Microbial cultivations play a key role in many different fields, such as in food, drug and bulk chemical production, as well as in waste-to-value concepts [1
]. Process monitoring, such as pH, dissolved oxygen (dO2
) and off-gas analysis, is state of the art in today’s industrial cultivations for guaranteeing product quality and safety. However, the most important parameter in bioprocesses, the biomass, can only be determined using offline methods or complex soft-sensor applications [2
]. These control systems are often dependent on inline/online/at-line detection systems, such as high-performance liquid chromatography (HPLC) for metabolites, off-gas balance, and/or dielectric spectroscopy measurements. The use of accurate and reliable biomass measurement systems [3
], especially of viable cell concentrations (VCCs), enables proper process control tools, which lead subsequently to more robust and reliable bioprocesses. The VCC is measured using offline measurement principles including marker proteins or fluorescence probes, such as flow cytometry or confocal microscopy [5
]. Because these control and analytical tools are cost intensive, classical bulk food products—such as yeast and beer—are produced in rather uncontrolled environments. Not only the complex raw material, but especially growth conditions of the yeast (propagation and fermentation) are of high importance for the quality of the final product. The implementation of online vitality measurements in the brewing industry has historically been hindered by affordable, simple, robust and reproducible tests [7
In general, online and inline biomass measurement approaches are rather scarce and are based on physical measurement principles. One principle generally applied is high-frequency alternating current (AC) impedance spectroscopy with high field amplitudes, used on the basis of the ß-dispersion [8
]. Cells with an integer cell membrane affect the relative permittivity between two electrodes and, therefore, this signal is used for the estimation of VCCs. A detailed description of the measurement principles can be found in [10
The model organism for the application of AC measurements in the ß-dispersion range is yeast, being a very important expression host for recombinant proteins [14
]. Additionally, approaches towards more complex expression systems, such as filamentous fungi and Chinese hamster ovary (CHO) cells, are already performed [17
]. These measurements show a strong dependence upon physical process parameters (such as aeration and stirring—causing gas bubbles, temperature shifts and pH gradients), and are furthermore highly affected by changes in the media’s composition during cultivation.
However, not only high-frequency impedance spectroscopy in the ß-range can be used for the determination of biomass, but changes of the electrical double layer by the adsorption/desorption of cells at the electrode surface (detectable at low frequencies in the mHz range; α-dispersion) can also provide valuable information. Besides the cell type itself (cell wall/membrane compositions, size and shape), many physical parameters, especially in the media (pH and ion concentrations), can influence the potential distribution in the double layer [20
]. Furthermore, the given method via α-dispersion detection is capable of detecting even very small numbers of bacteria in soil, food and feces-polluted water using interdigitated microelectrode designs [22
]. These studies were only performed at a very small scale and with a low cell concentration. In general, a threshold in the measurement was present at low cell concentrations. Exceeding this limitation, over time, very stable signals were achieved in these studies. Besides direct measurements in the broth, a modified electrode system in an interdigitated design can be used [28
]. First approaches towards process monitoring were shown by Kim et al. [31
], who worked with an inline sensor used in the lower frequency range between 40 Hz and 10 kHz for the real-time monitoring of biomass. Kim et al. showed the feasibility for measuring changes in the double-layer capacitance (CDL
), but no analysis of the CDL
itself was performed; only discrete extracted values for distinct frequency values were used. Recent studies on Escherichia. coli
showed reasonable results for VCC determination not only in the batch phase, but also in the fed-batch approaches, leading to far higher cell densities [32
In this study, impedance measurements in the α-dispersion range were performed during the batch-based cultivation of Saccharomyces cerevisiae aimed for usage in brewing applications. Different state-of-the-art methods were applied for determination of the corresponding total biomass—dry cell weight (DCW) and optical density (OD610) offline. Flow cytometry (FCM) in combination with the fluorescence dye (bis-(1,3-dibutylbarbituricacid)trimethineoxonol) (DiBAC) was used for a cell physiology evaluation to account for changes in the viability during cultivation. With this knowledge, we were able to correlate the total biomass to the extracted CDL.
A prototype inline probe was designed and built for easy plug-in measurements of the biomass. Online and new inline probes were tested using defined media with glucose and maltose in different concentrations and with malt extract as the complex base material in brewing.
2. Materials and Methods
2.1. Expression Host and Cultivation
All cultivations were performed using the S. cerevisiae strain, supplied by Brauerei GUSSWERK (Salzburg, Austria). For the preculture, 500 mL of sterile Delft medium was inoculated from frozen stocks (1.5 mL; −80 °C) and incubated in a 2500 mL High-Yield shake flask for 20 h (230 rpm; 28 °C). Batch cultivations were performed in a stainless-steel Sartorius Biostat Cplus bioreactor (Sartorius, Göttingen, Germany) with a 10 L working volume, and in an Infors Techfors-S bioreactor (Infors HT; Bottmingen, Switzerland) with a 20 L working volume. Aerobic batches were cultivated using 1000 to 1400 rpm stirrer speeds with an aeration of 2 vvm. Anaerobic batches were cultivated at 600 rpm and with a 2 to 4 L/min N2 flow. The composition of the defined Delft medium used was as follows: 7.5 g/L (NH4)2SO4, 14.4 g/L KH2PO4, 0.5 g/L MgSO4·7H2O, 2 mL of trace metal stock, 1 mL of vitamins, 50 µL of polypropylenglycol (PPG) as Antifoam, and maltose and glucose in different concentrations as a carbon source. For the malt extract-based fermentation, a preculture with Delft media was cultivated, which was afterwards inoculated into the malt extract solution (150 g/L malt extract in deionized water; Weyermann, Bavarian Pilsner, Bamberg, Germany).
2.2. Analytical Procedures
For the DCW measurements, 1 mL of the cultivation broth was centrifuged at about 9000 g, subsequently washed with 0.9% NaCl solution, and centrifuged again. After drying the cells at 105 °C for 48 h, the pellet was evaluated gravimetrically. DCW measurements were performed in five replicates and the mean error for DCW was about 3%. Offline OD610 measurements were performed in duplicates in a UV/VIS photometer, Genisys 20 (Thermo Scientific, Waltham, MA, USA).
Verification of the cell viability in defined medium samples was performed using FCM measurements. After the addition of DiBAC (Thermo Scientific, Waltham, MA, USA), the diluted cultivation broth was measured using a CyFlow Cube 8 flow cytometer (Sysmex-Partec, Bornbach, Germany). DiBAC is sensitive to the plasma membrane potential, and therefore a distinction between viable and non-viable cells can be achieved. Detailed information on the viability assay can be found elsewhere [33
]. The overall errors for this method were in the range of 0.5% to 1%.
Sugar concentrations in the fermentation broth were determined using a Supelco C-610H HPLC column (Supelco, Bellefonte, PA, USA) on an Ultimate 300 HPLC system (Thermo Scientific, Waltham, MA, USA) using 0.1% H3PO4 as a running buffer at 0.5 mL/min. Ethanol concentrations were determined using an Aminex HPLC column (Biorad, Hercules, CA, USA) on an Agilent 1100 System (Agilent Systems, Santa Clara, CA, USA) with 40 mM H2SO4 as a running buffer at 0.6 mL/min.
Cultivation off-gas was analyzed by gas sensors: IR for CO2 and ZrO2-based for O2 (Blue Sens Gas analytics, Herten, Germany).
2.3. Impedance Measurements
Physical analysis of VCCs in state-of-the-art capacitance probes, which rely on β-dispersion (107
Hz), show a high dependence on process parameters (e.g., stirring, temperature, pH, salt and substrate concentration, etc.) and the cultivation phase (exponential growth phase, starvation phase, etc.) [12
]. We focused the measurement on a different physical phenomenon (α-dispersion), which yields valuable information mainly regarding the biomass concentration. The “α-dispersion effect”, at frequencies below 10 kHz, which is most likely a result of deformation of ionic species around the cell membranes, was used for these measurements. The dielectric response was therefore proportional to the ionic charge gathered around the membrane of adsorbed cells on the electrode [20
]. Impedance measurements were recorded in the range of 106
Hz with amplitudes of 100 to 250 mV using the Alpha-A high-resolution dielectric analyzer (Novocontrol, Montabaur, Germany). Because measurements in this frequency range are largely determined by the double-layer region between the electrode and the media, rather minor interferences with the process parameters (aeration and stirring) were to be expected. Online flow cells showed the benefit of a laminar flow through the cell and minor turbulence, but they generally had the problems of differences in the process state (side stream) and of performing sterilization procedures. Inline probes should overcome these problems, but they may be strongly affected by the process parameters. Details on the fitting procedure and data evaluation are given in [32
2.4. Inline Probe Construction
As online probes are not directly situated inside the reactor but are often supplied by a side stream of the fermentation broth, changes in the metabolism in this time interval may be highly possible, but less disruption of the signal is also observed by the stirring and aeration of the system. Furthermore, online probes always pose the danger of contaminating the process, as the sterile barrier is not kept inside the fermenter. Therefore, for sterile processes without constant streams of broth, the assembly of an inline probe prototype used a commonly used 25 mm B. Braun safety port with O-ring (Ingold connector). Materials were chosen to be permanently stable at 130 °C, and could easily sustain in situ autoclavation procedures. The physical analysis of VCCs was monitored and investigated by the inline probe sketched in Figure 1
The body as well as the electrodes of the inline probe consist of high-grade steel, that is, austenitic stainless steel, which is approximately 140 mm in length and at least 12 mm in diameter. Each electrode has a diameter of 10 mm. The gap between the electrodes is approximately 2 mm.