Use of an optical sensor to directly monitor the metabolic activity of growing bacteria.

The metabolic activity of growing bacteria was directly monitored by using an electro-optical (EO) sensor. The sensor enables examination of bacteria in batch and continuous cultures. As examples, we report studies with Еscherichia coli, a bacterium with an aerobic type of metabolism, and Lactobacillus plantarum, a bacterium with an anaerobic type of metabolism. Bacterial growth was accompanied by a simultaneous change in both the hydrodynamic mean size (HMS) of the bacteria and the concentration of ions in the cytoplasm (CIC). Both variables were associated with the regulation of cellular metabolic activity, which can be cyclic during intense bacterial growth. A simultaneous change in metabolic activity and osmotic regulation was also found. For СIC and HMS measurements, we used online results of the EO analysis of cells suspended in water. The measured results for the CIC and HMS can be used to directly monitor bacterial metabolism. The results of this study are of practical importance for the real-time EO monitoring of the metabolic activity of growing bacteria without preliminary sample preparation.

Electrooptical Determination of Polarizability for On-Line Viability and Vitality Quantification of Lactobacillus plantarum Cultures.

The rapid assessment of cell viability is crucial for process optimization, e.g., during media selection, determination of optimal environmental growth conditions and for quality control. In the present study, the cells' electric anisotropy of polarizability (AP) as well as the mean cell length in Lactobacillus plantarum batch and fed-batch fermentations were monitored with electrooptical measurements coupled to fully automated sample preparation. It was examined, whether this measurement can be related to the cells' metabolic activity, and thus represents a suitable process analytical technology. It is demonstrated that the AP is an early indicator to distinguish between suitable and unsuitable growth conditions in case of a poor energy regeneration or cell membrane defects in L. plantarum batch and fed-batch cultivations. It was shown that the applied method allowed the monitoring of physiological and morphological changes of cells in various growth phases in response to a low pH-value, substrate concentration changes, temperature alterations, exposure to air and nutrient limitation. An optimal range for growth in batch mode was achieved, if the AP remained above 25·10-28 F·m2 and the mean cell length at ~2.5 µm. It was further investigated, in which way the AP develops after freeze-drying of samples, which were taken in different cultivation phases. It was found that the AP increased most rapidly in resuspended samples from the retardation and late stationary phases, while samples from the early stationary phase recovered slowly. Electrooptical measurements provide valuable information about the physiologic and morphologic state of L. plantarum cells, e.g., when applied as starter cultures or as probiotic compounds.

Electrooptical monitoring of cell polarizability and cell size in aerobic Escherichia coli batch cultivations.

The time-dependent development of cell polarizability and length in Escherichia coli batch fermentations were observed at-line with electrooptical measurements. While using a measurement system with fully automated sample preparation, the development of these properties can be observed with a comparable high frequency (six measurements per hour). The polarizability as well as the mean cell length both increase soon after inoculation and then decline from the growth phase on until the stationary phase is reached. Based on the dynamic behavior of polarizability, the growth phase can be divided into four distinct stages. Changes in the cultivation temperature or the pre-cultivation conditions lead to alterations in the development of the polarizability and mean cell length. Based on the frequency disperse of polarizability measured at four different frequencies from 210 to 2,100 kHz, a prediction model is established that is based on the relation of the polarizability to the metabolic activity. Applying multi-linear partial least squares methods (N-PLS), the model is able to predict the specific acetate synthesis and uptake with a root mean square error of prediction of 0.19 (6% of the mean). The method represents a tool for characterization of different stages with respect to microbial metabolic activity and the energy balance during batch cultivations.

Electric dipole moments of Escherichia coli HB 101.

The theoretical and experimental studies of the particles' electric dipole moments in the microscopic and submicroscopic size range show that in the case of polar and conductive media the interfacial components of the dipole moments are of greatest importance. While in the range of manometer's sizes there seems to be no important problems in the identification and in the estimation of the values of the dipole moments at present, in the micrometer range there are serious problems. In this communication these problems are considered and illustrated by electro-optic investigations of Escherichia coli HB 101.

Electrooptical measurements for monitoring metabolite fluxes in acetone-butanol-ethanol fermentations.

Anisotropy of electrical polarizability in Clostridium acetobutylicum cells during pH 5 controlled acetone butanol ethanol fermentations was observed. Cell length was determined from the electrooptical data. Mean length was determined as being 2.5 microm in the growth phase and 3.5 microm in the early stationary phase. Based on the obtained frequency dispersion of polarizability anisotropy (FDPA) in the range of 190 to 2,100 kHz, the switch from the acidogenic to the solventogenic phase could be monitored. The slope of polarizability versus the frequency made it possible to differentiate between phases of dominating acid and solvent production. Metabolite fluxes determined from concentration measurements correlated well to the polarizability. A partial least-squares (PLS) model was established and validated by applying data from several fermentations. The root mean square error of calibration (RMSEC) was 0.09 for the acid fluxes and 0.11 for the solvent fluxes. The root mean square error of prediction (RMSEP) was 0.20 for acid fluxes and 0.24 for solvent fluxes. The ratio of polarizability at high and low frequencies correlated to the ongoing sporulation process. At ratios below 0.25, spore formation in the cells became visible under the microscope. The advantage of using electrooptical measurements is the ability to observe metabolite fluxes rather than concentrations, which provides useful information on productivity during a bioprocess.

Evaluation of process-induced dimensional changes in the membrane structure of biological cells using impedance measurement.

The impact of high intensity electric field pulses, high hydrostatic pressure, and freezing-thawing on local structural changes of the membrane was determined for potato, sugar beet tissue, and yeast suspensions. On the basis of the electrophysical model of cell systems in biological tissues and suspensions, a method was derived for determining the extent of local damage of cell membranes. The method was characterized by an accurate and rapid on-line determination of frequency-dependent electrical conductivity properties from which information on microscopic events on cellular level may be deduced. Evaluation was based on the measurement of the relative change in the sample's impedance at characteristically low (f(l)) and high (f(h)) frequencies within the beta-dispersion range. For plant and animal cells the characteristic frequencies were f(l) approximately 5 kHz and f(h) > 5 MHz and for yeast cells in the range f(l) approximately 50 kHz and f(h) > 25 MHz. The observed phenomena were complex. The identification of the underlying mechanisms required consideration of the time-dependent nature of the processing effects and stress reactions of the biological systems, which ranged from seconds to several hours. A very low but significantly detectable membrane damage (0.004% of the total area) was found after high hydrostatic pressure treatment of potato tissue at 200 MPa. The membrane rupture in plant tissue cells was higher after freezing and subsequent thawing (0.9% of total area for potato cells and 0.05-0.07% for sugar beet cells determined immediately after thawing), which increased substantially during the next 2 h.