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

Abstract

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.

Influence of nutrient feeding variations on AbrB accumulation, sporulation and cry1Ac expression during fed-batch cultures of Bacillus thuringiensis

“…Bacilli and spores were counted by quadruplicate, diluting culture samples in saline solution, followed by vigorous mixing to avoid cell clumping and direct microscopic visualization in a Neubauer chamber (Dinorín et al, 2015;Farrera et al, 1998). Sporulation efficiency was calculated as the ratio of free spores (thermoresistant spores obtained from incubation 10 min, 85°C) at the end of the fed batch: bacilli cells at the onset of sporulation multiplying by 100 (López y López & de la Torre 2005).…”

“…Expression of cry1Ac gene was determined by β-galactosidase assay (López y López & de la Torre 2005;Dinorín et al, 2015), where 34 samples were taken at determined times in both fermentations (t0 -t34). To achieve this, samples of 1-ml were centrifuged (12,800 g, 4°C, 7 min) and supernatants were discarded.…”

“…The impedance parameters Reactance (R) and Resistance (X) were obtained by the Hioki 3532-50 LCR HiTester impedance meter, by applying both a fixed current of 20 mA (milliamps) and 5 V (volts) at 1.225 MHz. The impedance probes were configured in accordance with Dinorín et al (2015).…”

“…In the case of FBC2, a value lower than 2.6 Ohms was reached, which was consistent with a higher Bt concentration. At 1.225 MHz Resistance and Reactance manifest more propitious behavior in relation to the growth phases, due to changes in their signal influenced by morphological and physiological cellular changes (Dinorín et al, 2015).…”

APROVECHAMIENTO DE ALMIDÓN DE CEBADA DE MALA CALIDAD PARA LA PRODUCCIÓN DE ENZIMAS AMILOLÍTICAS POR Aureobasidium pullulans

“…Los matraces se incubaron orbitalmente a 180 rpm a 30 ºC, pH 5.5. Se tomaron muestras cada 12 h, para realizar el conteo celular durante 48 h. El crecimiento celular se cuantificó microscópicamente por duplicado en la cámara de Neubauer, diluyendo las muestras del medio de cultivo en solución salina, seguida de una agitación vigorosa en orden de evitar la sedimentación celular (Dinorín et al 2015).…”