring bar and a 15 min test). For plant cells, cell damage inside the measurement chamber was reduced by optimizing the stirring bar shape and reducing the test length to 5 min or less. This very simple design was shown to provide reliable and low-cost quantification of the oxygen uptake rate of plant and animal cells and can be use even for more demanding measurements such as oxygen affinity studies.     

Modelling and dynamic simulation of a moving bed bioreactor using respirometry for the estimation of kinetic parameters

Respirometry was used for the characterization of active autotrophic and heterotrophic biomass in a pilot scale moving bed bioreactor (MBBR). For this purpose biofilm samples attached to the carrier elements of the MBBR were transferred to a static gas/static liquid type respirometer with intermittent aeration. Known amounts of ammonia nitrogen and acetate were added to the respirometer. The dissolved oxygen (DO) decrease during the non-aeration phase was measured and used to calculate the oxygen uptake rate (OUR) of the active biomass. The resulting respirograms featured the typical endogenous and exogenous respiration phases and the shape of the respirogram was as expected from analogous respirometry with activated sludge. The OUR response was modelled with the activated sludge model ASM1 and the relevant kinetic parameter values for autotrophic and heterotrophic growth were adjusted. The adjusted parameter set and data originating from a 4-day long intensive measurement campaign were used for modelling and simulation of the pilot scale MBBR. The parameter estimation resulted in a good dynamic simulation of ammonia and nitrate variations in the effluent of the MBBR. Important MBBR properties including biofilm age, biofilm composition, and both attachment and detachment rate were extracted from the model.     

Cardiorespiratory performance during prolonged swimming tests with salmonids: a perspective on temperature effects and potential analytical pitfalls

A prolonged swimming trial is the most common approach in studying steady-state changes in oxygen uptake, cardiac output and tissue oxygen extraction as a function of swimming speed in salmonids. The data generated by these sorts of studies are used here to support the idea that a maximum oxygen uptake is reached during a critical swimming speed test. Maximum oxygen uptake has a temperature optimum. Potential explanations are advanced to explain why maximum aerobic performance falls off at high temperature. The valuable information provided by critical swimming tests can be confounded by non-steady-state swimming behaviours, which typically occur with increasing frequency as salmonids approach fatigue. Two major concerns are noted. Foremost, measurements of oxygen uptake during swimming can considerably underestimate the true cost of transport near critical swimming speed, apparently in a temperature-dependent manner. Second, based on a comparison with voluntary swimming ascents in a raceway, forced swimming trials in a swim tunnel respirometer may underestimate critical swimming speed, possibly because fish in a swim tunnel respirometer are unable to sustain a ground speed.     

Evaluation of the Total Carbonaceous Biochemical Oxygen Demand Method, Using a Compensated Recording Respirometer

The total carbonaceous biochemical oxygen demand test is described, and experimental data demonstrating its stoichiometry, precision, and accuracy are presented. The test is more reproducible and faster than the current 5-day biochemical oxygen demand test procedure, and if a respirometer is used, the effects of toxic chemicals, pH changes, and nutrient imbalances can be routinely monitored. The design principles of a multichannel compensated recording respirometer suitable for this test are described.     

Development of respirometry methods to assess the microbial activity of thermophilic bioleaching archaea

Respirometry methods have been used for many years to assess the microbial activity of mainly heterotrophic bacteria. Using this technique, the consumption of oxygen and evolution of carbon dioxide for heterotrophic carbon catabolism can be used to assess microbial activity. In the case of autotrophic bioleaching bacteria, carbon dioxide is used as a carbon source resulting in the consumption of both oxygen and carbon dioxide. The use of such respirometry techniques at high temperatures (up to 80 degrees C) for the investigation of bioleaching Archaea, however, poses particular difficulties. At these elevated temperatures, the solubility of oxygen into the liquid phase is particularly poor. This work details specific methods by which high temperature constraints are overcome while monitoring the activity of thermophilic Archaea using a Micro-Oxymax respirometer (Columbus Instruments). The use of elevated headspace oxygen concentrations, in order to overcome low oxygen solubility, is demonstrated as well as the effect of such elevated oxygen concentrations on microbial oxygen consumption rates. The relative rates of oxygen and carbon dioxide consumption are also illustrated during the oxidation of a chalcopyrite concentrate. In addition, this paper details generic methods by which respirometry data can be used to quantify inhibitory effects of a compound such as Na(2)SO(4). The further use of such data in predicting minimum hydraulic reactor retention times for continuous culture bioleaching reactors, as a function of concentration of potentially inhibitory compounds, is also demonstrated.     

An experimental comparison of respiration measuring techniques in fermenters and shake flasks: exhaust gas analyzer vs. RAMOS device vs. respirometer

Respiration measurement is applied as a universal tool to determine the activity of biological systems. The measurement techniques are difficult to compare, due to the vast variety of devices and analytical procedures commonly in use. They are used in fields as different as microbiology, gene engineering, toxicology, and industrial process monitoring to observe the physiological activity of living systems in environments as diverse as fermenters, shake flasks, lakes and sewage plants. A method is introduced to determine accuracy, quantitation limit, range and precision of different respiration measurement devices. Corynebacterium glutamicum cultures were used to compare an exhaust gas analyzer (EGA), a RAMOS device (respiration measurement in shake flasks) and a respirometer. With all measuring devices it was possible to determine the general culture characteristics. The EGA and the RAMOS device produced almost identical results. The scatter of the respirometer was noticeably higher. The EGA is the technique of choice, if the reaction volume is high or a short reaction time is required. The possibility to monitor cultures simultaneously makes the RAMOS device an indispensable tool for media and strain development. If online monitoring is not compulsive, the respiration of the investigated microbial system extremely low, or the sample size small, a respirometer is recommended.     

Modeling aerobic carbon source degradation processes using titrimetric data and combined respirometric-titrimetric data: experimental data and model structure

Experimental data are presented that resulted from aerobic batch degradation experiments in activated sludge with simple carbon sources (acetate and dextrose) as substrates. Data collection was done using combined respirometric-titrimetric measurements. The respirometer consists of an open aerated vessel and a closed non-aerated respiration chamber for monitoring the oxygen uptake rate related to substrate degradation. The respirometer is combined with a titrimetric unit that keeps the pH of the activated sludge sample at a constant value by addition of acid and/or base. The experimental data clearly showed that the activated sludge bacteria react with consumption or production of protons during aerobic degradation of the two carbon sources under study. Thus, the cumulative amount of added acid and/or base could serve as a complementary information source on the degradation processes. For acetate, protons were consumed during aerobic degradation, whereas for dextrose protons were produced. For both carbon sources, a linear relationship was found between the amount of carbon source added and the amount of protons consumed (in case of acetate: 0.38 meq/mmol) or produced (in case of dextrose: 1.33 meq/mmol) during substrate degradation. A model taking into account substrate uptake, CO(2) production, and NH(3) uptake for biomass growth is proposed to describe the aerobic degradation of a C(x)H(y)O(z)-type carbon source. Theoretical evaluation of this model for reference parameters showed that the proton effect due to aerobic substrate degradation is a function of the pH of the liquid phase. The proposed model could describe the experimental observations with both carbon sources.     

A continuous flow automatically recording respirometer

A respirometer suitable for continuous operation over extended periods of time is described. Oxygen is supplied to the culture by the electrolysis of water. The quantity of oxygen produced by electrolysis within any specified time interval is automatically printed on a tape. This record is combined with a continuous recording of the concentration of dissolved oxygen entering and leaving the reactor to produce a mass balance for oxygen in the system. Details on respirometer construction and operation are presented.     

PHOTOPHYSIOLOGY OF A TURF ALGAL COMMUNITY: INTEGRATED RESPONSES TO AMBIENT LIGHT AND STANDING BIOMASS1

This study investigated the variation in the relationship between photosynthesis and ambient light (P‐E curves) for turf algal communities on a temperate reef off the coast of South Australia, analyzing the integrated effects of ambient light and standing biomass. The photophysiology of turfs was studied in situ on a seasonal basis, examining algal communities growing on artificial substrate (plates) at depths of 4 m and 10 m. P‐E curves and estimates for the photokinetic parameters (P_m, R_d, α, E_k, and E_c) were obtained through oxygen evolution methods, using an automated underwater respirometer. Photoacclimation responses to changes in ambient light were strongly affected by the biomass of the community. P_m showed an inverse relationship to standing biomass, irrespective of depth and season, which was considered to be a response to self‐shading and boundary layer effects. Biomass effects imposed a high variance on estimates for all photosynthetic parameters, overshadowing differences observed for season and depth. Biomass also affected photoinhibition on turf communities, where significant afternoon depression of photosynthesis was observed in sparse turf patches when compared to denser patches. High areal productivity rates were maintained across all seasons with a significant decrease only being observed during winter.     

Aerobic metabolic scope and swimming performance in juvenile cod, Gadus morhua L.

Juvenile cod (Gadus morhua) were made to swim in a tunnel respirometer to determine the oxygen consumption during swimming at different speeds. Results were compared with measurements of standard and active metabolic rates in static respirometers before and after intense exercise. The oxygen consumption at maximum sustainable swimming speed was considerably lower than the peak oxygen consumption following exhausting exercise. It is suggested that these fish have a poorly developed system of aerobic (red) locomotor muscles which do not normally make a major demand upon oxygen consumption. Apparent specific dynamic action following feeding and repayment of oxygen debt following anaerobic exercise can each give rise to greater rates of oxygen consumption. Following exhausting exercise there is a delay of about 1 h before oxygen consumption reaches a peak level some 40% higher than the peak level observed during sustained swimming.     

Energetics of osmoregulation: I. Oxygen consumption by Fundulus heteroclitus

We have developed a flow-through method for measuring oxygen consumption in fish which allows continuous monitoring over periods of days with good accuracy. Our goal was to determine the changes in basal metabolic rate in estuarine fish as a function of salinity. We show that in Fundulus heteroclitus, the oxygen consumption drops by 50% during the first 12 hr in the respirometer, as the fish cease exploratory movements. We have determined the influence of temperature and body size on resting respiratory rate, but failed to find any circadian or tidal rhythm in aerobic respiration. With these variables controlled, we determined that changing from 10 to 30 ppt water had no demonstrable effect on oxygen uptake. Since there must be a large change in osmotic flux due to this change in salinity, it appears that the fish might be diverting energy from other uses rather than increasing aerobic energy production to meet the increased osmoregulatory work load.     

Population metabolism of Nebria brevicollis (F.) (Coleoptera: Carabidae)

Summary The respiratory metabolism of all life stages of a predatory beetle was investigated. Measurements were made throughout the year in a continuously recording respirometer. The Warburg respirometer was used to examine the relationship between oxygen cosumption and temperature.Respiration was affected by several factors. No periodicity in respiration was observed during the 24 hour period. During the breeding season the males showed a significantly higher respiratory rate than the females. Temperature influence was slightly greater on Instar I and adults in a breeding condition. Laboratory data were adjusted to field rates by using field temperature data and the regressions obtained from respiratory measurements made at different temperatures. Population estimates per m² were low. The seasonal trend in population metabolism was related to temperature. Population density and population production did not follow temperature closely.Population metabolism totalled 1735.95 Joules/m², of which 55.0% was due to adults. The production/respiration ratio (P/R) was 1:1. Total annual energy flux through the beetle population was 3365.13 Joules/m². The larvae accounted for 65% of this total.     

A reappraisal of activity metabolism in Atlantic cod (Gadus morhua)

Atlantic cod ( Gadus morhua ) were forced to swim in a swim tunnel respirometer until fatigued; oxygen consumption rate (O₂) was measured during swimming at incremental speeds until the fish was exhausted and during recovery from exhaustion. Maximal oxygen consumption (O_2max) occurred during maximal activity as has been found for other fish species, but at odds with the current paradigm for Atlantic cod. Earlier experiments had drawn the conclusion that O_2max in Atlantic cod occurs during recovery from exhaustive exercise. We found no support for this paradigm in our experiments and we propose that the respiratory physiology of Atlantic cod is not unlike that of other fishes.     

A novel in situ probe for oxygen uptake rate measurement in mammalian cell cultures

The newly developed in situ oxygen uptake rate (in situ OUR) probe presented in this article is based on the in situ microscope technology platform. It is designed to measure the oxygen uptake rate (OUR) of mammalian cells, an important parameter for metabolic flux analysis, inside a reactor (in situ) and in real-time. The system isolates a known volume of cell culture from the bulk inside the bioreactor, monitors the oxygen consumption over time, and releases the sample again. The sample is mixed during the measurement with a new agitation system to keep the cells in suspension and prevent oxygen concentration gradients. The OUR measurement system also doubles as a standard dissolved oxygen (DO) probe for process monitoring when it is not performing OUR measurements. It can be equipped with two different types of optical sensors (i.e., DO, pH) simultaneously or a conventional polarographic DO-probe (Clark type). This new probe was successfully tested in baby hamster kidney perfusion cell cultures.     

Steady-state measurements of respiration rate with an oxygen electrode.

An oxygen electrode was developed which measures steady-state respiration rates in a volume of 0.25 ml and at oxygen concentrations as low as 0.1 μm. The steady state was achieved by pumping air-equilibrated buffer into the respirometer at various rates. The method is most suitable for tissue slices.     

Treatment of obesity by exercise in the cold.

Six obese men aged 25 to 46 years exercised vigorously for 3 1/2 hours in a cold chamber (-34 degrees C) on 10 successive days. There was an early water loss of about 1 kg, probably due to mobilization of glycogen stores, and a subsequent fat loss of about 4 kg (as estimated from readings of skinfold thickness and confirmed by underwater weighing). The total fat loss was much greater than the calculated external energy deficit (as estimated from dietary records, an activity diary and use of a Kofranyi-Michaelis respirometer). Although some energy was "wasted" through ketosis, much of the additional energy was apparently used for synthesis of additional muscle protein. Both readings of skinfold thickness and underwater weighing indicated that the loss of adipose tissue was well sustained in the first 2 months after the exercise program. Further experiments are recommended to determine whether cold exposure makes a specific contribution to the observed weight loss, and whether this approach would be equally effective in a larger and less selected sample of obese patients.     

An automatic respirometer for determining oxygen uptake in crayfish (Austropotamobius pallipes (Lereboullet)) over periods of 3-4 days.

1. An automatic respirometer has been developed for continuous measurements over 3-4 days on 1-15 g crayfish. The sensor is a modified Mackereth oxygen electrode. Respiration is recorded on a millivolt potentiometric pen recorder during closed periods when the O2 concentration in the medium falls to a predetermined level. A solenoid-operated valve is then opened via a relay circuit energized by a reed switch mounted on the recorder. Medium flows through the respirometer until the O2 concentration is raised back to another predetermined level. Artificial media containing little or no nutrient salts are used to restrict the growth of microbes. 2. Respiration was determined chiefly on crayfish (Austropotamobius pallipes) with body wet weights of 7-0-12-5 g at 10-0 degrees C. In unrestrained but relatively quiescent animals, standard metabolism is described by the regression equation, ln O2 uptake = 3-3037 + 1-002 ln body wt. In restless crayfish active metabolism is described by the equation, ln O2 uptake = 4-4412 + 0-861 ln body wt.     

Microbial metabolic activity in soil as measured by dehydrogenase determinations.

The dehydrogenase technique for measuring the metabolic activity of microorganisms in soil was modified to use a 6-h, 37 degrees C incubation with either glucose of yeast extract as the electron-donating substrate. The rate of formazan production remained constant during this time interval, and cellular multiplication apparently did not occur. The technique was used to follow changes in the overall metabolic activities of microorganisms in soil undergoing incubation with a limiting concentration of added nutrient. The sequence of events was similar to that obtained by using the Warburg respirometer to measure O2 consumption. However, the major peaks of activity occurred earlier with the respirometer. This possibly is due to the lack of atmospheric CO2 during the O2 consumption measurements.     

A simple (14)C-respirometric method for assessing microbial catabolic potential and contaminant bioavailability

This paper describes the validation and application of a simple flask-based (14)C-respirometer system designed to assess mineralisation of (14)C-labelled substrates under defined conditions. Validation of this respirometer system indicated stoichiometric CO(2) trapping up to a maximum of 400 micromol of CO(2) (in a single trap). Polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were used to measure growth-linked biodegradation of [(14)C]naphthalene to (14)CO(2). A (14)C activity balance of 101.7+/-8.9% (n=6), after 74 h incubation time and 10 respirometer-opening events, indicated the suitability of the system for monitoring substrate mineralisation. This respirometric apparatus was then successfully applied to assess: (i) the PAH catabolism of microbes in a field contaminated soil, where naphthalene and phenanthrene were rapidly mineralised and (ii) soil-associated organic contaminant bioavailability, where increased soil-phenanthrene contact time resulted in a reduction in phenanthrene mineralisation in the soil. The described respirometer system differs from existing respirometer systems in that the CO(2) trap can be removed and replaced quickly and easily. The system is efficient, reproducible, adaptable to many situations, easy to construct and simple to use, it therefore affords advantages over existing systems.