The application of heat conduction microcalorimetry to study the metabolism and pharmaceutical modulation of cultured mammalian cells

The heat produced by animal cells in culture can be used as the primary indicator of the kinetics of their metabolism because the scalar flux of it is a function of the metabolic flux. The validity of the relationship between heat and metabolism was demonstrated theoretically through the concept of thermal advancement and in experiments by the use of continuous cultures. This validation permitted the application of heat flux as a probe of the metabolic state of cells in culture. It consisted of an on-line heat conduction microcalorimeter that measures the instantaneous heat flow and dividing the smoothed signal with one obtained simultaneously using a dielectric spectrometer that records the change in capacitance as an estimate of the amount of viable biomass. In this mini-review, it is shown with Chinese hamster ovary cells (CHO320) genetically engineered to produce interferon-γ (IFN-γ) that heat flux is an early signal of deteriorating metabolism in cultures that produce considerable amounts of toxic lactate under fully aerobic conditions. The early detection favours the use of heat flux as the control variable in fed-batch cultures. This is a particularly useful finding in the context of the pharmaceutical industry because it will help to ensure the high fidelity of the cytokines, antibodies and vaccines produced in large-scale cultures. The monotonic relationship between the fluxes for heat and metabolism means that the enthalpy balance method can be employed to test the validity of the growth reaction for cells in culture. This showed that the crucial ratio between the substrates, glucose and glutamine, in the culture medium was incorrect at 5.5:1 instead of about 3:1, depending on the phase of the culture. Together with other changes to the medium composition, an improved formulation was made that ensured faster cell growth and greater specific rate (flux) of IFN-γ constitutive secretion while decreasing glucose utilisation and, most importantly, halving the excretion of lactate, that is toxic to the cells and harmful to the fidelity of their secondary products. Indirect calorimetry (oxygen uptake rate, OUR) is often favoured over the direct technique, but the former only measures aerobic metabolism. The environmental conditions in cultures favours lactate production even under fully aerobic conditions. Developments in measuring OUR mean that the stationary liquid phase balance can be used successfully to make the calorimetric:respirometric (CR) ratio a valuable tool in optimising cell culture to grow cells that synthesise the maximum amounts of the high fidelity secondary products.

Besides the value of heat flux in improving the cultures of animal cells producing heterologous products, three different techniques are examined that should be valuable in the testing the many compounds that are produced on a speculative basis as potential drugs. They are: (i) a thin-film thermopile transducer as an immunosensor; (ii) infra-red imaging of cells cultured in multi-well microtitre plates and (iii) integrated circuit (IC) calorimetry for small samples and low detection limit. One or more of these methods could well find favour with industry in the near future.     

Importance of growth form on production of hybrid antibiotic byStreptomyces lividans TK21 by fed-batch and continuous fermentation

A fermentation strategy, based on the controlled feeding of growthlimiting nutrients in order to maintain metabolic activity for extended periods, has been examined in the case of the production of a hybrid antibiotic by a transformed strain ofStreptomyces lividans TK21. The fed-batch operation did not improve the results obtained with batch operation. Continuous cultures on defined medium showed stable levels of biomass concentration, but antibiotic production ceased when continuous operation was started. The results obtained indicate the critical influence that morphology of the cell aggregates has on metabolic activity. The antibiotic is produced only in culture conditions providing growth in compact mycelial pellets.     

Caloric properties of mixtures of refrigerants R22 / R114

The nonazeotropic refrigerant mixture chlorodifluoromethane (R22) and 1,2-dichlorotetrafluoroethane (R114) has been frequently suggested as a working fluid in cooling systems and heat pump applications. However, especially for mixtures exact and reliable measurements of the caloric properties are often missing, so that calculations with equations of state yield results of great uncertainty. In spite of the CFC-ozone problem of this mixture it can be considered as an exemplary mixture to set up accurate equations of state.

Therefore measurements with an isenthalpic throttle calorimeter were carried out for three different compositions of the mixture. The measured isenthalps could be reproduced within the experimental accuracy by polynomials. Together with the specific heat capacity of the pure components the measurements lead to several caloric properties in the liquid-, vapour- and critical region. The caloric properties can also be calculated by equations of state (EOS). It turned out that the results obtained from Bender's EOS with interaction parameters fitted to the experiments lead to a good agreement with the experimental data.     

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.     

Optimization of l-(+)-lactic acid production using pelletized filamentous Rhizopus oryzae NRRL 395

Lactic acid is used as a food additive for flavor and preservation and a precursor in the development of poly-lactic acid, a product used to make biodegradable plastics and textiles. Rhizopus oryzae NRRL 395 is known to be a strain that produces optically pure L: -(+)-lactic acid. The morphology of Rhizopus cultures is complex, forming filamentous, clumps, and pellet mycelia. Different morphology growth has significant effects on lactic acid production. In bioreactors, the filamentous or clump mycelia increase the viscosity of the medium, wrap around impellers, and block the nutrient transportation, leading to a decrease in production efficiency and bioreactor performance. Growing fungi in pellet form can significantly improve these problems. In this study, factors that affect lactic acid production in pelletized flask cultures using R. oryzae NRRL 395 were investigated in detail. Completely randomized designs were used to determine the influence of culture temperature, time, concentration of glucose, and inoculum size. Lactic acid fermentation using clump and pellet morphologies were performed in a 5 L fermentor at the optimal values obtained from flask culture. Finally, fed-batch culture was used to enhance the lactate concentration in broth. The final lactate concentration of fed-batch culture reached 92 g/L. The data presented in the article can provide useful information on optimizing lactic acid production using alternative source materials.     

Estimation of the optimal concentrations of residual sugar and cell growth rate for a fed-batch culture ofSaccharomyces cerevisiae

Estimation of the optimal concentrations of residual sugar in medium for a fed-batch culture of Baker’s yeast has been studied and practiced. The concentrations, however, depended on different species and targets of the biomass, which was expected to be made. Kinetic changes of the residual phosphate salt in the medium conformed to a logarithmic process until the fourth hour during an 11-h culture. The parabolic method (see ref. 9 later in article) might be qualified to maintain the concentrations of residual sugar around 0.15 g/L. It was demonstrated that cell growth followed a sigmoid process during a fed-batch culture, because the cells consumed the nutrient with two metabolic pathways, one was for cell conversion and another was for non-cell conversion. With the parabolic method, we can estimate kinetics of cell growth and cell growth rate during the culture.     

LC-HRMS-based targeted metabolomics for high-throughput and quantitative analysis of 21 growth inhibition-related metabolites in Chinese hamster ovary cell fed-batch cultures

Chinese hamster ovary (CHO) cells have been widely used in the biopharmaceutical industry for production of therapeutic proteins. CHO cells in fed-batch cultures produce various amino acid–derived intermediate metabolites. These small molecule metabolic byproducts have proven to be critical to cell growth, culture performance, and, more interestingly, antibody drug productivity. Herein, we developed an LC-HRMS-based targeted metabolomics approach for comprehensive quantification of total 21 growth inhibition-related metabolites generated from 14 different amino acids in CHO cell fed-batch cultures. High throughput derivatization procedures, matrix-matched calibration curves, stable isotope-labeled internal standards, and accurate mass full MS scan were utilized to achieve our goal for a wide range of metabolite screening as well as validity and reliability of metabolite quantification. We further present a novel analytical strategy for extending the assay's dynamic range by utilizing naturally occurring isotope M + 1 ion as a quantification analog in the circumstances where the principal M ion is beyond its calibration range. The integrated method was qualified for selectivity, sensitivity, linearity, accuracy, precision, isotope analysis, and other analytical aspects to demonstrate assay robustness. We then applied this metabolomics approach to characterize metabolites of interest in a CHO cell-based monoclonal antibody (mAb) production process with fed-batch bioreactor culture mode. Absolute quantification combined with multivariate statistical analysis illustrated that our target analytes derived from amino acids, especially from branched-chain amino acids, closely correlated with cell viability and significantly differentiated cellular stages in production process.     

Optimization of l -(+)-lactic acid production using pelletized filamentous Rhizopus oryzae NRRL 395

Lactic acid is used as a food additive for flavor and preservation and a precursor in the development of poly-lactic acid, a product used to make biodegradable plastics and textiles. Rhizopus oryzae NRRL 395 is known to be a strain that produces optically pure l-(+)-lactic acid. The morphology of Rhizopus cultures is complex, forming filamentous, clumps, and pellet mycelia. Different morphology growth has significant effects on lactic acid production. In bioreactors, the filamentous or clump mycelia increase the viscosity of the medium, wrap around impellers, and block the nutrient transportation, leading to a decrease in production efficiency and bioreactor performance. Growing fungi in pellet form can significantly improve these problems. In this study, factors that affect lactic acid production in pelletized flask cultures using R. oryzae NRRL 395 were investigated in detail. Completely randomized designs were used to determine the influence of culture temperature, time, concentration of glucose, and inoculum size. Lactic acid fermentation using clump and pellet morphologies were performed in a 5 L fermentor at the optimal values obtained from flask culture. Finally, fed-batch culture was used to enhance the lactate concentration in broth. The final lactate concentration of fed-batch culture reached 92 g/L. The data presented in the article can provide useful information on optimizing lactic acid production using alternative source materials.     

Biodegradation of PVA-based formulations

Investigation of the biodegradability of water soluble poly(vinyl alcohol) (PVA) based blown films was carried out under different lab-scale environmental conditions. In particular respirometric tests were utilized in order to evaluate the biodegradability of PVA films in composting, in modified Sturm test and in soil burial simulation tests. Several microbial inocula present in river water, mature compost, forest and farm soils as well as sewage sludge from municipal and paper mill wastewater treatments plants were utilized for the relevant tests. A mixed PVA-degrading microbial culture was obtained by a common enrichment procedure by using sewage sludge from paper mill as inoculum; this culture was tentatively utilized for the isolation of single PVA-degrading microorganisms. As a first result we can stress that significant biodegradation extent in fairly low incubation time can be obtained only in the presence of acclimated microbial populations such as those deriving from paper mill sewage sludge, in liquid cultures. Nevertheless separation of single degrading microbial species was impossible most likely due to the establishment of symbiotic or commensal interactions between the single components of the PVA-degrading mixed cultures. On the other hand, limited mineralization rates were recorded in solid cultures in the presence of soil or compost. Finally, a mechanism of degradation of polymer chains unlike random or unzipping was suggested in the presence of either PVA-degrading mixed culture and its filtrate by means of viscometric determinations of molecular weight within the time.     

Evaluation of microcalorimetric measurements in terms of information content for decomposition reactions

Decomposition reactions of liquids and solids can be observed by the heat development using microcalorimetric methods. By determination of the released heat flow for heating up a sample, it is possible to get details to answer safety relevant questions.For reactions nth order the overall activation energy and the accompanying frequency factor can be determined, provided that the heat release is determined by the rate of a single reaction step. Researches have been carried out whether these parameters are useable for safety technical specifications.Autocatalytic affected decomposition reactions are connected with special problems. This affects the experimental examination or interpretation of results, and also the precise identification of beginning decomposition reactions in technical reactors. The application of microcalorimetric measurements on decomposition reactions is described and associated problems are pointed out.The conclusions from thermoanalysis data alone are not sufficient in the final consequence for safety technical assessments.     

Metabolic fingerprinting as a tool to monitor whole-cell biotransformations

Fourier transform infrared (FT-IR) spectroscopy was employed as a rapid high-throughput phenotypic typing technique to generate metabolic fingerprints of Escherichia coli MG1655 pDTG601A growing in fed-batch culture, during the dioxygenase-catalysed biotransformation of toluene to toluene cis-glycol. With toluene fed as a vapour, the final toluene cis-glycol concentration was 83 mM, whereas the product concentration was only 22 mM when the culture was supplied with liquid toluene. Multivariate statistical analysis employing cluster analysis was used to analyse the dynamic changes in the data. The analysis revealed distinct trends and trajectories in cluster ordination space, illustrating phenotypic changes related to differences in the growth and product formation of the cultures. In addition, partial least squares regression was used to correlate the FT-IR metabolic fingerprints with the levels of toluene cis-glycol and acetate, the latter being an indicator of metabolic stress. We propose that this high-throughput metabolic fingerprinting approach is an ideal tool to assess temporal biochemical dynamics in complex biological processes, as demonstrated by this redox biotransformation. Moreover, this approach can also give useful information on product yields and fermentation health indicators directly from the fermentation broth without the need for lengthy chromatographic analysis of the products.     

Modulated differential scanning calorimetry in the glass transition region

Temperature-modulated differential scanning calorimetry (TMDSC) is based on heat flow and represents a linear system for the measurement of heat capacity. As long as the measurements are carried out close to steady state and only a negligible temperature gradient exists within the sample, quantitative data can be gathered as a function of modulation frequency. Applied to the glass transition, such measurements permit the determination the kinetic parameters of the material. Based on either the hole theory of liquids or irreversible thermodynamics, the necessary equations are derived to describe the apparent heat capacity as a function of frequency.Presented in part at the 24th Conference of the Northamerican Thermal Analysis Society, San Francisco, CA, September 10–13, 1995.     

Potentiometric measurement of lipoic acid reduction in microbial cultures: Part I. Influence of bacterial growth on the potential-time signals

In this paper, we investigate the theoretical relations between microbial growth and the time evolution of the zero-current potential of a gold electrode in microbial cultures provided with exogenic lipoic acid. A linear relationship was elicited by computer calculation between simple potential versus time data and inverses of apparent growth rates of organisms. To verify this relation experimentally, we carried out potential-time measurements in cultures supplied with increasing concentrations of different sugars, and followed the kinetics of bacterial growth by classical photometric measurements. Judging from the theory, the potentiometric and growth photometric data were in good agreement. These results show that the potentiometric data may be used as reliable indexes for bacterial growth.     

Potentiometric measurement of lipoic acid reduction in microbial cultures: Part II. Evaluation of the bactericidal efficacity of drugs affecting bacterial growth

Multichannel potential-time measurements were carried out in cultures of Escherichia coli growing in the presence of increasing amounts of aminoglycoside antibiotics. The potentiometric lag times were an increasing function of the drug concentration.Theoretical apparent growth rates of organisms in the presence of the drug could be calculated from these potential-time data, and were related to the antibiotic concentration. For each antibiotic tested, it was possible to determine an inhibition rate constant as a function of the drug concentration, which characterized the bactericidal efficacity of the drug.A potentiometric MIC (minimal inhibitory concentration) was defined as the concentration at which the inhibition rate constant would be equal to the specific growth rate of organisms with no drugs added to the culture broth: the potentiometric MIC values were consistent with the conventional MIC's determined by a standard diffusion technique.     

Biological Hydrogen Production Using Chloroform-treated Methanogenic Granules

In fermentative hydrogen production, the low-hydrogen-producing bacteria retention rate limits the suspended growth reactor productivity because of the long hydraulic retention time (HRT) required to maintain adequate bacteria population. Traditional bacteria immobilization methods such as calcium alginate entrapment have many application limitations in hydrogen fermentation, including limited duration time, bacteria leakage, cost, and so on. The use of chloroform-treated anaerobic granular sludge as immobilized hydrogen-producing bacteria in an immobilized hydrogen culture may be able to overcome the limitations of traditional immobilization methods. This paper reports the findings on the performance of fed-batch cultures and continuous cultures inoculated with chloroform-treated granules. The chloroform-treated granules were able to be reused over four fed-batch cultures, with pH adjustment. The upflow reactor packed with chloroform-treated granules was studied, and the HRT of the upflow reactor was found to be as low as 4 h without any decrease in hydrogen production yield. Initial pH and glucose concentration of the culture medium significantly influenced the performance of the reactor. The optimum initial pH of the culture medium was neutral, and the optimum glucose concentration of the culture medium was below 20 g chemical oxygen demand/L at HRT 4 h. This study also investigated the possibility of integrating immobilized hydrogen fermentation using chloroform-treated granules with immobilized methane production using untreated granular sludge. The results showed that the integrated batch cultures produced 1.01 mol hydrogen and 2 mol methane per mol glucose. Treating the methanogenic granules with chloroform and then using the treated granules as immobilized hydrogen-producing sludge demonstrated advantages over other immobilization methods because the treated granules provide hydrogen-producing bacteria with a protective niche, a long duration of an active culture, and excellent settling velocity. This integrated two-stage design for immobilized hydrogen fermentation and methane production offers a promising approach for modifying current anaerobic wastewater treatment processes to harvest hydrogen from the existing systems.     

Microcalorimetric study of the growth of <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> in an enriched culture medium

Enterococcus faecalis is a Gram-positive bacteria, considered one of the most common causes of nosocomial infections. Bacterial cultures produce an exchange of energy as a result of the bacteria metabolisms. The rate of heat production is an adequate measure of the metabolic activity of the organisms and their constituent parts. Microorganisms produce small amounts of heat: 1–3 pW per cell. Although the heat produced by bacteria is very small, their exponential reproduction in a culture medium permits heat detection through microcalorimetry. In this study, we analyzed the microcalorimetric behavior of Enterococcus faecalis. A thermal Calvet microcalorimeter was used. The inside of the calorimeter contains two stainless steel cells (experimental and reference). Experiments were carried out at final concentrations of 10⁶,10⁵,10³, and 10 CFU/mL and a constant temperature of 309.65 K was maintained within the microcalorimeter. Recording the difference in calorific potential over time we obtained E. faecalis’s growth curves. Thermograms were analyzed mathematically allowing us to calculate the constant growth, generation time and the amount of heat exchanged over the culture time.     

Optimized Fed-Batch Fermentation of Scheffersomyces stipitis for Efficient Production of Ethanol from Hexoses and Pentoses

Scheffersomyces stipitis was cultivated in an optimized, controlled fed-batch fermentation for production of ethanol from glucose–xylose mixture. Effect of feed medium composition was investigated on sugar utilization and ethanol production. Studying influence of specific cell growth rate on ethanol fermentation performance showed the carbon flow towards ethanol synthesis decreased with increasing cell growth rate. The optimum specific growth rate to achieve efficient ethanol production performance from a glucose-xylose mixture existed at 0.1 h^?1. With these optimized feed medium and cell growth rate, a kinetic model has been utilized to avoid overflow metabolism as well as to ensure a balanced feeding of nutrient substrate in fed-batch system. Fed-batch culture with feeding profile designed based on the model resulted in high titer, yield, and productivity of ethanol compared with batch cultures. The maximal ethanol concentration was 40.7 g/L. The yield and productivity of ethanol production in the optimized fed-batch culture was 1.3 and 2 times higher than those in batch culture. Thus, higher efficiency ethanol production was achieved in this study through fed-batch process optimization. This strategy may contribute to an improvement of ethanol fermentation from lignocellulosic biomass by S. stipitis on the industrial scale.     

Calorimetry of dual limitations in yeast cultures

Chemostat cultures of one aerobic fermenting yeast (Saccharomyces cerevisiae) and one aerobic respiring yeast (Kluyveromyces fragilis) have been grown under dual C + N limitation in an isothermal reaction calorimeter. The dual limitations resulted in uncoupled oxidation of part of the glucose, which enabled the culture to adapt the ratio of nitrogen to carbon consumption rates precisely to the N/C ratio that was fed into the calorimeter. This conclusion has been reached based on the calorimetric measurements, which reflect uncoupled respiration conspicuously as abnormal heat yields or ratios of heat release per biomass grown.     

Independent exponential feeding of glycerol and methanol for fed-batch culture of recombinant Hansenula polymorpha DL-1

As a novel feeding strategy for optimizing human epidermal growth factor (hEGF) production with a recombinant Hansenula polymorpha DL-1 using the methanol oxidase (MOX) promoter in H. polymorpha DL-1, independent exponential feeding of two substrates was used. A simple kinetic model considering the cell growth on two substrates was established and used to calculate the respective feeding rates of glycerol and methanol. In the fedbatch culture with methanol-only feeding, the optimal set point of specific growth rate on methanol was found to be 0.10 h-1. When the fed-batch cultures were conducted by the independent feeding of glycerol and methanol, the actual specific growth rate on glycerol and methanol was slightly lower than the set point of specific growth rate. By the uncoupled feeding of glycerol and methanol the volumetric productivity of hEGF increased from 6.4 to 8.0 mg/(L.h), compared with methanol-only feeding.     

Effect of heating rate on the thermal properties and devolatilisation of coal

The apparent specific heat of coal was measured by employing a computational calorimetric technique during continuous pyrolysis at heating rates of 10, 25 and 100°C min^-1. For all of the examined heating rates, the apparent specific heat was found to be approximately 1.4 kJ kg^-1 K^-1 at room temperature. When the sample reached decomposition temperature (~410°C), the specific heat increased to 1.9 kJ kg^-1 K^-1. From this point, the apparent specific heat was greatly influenced by the coal reaction mechanism. For this purpose a detailed gas analysis was carried out for the three examined heating rates. It was found that with increased heating rates, the devolatilisation reactions were shifted to higher temperatures, as reflected in the measured apparent specific heat. This revised version was published online in July 2006 with corrections to the Cover Date.