Calorimetric analysis of microorganisms in transient growth states to quantify changes of metabolic fluxes in response to nutrient deficiencies and osmostress

The catalytic potential of microorganisms is often limited by sub- or supra-optimal environmental factors, which frequently fluctuate with varying amplitude and frequency. In chemoorganoheterotrophic growth, microorganisms have to adjust their utilisation of Gibbs energy, and thus their heat flux and network of reaction fluxes, to cope with such fluctuations. A transient-state technique was developed to quantify changes of metabolism and to compare the enthalpy balance with the heat flux. This type of comparison can be used to assess the completeness of flux analysis. A special feeding system was designed to identify responses to specific environmental shortcomings. For this purpose, microorganisms were grown continuously in a bench-scale calorimeter and the concentration of a single component in the feeding stream was increased gradually, while all the other growing conditions were kept constant. The mathematical tools describing the concentration gradient and the microbial responses (i.e. key changes in metabolic rates) are presented. These equations were derived by solving the differential balances describing the concentrations of the substances varied in the feeding system, or similarly considering elements, substances and enthalpy in the calorimeter. The proposed method was tested on Variovorax paradoxus DMSZ 4065 and Halomonas elongata DMSZ 2581T subjected to nitrogen shortages and halo-stress, respectively. The major responses considered were the synthesis of poly-3-hydroxybutyrate (PHB) in the first case, and the synthesis of a compatible solute, 1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid (ectoine), in the latter. The measured rates and fluxes were consistent with the enthalpy and elemental balances as well as theoretical expectations, indicating that this transient-state technique is valid for quantifying changes of metabolic fluxes in response to specific stresses.