Two-methods approach to follow up biomass by impedance spectroscopy: Bacillus thuringiensis fermentations as a study model.

Impedance spectroscopy is used for the characterization of electrochemical systems as well as for the monitoring of bioprocesses. However, the data obtained using this technique allow multiple interpretations, depending on the methodology implemented. Hence, it is necessary to establish a robust methodology to reliably follow-up biomass in fermentations. In the present work, two methodological approaches, mainly used for the characterization of electrochemical systems, were employed to characterize and determine a frequency that allows the monitoring of biomass in Bacillus thuringiensis fermentations by impedance spectroscopy. The first approach, based on a conventional analysis, revealed a single distribution with a characteristic frequency of around 2 kHz. In contrast, the second approach, based on the distribution of relaxation times, gave three distributions (A, B, and C). The C distribution, found near 9 kHz, was more related to the microbial biomass than the distribution at 2 kHz using the equivalent circuits. The time course of the B. thuringiensis fermentation was followed; bacilli, spores, glucose, and acid and base consumption for pH were determined out of line; and capacitance at 9 kHz was monitored. The correlation between the time course data and the capacitance profile indicated that the monitoring of B. thuringiensis at 9 kHz mainly corresponds to extracellular activity and, in a second instance, to the cellular concentration. These results show that it is necessary to establish a robust and reliable methodology to monitor fermentation processes by impedance spectroscopy, and the distribution of relaxation times was more appropriate. KEY POINTS: • Application of impedance spectroscopy for bioprocess monitoring • Low-frequency monitoring of biomass in fermentations • Analysis of impedance data by two methodological approaches.

On-line monitoring of industrial interest Bacillus fermentations, using impedance spectroscopy.

Impedance spectroscopy is a technique used to characterize electrochemical systems, increasing its applicability as well to monitor cell cultures. During their growth, Bacillus species have different phases which involve the production and consumption of different metabolites, culminating in the cell differentiation process that allows the generation of bacterial spores. In order to use impedance spectroscopy as a tool to monitor industrial interest Bacillus cultures, we conducted batch fermentations of Bacillus species such as B. subtilis, B. amyloliquefaciens, and B. licheniformis coupled with this technique. Each fermentation was characterized by the scanning of 50 frequencies between 0.5 and 5 MHz every 30 min. Pearson's correlation between impedance and phase angle profiles (obtained from each frequency scanned) with the kinetic profiles of each strain allowed the selection of fixed frequencies of 0.5, 1.143, and 1.878 MHz to follow-up of the fermentations of B. subtilis, B. amyloliquefaciens and B. licheniformis, respectively. Dielectric profiles of impedance, phase angle, reactance, and resistance obtained at the fixed frequency showed consistent changes with exponential, transition, and spore release phases.

Reactance and resistance: main properties to follow the cell differentiation process in Bacillus thuringiensis by dielectric spectroscopy in real time.

During growth, Bacillus thuringiensis presents three phases: exponential phase (EP), transition state (TS), and sporulation phase (SP). In order to form a dormant spore and to synthesize delta-endotoxins during SP, bacteria must undergo a cellular differentiation process initiated during the TS. Dielectric spectroscopy is a technique that can be utilized for continuous and in situ monitoring of the cellular state. In order to study on-line cell behavior in B. thuringiensis cultures, we conducted a number of batch cultures under different conditions, by scanning 200 frequencies from 42 Hz to 5 MHz and applying fixed current and voltage of 20 mA and 5 V DC, respectively. The resulting signals included Impedance (Z), Angle phase (Deg), Voltage (V), Current (I), Conductance (G), Reactance (X), and Resistance (R). Individual raw data relating to observed dielectric property profiles were correlated with the different growth phases established using data from cellular growth, cry1Ac gene expression, and free spores obtained with conventional techniques and fermentation parameters. Based on these correlations, frequencies of 0.1, 0.5, and 1.225 MHz were selected for the purpose of measuring dielectric properties in independent batch cultures, at a fixed frequency. X and R manifest more propitious behavior in relation to EP, TS, SP, and spore release, due to particular changes in their signals. Interestingly, these profiles underwent pronounced changes during EP and TS that were not noticed when using conventional methods, but were indicative of the beginning of the B. thuringiensis cell differentiation process.