Simultaneously Monitoring the Metabolic Activity of Three Bacteria Utilizing a LAPS-Based Differential Sensor Set-up

Introduction On-line monitoring of the metabolic response of microorganisms can avoid costly and time-consuming disturbances at different stages in biofermentation processes. For instance, process downtimes in biogas plants caused by metabolically inactive microorganisms can induce irreversible hindrances and cost-intensive interventions, which can be prevented by utilizing an efficient monitoring system. A LAPS (light-addressable potentiometric sensor) can be a promising candidate for those monitoring systems, since it combines several advantages such as small size, possibility of mass fabrication, solid-state nature and short response time [1,2]. In general, LAPS with an appropriate recognition layer provides a spatially resolved detection of a certain analyte and can record 2D-chemical images of concentration changes of bio/chemical species on its flat surface [3]. In this work, a LAPS-based multi-chamber measuring system with 16 laser-diode modules was utilized as shown in Fig. 1 (left), which allows to simultaneously determine the extracellular acidification of three microorganisms: Escherichia coli (E. coli) K12, Corynebacterium glutamicum (C. glutamicum) ATCC13032, and Lactobacillus brevis (L. brevis) ATCC14869. With the knowledge about the acidification behavior of each microorganism in a multivariate-type analysis, a signal-response pattern can be obtained. In this context, the sensor signal and the status of the biofermentation process can be correlated.Materials and MethodsBacterial cultures were prepared and resuspensioned in phosphate-buffered saline (PBS) solution (0.2 mM/pH, pH 7.4). Different glucose concentrations were adjusted. A four-chamber LAPS measuring system was deployed for differential measurements, as assembled in Fig. 1. First, all four chambers were loaded with 100 μl of glucose solution, varying in each experiment between 0.042 mM and 5.0 mM. The salt bridge chamber was filled with 1 ml of 3 M KCl and was immersed into the four chambers, passing the bias voltage through a Ag/AgCl reference electrode to the sensor surface, containing the glucose solution. After a conditioning phase of 10 min with the particular glucose concentration, cells (200 µl) were added to the respective chambers. Chamber 1 was loaded with E. coli, chamber 2 with C. glutamicum and chamber 3 with L. brevis bacteria. Chamber 4 served as the reference chamber without cells containing only the diluted PBS solution. Four LDMs were considered for each chamber to read-out the sensor signals. The mean value of each chamber was used for further evaluations [4].Results and Conclusions Fig. 1 (right) depicts exemplary the metabolic responses as correlation curves between the LAPS signal and various glucose concentrations for the three types of investigated bacteria at a constant cell number of 4.8 × 109 cells/ml. The black curve refers to the extracellular acidification of E. coli, the red curve to C. glutamicum, and the green one to L. brevis bacteria. It was observed that by increasing the glucose concentration, the overall cellular metabolism increases due to an increase of H+-ion activity on the pH-sensitive transducer layer of the LAPS chip during the acidification phase. The obtained results will be discussed in context of varying cell numbers as well as status of a selected biofermentation process.Fig. 1. Schematic illustration of four-chamber differential LAPS set-up (left). RE: Ag/AgCl reference electrode, SC: sealing cap, SB: salt bridge chamber, MC: multi-chamber structure fixed on the sensor surface; LAPS consisting of layer structure of Al/p-Si/SiO2/Ta2O5 and modulated light source (LS) based on 16 laser-diode modules; 1–3: active sensor chambers with cells; 4: reference sensor chamber without cells; Iphoto: photocurrent; Vbias: bias voltage; AC: alternating current; LDMs: laser-diode modules. Correlation curves between LAPS signal response and glucose concentration (right). Mean values and standard deviations of potential change values of E. coli (black), C. glutamicum (red), L. brevis (green) at different glucose concentrations (0.042, 0.085, 0.17, 0.20, 0.33, 0.40, 0.50, 0.68, 0.83, 1.20, 1.67, 2.50, 3.33, and 5 mM) at constant cell number (4.8 × 109 cells). Three independent measurements were performed to calculate mean potential change rate values. The blue arrow in the diagram indicates a particular glucose concentration (1.67 mM), which was exemplarily chosen to compare calibration curves of metabolic responses of the three microorganisms by cell number variations.References [1] J.C. Owicki, L.J. Bousse, D.G. Hafeman, G.L. Kirk, J.D. Olson, H.G. Wada, J.W. Parce, The light-addressable potentiometric sensor: Principles and biological applications, Annu. Rev. Biophys. Biomol. Struct. 23 (1994) 87–113; doi: 10.1146/annurev.bb.23.060194.000511.[2] D.G. Hafeman, J.W. Parce, H.M. McConnell, Light-addressable potentiometric sensor for biochemical systems, Science 240 (1988) 1182–1185; doi: 10.1126/science.3375810.[3] T. Yoshinobu, K.I. Miyamoto, T. Wagner, M.J. Schoning, Recent developments of chemical imaging sensor systems based on the principle of the light-addressable potentiometric sensor, Sens. Actuators B 207 (2015) 926–932; doi: 10.1016/j.snb.2014.09.002.[4] S. Dantism, S. Takenaga, P. Wagner, T. Wagner, M.J. Schoning, Determination of the extracellular acidification of Escherichia coli K12 with a multi-chamber-based LAPS system, Phys. Status Solidi A 213 (2016) 1479–1485; doi: 10.1002/pssa.201533043.Figure 1