Automated Electrochemical Glucose Biosensor Platform as an Efficient Tool Toward On-Line Fermentation Monitoring: Novel Application Approaches and Insights

Abstract

Monitoring and control of fermentation processes remain a crucial challenge for both laboratory and industrial-scale experiments. Reliable identification and quantification of the key process parameters in on-line mode allow operation of the fermentation at optimal reactor efficiency, maximizing productivity while minimizing waste. However, state-of-the-art fermentation on-line monitoring is still limited to a number of standard measurements such as pH, temperature and dissolved oxygen, as well as off-gas analysis as an advanced possibility. Despite the availability of commercial biosensor-based platforms that have been established for continuous monitoring of glucose and various biological variables within healthcare, on-line glucose quantification in fermentation processes has not been implemented yet to a large degree. For the first time, this work presents a complete study of a commercial flow-through-cell with integrated electrochemical glucose biosensors (1^st generation) applied in different media, and importantly, at- and on-line during a yeast fed-batch fermentation process. Remarkably, the glucose biosensor-based platform combined with the developed methodology was able to detect glucose concentrations up to 150 mM in the complex fermentation broth, on both cell-free and cell-containing samples, when not compromised by oxygen limitations. This is four to six-fold higher than previously described in the literature presenting the application of biosensors predominately toward cell-free fermentation samples. The automated biosensor platform allowed reliable glucose quantification in a significantly less resource and time (<5 min) consuming manner compared to conventional HPLC analysis with a refractive index (RI) detector performed as reference measurement. Moreover, the presented biosensor platform demonstrated outstanding mechanical stability in direct contact with the fermentation medium and accurate glucose quantification in the presence of various electroactive species. Coupled with the developed methodology it can be readily considered as a simple, robust, accurate and inexpensive tool for real-time glucose monitoring in fermentation processes.

Keywords: bioprocesses; flow-through-cell; glucose biosensor; on-line monitoring; yeast fermentation.

FIGURE 1

Biosensor platform. (A) The biosensor chip B.LV5 designed as a flow-through-cell with connection to the SIX transmitter and luer fittings as connections for inlet and outlet. (B) The biosensor connected to the SIX transmitter.

FIGURE 2

(A) Steady-state current in nA as a function of the glucose concentration in mM obtained in the different media applying the biosensor platform under dynamic (flow of 0.2 ml/min) and static (no-flow) operation. (B) Raw signal development (current in nA as a function of time in min) obtained for different glucose concentrations in YP medium under dynamic operation. The lowest current curve (yellow) was obtained in buffer, followed by the response obtained in YP medium and at different glucose concentrations starting from 1 mM, over 5 mM to 10 mM. The steps between the indicated concentration values of 10 mM, 50 mM, 100 mM and 150 mM are 10 mM. (C) Average standard deviation in % per data point of glucose concentrations measured with the biosensor platform (internal duplicate) in the three media under investigation. (D) Average time until signal stabilization of biosensor measurements in min in the three media under investigation.

FIGURE 3

Calibration curves ranging from 1 to 150 mM glucose concentration obtained in YP medium applying a flow rate of 0.2 ml/min for biosensor chip 1 (A) and biosensor chip 2 (B). The red dotted lines indicate the division points between the low (1) and high (2) glucose concentration range. The decision, which section of the curve to apply was based on the current value. For both calibration curves, the critical current corresponds to 10 nA.

FIGURE 4

Data collected from fermentation 1 during at-line application of biosensor chip 1. (A) Biomass concentration as a function of time measured by optical density (OD600, at-line) and dry weight (DW, off-line) as well as continuously on-line via the CGQBIOR (backscatter units). (B) Glucose concentration measured at-line with and without cells by means of the biosensor platform as well as off-line (cell-free samples) by HPLC. The addition of glucose during the fermentation is indicated by black arrows. Besides, the increase in biomass over time is demonstrated by backscatter measurements as in Figure 4A. (C) Dissolved oxygen (DO) profile over the fermentation course. The red square must be considered as an outlier. (D) Average st. dev. and average time until signal stabilization (time until measurement result) of the different glucose measurements performed.

FIGURE 5

Data collected from fermentation 2 during on-line application of biosensor chip 2. (A) Continuous raw signal of the biosensing platform, current in nA as a function of the fermentation time in min, as well as the smoothed raw current signal obtained from a Matlab^® in-house smoothing function. (B) Comparison of the glucose concentrations measured with the biosensor platform (based on the smoothed raw current signal and the calibration curve obtained in YP applying flow (Figure 3B) and HPLC analysis of distinct fermentation samples. (C) Dissolved oxygen concentration over time measured with the optical minisensor off-line for manually withdrawn, cell-containing fermentation samples. The probes are numbered from 1 to 10 corresponding to their acquisition time as indicated in the OD600 profile [panel (D)]. (D) Yeast growth indicated as OD600 together with the DO as a function of the fermentation time in hours. The numbers above the OD600 data points indicate the time point of probes 1–10 in panel (C).

FIGURE 6

Different calibration profiles ranging from 1 to 150 mM glucose concentration obtained with biosensor chip 1 and 2, presenting the current [nA] as a function of the glucose concentration measured by HPLC [mM]. (A) Calibration profiles obtained with the electrochemical platform using biosensor chip 1 in buffer and YP medium before (1) and after (2) at-line application on fermentation samples, and after a 3 months storage period (3). (B) Calibration profiles obtained with the electrochemical platform using biosensor chip 2 in buffer and YP medium before (1) and after (2) on-line application during the fermentation. Moreover, the results of biosensor chip 1 and 2 are compared regarding the initial calibration in buffer and YPD medium.