Introducing a bioelectrochemical method for highly selective enumeration of magnetotactic bacteria

In this study, we employed an electrochemical (potentiometric) method to enumerate magnetotactic bacteria (MTB) during its coupling with iodometric titration to obtain a selective, precise and rapid counting system. Oxygen was considered as an important factor for the orientation and movement of MTB towards the magnet-modified indicator electrode. In the direct potentiometry, a linear correlation was detected between potentiometric response and dissolved oxygen (DO) concentrations. By the increase of the DO concentration, potential difference would increase in the range of 4.0 to 20.0 parts per million (ppm) at different pressure conditions. The reliability of the O2 bio-sensing feature provides a selective MTB-based cell enumeration methodology based on indirect potentiometric titration. Furthermore, a five-minute H2-purging resulted in an increase of potentiometric response sensitivity arising from the decrease in DO concentration of the electrolyte solution. Results were also investigated by zeta potential difference, which show the effect of charge density of MTB in presence of DO. Zeta potential was increased proportionally by addition of the MTB population. Regarding the reliability of the suggested method, data obtained by the designed system showed no statistical difference from those obtained by the most common procedure in microbiology for enumeration of bacteria, known as colony forming unit (CFU) method.

utilized to measure the proliferation and viability of mammalian cells, MTT and other relative materials can also be employed for measuring bacterial viability 7 . Another procedure to quantify bacterial growth is based on the measurement of the light scattering originating from the turbidity brought about by bacteria measured at 600 nm, referred to as optical density (OD 600 ) 13 . This method is often used as a rapid and cost-effective technique to detect bacterial growth and throughout the culture in broth media. At a quick glance, however there are numerous methods for counting bacteria. Several investigations are being carried out to introduce highly selective, inexpensive, more accurate, and facile procedures. Among these, electrochemical sensing methods can be considered to overcome the limitations of culturing and biochemical procedures like sensitivity and specificity. Furthermore, they need lower technical knowledge than highly specific microbial or immunological methods.
MTB can easily be oriented towards the Earth's magnetic field 14 . Magnetic and physical features of MTB make them valuable candidates in different fields including medicine like drug delivery 15 , treatment of infectious diseases, cancer therapy, and bioremediation [15][16][17] , cell separation 18,19 , enzyme immobilization 20 , biomineralization of metal ions 21 , waste water treatment, and for removing heavy metals [22][23][24] . However, although a wide variety of MTB's applications have been reported so far, there are no reports available for using this type of bacteria in manufacturing a bacterial enumeration system.
The aim of our study is to present a highly selective, precise, and cost-effective method for enumeration of the MTB without requiring any time-consuming steps of bacterial purification. Our proposed method briefly employs an electrochemical (potentiometric) method, to detect the interaction of dissolved oxygen (DO) with the MTB. For measuring the DO parameter, iodometric titration is coupled with a potentiometric method. Based on the end point of titration (color of indicator and potential difference), the correlation of the magnetotactic bacteria population and potential differences are determined. Our results show that, the designed system can provide a simple and accurate chemical method for selective enumeration of magnetotactic bacteria without requiring their isolation from non-magnetotactic species.

Results and Discussion
Dual o 2 -H 2 recognition biosensor based on the immobilized MtB. Potentiometry has proved in many studies to be a powerful tool for designing biosensors 25,26 and evaluate the capacity of microorganisms for biosensing purposes 27,28 . In our study, oxygen was considered as an important factor for movement and orientation of the MTB towards the magnet-modified indicator electrode. The relationship between DO and the potential difference in presence of MTB is shown in Fig. 1A. This shows that there is a linear correlation between the potentiometric gradient (ΔE) and the DO concentration.
Purging H 2 gas for 5 min. increased the sensitivity of the electrochemical response. The effect of H 2 gas was investigated by Thermogravimetric analysis (TG), which showed improvement of sensitivity. In TG analysis H 2 absorbed /adsorbed by MTB indicates the main effect on performance of MTB. The maximum potentiometric response was obtained when DO concentration was set to 20.0 mg L -1 , although salting-out effect has lowered this parameter in ecosystems 29 . Furthermore, studying the effect of different pH values demonestrated that the highest potentiometric sensitivity can be obtained at pH 9.0 prepared by Tris buffer (data not shown). Because of the presence of functional groups such as -OH and -NH 2 in the structure of the Tris buffer as well as its buffer capacity (β), it behaved as the axillary complexing agent with cationic species such as Fe(II) and Fe(III). These cations are critical for the bacterial growth. They are mineralized inside the cell as Fe 3 O 4 . The optimum concentration of Fe(II) and Fe(III) to obtain a potentiometric response with maximum sensitivity was estimated to be 5.0 ppm. Using direct potentiometry, the potentiometric response and DO concentrations showed a linear correlation, ranging from 4.0 to 20.0 ppm (Fig. 1A). Furthermore, in the optimum DO study, the effect of H 2 gas on potentiometric response of the bacterium showed significant enhancement in different potentials during H 2 purging. Figure 1B shows the increased sensitivity of the potentiometric response by H 2 purging for 5.0 min. and the reversed effect by prolonging H 2 purging time. This observation can be related to the decrease in the DO concentration of the electrolyte solution.
cell enumeration by the immobilized MtB. The reliability of the O 2 biosensing enabled us to introduce a selective MTB-based cell enumeration methodology using indirect potentiometric titration. In a typical experiment the titrant (Na 2 S 2 O 3 ) is added to the analyte solution and the potential would begin to change until reaching titration endpoint ( Fig. 2A). Two titration end points were detected belonging to free DO and MTB-interacted DO. However, only one end point was observed after deaeration and DO removal by N 2 purging (Fig. 2B).
Two independent endpoints were observed that correlated to i) the free DO in the electrolyte solution and ii) the DO adsorbed on the surface of MTB. Strong interaction of DO by MTB can probably lead to higher titrant consumption and subsequently observe two independent endpoints. Acceptable linear correlation was observed for the MTB population and the difference in potential (ΔE) and thus a calibration curve was obtained ( Fig. 3A and Table 1).
This method can be presented as a simple, selective, rapid, and reliable chemical technique to number MTB indirectly during following the correlation between potential difference and the MTB population. In the presence of (non-MTB) no interference was detected in the enumeration of MTB population (Table 2).
Three independent experiments were carried out and estimated via extrapolation of the calibration curve. Average population of the MTB used as the reference sample was 92753 ± 25 in all the tested samples. This method displayed a good selectivity towards MTB so that, after adding an excess population of different non-MTB (vs. the control sample), no statistical difference was detected. The presence of magnetosomes analog  with MTB flagella will enable them to swim, orient and subsequently making mass transfer towards a special magnetic pole when they are subjected to an external magnetic field. This intrinsic feature will differentiate MTB mainly from non-magnetotactic bacteria and even from the cell debris of dead magnetotactic bacteria. This property makes them potential therapeutic carriers 30 . In addition, the affinity of the isolated MTB for adsorbing or absorbing O 2 and H 2 can be considered as one of the other distinguishable characteristics of these bacteria from the non-magnetotactic ones. In our study, these features were considered as the key factors for reliable MTB enumeration by the method used. Accordingly, the designed enumeration system will be able to differentiate magnetotactic bacteria from other non-magnetotactic ones. The reliability of this method was evaluated via comparison data obtained by calibration curve and number of MTB in the real sample. Maximum and minimum percentages of relative errors range from 0.15 to 10.53% (Table 3), which is acceptable for potentiometric methods.
It has been reported that the oxygen charge density is remarkably enhanced after coordination with other ion species 26 . Therefore, the charge density of the MTB was significantly changed after adsorption of O 2 . It is expected that, physicochmeical absorption/adsorption on the colloidal surfaces with high active surface area may provide extra charge density 31 . A fixed charge (Δq) was considered during the interaction between MTB and O 2 . In thermodynamics 32 , Δq is divided in two parts including static and dynamic contributions. The static part of Δq static can be reported as dq static = idt. The potentiometric technique is operated at open circuit conditions; therefore the share of this kind of electrical charge was negligible and was experimentally equal to zero 32 . Whereas about the dynamic part of Δq (i.e. Δq dynamic ), this term is individually correlated to the capacitance behavior of MTB.  www.nature.com/scientificreports www.nature.com/scientificreports/ This finding can be reported as dV = dq dynamic /dC, which is correlated to the surface charge (i.e. Zeta potential) of the microorganism 33 . In other words, reaction between titrant and O 2 interacting with MTB changed the ratio of dq dynamic /dC, which is detected via their mass transfer (swimming) towards the indicator electrode. Eventually, these results were employed to measure the charge density (zeta potential) at open circuit conditions. To confirm this, the zeta potential of fresh MTB-containing solution was measured and compared to those estimated after reaching the endpoint of titration. As a result, a linear correlation was observed between MTB population and zeta potential difference (Fig. 3B). It was concluded that, the zeta potential difference was statistically affected by increasing the population of MTB and subsequently higher adsorption of O 2 .

conclusions
In the current study, a specified procedure for enumoration of magnetotactic bacteria was introduced by combining potentiometry and iodometric titration. Potentiometry is widely used as a powerful tool to assess the bio-sensing behavior of microorganisms. Using potentiometry, DO was detected as an affecting element on bacteria movement towards the magnet-modified indicator electrode and sunsequent potential difference so that a two or three-fold increase of DO concentration resulted in an enhanced accumulation of bacteria onto the elecrtode's surface which was quantified by a two or three fold increase in potential difference. A significant increase of zeta potential difference was detected by enhancing MTB population. Furthermore, according to the TG analyses, a five minute H 2 -purching increased the potential difference to near two-fold displaying the effect of H 2 absorption or adsorption behavior of bacteria on their movements and attachment onto the electrode's surface. It has been concluded that, the introduced method provides a seletcive, fast, cost-effective, precise, and reliable tool for enumoration of magetotactic bacteria without the necessity of their isolation from other non-MTB.

Methods
Reagents and Materials. All reagents with purities more than 99% including starch, H 2 SO 4 (96%) and also different salts including construction of cell enumeration system. The obtained data from direct potentiometry was further confiremed by using the iodometry titration as an indirect method 24 . The ΔE of indirect potentiometry was calculated in the titration endpoint. In this method, the DO concentration is measured indirectly through an estimation of the end point of the potentiometric titrimetry. In this titration, 3.64 g MnSO 4 .H 2 O, 0.50 g NaOH, 0.10 g NaN 3 , 0.15 g KI and 1 mL H 2 SO 4 (96%) were used as reagents, which were added to the analyte solutions (Fe(II) 0.078 mM and Fe(III) 0.038 mM and the MTB solution 1:1:2, V/V, in Tris buffer; 10.0 mM, optimum pH = 9.5), Na 2 S 2 O 3 (0.02 M) and starch (average molecular weight: 342.30 g . mol -1 ) used as titrant and color indicator. The interfering effect of Fe(II) in titration was removed using sodium azide according to the established procedure 34 . Indirect potentiometry was carried out based the same procedure of direct method except for the addition of iodometric titration reagents, agitation and titration of the solution by Na 2 S 2 O 3 until the end point. Furthermore, the selectivity of the presented method for exclusive enumeration of magnetic bacteria cells was investigated by using non-magnetic bacteria such as the gram-negative bacteria like Escherichia coli (ATCC 10536), Salmonella enterica subsp. enterica (ATCC 13076), Pseudomonas aeruginosa (ATCC 9027), the gram-positive bacteria Staphylococcus epidermidis (ATCC 12228), Streptococcus vestibularis (ATCC 49124), and Lactobacillus amylovorus (ATCC 33620) and the probiotic bacteria Lactobacillus acidophilus (KCTC 3164) and Lactobacillus gasseri (ATCC 33323) (all provided by the National Center for Genetic and Biological Reserves, Tehran, Iran). All these bacterial strains were tested as negative controls.
The net charge of the MTB surface is negative and can be balanced by opposite ions presented in the surrounding media 35 . Zeta potential has been considered as a useful technique to investigate the activity and mechanism of microorganisms 35,36 . In the current study, to determine the charge of the MTB cell surface in the presence of oxygen (100.0 parts per million, ppm), zeta potential was measured directly and indirectly. Direct method was carried out using a zeta potential analyzer (Svarovsky, Zeta Meter Inc., USA) via a two-microelectrode stainless steel plate (type: 316, 1.0×1.0 cm) with a distance of 2 cm. The capacitance current change was monitored using a galvanometer (United Scientific MGV002 DC Galvanometer) to reach the end point using a magnet (16.5