Yeast Propagation Control: Low Frequency Electrochemical Impedance Spectroscopy as an Alternative for Cell Counting Chambers in Brewery Applications
Round 1
Reviewer 1 Report
The manuscript of Brunauer et al. describes the use of low frequency electrochemical impedance spectroscopy as an alternative for cell counting chambers in industrial brewery sites that could act in substitution to traditional methods such as dry cell weight and optical density. I consider that manuscript reports an interesting topic but the discussion can be considered as preliminary.
The reported complex system is hardly fitted by a simple series R-C circuit (C defined as an ideal capacitor). Deviation form linear curve in Fig. 4 suggests that more elaborated circuit could be explored to describe more precisely the nature of the process.
It is important to optimize the best frequency for measurement of cells and also to analyze the possible deviation from ideal behavior in capacitor (I suggest to fit these results from a constant phase element disposed at different configuration – different model circuits).
Based on these modifications, it will be possible to determine the best frequency, the most adequate circuit and the influence of these parameters on overall precision (in comparison with standard techniques).
Author Response
The manuscript of Brunauer et al. describes the use of low frequency electrochemical impedance spectroscopy as an alternative for cell counting chambers in industrial brewery sites that could act in substitution to traditional methods such as dry cell weight and optical density. I consider that manuscript reports an interesting topic but the discussion can be considered as preliminary.
The reported complex system is hardly fitted by a simple series R-C circuit (C defined as an ideal capacitor).
You are absolutely right that a CPE fits the data to a way better extend. From our point of view the issues result in the comparability between CPE fits with different n values. As the resistance of the R-CPE element has a high error, we cannot calculate the correct double layer capacitance. As to clarify this point we added Figure 2 with raw data consideration and further text parts as given below:
In Figure 2 the raw data for impedance measurement during the lab scale cultivation are presented, right after inoculation and at elevated batch times. A presents the Nyquist plot, while B shows the Bode plot with absolute value of impedance.
Figure 2: exemplary raw data for two time points of the lab scale cultivation, a) Nyquist plot, b) Bode plot using absolute value of the impedance; c) fit with ideal capacity model.
As clearly visible in the raw data, a small shoulder is visible for the media contribution at high frequencies, while at lower frequencies the contribution of the double layer is predominant. In general, such data are fitted using complex circuit models. As already given in recent publications [38,39] the data can be fitted using constant phase elements (CPE) in combination with a resistance (R), so called R-CPE element, and resulted in an excellent fit of the raw data. Using CPE n and Q fitting parameters are used to calculate the sample capacitance (C) according to Eq. 2. [43], with C being the sample capacitance and R the real part of the R-CPE element.
However, we experienced problems in correct fitting of the R element since the error was tremendous high and this makes it impossible to determine the accurate capacitance in this case via Eq.2. These problems were also experienced in recent studies using low cell densities [38,39]. Therefore, we fitted our data using in a straightforward way: an offset resistance for cable and contact resistance and an ideal capacitor equivalent circuit element for the double layer capacitance were applied, see Eq. 3
Deviation form linear curve in Fig. 4 suggests that more elaborated circuit could be explored to describe more precisely the nature of the process.
From our point of view the overall quality in Figure 4 is based on metabolic switching of the cells, which effects the outer membrane and hence the ionic cloud. As we see these metabolic effects also for other cells, we believe that a rather more complex model for the biological system has to be used and that linear fits do not reflect the reality sufficiently. However, based on the aim of this work, we wanted to estimate the cell numbers roughly in atline/inline mode. Therefore, we think that the quality is in the moment enough for a suitable process analytical tool.
It is important to optimize the best frequency for measurement of cells and also to analyze the possible deviation from ideal behavior in capacitor (I suggest to fit these results from a constant phase element disposed at different configuration – different model circuits).
Our results indicate that the whole double layer is affected by the cells. In recent publications only one frequency was fitted and compared for cell numbers Kim et al (Sensors and Actuators B 138 (2009) 270–277). However, we believe that fitting the whole spectrum (at least in a range from 1000 Hz to 0.1 Hz in the alpha regime) results in far more stable data analysis. The deviations from the ideal capacitor is given now in Figure 2 C.
Reviewer 2 Report
Please see and make minor corrections highlighted in the attached pdf.
Comments for author File: 
Comments.pdf
Author Response
We thank the reviewer for the value comments.
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Online and inline biomass measurement approaches are rather scarce and are based on physical measurement principles. Beside the optical measurements, many biosensors use a change of an electrical signal for analysis. Many applications in this branch use impedance spectroscopy or cyclovoltammetry for detection of low amounts of a target proteins, cells, virus etc. [9-13]. In general, these systems use recognition elements like antibodies, aptamers or DNA to bind the target protein, cell, etc. The benefit of these systems is generally the very low limit of detection, but the drawback is the early saturation of such probes. In contrast, sensors used in process analytics would need not a low limit of detection and a high sensitivity for low amounts of the target, but a high linearity from cell densities of about 1 g/L to 100 g/L of cell dry weight. Many commercial sensors rely on high frequency alternating current (AC) impedance spectroscopy with high field amplitudes based. This relaxation phenomenon is referred to as ß-dispersion [14,15].
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However, the ß-relaxation in the frequency range from 107 to 104 Hz is not the only relaxation phenomenon which can be exploited for the determination of biomass. Changes of the electrical double layer by the adsorption/desorption of cells at the electrode surface, the so-called α-dispersion in a frequency range from 104 to 10-2 Hz, can provide valuable information. While ß-dispersion effects the entire cells through Wagner-Maxwell polarization, α-dispersion is preliminary based on ionic interactions and relaxation phenomena on the cell membrane [15].
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Chapter 2.3. In order to avoid self-plagiarism, we only cited the used setup in Ref. Slouka, C.; Wurm, D.J.; Brunauer, G.; Welzl-Wachter, A.; Spadiut, O.; Fleig, J.; Herwig, C. A Novel Application for Low Frequency Electrochemical Impedance Spectroscopy as an Online Process Monitoring Tool for Viable Cell Concentrations. Sensors 2016, 16, 1900.
Table 1. we changed the german wording to english. Table 1 is also presented in graph Figure 3 now, however we thought that doing it in both ways help the reader since picture 3 is quite crowded.
Changed Figure 3 now, accordingly
Data evaluation Eq 2. We do not see any diffusion limitation in this experimental setup and we do not use any redox probes, which would make these limitations accessible. The deviations in the capacitance are quite small and therefore highly sensitive. I understand that the discussion is maybe too short, therefore we added graph 2 and further parts in the manuscript. Detailed analysis of the data received during this measurement are already given in and accordingly cited Slouka, C.; Wurm, D.J.; Brunauer, G.; Welzl-Wachter, A.; Spadiut, O.; Fleig, J.; Herwig, C. A Novel Application for Low Frequency Electrochemical Impedance Spectroscopy as an Online Process Monitoring Tool for Viable Cell Concentrations. Sensors 2016, 16, 1900.
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Recent Figue 4: I added raw data now in Figure 2
Round 2
Reviewer 1 Report
The authors considered the hypothesis of use of most promising components in equivalent circuit (R-CPE circuit) and observed some discrepancies for value of resistance – all of these aspects are highlighted in the text. Deviation from the ideal capacitance is presented in the revised version of the manuscript.
Based on these adequate modifications and clarifications provided by the authors, I consider that manuscript can be accepted as is.
