Calorimetrie Study of Escherichia coli Growth on Bouillon Medium

Heat evolution during the growth of Escherichia coli in a stationary culture on bouillon medium was continuously monitored with a conduction-type batch calorimeter at different pHs and temperatures. The growth thermograms were found to be reproducible within percent errors of ±2.1%, ±0.8% and ±1.5% for the peak time of a thermogram, the peak height and the total heat evolution, respectively. The mean heat evolution for the formation of a unit E. coli cell was co = (1.69 ±0.08) X 10^—8 J cell^{— 1} at pH 6.2 and 37°C. The mean heat evolution rate per unit cell during growth was </ = 3.6ρwcell^_1, which was consistent with values calculated for other microbial cells. The growth rate constant calculated on kinetic analysis of the growth thermograms was μ = 0.532 ± 0.068 hr ^-1 at pH 6.2 and 37°C. The pH dependence of the growth rate constant showed that the growth activity of E. coli cells on bouillon medium is maximum in the pH range of 5.5 to 6.5. From the temperature-dependent variations in the growth rate constant, the apparent activation energy of E. coli growth was found to be E_a =65.3 ±7.1 kJ mol^-1.     

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.     

Application of dynamic calorimetry for monitoring fermentation processes

The rate of heat evolution (kcal/liter-hr) in mycelial fermentations for novobiocin and cellulase production with media containing noncellular solids was measured by an in situ dynamic calorimetric procedure. Thermal data so obtained have proved significant both in monitoring cell concentration during the trophophase (growth phase) and in serving as a physiological variable in the fermentation process. The validity of this technique has been demonstrated by closing the overall material and energy balances. The maintenance energy in a batch fermentation can also be calculated by integrating heat evolution data. This integration method is applicable to a fermentation lacking a precise cell growth curve. The maintenance coefficient, obtained for the novobiocin fermentation by Streptomyces niveus, is equal to 0.028 g glucose equivalent/g cell-hr. The production of novobiocin in the idio-phase (production phase) also correlates well with the amount of energy catabolixed for maintenance and this results in an observed conversion yield of glucose to novobiocin of 11.8 mg of novobiocin produced per gram of glucose catabolized. A new physiological variable, kilocalories of heat evolved per millimole of oxygen consumed, has been proposed to monitor the state of cells during the fermentation. This method may provide a simple way to monitor on-line shifts in the efficiency of cell respiration and changes in growth yields during a microbial process.     

Investigation of the potential of biocalorimetry as a process analytical technology (PAT) tool for monitoring and control of Crabtree-negative yeast cultures

Biological reaction calorimetry, also known as biocalorimetry, has led to extensive applications in monitoring and control of different bioprocesses. A simple real-time estimator for biomass and growth rate was formulated, based on in-line measured metabolic heat flow values. The performance of the estimator was tested in a unique bench-scale calorimeter (BioRC1), improved to a sensitivity range of 8 mW l^− 1 in order to facilitate the monitoring of even weakly exothermic biochemical reactions. A proportional–integral feedback control strategy based on these estimators was designed and implemented to control the growth rate of Candida utilis, Kluyveromyces marxianus and Pichia pastoris by regulating an exponential substrate feed. Maintaining a particular specific growth rate throughout a culture is essential for reproducible product quality in industrial bioprocesses and therefore a key sequence for the step from quality by analysis to quality by design. The potential of biocalorimetry as a reliable biomass monitoring tool and as a key part of a robust control strategy for aerobic fed-batch cultures of Crabtree-negative yeast cells in defined growth medium was investigated. Presenting controller errors of less than 4% in the best cases, the approach paves the way for the development of a generally applicable process analytical technology platform for monitoring and control of microbial fed-batch cultures.     

Mass production of lipase by fed-batch culture of Pseudomonas fluorescens

Summary High concentration production of an extracellular enzyme, lipase, was achieved by a fed-batch culture of Pseudomonas fluorescens. During the cultivation, temperature, pH and dissolved oxygen concentration wwre maintained at 23°C, 6.5 and 2–5 ppm, respectively. Olive oil was used as a carbon source for microbial growth. To produce lipase effectively the specific feed rate of olive oil had to be maintained in a range of 0.04–0.06 (g oil) · (g dry cell)^-1 · h^-1. The CO₂ evolution rate was monitored to estimate the requirement of olive oil. The ratio of feed rate of olive oil to the CO₂ evolution rate was varied in the range of 20–60 g oil/mol CO₂. The higher value of the ratio accelerated microbial growth, but did not favour lipase production. Once the high cell concentration of 60 g/l had been achieved, the ratio was changed from 50 to 30 g oil/mol CO₂ to accelerate the lipase production. By this CO₂-dependent method a very high activity of lipase, 1980 units/ml, was obtained. Both the productivity and yield of lipase were prominently increased compared with a conventional batch culture.     

Studies on Utilization of Hydrocarbons by Yeasts

The production of microbial cell substances from hydrocarbons has been attracting attention of people for many years. Production of bacterial cell from hydrocarbons is disadvantageous because of the difficulty in separating cell from the broth.

We have tested hydrocarbon-utilizing yeasts isolated from garden soil for cell production. The effect of medium composition on yeast growth and the utilization of individual hydrocarbon by yeast, strain Y-3, were investigated.

As a nitrogen source, urea was more effective than ammonium nitrate. When a very smal! amount of corn steep liquor was added, yeast growth was very improved. Aliphatic series of hydrocarbon lower than C₉ were not or very slightly assimilated by this yeast.

Generally speaking, series of even-number hydrocarbons were more effective than those of odd-number hydrocarbons.

We found that the yeast Y-3 strain reported in the previous paper¹⁾ has a diterminal oxidation system of hydrocarbon.

This yeast capable of growing in mineral-salts solution with hydrocarbons as sole source of carbon produced a series of dioic acid from n-undecane. These acids are 1,11-undecane dioic acid, 1,9-nonane dioic acid (azelaic acid), 1,7-heptane dioic acid (pimelic acid) and 1,5-pentane dioic acid (glutaric acid). 1,10-Decane dioic acid (sebacic acid) was also isolated from n-decane cultures.

Azelaic acid was partially transformed into pimelic acid and glutaric acid by treating it with resting cells of this yeast.

1,11-Undecane dioic was also transformed into azelaic acid pimelic acid, and glutaric acid by the same treatment as described above.     

Measurement of heat evolution and correlation with oxygen consumption during microbial growth

A procedure for measuring the rate of heat production from a fermentation has been developed. The method is based on measuring the rate of temperature rise of the fermentation broth resulting from metabolism, when the temperature controller is turned off. The heat accumulation measured in this manner is then corrected for heat losses and gains. A sensitive thermistor is used to follow the temperature rise with time. This procedure is shown to be as accurate as previous methods but much simpler in execution. Using this technique, the rate of heat production during metabolism was found to correlate with the rate of oxygen consumption. Experiments were performed using bacteria (E. coli and B. subtilis), a yeast (C. intermedia), and a mold (A. niger). The substrates investigated included glucose, molasses, and soy bean meal. The proportionality constant for the correlation is independent of the growth rate, slightly dependent on the substrate, and possibly dependent On the type of organism growth. This correlation has considerable potential for predicting heat evolution from the metabolism of microorganisms on simple or complex substrates and providing quantitative parameters necessary for heat removal calculations.     

Primi Dati Sull'andamento Stagionale delle Temperature del Troneo di Olivo

Abstract

Temperatures recorded in a olive-tree stem in 1961–1962. — In the Botanical Garden of Bari a temperature recorder has been placed, bearing 6 electrothermometers with platinum resistences having a sensitivity of 2,4 mm/C[ddot], with a recording approximation of 0,1 C[ddot].

The olive tree under examination is a grown-up tree, which has been pruned according to the local methode, and it belongs to a local breed named « Ogliarola leccese ».

— Five thermometers have been employed as follows: 1^rst in the air, hanging from the stem, m. 1,30, high; 2^nd into the outer bark, cm, 0,5 deep; 3^rd into the cambium, cm. 1,5 deep; 4^th into the sap-wood, cm. 6 deep; 5^th into the hearth-wood, cm. 22 deep.

— The thermometers were all placed on the east side of the stem.

— The figures and the graphs of the paper show that:

a) The whole stem of the specimen under examination has a mean temperature which is higher than that of the surrounding air.

b) The outer bark gets warmer than the air, during the day-time; during the night it gives up heat outside and inside, namely towards the stem tissues, particularly to the bark-cambium-sapwood zone, and also as far as the hearth wood zone.

c) The whole living zone of the stem (bark-cambium-sapwood) generally keeps temperature values intermediate between those of the outer bark and those of the air. The temperature values vary along the year.

d) In spring the cambium is warmer than the outer bark.

e) In winter cambium and sapwood generally have the same temperature. The temperature values recorded in this season are much lower than those of the outer bark and almost the same of those of the air.

f) The behaviour of temperature in cambium and sapwood is probably ruled by biological factors.

g) The hearth-wood keeps generally warmer than the air and cooler than other outer tissues. Its thermic rhytm is almost reversed in comparison with that of the outer tissues.

Il seems that hearth wood acts as an useful heat collector and distributor towards the living outer tissues during the diurnal and seasonal temperature remissions, at least in mild climates as that of Bari.     

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.     

Use of Azolla as a test organism in a growth chamber of simple design

Summary The construction and application of a new type of growth chamber, in which different growth conditionsi.e.: temperature, humidity, pH, light intensity, light colour, change in nutrient composition and gas exchange can easily be controlled, are presented. The method has previously been applied to twoAzolla speciesviz. Azolla filiculoides, which is cold tolerant andAzolla pinnata (distinguished in Vietnam as the form Xanh), which is heat tolerant. In the growth chamber natural growth conditions of the Azolla —Anabaena azollae symbiotic association were imitated as much as possible. For testing the system, methods discussed earlier^8,14 and some previously presented data, concerning photosynthetic activities, such as oxygen evolution and nitrogen fixation (acetylene reduction) of twoAzolla species³⁹, were partially used. Biomass ofA. filiculoides was measured and reactions to its environment at conditions when grown in the field and in the growth chamber, were studied. Growth and photosynthesis measurements were performed under special light conditions and with whole plants grown under laboratory conditions. Anthocyanin synthesis was studied in relation with humidity. Anthocyanin spectra were analyzed by means of a spectrum-deconvolution method. On leave from the Department of Plant Physiology of the University of Hanoi, Vietnam.     

Construction of a Promoter-cloning Vector in Pseudomonas aeruginosa

Computer simulations were employed to reconstruct the growth thermograms that are observable for microbial cultures in batch calorimeters having a culture vessel in the calorimetric unit. Differential equations were derived to characterize the heat evolution process on the basis of Monod’s growth equation with some modification. Theoretical growth thermograms were compared with actually observed calorimetric recordings and it was shown that the heat effect due to the microbial cells can be well reproduced for both non-growing and growing cultures of bakers’ yeast. It is concluded that the method used here gives a reasonable characterization of the thermograms observed for microbial cultures in a batch calorimeter, indicating the possibility of determining the appropriate kinetic parameters from calorimeter recordings by a parameter-fitting method.     

Engineering self-flocculating Halomonas campaniensis for wastewaterless open and continuous fermentation

Halomonas has been developed as a platform for the next generation industrial biotechnology allowing open and nonsterile growth without microbial contamination under a high-salt concentration and alkali pH. To reduce downstream cost associated with continuous centrifugation and salt containing wastewater treatment, Halomonas campaniensis strain LS21 was engineered to become self-flocculating by knocking out an etf operon encoding two subunits of an electron transferring flavoprotein in the predicted electron transfer chain. Self-flocculation could be attributed to the decrease of the surface charge and increase of the cellular hydrophobicity resulted from deleted etf. A wastewaterless fermentation strategy based on the self-flocculating H. campaniensis was developed for growth and the production of poly-3-hydroxybutyrate (PHB) as an example. Most microbial cells flocculated and precipitated to the bottom of the bioreactor within 1 min after stopping the aeration and agitation. The supernatant can be used again without sterilization or inoculation for the growth of the next batch after collecting the precipitated cell mass. The wastewaterless process was conducted for four runs without generating wastewater. PHB accumulation by the self-flocculent strain was enhanced via promoter and ribosome binding site optimizations, the productivities of cell dry weight and PHB were increased from 0.45 and 0.18 g·L ⁻¹·hr ⁻¹ for the batch process compared to 0.82 and 0.33 g·L ⁻¹·hr ⁻¹ for the wastewaterless continuous process, respectively. This has clearly demonstrated the advantages of the wastewaterless process in that it not only reduces wastewater but also increases cell growth and product formation efficiency in a given period of time.     

Energetics of growth of <Emphasis Type="Italic">Aspergillus tamarii</Emphasis> in a biological real-time reaction calorimeter

Fungal cultivation in a biological real-time reaction calorimeter (BioRTCal) is arduous due to the heterogeneous nature of the system and difficulty in optimizing the process variables. The aim of this investigation is to monitor the growth of fungi Aspergillus tamarii MTCC 5152 in a calorimeter. Experiments carried out with a spore concentration of 10⁵ spores/mL indicate that the growth based on biomass and heat generation profiles was comparable to those obtained hitherto. Heat yield due to biomass growth, substrate uptake, and oxygen uptake rate was estimated from calorimetric experiments. The results would be useful in fermenter design and scale-up. Heat of combustion of fungal biomass was determined experimentally and compared to the four models reported so far. The substrate concentration had significant effects on pellet formation with variation in pellet porosity and apparent density. Metabolic heat generation is an online process variable portraying the instantaneous activity of monitoring fungal growth and BioRTCal is employed to measure the exothermic heat in a noninvasive way.     

Ratio of heat production to oxygen consumption during the cell cycle of candida maltosa EH 15 grown on ethanol

The experimental technique for measurement of microbial culture heat evolution directly in fermenter has been described and its correctness analysed. Heat-to-oxygen ratio, Q₀, of synchronized yeast culture in the absence of fermentative metabolism has been found to be practically independent of a cell cycle phase and close to the theoretical constant predicted by the mass-energy balance theory. The collection of literature data on the heat-to-oxygen ratio is given. Energetic properties of cell biomass are discussed on the basis of the obtained and the surveyed values of Q₀.     

Microcalorimetric investigation of metabolism in rat hepatocytes cultured on microplates and in cell suspensions

In the present work, heat production rate in rat hepatocytes has been measured by use of thermopile heat conduction calorimeters. Both hepatocytes cultured in monolayers on microplates and hepatocytes in suspensions were used for microcalorimetric measurements. The highest heat production rate was found in newly cultured cells; thereafter, a gradual decrease was noted. After 1 day of culture, metabolic activity had reached a steady state that lasted about 4 days. A cell-density dependence of heat production was found, both in cell suspensions and in cultured hepatocytes on microplates. Higher cell concentration in the calorimeter ampoule was accompanied by decreasing heat production per cell. The heat output recorded for hepatocytes cultured on microplates (25 X 10(3) cells) was found to be 0.327 +/- 0.13 nW per cell after 24-48 h. Addition of sodium azide and sodium fluoride to tissue culture medium reduced heat production rate in cultured hepatocytes by 60 and 20%, respectively. Recording of heat production with the present calorimetric technique is relatively simple and fast, and offers the possibility to perform measurements in small samples of cultured hepatocytes on microplates, thus allowing long-term as well as repeated measurements on the same cell population.     

Thermodynamic theory explains the temperature optima of soil microbial processes and high Q10 values at low temperatures

Our current understanding of the temperature response of biological processes in soil is based on the Arrhenius equation. This predicts an exponential increase in rate as temperature rises, whereas in the laboratory and in the field, there is always a clearly identifiable temperature optimum for all microbial processes. In the laboratory, this has been explained by denaturation of enzymes at higher temperatures, and in the field, the availability of substrates and water is often cited as critical factors. Recently, we have shown that temperature optima for enzymes and microbial growth occur in the absence of denaturation and that this is a consequence of the unusual heat capacity changes associated with enzymes. We have called this macromolecular rate theory – MMRT (Hobbs et al., 2013 , ACS Chem. Biol. 8:2388). Here, we apply MMRT to a wide range of literature data on the response of soil microbial processes to temperature with a focus on respiration but also including different soil enzyme activities, nitrogen and methane cycling. Our theory agrees closely with a wide range of experimental data and predicts temperature optima for these microbial processes. MMRT also predicted high relative temperature sensitivity (as assessed by Q₁₀ calculations) at low temperatures and that Q₁₀ declined as temperature increases in agreement with data synthesis from the literature. Declining Q₁₀ and temperature optima in soils are coherently explained by MMRT which is based on thermodynamics and heat capacity changes for enzyme‐catalysed rates. MMRT also provides a new perspective, and makes new predictions, regarding the absolute temperature sensitivity of ecosystems – a fundamental component of models for climate change.     

Effect of carbon:nitrogen ratio on kinetics of phenol biodegradation byAcinetobacter johnsonii in saturated sand

In polluted soil or ground water, inorganic nutrients such as nitrogen may be limiting, so that Monod kinetics for carbon limitation may not describe microbial growth and contaminant biodegradation rates. To test this hypothesis we measured¹⁴CO₂ evolved by a pure culture ofAcinetobacter johnsonii degrading 120 µg¹⁴C-phenol per ml in saturated sand with molar carbon:nitrogen (CN) ratios ranging from 1.5 to 560. We fit kinetics models to the data using non-linear least squares regression. Phenol disappearance and population growth were also measured at CN1.5 and CN560.After a 5- to 10-hour lag period, most of the¹⁴CO₂ evolution curves at all CN ratios displayed a sigmoidal shape, suggesting that the microbial populations grew. As CN ratio increased, the initial rate of¹⁴CO₂ evolution decreased. Cell growth and phenol consumption occurred at both CN1.5 and CN560, and showed the same trends as the¹⁴CO₂ data. A kinetics model assuming population growth limited by a single substrate best fit the¹⁴CO₂ evolution data for CN1.5. At intermediate to high CN ratios, the data were best fit by a model originally formulated to describe no-growth metabolism of one substrate coupled with microbial growth on a second substrate. We suggest that this dual-substrate model describes linear growth on phenol while nitrogen is available and first-order metabolism of phenol without growth after nitrogen is depleted.     

Effect of physical factors on the production of bacteriocin from Pediococcus acidilactici ITV 26

The effect of pH, temperature and agitation on growth and bacteriocin production by Pediococcus acidilactici ITV 126 was investigated. Experiments were made in flasks containing MRS medium at 30 to 40°C, pH 5 to 7 and agitation 0 to 200 rpm. Factor levels were arranged in a 2³ factorial design with central and axial points. Anova and Tukey paired comparison tests showed that a temperature of 35°C favored bacteriocin production, whereas 40°C was best for cell growth. A statistical interaction of temperature and agitation was observed affecting microbial growth. pH 5 favored both cell growth and bacteriocin production. Journal of Industrial Microbiology & Biotechnology (2001) 26, 191–195. Received 30 October 1999/ Accepted in revised form 31 January 2001     

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.