ORIGINAL ARTICLE Ion-selective electrode integrated in small-scale bioreactor for continuous intracellular pH determination in Lactobacillus plantarum Duried Alwazeer 1 & Rémy Cachon 2 Received: 20 April 2019 /Accepted: 20 August 2019 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2019 Abstract The aim of the present study was to develop an ion-selective electrode method for the continuous determination of the intracel- lular pH in Lactobacillus plantarum using a small-scale bioreactor. This method employed a salicylate-selective electrode basing on the distribution of salicylic acid across the cytoplasmic membrane. This developed electrode responded to salicylate concen- trations above 20 μmol/L with a Nernstian sensitivity. The energized and concentrated cells were added into a thermostated small-scale bioreactor that contained the salicylate anions dissolved in a 100 mmol/L potassium phosphate buffer at different pH values. The changes in salicylate concentration that occurred in the medium containing bacterial suspension were measured as a voltage change. The cells of Lactobacillus plantarum showed maintenance of pH homeostasis at the studied pH ranging from 4.0 to 7.0, and they kept a neutral intracellular pH up to 5.8. The simplicity of the measuring preparation and the relatively low cellular concentration, as well as the advantages of the small-scale bioreactor, lead us to believe that the described method can facilitate the study of the physicochemical factors on the intracellular pH of lactic acid bacteria using a single pH probe in one method. Introduction The intracellular or cytoplasmic pH (pH in ) is an essential fac- tor for both metabolism and growth parameters due to its significant effect on different cellular processes during fer- mentation such as enzymatic functions, proton motive force, transport kinetics, and ATP generation (Hansen et al. 2016). In bacteria, the pH in is the relevant factor for most of the cell’ s enzymatic machinery, and the pH gradient (ΔpH = pH ex - pH in ) across the cytoplasm membrane is the major component of the proton motive force in acidic medium (Kashket 1987; Hansen et al. 2016). The different methods used to determine the cytoplasmic pH of bacteria are generally divided into two classes: ion distribution methods such as flow dialysis and ion-selective electrodes and internal pH indicator methods such as 31 P nuclear magnetic resonance spectroscopy (NMR) and fluorescent pH probes (Rottenberg 1979; Kashket 1985; Padan and Schuldiner 1986; Zhang et al. 2012; Wu et al. 2014; Bouix and Ghorbal 2015; Hansen et al. 2016; Kudo and Sasaki 2019; Xu et al. 2019). The fluorescent biosensors are generally divided into two groups: fluorescent dyes (probes) that possess several drawbacks in- cluding cytotoxic effects, limited cellular uptake, or leakage of the dye from cells and the genetically encoded fluorescent protein (FP)–based pH biosensors, which were developed to overcome some of the problems that are specifically associat- ed with fluorescent dyes (Rupprecht et al. 2017). The radio- activity technique, e.g., accumulation of [ 14 C] radioactive or- ganic acid probe, was also reported for measuring intracellular pH in Lactococcus lactis subsp. cremoris (Mercade et al. 2000) and Leuconostoc oenos (Loubiere et al. 1992). Some of these methods are not available for every researcher be- cause they depend on complex and expensive instruments such as NMR and radioactive and fluorometric methods. Over the last two decades, the fluorescent probe method was proposed as a new and alternative method to measure the intracellular pH of lactic acid bacteria (Breeuwer et al. 1996; Glaasker et al. 1996; Marty-Teysset et al. 1996; Belguendouz * Duried Alwazeer alwazeerd@gmail.com; duried.alwazeer@igdir.edu.tr 1 Research Center for Redox Applications in Foods (RCRAF), Department of Food Engineering, Faculty of Engineering, Iğdır University, 76000 Iğdır, Turkey 2 UMR Procédés Alimentaires et Microbiologiques, AgroSup Dijon, 1 Esplanade Erasme, 21000 Dijon, France Folia Microbiologica https://doi.org/10.1007/s12223-019-00749-5