Elsevier

Thermochimica Acta

Volume 544, 20 September 2012, Pages 10-16
Thermochimica Acta

A chip calorimeter for the monitoring of conventional bioreactors at elevated cell concentrations

https://doi.org/10.1016/j.tca.2012.06.004Get rights and content

Abstract

In conventional bioreactors, the assessment of metabolic heat via heat balancing is difficult. The reason is the influence of inherent variables such as power input due to stirring, environmental heat loss and evaporation. Thus, the aim of this study is to present a newly designed chip calorimeter which can be linked to any kind of bioreactor. This chip calorimeter with a new integrated real-time sample dilution was developed and tested during batch fermentation of the bacteria Escherichia coli VH33 and the yeast Arxula adeninivorans CBS8244. The presented device is operable up to an upper limit of 4 W L−1 for the volumetric metabolic heat production rate of these biological systems.

Highlights

► Online monitoring of microbial growth in bioreactors by a new chip calorimeter. ► Measurement of heat production rates up to 4 W L−1. ► Extended application range regarding biomass concentration due to real-time dilution of the sample.

Introduction

Growth, multiplication, maintenance and product formation of any living production system is inevitable connected to the continuous dissipation of Gibbs energy. In case of aerobic metabolism the process is enthalpy driven [1]. Therefore, the heat production rate can be used to quantitatively asses the overall metabolic activity [2]. Furthermore, applying Hess's law, the comparison of heat production rates and metabolic mass flows allows the proof of presumed models and the recognition of unknown metabolic pathways [3]. Assuming aerobic conditions, the heat production rate mainly reflects the catabolic part of the metabolism. Since the enthalpy of combustion of organic compounds is linearly correlated to the relative degree of reduction (Thornton's rule: −115 kJ per mole accepted electrons [4]), an exchange of electrons between carbon atoms does not provide a significant contribution to the overall enthalpy as it is the case for anabolic processes. Consequently, as the enthalpy of the anabolic processes is generally negligible, the metabolic heat production can be calculated based on the oxygen transfer rate (OTR). Since four moles electrons are absorbed by one mole oxygen, a nearly constant value for the oxygen related enthalpy ΔHox of about −460 kJ mol−1 results. This is denoted as the oxycaloric equivalent [5].

Differences between the heat flow directly measured and calculated from the OTR can be caused by pronounced deviations from Thornton's rule for substrate, biomass, or metabolic products (e.g. growth of yeast cells with ΔHox = −378 kJ mol−1 due to remarkably strong endothermal anabolism [6]). The decoupling of anabolic processes from aerobic catabolism, for example caused by anoxia, can be an additional reason for a significantly higher heat production compared to that calculated from the OTR [7]. Therefore, the simultaneous measurement of heat production and oxygen consumption can be a valid method for controlling biotechnological processes, in particular, if strong deviations from Thornton's rule allow the detection of product formation.

Up to now, two strategies have been applied for the online measurement of the metabolic heat production in bioreactors. Bioreactors can be intrinsically operated as (bioreactor-) calorimeters if all heat sources and sinks (e.g. heat loss to the environment, mixing enthalpies, neutralization, evaporation, and power input by the stirrer) are carefully balanced and, thus, the metabolic heat can be estimated in situ. Due to the high ratio of heat producing volume to heat exchanging surface can this strategy be generally applied to large-scale bioreactors [8], [9]. However, also bioreactor-calorimeters at small-scale have been developed [10]. Although commercially available bioreactor-calorimeters exist (RC-1 Bio, Mettler Instruments AG, CH), in general, custom-made bioreactors are applied that are expensive to modify and require many information for heat balance analysis. Alternatively, fermentation broth can be transfer through a sample loop from the bioreactor to a calorimeter. In this case, standardized calorimetric devices are useful as, for example, commercially available microcalorimeters (e.g. TAM, TA Instruments, USA [11]). A sample loop can be problematic due to clogging of the tubes and sedimentation of biomass inside the measuring cell. However, most serious limitations arise from the depletion of oxygen or carbon substrate during sample transfer, even if very high flow rates are applied [12]. The immersion of a calorimetric probe into the bioreactor would lead to considerably shorter sample transfer lines. However, the development of such a device by LKB, Sweden about 40 years ago was not successful [13]. Therefore, increasing attention has been payed to the application of chip calorimeters which seem to be promising for overcoming these problems.

The chip calorimeter is a miniaturized heat power meter based on silicon solid-state devices and manufactured using MEMS (micro-electromechanical system) technology. A crucial feature of this device is a chip membrane of several tens of nanometers to a few micrometers thick which serves as sample holder. Thin film calibration heaters and temperature sensors for heat and heat flow measurement are integrated therein [14]. The extremely low heat capacity and high thermal resistance of the chip membrane are essential preconditions to detect heat dissipation from small sample amounts. In the case of biological samples, nearly steady-state or slowly changing processes take place. Therefore, the measurement principle is based on determining a heat flux toward a heat sink. Consequently, the magnitude of the heat flow signal results from the overall heat rate of the sample, meaning the specific heat rate of the species, the number of species and their metabolic activity [15]. Additional advantages of chip calorimeters arise from their low thermal time constants due to the strongly reduced sample amount. Thermal time constants of only a few seconds allow enhanced sample throughput and decreases the impact of external temperature variations on the signal noise [16]. Both, the increased sample throughput and the improved technical compatibility to the reactor periphery is relevant for the use of the chip calorimeter in bioreactor control. Sensitivity and sample amount are not the challenge here, but rather its application in cases of increased particle density caused by higher biomass concentration and where gas bubbles arise by increased aeration inside the reactor. Results of heat rates from a batch culture of Escherichia coli growing in a laboratory-scale bioreactor and monitored using a chip calorimeter were reported by Maskow et al. [17]. They demonstrated that at low cell density (OD < 1) the applied chip calorimeter provides precise information on the status of the growing bacterial culture.

This current study presents a newly designed chip calorimeter with integrated, real-time sample dilution. The main objective of the development of the chip calorimeter was to measure the heat production rate of samples from conventional bioreactors at elevated cell densities.

Section snippets

Microbial strains

E. coli VH33 was used in this study (provided by the Instituto de Biotecnología of Universidad Nacional Autónoma de México). For this strain, the genetic sequence for the phosphotransferase system (PTS) in the bacterial chromosome was deleted. In order for glucose to pass the plasma membrane, the galactose permease (GalP), as an alternative uptake mechanism, was amplified on a genetic level [18]. Consequently, this strain has a strictly reduced overflow metabolism. Even in batch cultivations

Signal forms

Fig. 3A shows several measurement cycles of the stopped-flow operation mode. During the injection of the sample, an endothermal distortion of the signal caused by insufficient temperature equilibration of the incoming liquid is seen. Then, after stopping the injection, an exothermal signal shift Δu appears which correlates with the metabolic heat production rate of the sample (see Appendix A). Thereafter, the sample is replaced by saline during a second injection step. Usually, more than 1 mL of

Conclusions

Recently it was shown that chip calorimeters can successfully be applied for the real-time measurement of the heat production of microorganisms in highly diluted suspensions. The main objective of the presented work was to study the possible application of chip calorimeters for the monitoring of conventional bioreactors at elevated biomass concentrations. It is well accepted that conventional microcalorimeters can be connected to bioreactors of different kinds via a sample loop to measure the

Acknowledgments

The authors would like to thank Prof. Guillermo Gosset Lagarda, and the Instituto de Biotecnología of Universidad Nacional Autónoma de México for providing the strain E. coli VH33 used in this study. Financial support of the German Federation of Industrial Research Associations (AiF BMWi, AiF-Nr. 244 ZGB) is gratefully acknowledged.

References (27)

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