Design and testing of a calorimeter for biological uses

A rotatory microcalorimeter of the conduction type has been designed for the study of microbial metabolic processes under both aerobic and anaerobic conditions. The instrument can be performed in either batch mode or flow mode by changing the calorimeter vessels and the tube connections. The sensitivity and the time constant were determined by electrical calibrations. The heat sensitivity was 0.12 mV/mW with both yeast and other fungi. Because of the sufficient aeration and agitation, the calorimeter is available for studies on the fungi growth experiments of biotechnical interest.     

Baker''s yeast: production of d- and l-3-hydroxy esters

Baker's yeast grown under oxygen limited conditions and used in the reduction of 3-oxo esters results in a shift of the stereoselectivity of the yeast towards -hydroxy esters as compared with ordinary baker's yeast. The highest degree of stereoselectivity was obtained with growing yeast or yeast harvested while growing. In contrast, the stereoselectivity was shifted towards -hydroxy esters when the oxo esters were added slowly to ordinary baker's yeast supplied with gluconolactone as co-substrate. The reduction rate with gluconolactone was increased by active aeration. Ethyl -(S)-3-hydroxybutanoate was afforded in>99% ee. Both enantiomers of ethyl 3-hydroxypentanoate, -(R) in 96% ee and -(S) in 93% ee, and of ethyl 4-chloro-3-hydroxybutanoate, -(S) in 98% ee and -(R) in 94% ee, were obtained. The results demonstrate that the stereoselectivity of baker's yeast can be controlled to a large extent without the use of inhibitors, heat treatment, etc.     

Heat flux and the calorimetric-respirometric ratio as measures of catabolic flux in mammalian cells

It is advocated that cellular heat flow rate (Ø = dQ/dt, where Q is heat) be expressed as an intensive quantity specific to cell size (X) and termed heat flux (J_Ø/X). It has been the practice to cite such data on a ‘per cell’ basis, but it would be preferable to use biomass (cellular volume or mass). This quantity is shown to be a measure of metabolic activity and, more accurately, catabolic rate coupled to the demand for ATP in anabolic processes and work in the cell. Recent developments in flow microcalorimetry and dielectric spectroscopy reveal that heat flux can be measured on-line, with the potential of industrial use as a control variable in the growth of hybridoma and genetically engineered cells. This is because the enthalpy change of growth can be regarded as a unique kind of stoichiometric coefficient directly related to the mass coefficients in the growth reaction. This can be verified by an enthalpy balance comparing data for material fluxes of catabolites with the value for heat flux. Information revealed by the stoichiometric growth equation can be used to improve medium design.

The ratio of heat flux to oxygen consumption (flux) is known as the calorimetric-respirometric (CR) ratio. It detects anaerobic processes when the value is more negative than −450 (±5%) kJ mol⁻¹ O₂. These processes are found in cells growing under fully aerobic conditions, because glycolysis provides biosynthetic precursors with lactate as the byproduct. It is suggested that the CR ratio would be a powerful on-line control variable for the growth of animal cells in bioreactors.     

计算机辅助氧弹量热计探索火箭燃料的推进性能

本实验基于现行“综合化学实验”进行创新性改进。将一定压力的氮气充入氧弹,在以硝酸钾为氧化剂、三氧化二铁为催化剂的条件下,混合一定比例的蔗糖得到硝糖燃料,进行燃烧,采用环境恒温量热计记录蔗糖燃烧过程中的温度变化,再通过雷诺作图法校正产生的DT偏差,最终计算得到硝糖燃烧过程的恒容热效应,即为硝糖燃料的爆热值。当蔗糖:硝酸钾:氧化铁的质量比为39:59:2时,其爆热值最大,比冲最大。在上述实验的基础上,选择最佳配比的硝糖燃料,用于计算机仿真模拟火箭发射系统。通过改变燃烧室压力和燃料流速,计算得到该最佳配比硝糖燃料的比冲和火箭飞行高度,进而得到采用单级火箭将东方红一号卫星送入预定轨道的发动机参数。改变每一级火箭的燃料类型与比例,设计得到适于推进不同卫星进入预定轨道的二级火箭和三级火箭。通过设计氧弹量热计的拓展应用与计算机仿真模拟相结合的实验,达到已有实验的创新设计、启发学生创造性和引起科研兴趣的目的。     

Kinetic study of growth,lipid and carotenoid formation in β-carotene producing <Emphasis Type="Italic">Rhodotorula glutinis</Emphasis>

The oleaginous and red yeast Rhodotorula glutinis CCY 20-2-26 is known for its high-storage lipids accumulation in cells as well as for β-carotene biosynthesis. The work is focused on the study of lipogenesis and carotenoid pigments formation by the yeast grown in media with four different C/N ratios (20:1, 50:1, 70:1 and 100:1). Total fatty acids accumulation in cells reached the maximal value of 48% and yielded up to 8.9 g fatty acid/L media under C/N 70:1. On contrary, while the highest pigment accumulation in the yeast (1268 μg/g of cells) was found at the C/N ratio of 20:1, the maximal carotenoid yield (mainly β-carotene) of 12.7 mg/L was obtained under 50:1 ratio. Calculation of kinetic parameters of metabolites production revealed the metabolic correlations among glucose consumption, lipogenesis and carotenoid biosynthesis. It was demonstrated that glucose exhaustion resulted in reduction of growth and lipid accumulation in cells. Then, storage fatty acids, especially palmitic and oleic acids, begun to be degraded by β-oxidations and formed acetyl-CoA which was especially used for biosynthesis of β-carotene. This is the first study providing the new hypothesis about the metabolic connection between fatty acids and β-carotene metabolism in the red yeasts.     

An isothermal heat flow calorimeter for large-volume applications

Design, construction, calibration, and testing of a new isothermal heat flow calorimeter suitable for investigation of large-volume specimens are presented. The measuring vessel has the volume of 1370?cm³, and the calorimeter allows for the measurement at surrounding air temperatures of 5?C60?°C. A practical application of the device is demonstrated at the determination of specific hydration heat of cement paste and concrete with silica-aggregate size of up to 16?mm, having the same water/cement ratio. The differences over the whole measuring time period of about 100?h are lower than 2% which indicates a good potential of the calorimeter for the measurement of total hydration heat of composite materials. A reference measurement of hydration heat of cement paste using common isothermal heat flow calorimeter with the measuring vessel of 1?cm³ shows an agreement within ±7%, which seems acceptable, taking into account the heat transport processes in the far larger specimens. The designed calorimeter may find use in future also in other applications where larger specimens are required, such as the measurement of adsorption heat, solution heat, various reaction heats, and enthalpy of liquid?Csolid transition in heterogeneous systems with large representative elementary volumes.     

Molecular characterization of a gene for aldose reductase (CbXYL1) from Candida boidinii and its expression in Saccharomyces cerevisiae

Candida boidinii produces significant amounts of xylitol from xylose, and assays of crude homogenates for aldose (xylose) reductase (XYL1p) have been reported to show relatively high activity with NADH as a cofactor even though XYL1p purified from this yeast does not have such activity. A gene coding for XYL1p from C. boidinii (CbXYL1) was isolated by amplifying the central region using primers to conserved domains and by genome walking. CbXYL1 has an open reading frame of 966 bp encoding 321 amino acids. The C. boidinii XYL1p is highly similar to other known yeast aldose reductases and is most closely related to the NAD(P)H-linked XYL1p of Kluyveromyces lactis. Cell homogenates from C. boidinii and recombinant Saccharomyces cerevisiae were tested for XYL1p activity to confirm the previously reported high ratio of NADH:NADPH linked activity. C. boidinii grown under fully aerobic conditions showed an NADH:NADPH activity ratio of 0.76, which was similar to that observed with the XYL1p from Pichia stipitis XYL1, but which is much lower than what was previously reported. Cells grown under low aeration showed an NADH:NADPH activity ratio of 2.13. Recombinant S. cerevisiae expressing CbXYL1 showed only NADH-linked activity in cell homogenates. Southern hybridization did not reveal additional bands. These results imply that a second, unrelated gene for XYL1p is present in C. boidinii.     

Microcalorimetric qualitative analysis of biofilm development in porous media used in wastewater treatment by constructed wetland

In wastewater treatment by constructed wetland, the biodegradation capability of the biomass developed in the soil is one of the most important factors. For this kind of treatment unit, soil properties are studied to improve its filtration capacity and hydraulic residence time of the wastewater. The impact of soil properties like porosity and soil components on biomass development and biodegradation capacity seem to be less studied certainly due to the complexity of microbial identification techniques currently used. The study presented here is a preliminary work to validate that calorimetric technique could be a tool in the understanding of biodegradation capacity of wastewater treatment processes. Biofilm is preliminary developed in columns filled with different porous materials of well known porosity and constitutive components. These columns are fed with the same continuous flow of synthetic solution (C, N, and P) as a substrate amending during 3 weeks. Then each week, 2 mL samples of porous media from these columns are analyzed in isothermal calorimeter for 48 h. Net heat flow is recorded before and after substrate injection. This work results in the definition of the procedure for batch experiments in calorimeter for wastewater process efficiency. The results of these experiments show that the microbial reaction due to substrate amendment is highly depending on the porous material used for biofilm growth. Indeed calorimetric signals recorded lead to conclude that biofilm grown on plastic beads has a faster and more intensive reaction to glucose amendment than biofilm grown on glass beads. At least, two glass beads samples analyzed in the calorimeter after the same duration of feeding with synthetic solution have very different response to glucose or synthetic solution.     

The effect of gas pressure on the melting behavior of compounds

The melting and crystallization behavior of pure compounds under gas pressure was experimentally investigated using a scanning transitiometer, which is a sensitive twin calorimeter combined with a computer driven PVT control. This technique allows to determine different thermodynamic effects occurring with changes of state. While one of the three state variables pressure P, volume V, or temperature T is kept constant, another one is changed with time, both the enthalpy effect and the change of the remaining third state variable can simultaneously be measured with high precision. Thermodynamic equilibrium can be achieved during the entire process because the state is changed very slowly. The solid–liquid phase change of dimethylterephthalate (DMT) and biphenyl was investigated under He, CO₂, and N₂O pressure up to 30 MPa, and the SLG three phase lines were determined. The solid–liquid phase change appeared unexpectedly as a pair of two different heat signals.     

N,N'-(对位取代苯基)乙二胺的质子化及其与Cu(Ⅱ)配位的滴定量热测定

The apparent thermodynamic functions of the protonation and coordination of N,N′-(para-substituted phenol) ethylenediamine with Cu (Ⅱ) has been determined at 25.0±0.1 ℃ and I=0.1 mol.dm~(-3) (NaClO_4) in 90% (V/V) ethanol by the constant temperature environment-continuous titration calorimeter which had been constructed in our laboratory. It has been found that the heat of protonation (H_(L1)) is linearly correlated with the Hammett constants and the formation of H~+ bridge occurs in ~H+ -N, N′-(p-RPh)_2en (1:1) systems.     

Biocalorimetry as a process analytical technology process analyser; robust in-line monitoring and control of aerobic fed-batch cultures of crabtree-negative yeast cells

Control of bioprocesses requires reliable and robust on- or in-line monitoring tools providing real-time information on process dynamics. Heat generation related to metabolic activity of living systems is currently gaining importance in bioprocess industry due to its non-invasive and essentially instantaneous characteristics. This study deals with monitoring and control of pure aerobic fed-batch cultures of three Crabtree-negative yeast strains, Kluyveromyces marxianus, Candida utilis and Pichia pastoris, based on in-line measured, metabolic heat flow signals. A high resolution biocalorimeter (BioRC1) was developed from a standard bench-scale heat flow calorimeter (RC1). The BioRC1 was equipped with in-line (dielectric spectroscopy, pH probe and dissolved oxygen probe) and at-line (exit gas analyser) sensors to characterise the growth behaviour of the yeast cells. Both metabolic heat flow and biomass profiles exhibited similar behaviour proving the significance of employing heat flow signal as a key-parameter for the system under investigation. A simple estimator for biomass concentration and specific growth rate was formulated based on heat flow values. In order to evaluate the potential of calorimetry as a reliable and powerful process monitoring tool, the robustness, reliability as well as the broad applicability of the developed estimators was assessed through comparison with off-line measurement techniques and showed promising results for general applicability with a wide range of bioprocesses.     

Cell surface acid-base properties of Escherichia coli and Bacillus brevis and variation as a function of growth phase, nitrogen source and C:N ratio

Potentiometric titration has been conducted to systematically examine the acid–base properties of the cell surfaces of Escherichia coli K-12 and Bacillus brevis as a function of growth phase, nitrogen source (ammonium or nitrate), and carbon to nitrogen (C:N) ratio of the growth substrate. The two bacterial species revealed four distinct proton binding sites, with pK_a values in the range of 3.08–4.05 (pK₁), 4.62–5.57 (pK₂), 6.47–7.30 (pK₃), and 9.68–10.89 (pK₄) corresponding to phosphoric/carboxylic, carboxylic, phosphoric, and hydroxyl/amine groups, respectively. Two general observations in the data are that for B. brevis the first site concentration (N₁), corresponding to phosphoric/carboxylic groups (pK₁), varied as a function of nitrogen source, while for E. coli the fourth site concentration (N₄), corresponding to hydroxyl/amine groups (pK₄), varied as a function of C:N ratio. Correspondingly, it was found that N₁ was the highest of the four site concentrations for B. brevis and N₄ was the highest for E. coli. The concentrations of the remaining sites showed little variation. Finally, comparison between the titration data and a number of cell surface compositional studies in the literature indicates one distinct difference between the two bacteria is that pK₄ of the Gram-negative E. coli can be attributed to hydroxyl groups while that of the Gram-positive B. brevis can be attributed to amine groups.     

三聚氰胺的生成焓、热容和熵

通过多种热化学方法研究了三聚氰胺的热力学性质. 首先用氧弹式燃烧热量计测定了三聚氰胺在298.15 K 时的燃烧热, 根据燃烧热结果, 计算出三聚氰胺的标准摩尔燃烧焓和标准摩尔生成焓, 分别为: △cHΘm=(-2455.17±4.65) kJ·mol-1; △fHΘm =(-763.38±5.16) kJ·mol-1. 然后根据键焓与燃烧焓之间的关系, 估算出三聚氰胺中的C≈N(此键介于单键与双键之间)键能为458.30 kJ·mol-1, 此值介于碳氮单键键能和双键键能之间. 通过绝热热量计测定了三聚氰胺从80到385 K的低温热容. 根据热容值, 计算了此温度区间的标准摩尔生成焓, 其与温度呈线性关系. 另外, 三聚氰胺的热稳定性也用热重-差示扫描量热(TG-DSC)法进行了分析, 确定其分解的DSC曲线的峰顶温度为603.37 K.     

Solution calorimetry to monitor swelling and dissolution of polymers and polymer blends

The observed rate of drug release from a polymeric drug delivery system is governed by a combination of diffusion, swelling and erosion. It is thus not a simple task to determine the effects of the polymer on the observed drug release rate, because the swelling characteristics of the polymer are inferred from the drug release profile. Here we propose to use solution calorimetry to monitor swelling. Powdered polymer samples (HPMC E4M, K4M, K15M and NaCMC, both alone and in a blend) were dispersed into water or buffer (pH 2.2 and 6.8 McIlvaine citrate buffers) in a calorimeter and the heat associated with the swelling phenomena (hydration, swelling, gelation and dissolution) was recorded. Plots of normalised cumulative heat (i.e. q_t/Q, where q_t is the heat released up to time t and Q the total amount of heat released) versus time were analysed by the power law model, in which a fitting parameter, n, imparts information on the mechanism of swelling.

For all systems the values of n were greater than 1, which indicated that dissolution occurred immediately following hydration of the polymer. However, while not suitable for determining reaction mechanism, the values of n for each polymer were significantly different and, moreover, were observed to vary both as a function of particle size and dissolution medium pH. Thus, the values of n may serve as comparative parameters. Properties of the polymer blends were observed to be different from those of either constituent and correlated with the behaviour seen for polymer tablets during dissolution experiments. The data imply that solution calorimetry could be used to construct quantitative structure–activity relationships (QSARs) and hence to optimise selection of polymer blends for specific applications.     

The use of advanced calorimetric techniques in polymer synthesis and characterization

Progress in the understanding of polymer synthesis, including the crucial step of initiation and undesired side reactions, and in characterization of polymers, especially their thermal behaviour, are directly related to advances in calorimetric technologies.

In polymer synthesis, since polymerization reactions are highly exothermic, reaction calorimetry (RC) is an appropriate technique for on-line process monitoring. Measurements are non-invasive, rapid, and straightforward. Viscosity increase and fouling at the reactor wall are typical features of many polymerizations. The global heat transfer coefficient, UA, also changes drastically when viscosity increases and affects the accuracy of calorimetric measurements. Our approach was focused on oscillating temperature calorimetry (TOC). Reactions were performed with two different reaction calorimeters, i.e. an isoperibolic calorimeter and a Calvet-type high sensitivity differential calorimeter, respectively. Special attention was paid to the interpretation of the measured signals to obtain reliable calorimetric data. The evolution of heat transfer coefficient was followed by performing two Joule effect calibration experiments, before and after the reaction, and the two values interpolated to obtain the desired profile of UA. A differentiation method based on the convolution of the measured heat flow by the generated one was used for determining the time constants and deconvoluting the measured heat flow.

With respect to polymer characterization, pressure-controlled scanning calorimetry, also called scanning transitiometry, is now a well established technique. The transitiometer was coupled to an ultracryostat to work at low temperature. The assembly was used to follow the pressure effect on phase change phenomena such as fusion/crystallization and glass transition temperature T_g of low molecular weight substances or high molecular weight polymers.     

Mn(Ⅲ)-mediated radical reaction of 2-isocyano-6-alkenyl(alkynyl)benzonitriles with arylboronic acids:Synthesis of pyrroloisoquinoline derivatives

A Mn(Ⅲ) mediated radical reaction of new designed multi-functionalized 2-isocyano-6-alkenyl(alkynyl)benzonitriles with arylboronic acids has been developed.This reaction provides a method for the synthesis of pyrroloisoquinoline derivatives through the formation of two C-C bonds and one C-N bond via radical cascade cyclization in one step.     

Measurements of excess enthalpies at high temperature and pressure using a new type of mixing unit

A flow mixing unit (calorimetric cell and auxiliary devices) has been designed for measuring the enthalpy of mixing or reaction of two fluids (gas+liquid or liquid+liquid). The indicator of the heat effect is a differential heat flux calorimeter, SETARAM C-80, allowing measurements at temperatures up to 300°C. The mixing cell is made of a stainless-steel capillary (o.d 1.6 mm, length 2.4m) which is coiled in a cylindrical form and tightly fitted in the thermopile well of the calorimeter. The fluids are delivered from the high pressure piston pumps and circulated through the system at controlled flow rates ranging from 100 to 1500 L-min^–1. The tests were carried out at pressures up to 20 MPa. Special care was taken to allow good thermostatting of fluids entering the mixing cell. Check measurements were made with one liquid-liquid system (C₂H₅OH+H₂O) and one gas-liquid system (CO₂+C₆H₅CH₃); our enthalpies of mixing agreed with the literature values in most cases to 2%. For the system ethanol+water the experiments have been also performed at temperature of 250°C and pressures of 15 and 20 MPa. The endothermal mixing effect was higher than expected indicating an increase in the excess heat capacity.     

Doped Graphene as a Metal‐Free Carbocatalyst for the Selective Aerobic Oxidation of Benzylic Hydrocarbons,Cyclooctane and Styrene

Nitrogen (N)‐, boron (B)‐, and boron,nitrogen (B,N)‐doped graphene (G) act as carbocatalysts, promoting the aerobic oxidation of the benzylic positions of aromatic hydrocarbons and cyclooctane to the corresponding alcohol/ketone mixture with more than 90 % selectivity. The most active material was the co‐doped (B,N)G, which, in the absence of solvent and with a substrate/(B,N)G ratio of 200, achieved 50 % tetralin conversion in 24 h with a alcohol/ketone selectivity of 80 %. An FT‐Raman spectroscopic study of a sample of (B,N)G heated at 100 °C in the presence of oxygen revealed new bands that disappeared upon evacuation and that have been attributed to hydroperoxide‐like species formed on the G sheet based on the isotopic shift of the peak from 819 to 779 cm^?1 when ¹⁸O₂ was used as the oxidizing reagent. Furthermore, (B)G and (N)G exhibited high catalytic activity in the aerobic oxidation of styrene to benzaldehyde (BA) in 4 h. However, the product distribution changed over time and after 10 h a significant percentage of styrene oxide (SO) was observed under the same conditions. The use of doped G as catalyst appears to offer broad scope for the aerobic oxidation of benzylic compounds and styrene, for which low catalyst loading, mild reaction temperatures, and no additional solvents are required.     

Calorimetric study on the effect of Co γ-rays on the growth of microorganisms

Using a calorimeter equipped with 24 sample units, the heat evolution from growing Saccharomyces cerevisiae, Escherichia coli and spores of Bacillus pumilus and Bacillus stearothermophilus was detected in the form of growth thermograms. Irradiation with ⁶⁰Co γ-rays affected the growth pattern, which was used for a quantitative analysis of the effect on microorganisms. Irradiation of B. pumilus and B. stearothermophilus spores led to dose-dependent delays in growth, indicating a bactericidal effect. In case of ⁶⁰Co γ-irradiated S. cerevisiae, a dose-dependent reduction of the growth rate constant was observed together with the retardation in growth, indicating a combination of bactericidal and bacteriostatic effects. An equation to determine the number of survivors on the basis of the retardation in growth t and the growth rate constant μ was developed, which proved the opportunity to use the calorimetric technique in predictive microbiology.