Electrochemical Investigation of Thiobacillus Denitrificans in a Bacterial Composite

Thiobacillus denetrificans from DSMZ laboratory, cultivated at mineral medium (N°113 DSMZ)¹⁵; preparing four solutions: A, B, C and D for 1 l. The four solutions are prepared separately: the solution of trace elements, the solution A (containing in addition to the base salts, the electron acceptor, NO₃⁻), the solution B containing the thiosulfate (electron donor), solution C containing the carbon source (bicarbonate), and solution D. The composition of these different media is trace element solution SL-4: 50 g Na₂–EDTA; 20 g FeSO₄.7 H₂O; 10 g ZnSO₄.7H₂O; 30 mg MnCl₂.4 H₂O; 0.3 g H₃BO₃; 0.2 g CoCl₂.6H₂0; 10 mg CuCl₂.2 H₂O; 20 mg NiCl₂.6H₂O; 30 mg Na₂MoO₄.2H₂O; 1000 ml water. Solution A¹⁶: 2 g KH₂PO₄; 2 g KNO₃; 1 g NH₄Cl;0.8 g MgSO₄.7H₂O; 2 ml elements trace solution SL-4 and 960 ml water. The pH adjusted at 7 with NaOH. Solution B¹⁷: 5 g Na₂S₂O₃.5H₂O, 20 ml water; Solution C¹⁸: 1 g NaHCO₃, 20 ml water; Solution D¹⁹: 20 mg FeSO₄.7H₂O; 10 ml 0.1 N H₂SO₄.

Media A, B and D are boiled and degassed under filtered N₂. Solution C is degassed under filtered N₂/CO₂ (80%/20%). Solutions A, B, C and D are sterilized separately by autoclaving at 121 °C for 15 min. The pH of solution A is checked after autoclaving with a drop taken aseptically and deposited on a piece of pH paper (it should remain around 7).²⁰

Five bottles of 100 ml containing 96 ml of solution A were prepared as described before and completed with 2 ml of solution B, 2 ml of solution C and 0.1 ml of solution D taken by syringe 2 to 3 ml of Thiobacillus denitrificans suspension are added in four medium bottles.²¹ The 5th bottle is used as a control. The five bottles are incubated at 30 °C in a stove for one week.^22,23 Bacteria growth was monitored by direct counting with an optical microscope of a drop put between two microscope glass slides at 100X magnification. At the last day of the growing period, pictures of bacteria were taken for epifluorescence analysis. After one week of incubation, DO₆₀₀ = 1 it was equivalent of 6.4 * 10⁸ cells ml⁻¹ and it was conserved at +4 °C.²⁴

The culture was harvested by centrifugation at 5000 g for 10 min at room temperature.²⁵ The pellet was washed twice with 1 mM KCl, and then suspended in 1 mM KCl in order to reach a cell density between 2 × 10⁹ and 5 × 10⁹ cell ml⁻¹ (determined by optical density measurement) as shown in Fig. S1 in the Supplementary Material (available online at stacks.iop.org/JES/167/135502/mmedia).²⁶

The biocomposite was prepared according do protocol from the literature⁸ by mixing one volume of bacterial suspension (5 * 10⁹ Cells ml⁻¹), one volume of MWCNT-COOH suspension (5 mg ml⁻¹ dispersed in water by sonication for 30 min), protamine (5 mg ml⁻¹, dispersed in water by vertexing for 30 min), and two volumes of 1 mM KCl for 15 min until a sedimentation was observed (Fig. S2 in the Supplementary Material). After that biocomposite has sedimented in the suspension, it was deposited it on the graphite felt by vacuum filtration as shown in Fig. S3 in the Supplementary Material.

In epifluorescence microscopy, the LIVE ™ Bac Light™ kit (Thermo Fischer Scientific) was used.²⁷ Briefly; samples were mixed with DNA dyes SYTO 9 (1.67 μM) and propidium iodide (1.67 μM) and incubated for 15 min before filtration (on 0.22 μm membrane filters).²⁸ The filtrate was afterwards dropped on microscope glass slide and observed with an epifluorescence microscope (OLYMPUS BX51) with a magnification factor ×1000.²⁹ A sample image is provided in Fig. S4 in the Supplementary Material. High magnification scanning electron microscopy (SEM) was performed with JSM-IT 500 HR from JEOL. Energy-dispersive X-ray spectroscopy (EDS) was performed with a table-top JEOL SEM equipped with a JEOL analyzer.³⁰

The experiments were carried out in a conventional electrochemical three electrodes cell (Fig. S5 in Supplementary Material). with a graphite felt as working electrode having 17 mm diameter and 5 mm thickness (GFD4.6 EA from SGL, Germany), stainless steel as a counter electrode and Ag/AgCl as a reference electrode (sat. KCl, SE11 Sensortechnik Meinsberg, Germany). The cell was connected to a bath water thermostat at 30 °C. Experiments were performed in 20 ml of PBS at pH 7 under nitrogen flow and slow magnetic agitation If not stated otherwise, all potentials in this work are provided vs Ag/AgCl (sat. KCl; +0.197 V vs Standard hydrogen electrode SHE). Cyclic voltammetry experiments were done under nitrogen atmosphere at a scan rate 5 mV s⁻¹, at room temperature and at 30 °C.³¹ Nitrate (20 or 40 mM) was added after the current response was stabilized; Amperometric measurements have been performed at constant potential at −0.46 V or −0.68 V vs Ag/AgCl. After decay of the capacitive current, nitrate or nitrite have been added in the solution.

Figure S5 in Supplementary Material shows the Thiobacillus denitrificans observed in epifluorescence microscopy. With a Bac Light Live/Dead assay, the green staining shows that bacteria display low membrane permeation and are potentially viable.³² After formation of the biocomposite of Thiobacillus denitrificans cells and carbon nanotube, the material is deposited on graphite felt. Figure 1 reports details of this biomaterial when immobilized on the graphite fibers (Figs. 1D–1G) and the electrode obtained by following the same protocol but without bacteria (Figs. 1A–1C). Aggregates are visible in the presence of bacteria. High resolution imaging allows to get a detailed view of the interaction between MWCNT with bacteria when protamine is present. At pH 7, protamine is positively charged (isoelectric point of 9.5) whereas bacterial cells and MWCNT are negatively charged.³³ Together, these elements self-assemble and MWCNTs surround the bacterial cell (see Figs. 1G and 1H).

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Figures 2A, 2B show the morphology of the graphite felt with two different magnifications as a bare electrode.³³ Figures 2C to 2D shows the MWCNT and protamine after deposition in graphite felt with different magnifications. Figure 2F shows complex of protamine and MWCNT-COOH on the carbon felt as white dots.³⁴ Figure 2G shows the Mapping EDX which show the distribution of the elements. The percentage of all C, N, O and S are 76, 4, 12 and 8% ensuring the presence of both MWCNT and protamine.³⁵

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Preliminary experiments have been directed to evaluate the response to 20 mM nitrate of Thiobacillus denitrificans in a biocomposite electrode. First, a control experiment with an electrode prepared by following a similar protocol but in the absence of the bacterial cells do not show any response to the introduction of nitrate (Fig. 3A). The bacterial cells were stored in 1 mM KCl for 5 (Fig. 3B), 10 (Fig. 3C), and 15 d (Fig. 3D) before to be introduced in the biocomposite for electrochemical characterization. With the fresher culture (5 d), a current increase in observed upon the introduction of nitrate in the solution. The signal is broad with a peak around—0.35 V vs Ag/AgCl. After 10 d, a change is still noticed in the voltammogram in the same potential region and a higher current is noticed at higher potential, close to −0.6 V vs Ag/AgCl. After 15 d, no current change was observed upon addition of nitrate. These results suggest that Thiobacillus denitrificans possesses membrane proteins responsible for the extracellular electron transfer linked to nitrate reduction but also that electron transfer chain is degrading with time. The response to nitrite was also evaluated with a relatively fresh Thiobacillus denritificans culture (Fig. 4). No response of the electrode is observed in the absence of bacterial cells (Fig. 4A) and a clear current response to nitrite is observed at low potential, below −0.6 V Fig. 4B. A hypothesis is that the low potential response (close to −0.35 V) is linked to nitrate reduction while the current increase at a lower potential (close to −0.6 V) is linked to nitrite reduction. To control that idea, constant potential amperometry have been performed and discussed in the next section.

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Figure 5B A reports the amperometry measurement performed at −0.46 V vs Ag/AgCl, first in the absence of nitrate and then after spiking 20 mM NO₃⁻. When the experiment was performed in the absence of bacterial cells (curve (a)), no current change was observed when nitrate was spiked but a clear response was visible in the presence of Thiobacillus denitrificans in the biocomposite electrode (curve b), and the current was increasing by about 1 μA cm⁻². A similar experiment was performed with nitrite. (Fig. 5B). A lower potential was applied to the biocomposite electrode, −0.68 V vs Ag/AgCl. No response was observed in the absence of bacterial cells (curve a) and a significant current increase was observed upon successive additions of 20 mM nitrite. Interestingly, the current increase was about 10 times larger with nitrite that with nitrate, about 10 μA cm⁻² increase after each addition of 20 mM nitrite.

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The purpose of the following experiments is to evaluate the influence of temperature on the electrochemical reactions that were identified in the previous section. Figure 6 reports the electrochemical response of Thiobacillus denitrificans in the biocomposite to 20 mM nitrate at room temperature.³⁶ The response is similar as the one observed before (compare it with Fig. 3B). Here, the next step was to increase the temperature of the electrochemical cell to 30 °C (curve c). In that conditions, the current response increases significantly and the catalytic signal becomes more visible. The peak potential was moved to a slightly higher value, from −0.35 V to −0.43 V vs Ag/AgCl. We explain this relation between the increase of the current and the temperature to the fact that the membrane proteins of the bacteria are more active at the temperature of 30 °C and as we have deprived the bacteria of all its extracellular metabolites.³⁷

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The influence of concentration of nitrate on the response of Thiobacillus denitrificans biocomposites at 30 °C was evaluated.³² It is presented in Fig. 7. When relatively small amount of nitrate was added (from 10 to 20 mM) a limited current increase was observed at −0.46 V vs Ag/AgCl and a second signal can be noticed at −0.61 V vs Ag/AgCl. In the presence of 40 mM nitrate, the current at −0.46 V increased significantly and the presence of second redox signal at −0.61 V is confirmed. The largest concentration (60 mM) leads only to a limited increase in current.

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Figure 8 shows the calibration curve as a linear relationship between different NO₃⁻ concentrations (10–60 mM) and the current density (absolute values).

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The linear equation³⁸ was J/μA cm⁻² = 0.32 C + 13.7 with a correlation coefficient of 0.94. This means that the steady state reduction current increases with gradual increase in NO₃⁻ concentration. The sensitivity of the proposed method was evaluated using both the limit of detection (LOD) and limit of quantification (LOQ) values. The LOD and LOQ were calculated using the following³⁹:

$$\begin{eqnarray*}&&{\rm{LOD}}={\rm{3}}\,{\rm{s}}/{\rm{b}}\end{eqnarray*}$$

$$\begin{eqnarray*}&&{\rm{LOD}}={\rm{3}}\,{\rm{s}}/{\rm{b}}\end{eqnarray*}$$

Where s is the standard deviation of the reduction peak current (three runs) and b is the slope (mA M⁻¹) of the related calibration curves; they were found to be 6.3 μM and 21 μM, respectively. This is a good detection limit which means that we can use this system as a sensor to know the current of any nitrate concentration.⁴⁰