The application of a novel heat flux calorimeter for studying growth of Escherichia coli W in aerobic batch culture

The modification and principle of a novel heat flux calorimeter for the in situ, on-line measurement of the heat generated during microbial growth is described. Data concerning the physical characterization of the calorimeter as a fermentor, including stability and sensitivity of the heat signal, are presented. The calorimeter has been successfully applied to the study of the aerobic batch culture of Escherichia coli W on glucose under carbon and nitrogen limitation. A direct correlation between growth and heat evolution was obtained. Quantitative analysis of the data suggests that the new calorimetric technique could be used for monitoring growth and specific metabolic events, for convenient medium optimization, and as a basis for a novel fermentation process control system.     

A sensitive direct calorimeter for small mammals

A sensitive direct calorimeter for small animals is presented. Its principle is based on the measurement of the heat transfer from the animal chamber to a heat sink. The system gives repetitive measurements with a high efficiency and allows a detailed time-related measurement of the heat production by the whole animal. Its low response time can be advantageously used for the study of post-prandial heat generation and diet-induced thermogenesis. Data on the heat production by Wistar and lean and obese Zucker rats is also included.     

Miniaturized calorimetry - a new method for real-time biofilm activity analysis

The partial dissipation of Gibbs energy as heat reflects the metabolic dynamic of biofilms in real time and may also allow quantitative conclusions about the chemical composition of the biofilm via Hess' law. Presently, the potential information content of heat is hardly exploited due to the low flexibility, the low throughput and the high price of conventional calorimeters. In order to overcome the limitations of conventional calorimetry a miniaturized calorimeter for biofilm investigations has been evaluated. Using four thermopiles a heat production with spatial and temporal resolutions of 2.5 cm(-1) and 2 s(-1) could be determined. The limit of detection of the heat flow measurement was 20 nW, which corresponds to the cell density of an early stage biofilm (approx. 3x10(5) cells cm(-2)). By separating biofilm cultivation from the actual heat measurement, a high flexibility and a much higher throughput was achieved if compared with conventional calorimeters. The approach suggested allows cultivation of biofilms in places of interest such as technological settings as well as in nature followed by highly efficient measurements in the laboratory. Functionality of the miniaturized calorimeter was supported by parallel measurements with confocal laser scanning microscopy and a fiber optic based oxygen sensor using the oxycaloric equivalent (-460 kJ mol-O2(-1)).     

A flow calorimeter for assay of hormone- and metabolite-induced changes in steady-state heat production by tissue

A compact, heat conduction, flow calorimeter for use in monitoring tissue response to metabolic regulators has been designed and constructed. The instrument operates as a perfusion apparatus. Heat production by tissue can be measured continuously, and nutrient and oxygen concentrations can be kept at optimum, constant levels. Introduction of substances of interest is made without production of attendant thermal mixing artifacts. The resolution time of events is less than 10 s. The thermal stability of the instrument is maintained by dynamic methods. The sensitivity of the instrument, 7.2 μW/μV, allows observation of changes in heat production as small as 3 × 10^?4 to 5 × 10^?4 cal/min, the equivalent of 3–5% of the heat production of a representative 1-g quantity of fresh tissue. The calorimeter was used to monitor changes in the steady-state heat production of 250-mg samples of corn coleoptile tissue in response to the plant growth substance, indoleacetic acid.     

An optimized differential heat conduction solution microcalorimeter for thermal kinetic measurements

Heat conduction calorimeters are widely used in biological sciences, but baseline instability, low resolution, electrical noise and motion artifacts have limited their utility. Two main sources of noise, baseline fluctuation or drift and a motion artifact, were traced to amplifier drift, a small (0.015°C) gradient within the constant temperature cylinder, and the method of installing the thermopiles. The addition of heaters to the top and bottom of the cylinder reduced the gradient to approximately 0.003°C and greatly reduced the slow component of the motion artifact. The drift error was reduced by proper mounting of the amplifier and its external components and the enclosure of the calorimeter in a temperature-controlled box.An R-C model of the heat flow in the calorimeter was developed which was employed to discover several means of increasing sensitivity without increasing the rise-time of the calorimeter. Analysis, also based on the model, showed that variations in the air gap between the cell holder can be a major source of error when the calorimeter is used to investigate the kinetics of a chemical reaction. This analysis also showed that the time for the heat to flow through the solution through the solution in the cell can be the dominant factor in determining the rise-time of the instrument.The heat conduction calorimeter described here has improved characterics: a baseline stability of 200 nJ · s^?1 (peak-to-peak) over a 48 h period; a resolution of 200 nJ · s^?1; a sensitivity of 6.504 ± 0.045 J · V^?1 · s^?1 referred to the sensor output; and a rise-time of 122 s for the 10–90% response.     

Synchronous direct gradient layer and indirect room calorimetry

Rumpler, James L., and William V. Seale. Synchronousdirect gradient layer and indirect room calorimetry.J. Appl. Physiol. 83(5):1775-1781, 1997.A dual direct/indirect room-sized calorimeter isused at the Beltsville Human Nutrition Research Center to measure heatemission and energy expenditure in humans. Because the response timesof a gradient layer direct calorimeter and an indirect calorimeter arenot equivalent, the respective rate of heat emission and energyexpenditure cannot be directly compared. A system of equations has beendeveloped and tested that can correct the respective outputs of thedirect gradient layer calorimeter and indirect calorimeter for delaysdue to the response times of the measurement systems. Performance testsusing alcohol combustion to simulate a human subject indicate accurate measurements of heat production from indirect (99.9 ± 0.4%),indirect corrected for response time (99.9 ± 0.5%), direct (99.9 ± 0.8%), and direct corrected for response time (99.9 ± 0.8%)calorimetry systems. Results from 24-h measurements in 10 subjectsindicate that corrected heat emission is equivalent to (99.8 ± 2.0%) corrected energy expenditure. However, heat emission measuredduring sleep was significantly greater (14%) than energy expenditure,suggesting a change in the energy stored as heat in the body. Thisdifference was reversed during the day. These results illustrate howthe simultaneous measurement of heat emission and energy expenditure provides insights into heat regulation.

     

Microcalorimetric methods for substrate determination in flow systems with immobilized enzymes.

The enthalpy of processes catalyzed by immobilized enzymes in the reaction cell of a LKB-flow calorimeter is used for determination of urea (0.5-5 mumol) and glucose (0.03-0.5 mumol). Accuracy is 2-5% and the time needed for one analysis is 20 min. A sensitive "enzyme thermistor" consisting of a flow through cell with an immobilized enzyme and two thermistors is described, which permits glucose determinations (0.05-1 mumol +/- 0.03 mumol) by means of temperature difference caused by reaction heat. Coupling of enzyme reactions for increasing reaction heat and consequently sensitivity in calorimetric determinations is demonstrated.     

A differential scanning calorimeter for ice nucleation distribution studies--application to bacterial nucleators

A differential scanning calorimeter has been developed for the automatic detection and measurement of dropwise freezing within a sample of 100-200 water drops. A typical drop size of 1 microliter is employed. The sample is distributed on flat, square (4-cm) thermoelectric sensors and the temperature is scanned downward by conductive cooling to a liquid nitrogen bath. The rate of cooling, typically 1 degree C/min, is set by the choice of a heat conduction rod between the calorimeter and the liquid nitrogen bath. The voltages from the thermopiles along with a system temperature-measuring thermocouple are continuously monitored by digital voltmeters and recorded every half-second in a computer memory. A freezing event in a drop is detected by a characteristic voltage signal whose integral with time is proportional to the size of the drop and its heat of fusion. The half-life of a freezing event signal is 10 s for a 1-microliter drop. The integrated signal produced from multiple freezing events is shown to provide a direct measure of the number of drops frozen at a given temperature. A distribution curve and its smoothed derivative can be constructed directly from these measurements. The instrument, which is termed an "ice nucleometer," is illustrated in determining the ice nucleation distribution in a population of Escherichia coli harboring cloned ice nucleation genes.     

Computer simulation for deconvolution of a heat conduction batch microcalorimeter by the D-B finite element technique

The method described here is a generla numerical analysis procedure which has been applied to a heat conduction Batch calorimeter for the deconvolution of its thermograms, and is based on a computer simulations of the heat conduction behavior of the instrument with time. We show by means of test signals that the method can deconvolute the signal with a resolving time that is about two orders of magnitude smaller than the time constant of the calorimeter itself. The method can be applied to time signals generally, provided that the instrument producing them can be stimulated.     

Exercise and postexercise energy expenditure in growing pigs

Young pigs (ca. 10 kg) were trained to run on a motor-driven treadmill for 1 h each day. After a 2-week training period the gas exchange of exercised and control animals was measured using an open circuit, indirect calorimeter. The exercised pigs ran for 2 h in the calorimeter, and then rested for 2 h. They received a day's allocation of feed and remained in the calorimeter for a total of 23 h. The total heat production of the exercised pigs was 523 kJ/kg, compared with 433 kJ/kg of the controls. Monitoring the heat production throughout the 23-h period showed that only 43% of the extra heat dissipated by the exercised pigs was lost during the 2 h of exercise, with a higher rate of heat production for the remaining 21 h accounting for the 57% of the extra energy dissipated as heat. The results suggest that exercise increases energy expenditure well beyond the time devoted to the activity itself.     

Neurotensin analog NT77 induces regulated hypothermia in the rat

The potential use of hypothermia as a therapeutic treatment for stroke and other pathological insults has prompted the search for drugs that can lower core temperature. Ideally, a drug is needed that reduces the set-point for control of core temperature (T(c)) and thereby induces a regulated reduction in T(c). To this end, a neurotensin analog (NT77) that crosses the blood brain barrier and induces hypothermia was assessed for its effects on the set-point for temperature regulation in the Sprague-Dawley rat by measuring behavioral and autonomic thermoregulatory responses. Following surgical implanation of radiotransmitters to monitor T(c), rats were placed in a temperature gradient and allowed to select from a range of ambient temperatures (T(a)) while T(c) was monitored by radiotelemetry. There was an abrupt decrease in selected T(a) from 29 to 16 degrees C and a concomitant reduction in T(c) from 37.4 to 34.0 degrees C 1 hr after IP injection of 5.0 mg/kg NT77. Selected T(a) and T(c) then recovered to control levels by 1.5 hr and 4 hr, respectively. Oxygen consumption (M) and heat loss (H) were measured in telemetered rats housed in a direct calorimeter maintained at a T(a) of 23.5 degrees C. Injection of NT77 initially led to a reduction in M, little change in H, and marked decrease in T(c). H initially rose but decreased around the time of the maximal decrease in T(c). Overall, NT77 appears to induce a regulated hypothermic response because the decrease in T(c) was preceded by a reduction in heat production, no change in heat loss, and preference for cold T(a)'s. Inducing a regulated hypothermic response with drugs such as NT77 may be an important therapy for ischemic disease and other insults.     

Design of a Calorimeter for the Continuous Study of Heat Production during Anaerobic Microbial Growth

A conduction type calorimeter has been designed to chase microbial growth in batch system. The calorimeter is of a twin structure having thermopile plates as a temperature sensor, The heat evolution during the microbial growth at a required temperature can be observed as an output-voltage generated at thermopile terminals with a sensitivity of 58.5 mV K^?1

A stainless steel cell with a volume of 300 cm³ serves as a culture cell which is capable of being autoclaved prior to the initiation of calorimetric run, taking out from the calorimeter body.

Because of the twin structure, the apparatus works with sufficient stability in detecting small heat evolution for long duration. Its operation has been demonstrated with the growth of Sacch. cerevisiae grown on liquid synthetic medium under anaerobic condition.     

Construction of a low cost and highly sensitive direct heat calorimeter suitable for estimating metabolic rate in small animals

Although the concept of a metabolic rate is readily understood, actual measurement of metabolism has proved much more difficult. The numerous strategies for estimation of metabolic rate all result in an incomplete accounting. Respirometry or gas exchange is the most widely used approach but mostly ignores the anaerobic component. Here, we describe a readily-built and low cost direct heat calorimeter that may be coupled with standard respirometry equipment to provide a more complete portrait of metabolism. The device is sensitive and provides a predictable measurement of heat flow from an organism.     

Calorimetric investigation of aerobic fermentations

A modified bench scale calorimeter has been employed to determine the heat generated by various microbial strains growing on a range of different substrates, covering degrees of reduction from 3 to 6.13. The results are analyzed, and interpreted in the light of coupled enthalpy and elemental balances. The heat released by the microbial cultures has been found to correlate linearly with other process variables, such as biomass generation and oxygen uptake. The ratio between the heat generated and the biomass formed, the so-called "heat yield" (Y(Q/x)), has been shown both on theoretical and experimental grounds to increase with increasing degree of reduction of the substrate and to decrease with increasing biomass yield. The two effects could be combined into a simple model which permits the amount of heat released per unit of biomass formed to be predicted from the degree of reduction of the substrate as the only independent variable. The ratio between the heat generated and the oxygen taken up was constant at 440 kJ (mol O(2))(-1) throughout all experiments as expected from theoretical considerations for strongly aerobic processes.     

Temperature regulation during acute heat loads in rats after short-term heat exposure.

Eleven rats were kept at an ambient temperature of 33.5 degrees C (HC) for 4-5 consecutive days, 9 additional rats were subjected to 33.5 degrees C for approximately 5 h daily (HI) for the same period, and 12 controls (Cn) were kept at 24 degrees C. After the exposure, the rats were placed in a direct calorimeter, where the wall temperature was set at 24 degrees C, and subjected to direct internal heating (6.2 W.kg-1, 30 min) through an intraperitoneal electric heater. After the first heat load and when thermal equilibrium had been attained again, the rats were subjected to indirect external warming by raising the jacket water temperature surrounding the calorimeter from 24.0 to 38.8 degrees C in 90 min. Hypothalamic (Thy) and colonic temperatures (Tco), evaporative and nonevaporative heat loss, and metabolic heat production (M) before the acute heat loads did not differ among the groups. During heat loads, the latent times for the onsets of the rises in tail skin temperature and evaporation were significantly longer, and Thy and Tco at the start of increases in heat losses tended to be higher, in the HC than in the Cn. M significantly decreased in all groups, but the magnitude and duration of reduction in M were significantly greater in the HC than in the Cn. There were no differences between the thermoregulatory responses to heat loads of the HI and Cn. These results suggest that in HC the threshold core temperature for heat loss response and the upper critical temperature have already shifted to a higher level and that HC respond to heat stress more strongly with the reduction of M than Cn. Short-term intermittent heat exposure had little effect on the thermoregulatory mechanisms in rats.     

Fast titration experiments using heat conduction microcalorimeters.

Thermopile heat conduction calorimeters normally have high time constants. Multistep titration experiments involving fast processes may then require several hours to perform. It is demonstrated that such experiments can be conducted about 10 times faster, without loss of accuracy, by use of a "dynamic correction method". For a new small vessel thermopile conduction calorimeter, injections can be made at 1.5-min intervals.     

A vitalistic model to describe the thermal inactivation ofListeria monocytogenes

Summary Thermal inactivation of microorganisms has traditionally been described as log-linear in nature, that is the reduction in log numbers of survivors decreases in a linear manner with time. This is despite a plethora of data that shows consistent deviations from such kinetics for a wide range of organisms and conditions and that cannot be accounted for by experimental artifacts. Existing thermal death models fail to take such deviations into account and also fail to quantify the effects of heating menstruum on heat sensitivity. The thermal inactivation ofListeria monocytogenes ATCC 19115 has been investigated using a factorially-designed experiment comparing 45 conditions of salt concentration, pH value and temperature. Heating was carried out using a Submerged Coil heating apparatus that minimized experimental artifacts. Low pH values increased, whilst high salt concentrations decreased heat sensitivity. Results showed a significant and consistent deviation from log-linear kinetics, particularly at low temperatures. A number of distributions were tested for suitability to describe the variability of heat sensitivity within the population of heated cells (vitalistic approach). The use of the logistic function and log dose (log time) allowed the development of an accurate unifying predictive model across the whole range of heating conditions. It is proposed that this approach should be tested as a generalized modeling technique for death kinetics of vegetative bacteria.     

Analytical grade C-phycocyanin obtained by a single-step purification process

C-phycocyanin (C-PC) is a phycobiliprotein that can be used as a natural blue dye in the food and cosmetic industries, as a biomarker or as an agent in medical treatments, depending on its purity grade. Here we described for the first time a single-step purification process of C-PC extracted from the wet biomass of Spirulina (Arthrospira) platensis LEB-52 using ion exchange chromatography with pH gradient elution. Different conditions varying the elution buffers and volumes, the loading pH and the addition of salt in the elution buffer were studied. The chromatographic condition that resulted in high recovery and purity consisted in equilibration and washing with 0.025 mol/L Tris-HCl buffer pH 6.5 and elution combining a step with 0.08 mol/L NaCl in 0.025 mol/L Tris-HCl buffer pH 6.5 and a pH gradient elution with 0.05 mol/L citrate buffer pH 6.2–3.0. This process resulted in C-PC with purities of 4.2 and 3.5 with recoveries of 32.6 and 49.5 %, respectively, in one purification step.     

Thermal inactivation of Listeria monocytogenes studied by differential scanning calorimetry

The effect of NaCl on the thermal inactivation of Listeria monocytogenes has been investigated by conventional microbiological techniques and by using differential scanning calorimetry (DSC). Addition of 1.5 M-NaCl to cells grown at lower NaCl concentrations significantly increases the tolerance of cells to mild heat stress (56-62 degrees C). DSC thermograms show five main peaks which are shifted to higher temperatures in the presence of 1.5 M-NaCl. Measurement of loss of viability in the calorimeter gave good correlation between cell death and the first major thermogram peak at two NaCl concentrations. The time course of the loss of this first peak when cells were heated and held at 60 degrees C in the calorimeter matched the loss of viability, whereas the peak attributable to DNA showed little change during this process. The use of DSC to investigate the mechanisms involved in thermal inactivation is discussed.