N-hexanoyl-l-homoserine lactone (C6-HSL) is a distinctive signal produced by Gram-negative bacterial strains. The signal is used to cell-to-cell communication. Oil and gas companies are suffering from Microbiologically-influenced corrosion (MIC) induced Sulfate-reducing bacteria (SRB. SRB induce severe pitting corrosion on the metal surface especially when attached and form biofilms. Nowadays, scientists are looking for an applicable method to detect SRB-biofilms. Metal oxides (MOx) intercalated into a polymers matrix, specifically conducting polymers (CPs), to sense different biological molecules effectively such as C6-HSL due to its ability to form a coordination bond and its high selectivity. Therefore, this work was directed to provide a novel quorum-signaling molecule, C6-HSL, sensing technique to distinguish invisible SRB-biofilms attached to a metal surface. Hence, two different MOx/Polyaniline-Dodecyl benzene sulfonic acid (PANI-DBSA) composites (ZnO/PANI-DBSA and Fe2O3/PANI-DBSA) were synthesized and structurally characterized. Afterwards, the composites were applied with carbon paste 1% by weight over a carbon working electrode (WE) to detect the C6-HSL qualitatively and quantitatively via an electrochemical analysis. The electrochemical impedance spectroscopy (EIS) verified the ability of the obtained composites to monitor the C6-HSL produced by SRB-biofilm compared to the standard material. The monitoring composites achieved the intended results where the observation swapped from 50 to 1000 ppm of the C6-HSL concentrations. The limit of detection (LOD) of the ZnO/PANI-DBSA and Fe2O3/PANI-DBSA was 624 and 441 ppm, respectively. Furthermore, the SRB-biofilm was confirmed by a calorimetric measurement in addition to EIS, where the outcomes were compatible.
Article
Quorum-Signaling Molecule Detection Based on Composite Sensors: Metal Oxides/Conducting Polymer
https://doi.org/10.21203/rs.3.rs-3227362/v1
This work is licensed under a CC BY 4.0 License
Journal Publication
published 02 May, 2024
You are reading this latest preprint version
Quorum Sensing
Sensors
Conducting polymers
Metal oxides
Nanocomposites
AHL
SRB-Biofilm
- Quorum-Sensing Based on CPs/MOx as active materials for screen-printed electrodes.
- Innovative sensitive ZnO/PANI-DBSA and Fe2O3/PANI-DBSA materials for C6-HSL detection.
- EIS is an efficient electrochemical approach for sensing SRB signals.
- Compatibility of C6-HSL recognition using the fabricated sensors and a colorimetric method.
Bacteria communicate with each other, in their surrounding medium, through the generation of chemical signaling molecules, chemotactic particles, or a pheromone known as quorum sensing (QS) molecules. It has been reported that there is a direct correlation between the chemical signaling molecules and bacterial concentration [1, 2]. In addition, the bacteria use the QS not only in communicating but also in regulating/synchronizing the expression of numerous genes that are necessary for bacteria to function as a community [3, 4]. Biofilms are defined as microbial communities characterized by bacterial cells that are attached to a substrate. These cells, when attached to a substrate, aggregate within a matrix of extracellular polymeric substances (EPS) that they produced, and exhibit variable phenotypes that influence the growth rate and gene replication [5, 6]. Key nutrients availability [7], chemotaxis in the surface direction [8], bacterial movement [9], attachment to the surface, and surfactant existence [10] are some impacts that affect the formation of the biofilm. Typically, Gram-negative bacteria generate a chemical signal molecule known as N-acylated homoserine lactones (AHLs). Recently, researchers discovered a valuable pathway to monitor bacterial activities and colony settlement size through the assessment of the AHLs [13–16]. Sulfate-reducing bacteria (SRB) as a group of Gram-negative bacteria have been recognized as one of the most harmful bacteria for iron metals utilized in oil and gas fields. [16, 17]. SRB induce microbially influenced corrosion (MIC) for iron metals by producing corrosive metabolites such as hydrogen sulfide gas (H2S), assumed electrochemical (cathodic depolarization) and microbial colonization (biofilm formation) [17]. SRB produce many AHLs including N-hexanoyl homoserine lactone (C6-HSL) and N-dodecanoyl homoserine lactone (C12-HSL [16]. Many efforts have been paid to detect SRB-AHLs signals to detect and inhibit such microbial communities. Many conventional techniques such as HPLC-MS/MS [18], HPLC/ESI-MS [19], LC-MS [20], GC–MS [21], and other chromatographic examinations are considered inadequate techniques due to they consume time and display a high cost [13, 14]. It has been noted that; few researchers were focused on the detection of SRB-biofilms using smart devices like sensors. In the last decade, a simple non-destructive technique like electrochemical impedance spectroscopy (EIS) was employed for this purpose [23]. The EIS has been employed to detect AHLs using a working electrode (WE) produced by a screen-printed electrode (SPE) technique [14]. The WE is fabricated based on metals [24], metal oxides (MOx) [13, 14] or metal/ metal oxide combination [25] which be utilized alone or embedded in a polymer matrix and/or one of the carbon forms materials. The research studies confirmed this method's accuracy, rapid response, and sensitivity for estimating the chemical signals resulting from bacterial assemblies compared with other electrochemical methods [14, 15, 26]. MOx including Zinc oxide (ZnO) [27], and Ferric oxide (Fe2O3) [1] displayed a good performance to sense the AHLs with high sensitivity, fast response, and ease to integrate into compact electronic devices like SPE. However, the MOx-based sensors suffered from low-limited selectivity [28] and confliction measurement with other dissolved ions in formation water as well as it is operated under a high-temperature process (> 100°C) [29]. Conductive polymers (CPs) are considered a successful alternative material to the MOx as a sensitizer of organic molecules [30, 31]. Indeed, the CPs present the advantages of easy preparation through chemical or electrochemical procedures, and they can sense the selective ion in various medium (basic, acidic, and neutral) at room temperature. Nevertheless, their long-term stability due to a moisture uptake need to be improved [32–35]. Conducting polyaniline (PANI) showed great potential in the field of electrochemical applications including sensors, biosensors, and supercapacitors. This can be attributed to its superlative characteristics, including its affordability, strong stability, electrical conductivity, and widespread availability [36–39]. In this article, promising nanomaterials with a high sensitivity to C6-HSL molecules were prepared depending on divalent nano metal oxides represented in ZnO and Fe2O3 doped in CPs matrix, polyaniline-dodecylbenzene sulfonic acid (PANI-DBSA). Afterwards, the synthesized composites were confirmed structurally and surface morphologically using several analyses including such as FT-IR, Raman, XRD, SEM, HR-TEM, GPC and DLS. The fabricated sensors were evaluated under the environmental conditions of oil wells infected with SRB by the EIS. In addition, the SRB-biofilm was detected by evaluating the corrosion rate (CR) of the carbon steel at ambient conditions and the results were confirmed with compatible colorimetric analysis.
2.1. Synthesis of MOx NPs and PANI-DBSA/MOx nanocomposites
ZnO NPs [40] and Fe2O3 [41] were synthesized by co-precipitation method according to the corresponding references, more details are illustrated in the supplementary materials (SM). The PANI-DBSA nanocomposites with MOx were prepared by an emulsion polymerization technique. Briefly, 10 mmol of the DBSA was prepared by diluting the concentrated acid with distilled water in a ratio of 1: 9, respectively. After that, 0.3 moles of the MOx (ZnO or Fe2O3) were added, and then the suspensions were mechanically stirred to obtain a homogeneous distribution of the MOx in the DBSA solution. Afterwards, the molecular equivalent by weight of aniline monomers was added followed by a satisfied stirring to get the aniline-DBSA salt. The aniline-DBSA salt was polymerized immediately after slowly adding 5 mmol of KPS to the emulsion at 0°C. The solution was kept at 0°C to complete the polymerization process. Finally, the prepared composites were washed with methanol and distilled water to eliminate any contamination, then dried in an oven at 50°C for 3 h to obtain ZnO/PANI-DBSA, Fe2O3/PANI-DBSA nanocomposites. The non-doped PANI-DBSA was also prepared according to the same procedure mentioned before except for the MOx NPs addition step.
2.2. Electrochemical sensing of enriched-SRB biofilm and C6-HSL
2.2.1. SRB media preparation and cultivation
The bacteria consortium in this study was obtained from a water sample with a salinity content (NaCl) of 2.6% that was collected from a water tank of North Bahria, Qarun Petroleum Company, western desert, Egypt. In the field, the water sample was enriched using an anaerobic selective media (modified Postgate´s-B medium) according to Postgate [42]. Modification in Postgate´s medium composition was done using the original water salinity (NaCl) of 2.6% and pH of 6.76 during media preparation. Media preparation, enrichment and cultivation were according to the modified Hungate's technique for anaerobes [43]. Afterwards, the enriched SRB sample was further enriched and used as inocula for the cultivated reactors at 37°C for 14 days and the black precipitation (Ferrous sulfide) was noticed [42]. The bacterial count was estimated for the inocula using the most probable number (MPN) [44]. A mild steel coupon AISI 1018 (mild/low carbon steel strip COSASCO's, Rohrback Cosasco Systems, Inc) with a dimension of 1.0 x 1.0 x 0.32 cm was used as a working electrode. The chemical composition of the coupons was carbon, C 0.14–0.20%, Iron, Fe 98.81–99.26% (as remainder), Manganese, Mn 0.60–0.90%, Phosphorous, P ≤ 0.040% and Sulfur, S ≤ 0.050%. The mild steal coupons were polished with a series emery paper with different grades (320, 400, 600, 800, 1000, 1200), followed by ethanol degreasing.
2.2.2. Assessment of enriched-SRB biofilm formation
In a conventional glass three-electrodes cell, the electrochemical measurements of open circuit potential (OCP) and electrochemical impedance spectroscopy (EIS) were tested by Origa Flex potentiostat-galvanostat system under anaerobiotic circumstances at an ambient temperature. The culture media, previously described, was used for all studies, either inoculated or not with the enriched SRB. The OCP was accomplished by allowing the WE to soak in the solution under investigation for 30 min before conducting the electrochemical tests.
2.2.3. Assessment of C6-HSL
The obtained composites: ZnO/PANI-DSBA and Fe2O3/PANI-DSBA were mixed with the carbon paste with a percentage not exceeding 1% and deposited as sensing materials for C6-HSL on the working electrode of the SPE sensor as shown in Figure S1a and b. The prepared sensors were evaluated using the EIS technique for measuring the bacterial signals by immersing them in customized anaerobic cells inoculated by enriched SRB with different C6-HSL concentrations. The SRB bacteria had allowed to grow in the incubator and then the SRB media were transferred to the measurement cell containing the sensor. The main target of the sensor that has the active materials (PANI-DBSA/ZnO or PANI-DBSA/Fe2O3) on the top of the working electrode reacts with the signal (C6-HSL) produced by the SRB bacteria assembly and detects its concentration as a function of changing the of EIS performance. The EIS measurements were fulfilled by the Origalys–signal multi-channel system (OrigaFlex 01A) with a frequency range of 0.01Hz-100 kHz with an amplitude of 150 mV. The recorded Nyquist plot of the impedance data was fitted by Zsimwin Version 3.0.1 software, see Figure S2.
2.2.4. Colorimetric detection of C6-HSL
The biosensor Agrobacterium tumefaciens KYC55 (pJZ372; pJZ384; pJZ410) was used for the detection of the C6-HSL production. This bacterial strain is responsible for the expression of a lacZ fusion in response to the C6-HSL. This leads to cleavage of 5-Bromo-4-chloro-3indolylgalactopyranoside (X-Gal) (Promega, Madison, WI, USA) and staining the agar plates with blue color as an indication for the C6-HSL and recorded as a positive result [45]. The C6-HSL production was assessed as (+ 1) for the positive medium production of the C6-HSL in comparison to the positive (+ ve) and negative (-ve) controls [45]. Further information describing the procedure of the colorimetric detection of the C6-HSL using the biosensor colorimetric technique was demonstrated in the supplementary materials.
3.1. Characterizations of the prepared PANI-DBSA/MOx nanocomposites
The XRD of the prepared PANI-DBSA (Fig. 1) displayed its amorphous structure via two peaks at 2θ = 20 and 25°. The (110) plane was indexed for this reflection.
The XRD patterns of the prepared materials were displayed in Fig. 1. The particles' average sizes were calculated by Debye-Scherrer’s equation as follows [46, 47]:
$$\text{D}=\frac{0.9\lambda }{\beta cos\theta }$$
1
Where D is the crystal size, λ is the x-ray wavelength, β is the full width at half maximum of the diffraction peak, and θ is the Bragg diffraction angle.
The data demonstrated that; all the peaks’ values can be referred to as the hexagonal structure of ZnO NPs. These peaks matched with those in the JCPDS card (Card No. 89-1397). The high intensity and the narrow width of the ZnO diffraction peaks confirmed the resulting product as it has a high crystallinity with a calculated average size of 20.95 nm. Moreover, the XRD pattern of the Fe2O3 NPs confirmed the formation of the Fe2O3 phase. All peaks were well matched with those in the JCPDS card for the Fe2O3 (ICDD card no. 330664).
The HR-TEM image (see Fig. 2a) provided visual evidence of the nanocrystalline particles which consisted of hexagonal and rod-like morphologies of ZnO NPs. The images revealed a slight agglomeration of these particles. In addition, the HR-TEM images, as depicted in Fig. 2b, provided additional confirmation of the spherical shape of the Fe2O3 nanoparticles which exhibited a slight agglomeration. The diameters of the nanoparticles range from 50 to 65 nm, and these estimated values closely align with those of XRD. This result confirmed the accuracy of the size determination based on the TEM analysis and supported the understanding of the Fe2O3 nanoparticle characteristics. Furthermore, the TEM image (see Fig. 2c) of the nanocomposite featuring ZnO nanostructures revealed the dispersion of the spherical ZnO nanoparticles with sizes ranging from 35 to 45 nm throughout the polymer matrix. Also, Fig. 2d provided the morphology of the Fe3O4/PANI-DBSA as an aggregate of very fine particles with uniform structure. Furthermore, it completely differs from the pure Fe3O4 particles (Fig. 2c) which exhibited a spherical shape that undergoes significant changes upon the PANI-DBSA doping. The presence of dark spots in the TEM image corresponded to these nanoparticles and indicated their presence within the composite material.
The molecular weight of the prepared PANI-DBSA was measured via GPC analysis. The average molecular weight (Mw) was 15080 g/mol and the polydispersity (PDI) was 3.6. Moreover, the structure confirmation and morphological characteristics of the PANI-DSBA were investigated using FT-IR (Figure S3) and SEM (Figure S4), respectively. The size distributions of the prepared MOx particles were determined by DLS (Figure S5). Additionally, Raman's analysis of all the prepared materials was presented in Figure S6.
3.2. Electrochemical assessment of enriched-SRB biofilm and C6-HSL
3.2.1. Electrochemical Assessment of enriched-SRB biofilm using SPE
The MIC behavior of the carbon steel specimens immersed in an anaerobic modified Postgate´s-C media (sterile uninoculated and enriched SRB-containing media) was evaluated using OCP, which is discussed in SM (see Figure S7), and EIS to inspect the influence of SRB-biofilm on the carbon steel. The measurements were investigated after 5 days of biofilm formation intervals for 30 days under static and anaerobic conditions.
3.2.1.1. EIS data analysis
Figures S8 a and b displayed the Nyquist curves with time for the carbon steel immersed in the control and enriched-SRB media under anaerobic conditions favorable for bacterial growth, respectively. To explain the experimental EIS spectra acquired in the case of a biofilm formed on the carbon steel surface, the acquired data were fitted and simulated with two suitable electrical equivalent circuit models Rs (Qdl Rct) and Rs (Cbf (Rbf (Cdl Rct)) for the sterile and enriched-SRB media, respectively as illustrated in Figure S9, where Rs denotes solution resistance, Qdl represented constant phase element (CPE), double-layer capacitance (Cdl), Rbf denotes biofilm resistance, and Rct signifies charge transfer resistance. The fitting EIS data are listed in Tables (1, 2). Figure S8 displayed that; by increasing the immersion time for the exposed metal coupons to the various media, the diameter of the Nyquist curves shifts a lot. In the presence of the enriched-SRB (Figure S8b), after 20 days of immersion, the diameter of the Nyquist curves reached its highest value which indicates that a protective coating made of the EPS and corrosion products has assembled on the surface of the carbon metal steel. Alternatively, the diameter of the Nyquist plots gradually decreased at 25 and 30 days of immersion times. It is worth mentioning that the Nyquist plot diameter amplified as a result of the biofilm-corrosion formation which offers more metal protection, while the diminishing behavior, indicated the presence of a permeable or discontinuous coating which stimulates the disintegration of the SRB-biofilm and the protective passive layer [48]. Inspection of Tables 1 & 2, it was noted that both media have good conductivity due to the solution resistance (Rs) being overly low and changing slightly with disclosure time, where the values of the Rs were significantly smaller in the enriched SRB-medium than in the sterile medium after 5 days of immersion as a consequence of the producing of bacterial solubilized metabolites such as pyruvic acid [49]. From the electrochemical point of view, the size of the capacitive circle at a low frequencies region was represented by the diameter of the semicircle, pointing out the difference in the charge transfer resistance (Rct) that expresses the anodic reaction growth was ruled by the charge transport process. Interestingly, as outlined in Table 1, the Rct values were observed to have a variability trend in the sterile medium which can be referred to the occurrence of chlorides ions from the oilfield water samples, causing pitting corrosion on the carbon steel surface (decreasing Rct, at periods of 1, 15 and 25 days being about 102.4, 114.7 & 99.22 ohm.cm2, respectively. On the contrary, at the immersion periods of 5, 10, 20, & 30 days, the Rct values increased to 790.9, 1706, 1216, and 1559 ohm.cm2, respectively, confirming the metallic passivation as shown in Figure S8a [50]. Further investigation (see Table 2) it is obvious that in the enriched SRB-medium, the increase of the Rct value was attained at an immersion time of 10 days and has a value of 149.7 ohm.cm2, attributing to enriched-SRB biofilms on the metal surface and certain areas of the surface were covered by microbial clusters, leading to the surface resistance of the metal to increase [51]. After 15 days of immersion, there was a decrease in the Rct value to 79.59 ohm.cm2 and then achieved a relatively stable state until 30 days, indicating an upsurge in charge transfer between the enriched-SRB biofilm and the electrode surface. With successive decreases in the Rct values, the enriched-SRB amplified the corrosion rate of the carbon steel through the production of biofilm, formation of sulfide, and consequent accumulation of conductive iron sulfide layers with the extent the immersion period from 15 days up to the last day at 30 days. These porous and conductive FeS films formed on the metallic surface of the electrode enhanced the corrosion kinetics in the presence of SRB. Figure 3a depicts the variation of charge transfer resistance (Rct) with the immersion time. Regarding the bio-film resistance (Rbf) values for SRB medium, it is apparent from Table 2, it is apparent that the Rbf value greatly decreases with immersion time, an embodiment that the charge transfers at the biofilm/the WE electrode surface interface become faster, which increases the MIC rate. Figure 3b presents a difference in the bio-electrochemistry behavior of the SRB system in terms of the biofilm resistance (Rbf) and the Cdl with time. What is more, the Rbf values in the SRB medium significantly decreased in the first 5 days of immersion of carbon steel starting from 15.11 to 5.420 ohm.cm2, implying that the initially formed biofilm lost some of its structural parts which exposed the metal surface to the corrosive species existing in the solution and confirming the instability of this bio-film [52]. Concerning the Cdl values, the results show that they increased during the periods of immersion which, assisted the presence of the bio-film porosity and accelerated the rate of carbon steel corrosion. Generally, the instability of the capacitance depends upon the formation of the bio-film at the interface and is related to the increase of active regions through the pores forming in the bio-film as a result of the microbiological growth phases.
| Time (d) | Sterile medium | |||
|---|---|---|---|---|
| Rs (Ω cm2) | Rct (Ω cm2) | Qdl | n | |
| 1 | 1.848 | 102.4 | 0.002593 | 0.7417 |
| 5 | 4.401 | 790.9 | 0.001032 | 0.8565 |
| 10 | 4.041 | 1706 | 0.001182 | 0.8626 |
| 15 | 3.490 | 114.7 | 0.001656 | 0.8312 |
| 20 | 2.913 | 1216 | 0.001519 | 0.8678 |
| 25 | 3.022 | 99.22 | 0.002009 | 0.8696 |
| 30 | 3.515 | 1559 | 0.001768 | 0.8764 |
| Time (d) | SRB medium | ||||
|---|---|---|---|---|---|
| Rs (Ω cm2) | Rct (Ω cm2) | Rbf (Ω cm2) | Cbf (F cm− 2) | Cdl (F cm− 2) | |
| 1 | 4.094 | 36.1 | 15.11 | 0.002883 | 0.008979 |
| 5 | 2.122 | 149.1 | 5.420 | 0.006763 | 0.02281 |
| 10 | 2.036 | 149.7 | 7.173 | 0.01914 | 0.0164 |
| 15 | 2.269 | 79.59 | 9.951 | 0.03845 | 0.02542 |
| 20 | 2.825 | 64.94 | 8.028 | 0.03266 | 0.03541 |
| 25 | 2.427 | 43.41 | 6.771 | 0.03878 | 0.05946 |
| 30 | 2.136 | 49.5 | 7.815 | 0.04295 | 0.06384 |
3.2.2. Electrochemical assessment of C6-HSL using SPE
To calibrate the C6-HSL amount per part per million (ppm), the known concentrations were dissolved in the prepared salty water then the electrochemical impedance spectroscopy (EIS) technique was implemented to measure the resistance for every C6-HSL dose. A water's salinity content was adjusted at 26000 ppm (NaCl concentration) to simulate the salinity of the formation water sample. The concentrations of the C6-HSL that were used for extrapolating the calibration curve varied from 50 to 1000 ppm. The total true resistance (ZR) was a sign of the C6-HSL concentration changing. The ZnO/PANI-DSBA and the Fe2O3/PANI-DSBA sensors were used for the calibration. The EIS spectra of the known concentrations of the C6-HSL were provided from the calibration curve. The electrical equivalent circuit (EEC) was obtained from the fitted EIS ( see Figs. 4a-d) The Electrochemical parameters of the EEC are defined as follows; RSM is sensing materials resistance, Rct signifies charge transfer resistance and n is CPE exponent and it has values ranging from − 1 to 1, and (W) is a Warburg impedance [53, 54].
The EEC parameters of the detected C6-HSL at different concentrations were obtained by the fabricated sensors based on both sensing materials, ZnO/PANI-DSBA and Fe2O3/PANI-DSBA (Table 3). The standard calibration curve was created by plotting the estimated Rct (Y-axis) against known concentrations of the C6-HSL (X-axis). According to the enclosed Figs. 4c and d, the presence of the C6-HSL with various concentrations (50-1000 ppm) for both sensors resulted in a quasi-linear correlation with Rct and the general equation was (Y = slope X + intercept), where the equations were (Y= -0.05406X + 66.3, r2 = 0.954) and (Y = Y=-0.06705x + 79.6, r2 = 0.961) for the ZnO/PANI-DSBA and the Fe2O3/PANI-DSBA-based sensors, respectively. The LOD for the C6-HSL was determined to be 739 and 484 ppm for the ZnO/PANI-DSBA and the Fe2O3/PANI-DSBA-based sensors, respectively. The used formula for LOD was equal to 3s/m where s represents the standard deviation of Rct in the blank sample (n = 3), and m corresponds to the slope of the relative calibration curves of the C6-HSL. Table S1 exhibits a comparison between our work and recently reported nanomaterial-based electrochemical methods for the determination of quorum sensing molecules. Through our investigation, we discovered that our sensor exhibits a greater accuracy and is more efficient to detect the C6-HSL molecules. As shown from the calibration curves of both sensors, the Rct values were decreased by increasing the C6-HSL concentrations [13, 14]. The theory of determining the concentration of bacterial signals mainly relies on the formation of a complex between the bacterial signals and the different metals included in the composition of the two prepared composites, ZnO/PANI-DSBA and Fe2O3/PANI-DSBA. Briefly; it is well known that the C6-HSL contains a lactone group, and this lactone group contains an oxygen atom that has unpair electrons. Simply, the oxygen atom donated these unshared electrons to the “d” or “f” orbitals of the metals embedded in the composites and form a coordination bond as follows:
| Parameters C6 – HSL conc. (ppm) | RS (Ω.cm2) | Cdl (F.cm− 2) | RSM (Ω.cm-2) | Q (F.cm− 2) | n | Rct (Ω.cm− 2) | W (Ω−1.cm− 2 s1/2) | RSE (%) |
|---|---|---|---|---|---|---|---|---|
| ZnO/PANI-DSBA-based sensor | ||||||||
| 0 | 1.97E + 01 | 3.20E-06 | 1.44E + 02 | 3.84E-04 | 4.07E-01 | 8.38E + 01 | 6.05E-03 | 11.31 |
| 50 | 2.17E + 01 | 6.93E-04 | 1.35E + 02 | 1.92E-04 | 5.76E-01 | 5.86E + 01 | 8.20E-03 | 13.50 |
| 125 | 1.09E + 01 | 5.32E-03 | 9.43E + 01 | 4.48E-04 | 5.41E-01 | 4.67E + 01 | 7.14E-03 | 8.55 |
| 250 | 7.87E + 00 | 1.72E-08 | 6.71E + 01 | 3.86E-04 | 5.10E-01 | 3.67E + 01 | 1.09E-02 | 10.54 |
| 500 | 7.68E + 00 | 2.20E-06 | 4.22E + 01 | 4.22E-04 | 5.10E-01 | 2.83E + 01 | 1.94E-02 | 8.29 |
| 750 | 7.13E + 00 | 3.94E-6 | 4.01E + 01 | 2.93E-04 | 5.01E-01 | 19.3E + 01 | 1.56E-02 | 10.15 |
| 1000 | 6.93E + 00 | 1.14E-04 | 3.79E + 01 | 1.81E-02 | 5.62E-01 | 1.34E + 01 | 1.09E-04 | 11.23 |
| Fe2O3/PANI-DSBA-based sensor | ||||||||
| 0 | 1.43E + 01 | 6.07E-08 | 1.28E + 02 | 1.04E-05 | 7.46E-01 | 1.04E + 02 | 1.04E-04 | 9.81 |
| 50 | 1.28E + 01 | 4.82E-09 | 9.76E + 01 | 1.26E-05 | 7.47E-01 | 8.67E + 01 | 1.25E-02 | 15.46 |
| 125 | 9.46E + 00 | 2.19E-05 | 9.83E + 01 | 1.54E-04 | 5.49E-01 | 6.31E + 01 | 1.17E-02 | 13.48 |
| 250 | 6.24E + 00 | 1.07E-06 | 5.29E + 01 | 3.10E-04 | 4.98E-01 | 5.43E + 01 | 1.18E-02 | 7.88 |
| 500 | 7.95E + 00 | 2.77E-06 | 5.29E + 01 | 1.88E-04 | 6.56E-01 | 2.82E + 01 | 1.33E-02 | 8.47 |
| 750 | 1.14E + 01 | 1.93E-05 | 4.21E + 01 | 1.56E-04 | 5.23E-01 | 1.69E + 01 | 1.76E + 02 | 11.42 |
| 1000 | 6.45E + 00 | 1.15E-04 | 3.79E + 01 | 1.72E-02 | 5.77E-01 | 1.19E + 01 | 1.17E-03 | 12.77 |
The formation of a coordination complex between the lactone group as a dentate, and the metals as ligands, led to an enhancement of the interaction at the working electrode/metals interface. That means the greater the bacterial growth, the greater the formation of the complex via the sensor/medium interface. Therefore, the Rct was diminished with the C6-HSL increment.
3.2.3. Real sample measurements
To determine the different concentrations of the C6-HSL after SRB inoculation for varied periods (2–4 weeks). The SRB bacteria were allowed to be enriched and then the produced C6-HSL signals were estimated by the fabricated sensors. The measured unknown concentrations (ppm) of the C6-HSL presented in the formation water of oil wells, containing SRB bacteria with the relative standard deviation (RSD), can be seen in Table 4, where every sample was tested three times. As seen in Figs. 5a and b for both ZnO/PANI-DSBA and Fe2O3/PANI-DSBA based fabricated sensors, the bacteria counts increased by time and relatively the C6-HSL signals as well increased, which means an increasing in the conductivity as well as diminishing the resistivity. These findings confirmed the occurrence of the naturally developed biofilm, in the collected formation water, and displayed its impact on carbon steel, as observed in the previous section. It was noted that the CR increased over time due to bacterial growth and biofilm formation. The results also confirmed the increment of the C6-HSL over time (from 2 to 4 weeks).
| Time of sensing by different sensors | Rct value (Ω.cm− 2) | C6-HSL concentration (ppm) | n | RSD |
|---|---|---|---|---|
| ZnO/PANI-DSBA after 2 weeks | 3.42E + 01 | 570.2 | 3 | ± 2.97 |
| ZnO/PANI-DSBA after 4 weeks | 0.94E + 01 | < 1000 | 3 | ± 1.54 |
| Fe2O3/PANI-DSBA after 2 weeks | 5.36E + 01 | 333.7 | 3 | ± 6.71 |
| Fe2O3/PANI-DSBA after 4 weeks | 0.53E + 01 | < 1000 | 3 | ± 1.86 |
The similar attitude of both sensors was attributed to the presence of metal oxide on the fabricated sensors where they follow the capacitance behaviour. On the other hand, when we measured the unknown concentrations of the actual sample after 2 and 4 weeks of growth time with the sensor fabricated only with PANI-DBSA, the performance of the sensor still followed the role of conductivity of SRB-solution that increased with the bacteria incubation period (see Figure S10). This is explanted by the ability of the PANI-DBSA to interact with a carbonyl group of the C6-HSL and form amide bonds. However, the pattern of Nyquist-EIS significantly differed from the sensors containing composites of the PANI-DSBA or the Fe2O3 or ZnO. In conclusion, both sensors observed the same attitude to detect the C6-HSL in the contaminated formation water with a slight superiority to the ZnO/PANI-DBSA, this may be attributed to the electrocatalytic activity and fast electron transfer properties of the ZnO as well as its nanomorphology.
3.3. Colorimetric confirmation of C6-HSL
The C6-HSL production from the enriched-SRB biofilm was calorimetrically calculated and displayed in Fig. 6. The result was calculated as the C6-HSL intensity i.e. +1 which indicates the medium quantity of the C6-HSL production [55]. It has been reported that SRB-biofilms use the C6-HSL signal molecules for metabolic activity and interspecies communication [56]. Furthermore, Sivakumar et al. (2019) described a potential correlation between the C6-HSL intensity and SRB-biofilm in a salinity medium [16]. Moreover, the increased expression of biofilm-related genes and SRB-enzyme`s productivity were linkages between the produced C6-HSL and transcriptomic sulfate [16].
Two different MOx/CPs nanocomposites; the ZnO/PANI-DBSA and the Fe2O3/PANI-DBSA were successfully synthesized and characterized chemically and morphologically using FT-IR, Raman, SEM, TEM, DLS, and XRD analyses. The prepared nanocomposites were mixed with 1% by-weight conductive carbon paste over the carbon working electrode to detect the C6-HSL via an electrochemical analysis. The OCP and EIS data corresponding to SRB showed that the rate of corrosion accelerated with the extension of the immersion time into the target media due to the SRB activity which further led to a stable phase as a result of the formation of the biofilm passive layer. The detection limit for C6-HSL was 624 and 441 ppm for the ZnO/PANI-DBSA and the Fe2O3/PANI-DBSA, respectively, with a linear range of 50-1000 ppm. Furthermore, the electrochemical assessment of the produced C6-HSL using SPE after SRB incubation periods (2–4 weeks) confirmed that the Rct values decreased with increasing the C6-HSL concentrations, meaning that with increasing the conductivity and decay of the resistivity as a result of SRB-biofilm formation on the interface of the SPE. The colorimetric assessment of C6-HSL confirmed that the growth of SRB-biofilm under salinity conditions was correlated to the intensity of C6-HSL produced. Considering these factors, the new method using the ZnO/PANI-DBSA and the Fe2O3/PANI-DBSA as sensing materials for the SPE presents a practical and advantageous approach for accurately measuring the C6-HSL concentration. This procedure was effective, simpler and cheap compared with other methods used to quantitative measure the C6-HSL. The ZnO/PANI-DBSA-based sensor showed the highest sensitivity towards C6-HSL than the Fe2O3/PANI-DBSA. This may be attributed to the higher electron transfer properties and the electrocatalytic activity of the ZnO as well as its nanomorphology. Overall, the utilization of the ZnO/PANI-DBSA and the Fe2O3/PANI-DBSA in the SPE presents an easy and sensitive approach for monitoring the C6-HSL released by SRB in oil & gas wells, contributing to improved understanding and management of microbial activity in these environments.
Acknowledgements
This work was financially supported by ASRT (Call 3, NO. 28).
Declaration of Interest
The authors do not have any conflicts of interest to declare.
Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.
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No competing interests reported.