Effect of Electro-Oil Acclimation of an Indigenous Strain on the Performance of Sediment Microbial Fuel Cells (SMFC)

Abstract

Sediment microbial fuel cell (SMFC) is a type of MFC without a proton exchange membrane. However, SMFC have had problems with low-power production performance. In this paper, the effects of native bacteria (K1) in oily sludge and their electro-oil-induced domestication on the power generation and oil removal performance of SMFC were studied. The results showed that K1 belonged to Ochrobactrum intermedium. During the domestication process, an upward trend was shown in the OD600 and ORP values in the culture medium, and it grown best at 0.7 V. Ochrobactrum intermedium K1 significantly increased the average output voltage, electromotive force, and maximum power density of SMFC and reduced the apparent internal resistance of the battery. The maximum power density was 169.43 mW/m³, which was 8.59 times higher than that of the control group. Ochrobactrum intermedium K1 improved the degradation of crude oil by SMFC. Ochrobactrum intermedium K1 enhanced the degradation of high-carbon alkanes and even-carbon alkanes in n-alkanes. Cyclic voltammetry and chronoamperometry tests showed that after acclimation, Ochrobactrum intermedium K1 improved the extracellular electron transfer efficiency (EET) mediated by c-Cyts and flavin by increasing the surface protein redox potential.

1. Introduction

Oil-bearing sludge is a pollutant generated during the process of oil exploration and the development and treatment of oil-bearing wastewater. Oil-bearing sludge contains a large amount of aged crude oil, waxes, colloids, asphaltenes, suspended solids, heavy metals, and other substances [1,2]. Due to its special properties, a large amount of oily sludge creates a serious burden to the surrounding ecosystem without treatment or improper treatment, which directly threatens the plants and animals living in this environment. Presently, oily sludge treatment involves oil sludge separation or a high-temperature pyrolysis process which has the risk of secondary pollution. Sedimentary microbial fuel cells (SMFC) can reduce the risk of secondary pollution and have high energy recovery [3,4].

SMFC is a green and environmentally friendly method, and it is widely used in the removal process of pollutants mainly based on organic matter, including water pollution treatment, soil pollution treatment, residual food treatment and other processes [5]. SMFC is a type of MFC with a simple structure in which the anode material is embedded in an anaerobic deposit and the cathode electrode is suspended in oxygen-rich water and connected to the external circuit by wires. SMFC does not require a proton exchange membrane and is less expensive than common MFCs, converting biomass energy into electricity while catabolizing sediments [5]. Aijie Wang [6] developed a three-dimensional floating biological cathode (FBC) sediment microbial fuel cell (SMFC) for power generation and biodegradation of sediment organic matter, with a maximum power density of 1.00 W·m⁻³. Tian-shun Song et al. [6] constructed the SMFC of activated carbon fiber felt nitric acid treatment electrode, and the maximum power density of the battery reached 74.5 mW m⁻². Xun Xu et al. [7] in order to enhance the biodegradation of organic matter in sedimentary microbial fuel cells (SMFCs), added iron oxide (III) as an alternative electron acceptor to the sediment, which yielded a maximum power density and maximum voltage of 87.85 mW/m² and 0.664 V, respectively. In summary, SMFCs effectively enhance the biodegradation and power generation of organic matter in sediments [8].

Electrochemically active bacteria are the key components of SMFC. Electrochemically active bacteria catalyze the degradation of organic pollutants such as petroleum hydrocarbons and convert the chemical energy from the degradation process into electrical energy for SMFC power generation [9]. The SMFC treatment of anode substrate was constructed by Guo et al. [10]. Mixed bacteria are currently the most common form of inoculation used in SMFC anode studies. The complex interactions between the mixed bacteria make it complicated to study the analysis of the mechanism of the individual action of electrochemically active bacteria in the anode substrate [11,12,13]. At the same time, studies have shown that the addition of pure electrochemically active bacteria can better promote battery power generation [14].

There is a competitive relationship between the degradation and utilization of fuel between electrogenic bacteria and non-electrogenic bacteria which makes the power production capacity of composite bacteria worse than that of electric-producing bacteria Q1 alone. Electrochemically active bacteria produce electricity through extracellular electron transfer (EET) in SMFC. EET involves the bidirectional flow and exchange of electrons between intracellular and extracellular redox electron donors and receptors. The outward flow of electrons is a fundamental process by which electrochemically active bacteria exert their electrically productive effects [15,16]. EET includes a direct electron transfer mode with OM c-Cyts or nanowires and a shuttle-mediated indirect electron transfer mode with endogenously secreted soluble flavins, including flavin mononucleotide (FMN) and riboflavin (RF) [17,18]. When heterologously expressed, the biosynthetic pathway of flavin from Bacillus subtilis in the model strain S.oneidensis MR-1 increased the concentration of secreted flavin and increased the maximum power of the battery [19]. It can be seen that the electricity production capacity of electrochemically active bacteria is usually determined by the kinetics and efficiency of EET [20,21]. Electrochemically active monocultures isolated from virgin oil-bearing sludge have an endogenous advantage [22,23]. The efficiency of the bidirectional conversion of chemical energy to electrical energy is greatly limited by weak substrate uptake, small intracellular electron flux, low EET efficiency, and poor biofilm formation [24]. The acclimation process can significantly improve the electricity production efficiency of electrochemically active bacteria [25,26]. In summary, an indigenous bacterium was isolated from the indigenous flora of oily sludge and domesticated by electro-oil induction. The SMFC of oily sludge with this indigenous strain was constructed, and the effects of electro-oil induction and domestication of this indigenous strain on the electricity generation performance and crude oil degradation performance of SMFC were analyzed. The effects of electro-oil-induced acclimation on the EET efficiency and electrochemical activity of this indigenous strain were tested and analyzed.

2. Experimental

2.1. Materials

2.1.1. Chemical Materials

The chemical reagents used, including NaCl, NH₄Cl, NaHCO₃, KCl, KH₂PO₄, CaCl₂, MgCl₂, KCl, NaCl, Na₂HPO₄, KH₂PO₄, HCl, NaOH, dichloromethane, chloroform, methanol, potassium bromide, anhydrous sodium sulfate, and n-hexane, were all analytically pure (≥99.8%) and were purchased from Tianjin Beilian Fine Chemicals Development Co., Ltd. (Tianjin, China). The crude oil used in this work was obtained from Shengli Oilfield. The BR biological reagents used, including peptone and yeast powder, were purchased from Beijing Aoboxing Biotechnology Co., Ltd. (Beijing, China). Various solutions were prepared with deionized water in this study.

2.1.2. Other Materials

The target used in this work was obtained from Shengli Oilfield (China). The water content of oily sludge was 5.48%, the inorganic salt content was 10.23%, and the oil content was 29.62%. The carbon cloth, carbon rod, and carbon felt were purchased from Tianjin Carbon Factory. The conductor was purchased from Casenhua Wire and Cable Factory. The nitrogen and helium were purchased from Tianjin Beilian Fine Chemicals Development Co., Ltd. (Tianjin, China). The Nafion membrane and three-electrode system were purchased from Shanghai Yidian Scientific Instrument Co., Ltd. (Shanghai, China).

2.2. Experimental Methods

2.2.1. Isolation and Identification of Indigenous Bacteria

A total of 10 g of oily sludge was taken in the culture medium, and pure bacteria derived from indigenous flora were isolated by the coating plate method and plate streaking method. The 16 SrDNA sequencing and strain tree alignment of pure bacteria were carried out [27,28]. The band of the DL5000 DNA Marker between 1500 bp was observed, and the sequencing results were spliced by DNAMAN 10.0 software. Finally, the sequence information in the seq file obtained from the sequencing results was input into the blast alignment system in the NCBI data, and similar sequences in the database were searched to obtain the sequence alignment results.

2.2.2. Electric-Oil-Induced Domestication

Pure bacteria derived from indigenous flora were isolated from 10 g oily sludge in a culture solution by the coating plate method and plate streaking method. They were then cultured at 35 °C and 120 r/min for 2 days. The supernatant was used as the material for electro-induced acclimation. Subsequently, 3 g of pure bacteria were added to the electrode solution. In addition to the first step, 10 mL of the bacterial solution after the last domestication stage was added to each domestication stage for culture. The culture medium was applied to the electrochemical workstation (CS150M, Cossite Instruments, Wuhan, China). The voltage was increased (0.6 V, 0.7 V, 0.8 V, 0.9 V, 1.0 V), and increased crude oil (2600 mg/L, 4100 mg/L, 5600 mg/L, 7100 mg/L, 8600 mg/L) was added to the culture medium. Each stage was cultured at 35 °C and 60 r/min for 1 d. Finally, the culture medium was taken out every 6 h, and the absorbance was measured using an ultraviolet spectrophotometer (UV-2600, Spectrum Analysis Detection Technology Co., Ltd., Shanghai, China), and the ORP was measured using a redox activity analyzer (ORPT-6000, Xuantian Environmental Technology Co., Ltd., Shanghai, China). Each group of data was measured three times, and the error was calculated.

2.2.3. Construction of SMFC

The schematic diagram of SMFC is shown in Figure 1. The reaction chamber was cylindrical, the effective volume was 2 L, the upper part was the cathode area, and the lower part was the anode area. The distance between the anode and cathode was 14 cm. The pretreated carbon felt was used as a wire for the anode and cathode. The anode was embedded in the anode substrate, and the cathode floated on the cathode liquid surface and contacted the air. The two poles were then connected by a wire and a 1000 Ω resistor element and connected to the data collector. The 800 g oily sludge was mixed evenly with 100 mL Anodic liquid and added to the bottom of the SMFC reaction chamber as an anode substrate. The 1 L catholyte was added to the SMFC reaction chamber and covered above the anode substrate. Finally, the SMFC was sterilized, and 10 mL of pure bacteria were added. The strain was not added as the control group, while the SMFC of the pure bacteria before domestication was added as the experimental group 1, and the SMFC of the domesticated pure bacteria was added as the experimental group 2. Next, the performance data of SMFC were recorded.

2.2.4. SMFC Electricity Generation and Oil Removal Test

The battery output voltage was recorded in real-time by the data collector (34970A, KEYSIGHT, Wuhan, China) and uploaded to the computer. The power density curve and polarization curve were obtained by a steady-state discharge experiment on the electrochemical workstation. The apparent internal resistance and electromotive force were calculated by polarization curve fitting. The SMFC power density was calculated by the following Formula (1) [29,30].

(1)$\mathrm{P}=\mathrm{U}\mathrm{I}/\mathrm{V}$

where P is power density, mW/m³; U is voltage, mV; I is current, mA; and V is anode chamber volume, m³.

After 21 days of SMFC operation, 3 g of the anode sediment sample was dried naturally at room temperature. The crude oil was separated from the sample by Soxhlet extraction. The mass was weighed and calculated to obtain the mass fraction of oil before and after [29,31]. Formula (2) is as follows:

(2)${\mathrm{R}}_1={\mathrm{m}}_1/{\mathrm{m}}_2$

where R₁ is oil content, %; m₁ is the mass of crude oil in the sample, g; and m₂ is the sample mass after dehydration, g.

2.2.5. Gas Chromatography-Mass Spectrometry n-Alkanes Component Test

Ten grams of oily sludge samples before and after SMFC degradation were dug and extracted by Soxhlet extraction. Then, 50 mL of the extract was extracted and dehydrated with anhydrous sodium sulfate. After the sample was concentrated and purified, it was diluted in a sample bottle with n-hexane for subsequent testing. The abundance of n-alkanes in the samples was tested using a chromatographic-mass spectrometer (LC-MS 1000, THERMO, Wuhan, China). The relative abundance of each component of n-alkanes was obtained by integrating the peak area of the component in the chromatogram. Gas chromatography-mass spectrometry analysis conditions included an injection temperature of 290 °C and an He carrier gas (flow rate 1.2 mL/min). The electron beam energy of the ion source was 70 eV, and the ion source temperature was 260 °C. The transmission line temperature was 280 °C. The mass scanning range was 50~650 (m/z), and the scanning period was 100 ms.

The parameters that can be used to characterize the biological evolution of n-alkanes mainly include the main peak carbon number, OEP (the mass ratio of odd and even carbon numbers of high carbon number n-alkanes (carbon number > 21)), W (∑ C21−)/W (∑ C22+), Pr/Ph (pristane (Pr) and phytane (Ph)), W (Pr)/W (C17), and the W (Ph)/W (C18) ratio [32,33]. The OEP Formula (3) is as follows:

(3)$\mathrm{O}\mathrm{E}\mathrm{P}={\left(\frac{{\mathrm{C}}_{\mathrm{k}-2}+6{\mathrm{C}}_{\mathrm{k}}+{\mathrm{C}}_{\mathrm{k}+2}}{4{\mathrm{C}}_{\mathrm{k}-1}+4{\mathrm{C}}_{\mathrm{k}+1}}\right)}^{(-1)(\mathrm{k}-1)}$

where k is the peak carbon number (the carbon number of n-alkanes with the largest mass fraction in the sample). w (∑ C21−)/W (∑ C22+) is the ratio of the sum of the mass fractions of n-heneicosane and its preceding n-alkanes to the sum of the mass fractions of n-docosane and its subsequent n-alkanes.

2.2.6. Cyclic Voltammetry and Chronoamperometry Test

At first, a three-electrode system was constructed and connected by an electrochemical workstation. In the three-electrode system, glassy carbon (diameter 3 mm), platinum wire, and Ag/AgCl (saturated KCl solution treatment) were used as the working electrodes, the counter electrode, and the reference electrode, respectively. The potential value was relative to the saturated Ag/AgCl reference electrode. The electrolyte was then treated with nitrogen for more than 15 min to remove oxygen from the electrolyte. After the strain grew to a stable stage, the cells were collected by centrifugation and washed three times with PBS buffer to remove the extracellular substances attached to the cell surface. Finally, the washed bacteria were immediately fixed on the glassy carbon electrode with Nafion membrane for a cyclic voltammetry test and chronoamperometry test. In addition, pure bacteria supernatant needed to be tested. In the cyclic voltammetry test, the potential scanning range was 0.6–1 V, and the scanning rate was 10 mV·s⁻¹. In the chronoamperometry test, 0.05 V and 0.1 V potentials were applied to the working electrode.

3. Results and Discussion

3.1. Strain Identification

A pure strain (K1) was isolated from the oily sludge by the coating plate method and plate streaking method. The 16SrDNA sequencing and species tree comparison of K1 were carried out in advance [34,35]. The band of the K1 DL5000 DNA Marker between 1500 bp was observed. The sequencing results were spliced by DNAMAN 10.0 software. Finally, the sequence data in the seq file obtained from the sequencing results were input into the Blast alignment system in the NCBI data to search for similar sequences in the database to obtain sequence alignment results. As shown in Figure 2 and Figure 3, it can be seen from Figure 2 that the DL5000 DNA Marker of K1 had a clear band between about 1500 bp, and the 16SrDNA of the sample was amplified. Figure 3 shows the phylogenetic tree and homology of K1. From Figure 3, it can be seen that K1 has the highest similarity with Ochrobactrum intermedium strain NBRC 15820, with a homology of 99.93%. Its similarity with other strains of the genus Ochrobactrum is more than 99%, so K1 can be classified as Ochrobactrum intermedium [36].

3.2. Electric-Oil-Induced Domestication

The change in the Ochrobactrum intermedium K1 cell concentration is reflected in the change in OD600 (absorbance at 600 nm wavelength) [4]. The ORP (oxidation-reduction potential) directly reflects the redox ability of the environment in which micro-organisms are located and are also characterized by the metabolism of micro-organisms [37]. The changes of OD600 and ORP (redox potential) in the Stage 1–Stage 5 medium induced by electro-oil are shown in Figure 4. In Stage 1 (the acclimation time was 0–24 h), the voltage and crude oil applied in the stress environment were 0.6 V and 2600 mg/L, respectively. OD600 and ORP increased slowly with time, and Ochrobactrum intermedium K1 increased slowly. The Ochrobactrum intermedium K1 began to withstand the stress of crude oil and the current environment. The ORP value had a downward trend in the early stage, indicating that Ochrobactrum intermedium K1 was reducing metabolism to ensure proliferation. In Stage 2 (the acclimation time was 24–48 h), the applied voltage and crude oil in the stress environment were 0.7 V and 4100 mg/L, respectively. The OD600 and ORP increased rapidly with time. In general, OD600 increased greatly in the later period, indicating that Ochrobactrum intermedium K1 needed a long time to adapt to the stressful environment. In Stage 3 (the acclimation time was 48–72 h), the applied voltage and crude oil in the stress environment reached 0.8 V and 5600 mg/L, respectively. OD600 increased rapidly in the middle and late stages of the early stage, while ORP increased rapidly in the early stage and decreased in the late stage. The metabolism of Ochrobactrum intermedium K1 in the early stage was rapidly used to adapt to the stressful environment, and the late adaptation was completed for proliferation. In Stage 4 (the acclimation time was 72–96 h), the voltage and crude oil applied in the stress environment had reached 0.9 V and 7100 mg/L, respectively. There were changes in OD600 and ORP over time, and there was an upward trend. Compared to other stages, the comprehensive effect of the fourth stage was the best, probably because the stress environment Ochrobactrum intermedium K1 was the most suitable. In Stage 5 (the acclimation time was 96–120 h), the applied voltage and crude oil in the stress environment reached 1 V and 8600 mg/L, respectively. OD600 and ORP increased slowly with time, indicating that Ochrobactrum intermedium K1 metabolism and proliferation slowed down because the high-stress conditions gradually approached the critical value of Ochrobactrum intermedium K1 short-term tolerance. Ochrobactrum intermedium K1 proliferation did not decline, indicating that Ochrobactrum intermedium K1 can still withstand the stressful environment. From Stage 1 to Stage 5, the OD600 and ORP in the culture medium have a phased upward trend which shows that the Ochrobactrum intermedium K1 cell concentration and redox ability of Ochrobactrum intermedium K1 in the culture medium increases, the metabolic activity becomes higher, and Ochrobactrum intermedium K1 gradually adapts to the increasing voltage and crude oil stress environment. Among them, the voltage of 0.7 V is the most suitable for strain growth. Therefore, it is expected to obtain Ochrobactrum intermedium K1 which adapts to higher stress conditions by extending the acclimation time while maintaining the unchanging stress conditions or adjusting the introduction of new stimulating strain growth conditions. EET efficiency is an important factor in Ochrobactrum intermedium K1 metabolism, and the EET efficiency of Ochrobactrum intermedium K1 may be improved in this process.

3.3. Analysis of the Effect of Strain K1 on SMFC

The optimum growth temperature of Ochrobactrum intermedium is about 30 °C in a weak alkaline environment. SMFC proton consumption becomes faster and more conducive to the electrochemical activity of micro-organisms, accelerating the transfer and consumption of electrons, thus accelerating its metabolism [37,38]. Figure 5 and Figure 6 show the experimental results of the SMFC electricity generation performance of the control group, experimental group 1, and experimental group 2. From Figure 5 and Figure 7, we can see that the average output voltage of the control group was 60.49 mV, and the battery electromotive force of the longitudinal axis intercept of the polarization curve was 189.68 mV. The apparent internal resistance of the slope was 716.6 Ω, and the maximum power density of the power density curve was 18.21 mW/m³. The original properties of SMFC under relatively sterile conditions were observed. The control group was set to observe the original performance of SMFC under relatively sterile conditions. The average output voltage of experimental group 1 was 236.72 mV. The longitudinal intercept of the polarization curve showed that the electromotive force of the battery was 398.54 mV. The slope showed that the apparent internal resistance was 683.6 Ω, and the power density curve showed that the maximum power density was 118.96 mW/m³. The average output voltage of experimental group 2 was 283.10 mV, which was 4.68 times higher than that of the control group. The polarization curve of the longitudinal axis intercept performance of the battery electromotive force was 654.4 mV, which was 3.45 times higher than the control group of the battery electromotive force. The apparent internal resistance of the slope was 664.5 Ω, which was slightly lower than that of the control group. The power density curve showed that the maximum power density was 169.43 mW/m³, which was 8.59 times higher than that of the control group. These data comprehensively reflect the power generation performance of the battery. The Ochrobactrum intermedium K1 was enriched on the anode of the SMFC, and the organic matter was oxidized for the metabolic process. The generated electrons were transmitted to the cathode through an external circuit and bind to the electron acceptor at the cathode. The EET process of the Ochrobactrum intermedium K1 continuously transfers the generated electrons and forms a current loop in the SMFC [39,40]. The experimental results showed that the electro-oil-induced acclimated Ochrobactrum intermedium K1 promoted the electrical performance of SMFC, and the electrochemical activity of Ochrobactrum intermedium K1 was improved after acclimation.

The oil removal performance of SMFC is positively correlated with the electricity production performance, and Ochrobactrum intermedia was widely used in petroleum hydrocarbon degradation [41,42]. Figure 8 shows the change in the oil content before and after SMFC treatment of oily sludge in the control group, experimental group 1, and experimental group 2. From Figure 8, it can be seen that the oil content of the anode sediment after SMFC treatment in the control group was 24.98%, and the oil removal rate of the battery was 15.65%. The oil content of the anode sediment in experimental group 1 was 18.03%, and the oil removal rate of the battery was 37.6%. The lowest oil content of the anode sediment after SMFC treatment in experimental group 2 was 14.23%, and the oil removal rate of the battery was 47.81%. The oil removal rate of the battery for experimental group 1 was 1.27 times. Ochrobactrum intermedium K1 significantly improved the oil removal effect of SMFC, and the electro-oil-induced domesticated Ochrobactrum intermedium K1 promoted the oil removal of SMFC more than the undomesticated strain.

Figure 9 and Figure 10 show the content of n-alkanes before and after SMFC treatment of oily sludge in the control group. The experimental groups 1 and 2 were valued before SMFC treatment. According to the experimental results in Table 1, the long-chain hydrocarbons were degraded, and the number of main carbon peaks moved to the left. The number of main peak carbons of n-alkanes in experimental group 1 decreased from 26 to 24 before and after the operation of the SMFC, and the number of main peak carbons of n-alkanes in experimental group 2 decreased from 26 to 19 before and after the operation of the SMFC, indicating that the Ochrobactrum intermedium K1 in the SMFC degraded n-alkanes with 24–26 main carbon peaks before domestication, and the Ochrobactrum intermedium K1 in the SMFC degraded n-alkanes with 19–24 main carbon peaks after domestication. The OEP value of experimental group 1 was 1.38 before treatment and 1.37 after treatment, and the OEP value of experimental group 2 was 1.68 after treatment, indicating that domestication improved the degradation of even carbon chain alkanes by Ochrobactrum intermedium K1. The parameter value of W ∑ (C21−)/W ∑ (C22+) in experimental group 1 was 0.72 before treatment and 0.79 after treatment. The parameter value of W ∑ (C21−)/W ∑ (C22+) in experimental group 2 was 0.95 after treatment, indicating that domestication improved the degradation of long-chain alkanes by Ochrobactrum intermedium K1 in SMFC. In the process of microbial degradation of crude oil, W ∑ (C21−)/W ∑ (C22+) will increase or first increase and then decrease [43]. This is due to the obvious demethylation of high-carbon alkanes during SMFC treatment where high-carbon alkanes were more easily degraded, resulting in the selective degradation of high-carbon alkanes by Ochrobactrum intermedium K1 [44]. The parameter value of W (Pr)/W (W17) was 1.79 before treatment and 1.96 after treatment in experimental group 1. The parameter value of W (Pr)/W (W17) in experimental group 2 was 2.25. The W (Ph)/W (C18) value of experimental group 1 was 0.78 before treatment and 0.82 after treatment. W (Ph)/W (C18) was 0.85 in experimental group 2. W (Pr)/W (C17) and W (Ph)/W (C18) are the correlation parameters of pristane (Pr) with the adjacent C17 alkanes and phytane (Ph) with the adjacent C18 alkanes, respectively. The greater the value is, the higher the degradation rate of n-alkanes is [45]. The two values of experimental group 1 and experimental group 2 of oily sludge increased after SMFC treatment, indicating that Ochrobactrum intermedium K1 improved the SMFC treatment of n-alkanes in oily sludge, and the domesticated Ochrobactrum intermedium K1 was better. The degradation products of n-alkanes also provide a sufficient available carbon source for micro-organisms which is more conducive to the growth of the Ochrobactrum intermedium K1 and the operation of SMFC.

3.4. Electrochemical Activity Analysis

The mechanism of electron transfer was tested by cyclic voltammetry. According to the experimental results in Figure 11, there were two oxidation peaks at −0.1 V and −0.4 V and one reduction peak at −0.2 V [45] in the cyclic voltammetry test of the three-electrode system for Ochrobactrum intermedium K1 before electro-oil acclimation. The direct extracellular electron transfer of the corresponding peak potential was caused by cytochrome c on the cell surface. There were redox active substances on the cell surface of Ochrobactrum intermedium K1, and electron transfer occured between the electrode. The redox potential of flavin produced by the Ochrobactrum intermedium K1 corresponds to −0.4 V by indirect extracellular electron transfer between flavin and electrode [38]. During Ochrobactrum intermedium K1 metabolism, flavin is transferred to the cell surface to contact cytochrome c, and ferrous ions in cytochrome c are converted into iron ions, releasing electrons into the electrode to form a current loop. Flavin can be synthesized by most micro-organisms, and its adsorption on Ochrobactrum intermedium K1 cells or electrode surfaces is not easily cleaned. Therefore, there is no redox peak current response in the cyclic voltammogram of the blank medium [39,40]. According to the experimental results in Figure 12, in the cyclic voltammetry test of the three-electrode system, Ochrobactrum intermedium K1 after electro-oil acclimation had two oxidation peaks at −0.1 V and −0.4 V and a reduction peak at −0.2 V. The area of the cyclic voltammogram increased significantly, and the electrochemical activity of Ochrobactrum intermedium K1 was significantly improved. Ochrobactrum intermedium is a Gram-negative bacterium which is generally an EET mode of endogenously secreted soluble flavins (including flavin mononucleotide (FMN) and riboflavin (RF)) for shuttle-mediated indirect electron transfer [40].

There were redox-active substances on the surface of Ochrobactrum intermedium K1. Chronoamperometry analysis verified that the redox-active substances on the surface of the bacteria participated in the electron transfer process of bacterial metabolism. The results of chronoamperometry are shown in Figure 12. Figure 13 and Figure 14 show that the current response curve of Ochrobactrum intermedium K1 before electro-oil acclimation was applied with 0.05 V and 0.1 V potential at the working electrode, respectively. The metabolic bacteria automatically adjusted the ratio of the oxidation state and the reduction state of the redox active protein on the cell surface according to the working electrode potential. That is, the combination of cytochrome c and the riboflavin secreted by the bacteria changed the potential of the final electron donor on the cell surface to be lower than the electrode potential, thereby promoting Ochrobactrum intermedium K1 metabolism to produce electrons that were transferred from the bacteria to the electrode surface, resulting in an increase in current response at both potentials [45,46]. The current response curve of the Ochrobactrum intermedium K1 after electro-oil acclimation increased more rapidly than that of the Ochrobactrum intermedium K1 before acclimation, and the peak current was higher; that is, the redox-active substance adjustment ability of Ochrobactrum intermedium K1 became stronger after acclimation, and the electrochemical activity was enhanced. When the working electrode potential was set to 0.05 V, the peak current response was caused by the oxidation reaction at −0.4 V, and when the working electrode potential was set to 0.1 V, the peak response of the current was caused by the oxidation reaction at about −0.4 V and−0.1 V. It was further confirmed that after domestication, Ochrobactrum intermedium K1 cells themselves could adjust the redox potential of surface proteins and adapt to the state of electrical stress, thereby improving the electrochemical activity.

4. Conclusions

This study focuses on the problem of low production voltage of SMFC. In this paper, the effects of protobacteria (Ochrobactrum intermedium K1) and their electro-oil-induced domestication on the power generation and de-oiling performance of SMFC were studied by gas chromatography-mass spectrometry and cyclic voltammetry. The conclusions are as follows:

Ochrobactrum intermedium K1 with electrochemical activity was isolated from oily sludge. The results of 16 SrDNA detection showed that Ochrobactrum intermedium K1 was Ochrobactrum intermedium. The OD600 and ORP values of the culture medium showed an upward trend at each stage. The electro-oil induction and domestication effect was good, and the Ochrobactrum intermedium K1 gradually adapted to the high-stress environment., It grows best at 0.7 V.

After electro-oil-induced domestication, the area of the cyclic voltammetry curve of Ochrobactrum intermedium K1 increased, and the peak response of the chronoamperometry current increased. The electro-oil-induced domestication improved the electrochemical activity of Ochrobactrum intermedium K1, which was mainly reflected in the fact that Ochrobactrum intermedium K1 changed the protein potential of cells and increased the EET efficiency of indirect electron transfer mediated by cytochrome C and flavin.

The electro-oil-induced acclimated Ochrobactrum intermedium K1 significantly increased the average output voltage, battery electromotive force, and maximum power density of the SMFC and reduced the apparent internal resistance of the battery. The maximum power density was 169.43 mW/m³, which was 8.59 times higher than that of the control group. The biological evolution of n-alkanes showed that the domestication process promoted the degradation of n-alkanes by Ochrobactrum intermedium K1 in SMFC, especially high-carbon alkanes and even-carbon alkanes.

Author Contributions

Conceptualization, Y.P. and C.H.; methodology, Y.P.; software, J.G.; validation, S.T., W.R. and Y.C.; formal analysis, Y.P.; investigation, K.F.; resources, S.T.; data curation, S.T.; writing—original draft preparation, Y.P.; writing—review and editing, Y.P.; visualization, Y.C.; supervision, S.T.; project administration, S.T.; funding acquisition, Y.P. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

Due to legal constraints, the data collected for this study cannot be made publicly available.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

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Figures and Table

Figure 1. The diagram of SMFC construction (1, data acquisition system; 2, external resistance; 3 and 4, battery cathode and anode, respectively).

Figure 2. DNA Marker bands of Ochrobactrum intermedium K1.

Figure 3. Phylogenetic tree and homology of Ochrobactrum intermedium K1.

Figure 4. Changes in OD600 and ORP values of electro-oil-induced acclimation in Stage 1–Stage 5 culture medium.

Figure 5. Power density curve and polarization curve of SMFC in the control group, experimental group 1, and experimental group 2.

Figure 6. The oil content of oily sludge before and after SMFC treatment in the control group, experimental group 1 and experimental group 2.

Figure 7. The output voltage of SMFC in the control group, experimental group 1, and experimental group 2.

Figure 8. The content of n-alkanes in the control group, experimental group 1, and experimental group 2 before and after SMFC treatment.

Figure 9. The contents of n-alkanes before and after SMFC treatment in the control group, experimental group 1, and experimental group 2.

Figure 10. Cyclic voltammetry curves of Ochrobactrum intermedium K1 before electro-oil-induced acclimation.

Figure 11. Cyclic voltammetric curves of Ochrobactrum intermedium K1 after Electro-Oil Induction.

Figure 12. The curve of chronoamperometric current of the Ochrobactrum intermedium K1 before electro-oil induction and domestication.

Figure 13. The curve of chronoamper of Ochrobactrum intermedium K1 after electro-oil induction and domestication.

Figure 14. The EET diagram of Ochrobactrum intermedium K1.

Table 1

Biological evolution parameters of n-alkanes before and after adding Ochrobactrum intermedium K1 and supernatant MFC and control group.

Parameter	Main Peak Carbon Number		OEP		W∑(C21−)/W∑(C22+)		W(Pr)/W(W17)		W(Ph)/W(C18)
	Before	After	Before	After	Before	After	Before	After	Before	After
Group 1	26	24	1.38	1.37	0.72	0.79	1.79	1.96	0.78	0.82
Group 2	26	19	1.38	1.68	0.72	0.95	1.79	2.25	0.78	0.85

References

1. Ge, C.; Lei, D.; Liu, S.; Shen, Y.; Liu, Z.; Shan, H. Pilot Studies on Treatment of Oily Solid Waste with Ex Situ Smoldering Remediation Technology. Chin. J. Environ. Eng.; 2022; 16, pp. 601-611. [DOI: https://dx.doi.org/10.12030/j.cjee.202108168]

2. Liu, F.; Liu, H.; Zhu, H.; Xie, Y.; Zhang, D.; Cheng, Y.; Zhang, J.; Feng, R.; Yang, S. Remediation of petroleum hydrocarbon contaminated groundwater by biochar immobilized bacterial consortia. Environ. Chem.; 2022; 41, pp. 3436-3447. [DOI: https://dx.doi.org/10.7524/j.issn.0254-6108.2021062601]

3. Pinedo, J.; Ibáñez, R.; Lijzen, J.P.A.; Irabien, A. Assessment of soil pollution based on total petroleum hydrocarbons and individual oil substances. J. Environ. Manag.; 2013; 130, pp. 72-79. [DOI: https://dx.doi.org/10.1016/j.jenvman.2013.08.048] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/24064142]

4. Yang, Y.; Ding, Y.; Hu, Y.; Cao, B.; Rice, S.A.; Kjelleberg, S.; Song, H. Enhancing bidirectional electron transfer of Shewanella oneidensis by a synthetic flavin pathway. ACS Synth. Biol.; 2015; 4, pp. 815-823. [DOI: https://dx.doi.org/10.1021/sb500331x]

5. Song, X.; Wang, W.; Cao, X.; Wang, Y.; Zou, L.; Ge, X.; Zhao, Y.; Si, Z.; Wang, Y. Chlorella vulgaris on the cathode promoted the performance of sediment microbial fuel cells for electrogenesis and pollutant removal. Sci. Total Environ.; 2020; 728, 138011. [DOI: https://dx.doi.org/10.1016/j.scitotenv.2020.138011]

6. Xia, C.; Xu, M.; Liu, J.; Guo, J.; Yang, Y. Sediment microbial fuel cell prefers to degrade organic chemicals with higher polarity. Bioresour. Technol.; 2015; 190, pp. 420-423. [DOI: https://dx.doi.org/10.1016/j.biortech.2015.04.072]

7. Wang, A.; Cheng, H.; Ren, N.; Cui, D.; Lin, N.; Wu, W. Sediment microbial fuel cell with floating biocathode for organic removal and energy recovery. Front. Environ. Sci. Eng.; 2012; 6, pp. 569-574. [DOI: https://dx.doi.org/10.1007/s11783-011-0335-1]

8. Song, T.; Tan, W.; Wu, X.; Zhou, C.C. Effect of graphite felt and activated carbon fiber felt on performance of freshwater sediment microbial fuel cell. J. Chem. Technol. Biotechnol.; 2012; 87, pp. 1436-1440. [DOI: https://dx.doi.org/10.1002/jctb.3764]

9. Xu, X.; Zhao, Q.; Wu, M.; Ding, J.; Zhang, W. Biodegradation of organic matter and anodic microbial communities analysis in sediment microbial fuel cells with/without Fe (III) oxide addition. Bioresour. Technol.; 2017; 225, pp. 402-408. [DOI: https://dx.doi.org/10.1016/j.biortech.2016.11.126]

10. Guo, H.; Xie, S.; Deng, H.; Geng, X.; Wang, P.; Huang, C.; Tang, S. Electricity production and bacterial communities of microbial fuel cell supplied with oily sludge. Environ. Prog. Sustain. Energy; 2020; 39, e13409. [DOI: https://dx.doi.org/10.1002/ep.13409]

11. Cai, T.; Zhang, Y.; Wang, N.; Zhang, Z.; Lu, X.; Zhen, G. Electrochemically active microorganisms sense charge transfer resistance for regulating biofilm electroactivity spatio-temporal distribution, and catabolic pathway. Chem. Eng. J.; 2022; 442, 136248. [DOI: https://dx.doi.org/10.1016/j.cej.2022.136248]

12. Rui, Z.; Yun, W.; Lutian, W.; Qiang, W.; Hongwei, Z. Cathode Denitrification of Microbial Fuel Cells. Prog. Chem.; 2020; 32, pp. 2013-2021. [DOI: https://dx.doi.org/10.7536/PC200332]

13. Yuan, Y.; Li, J.; Zhang, Z.; Song, B.; Liu, F.; Zhang, J. Effect of carbon cloth-loaded Co₃O₄ nanosheet array anode on the power generation performance of microbial fuel cell. Chin. J. Environ. Eng.; 2022; 16, pp. 2447-2456. [DOI: https://dx.doi.org/10.12030/j.cjee.202104154]

14. Qian, Z.; Yang, L.; Xie, B.; Liu, H.; Liu, H. Isolation and identification of a simultaneous electricity production and denitrification strain in a microbial fuel cell with biocathode and its characteristics. Chin. J. Environ. Eng.; 2019; 13, pp. 1986-1994. [DOI: https://dx.doi.org/10.12030/j.cjee.201811110]

15. You, Z.; Li, F.; Hao, S. Design and construction of electroactive cells by synthetic biology strategies. Synth. Biol. J.; 2022; 3, 1031. [DOI: https://dx.doi.org/10.12211/2096-8280.2022-014]

16. Lin, T.; Ding, W.; Sun, L.; Wang, L.; Liu, C.-G.; Song, H. Engineered Shewanella oneidensis-reduced graphene oxide biohybrid with enhanced biosynthesis and transport of flavins enabled a highest bioelectricity output in microbial fuel cells. Nano Energy; 2018; 50, pp. 639-648. [DOI: https://dx.doi.org/10.1016/j.nanoen.2018.05.072]

17. Yang, Y.; Xu, M.; Guo, J.; Sun, G. Bacterial extracellular electron transfer in bioelectrochemical systems. Process Biochem.; 2012; 47, pp. 1707-1714. [DOI: https://dx.doi.org/10.1016/j.procbio.2012.07.032]

18. Liu, L.; Xiao, Y.; Wu, Y.; Chen, B.; Zhao, F. Electron transfer mediators in microbial electrochemical systems. Prog. Chem.; 2014; 26, 1859. [DOI: https://dx.doi.org/10.7536/PC140740]

19. Shao, Y.-Z.; Che, J.; Cheng, C.; Jiang, Z.-Y.; Xue, C. Advances in Molecular Biological Methods to Improve Extracellular Electron Transport Efficiency of Electroactive Microorganisms. China Biotechnol.; 2021; 41, pp. 50-59. [DOI: https://dx.doi.org/10.13523/j.cb.2102020]

20. Huang, Y.; He, Z.; Xu, L.; Yang, B.; Hou, Y.; Lei, L.; Li, Z. Alternating current enhanced bioremediation of petroleum hydrocarbon-contaminated soils. Environ. Sci. Pollut. Res.; 2021; 28, pp. 47562-47573. [DOI: https://dx.doi.org/10.1007/s11356-021-13942-2]

21. Zhang, Z.; Zhong, W.; Mu, H. Refinery residual sludge reduction technologies by using multi-function microbe. J. Petrochem. Univ.; 2010; 23, 23.

22. Cerqueira, V.S.; Hollenbach, E.B.; Maboni, F.; Vainstein, M.H.; Camargo, F.A.O.; Maria do Carmo, R.P.; Bento, F.M. Biodegradation potential of oily sludge by pure and mixed bacterial cultures. Bioresour. Technol.; 2011; 102, pp. 11003-11010. [DOI: https://dx.doi.org/10.1016/j.biortech.2011.09.074] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/21993328]

23. Da Silva, L.J.A.; Alves, F.C.; de França, F.P. A review of the technological solutions for the treatment of oily sludges from petroleum refineries. Waste Manag. Res.; 2012; 30, pp. 1016-1030. [DOI: https://dx.doi.org/10.1177/0734242X12448517] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/22751947]

24. ElMekawy, A.; Hegab, H.M.; Dominguez-Benetton, X.; Pant, D. Internal resistance of microfluidic microbial fuel cell: Challenges and potential opportunities. Bioresour. Technol.; 2013; 142, pp. 672-682. [DOI: https://dx.doi.org/10.1016/j.biortech.2013.05.061] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/23747174]

25. Ma, D.; Jiang, Z.-H.; Lay, C.-H.; Zhou, D. Electricity generation from swine wastewater in microbial fuel cell: Hydraulic reaction time effect. Int. J. Hydrog. Energy; 2016; 41, pp. 21820-21826. [DOI: https://dx.doi.org/10.1016/j.ijhydene.2016.08.019]

26. Hirose, A.; Kouzuma, A.; Watanabe, K. Towards development of electrogenetics using electrochemically active bacteria. Biotechnol. Adv.; 2019; 37, 107351. [DOI: https://dx.doi.org/10.1016/j.biotechadv.2019.02.007]

27. Modestra, J.A.; Mohan, S.V. Bio-electrocatalyzed electron efflux in Gram positive and Gram negative bacteria: An insight into disparity in electron transfer kinetics. RSC Adv.; 2014; 4, pp. 34045-34055. [DOI: https://dx.doi.org/10.1039/C4RA03489A]

28. Yu, Y.; Wu, Y.; Cao, B.; Gao, Y.-G.; Yan, X. Adjustable bidirectional extracellular electron transfer between Comamonas testosteroni biofilms and electrode via distinct electron mediators. Electrochem. Commun.; 2015; 59, pp. 43-47. [DOI: https://dx.doi.org/10.1016/j.elecom.2015.07.007]

29. Chen, L.; Xiao, C.; Luo, X.; Sun, W. Study on biological degradation and transform characteristics of different components in petroleum hydrocarbon used by bacterial consortium. Environ. Earth Sci.; 2016; 75, 816. [DOI: https://dx.doi.org/10.1007/s12665-016-5599-0]

30. Hobday, A.J.; Smith, A.D.M.; Stobutzki, I.C.; Bulman, C.; Daley, R.; Dambacher, J.M.; Deng, R.A.; Dowdney, J.; Fuller, M.; Furlani, D. et al. Ecological risk assessment for the effects of fishing. Fish. Res.; 2011; 108, pp. 372-384. [DOI: https://dx.doi.org/10.1016/j.fishres.2011.01.013]

31. Velho-Pereira, S.; Kamat, N. Antimicrobial screening of actinobacteria using a modified cross-streak method. Indian J. Pharm. Sci.; 2011; 73, 223. [DOI: https://dx.doi.org/10.4103/0250-474x.91566]

32. Wolcott, R.D.; Hanson, J.D.; Rees, E.J.; Koenig, C.D.; Wolcott, R.A.; Cox, S.B.; White, J.S. Analysis of the chronic wound microbiota of 2,963 patients by 16S rDNA pyrosequencing. Wound Repair Regen.; 2016; 24, pp. 163-174. [DOI: https://dx.doi.org/10.1111/wrr.12370]

33. Narisetty, V.; Prabhu, A.A.; Bommareddy, R.R.; Cox, R.; Agrawal, D.; Misra, A.; Haider, M.A.; Bhatnagar, A.; Pandey, A.; Kumar, V. Development of Hypertolerant Strain of Yarrowia lipolytica Accumulating Succinic Acid Using High Levels of Acetate. ACS Sustain. Chem. Eng.; 2022; 10, pp. 10858-10869. [DOI: https://dx.doi.org/10.1021/acssuschemeng.2c02408]

34. Carmona-Martinez, A.A.; Harnisch, F.; Fitzgerald, L.A.; Schröder, U. Cyclic voltammetric analysis of the electron transfer of Shewanella oneidensis MR-1 and nanofilament and cytochrome knock-out mutants. Bioelectrochemistry; 2011; 81, pp. 74-80. [DOI: https://dx.doi.org/10.1016/j.bioelechem.2011.02.006]

35. Lemos, J.G.; Stefanello, A.; Garcia, M.V.; Furian, A.F.; Cichoski, A.J.; Copetti, M.V. Potential of electrolyzed water to inactivate bread and cheese spoilage fungi. Food Res. Int.; 2022; 162, 111931. [DOI: https://dx.doi.org/10.1016/j.foodres.2022.111931]

36. Light, S.H.; Su, L.; Rivera-Lugo, R.; Cornejo, J.A.; Louie, A.; Iavarone, A.T.; Ajo-Franklin, C.M.; Portnoy, D.A. A flavin-based extracellular electron transfer mechanism in diverse Gram-positive bacteria. Nature; 2018; 562, pp. 140-144. [DOI: https://dx.doi.org/10.1038/s41586-018-0498-z]

37. Kokko, M.E.; Mäkinen, A.E.; Sulonen, M.L.K.; Puhakka, J.A. Effects of anode potentials on bioelectrogenic conversion of xylose and microbial community compositions. Biochem. Eng. J.; 2015; 101, pp. 248-252. [DOI: https://dx.doi.org/10.1016/j.bej.2015.06.007]

38. Kang, M.; Ren, L.; Ren, H.; Zhao, Y.; Kawamura, K.; Zhang, H.; Wei, L.; Sun, Y.; Wang, Z.; Fu, P. Primary biogenic and anthropogenic sources of organic aerosols in Beijing, China: Insights from saccharides and n-alkanes. Environ. Pollut.; 2018; 243 Pt B, pp. 1579-1587. [DOI: https://dx.doi.org/10.1016/j.envpol.2018.09.118]

39. Fan, J.; Okyay, T.O.; Rodrigues, D.F. The synergism of temperature, pH and growth phases on heavy metal biosorption by two environmental isolates. J. Hazard. Mater.; 2014; 279, pp. 236-243. [DOI: https://dx.doi.org/10.1016/j.jhazmat.2014.07.016]

40. Zhai, Q.; Gong, S.; Wang, Y.; Lyu, Q.; Liu, Y.; Ling, Y.; Wang, J.; Simon, G.P.; Cheng, W. Enokitake mushroom-like standing gold nanowires toward wearable noninvasive bimodal glucose and strain sensing. ACS Appl. Mater. Interfaces; 2019; 11, pp. 9724-9729. [DOI: https://dx.doi.org/10.1021/acsami.8b19383]

41. Wang, W.; He, Y.; Wang, B.; Dong, M.; Zhang, H.; Shen, C. Experimental study on wax removal and viscosity reduction of waxy crude oil by Ochrobactrum intermedium. J. Pet. Sci. Eng.; 2022; 213, 110445. [DOI: https://dx.doi.org/10.1016/j.petrol.2022.110445]

42. Cai, L.; Zhang, H.; Feng, Y.; Dong, B.; Wang, Y.; Ge, C. Iron and carbon granules added to anode enhanced the sludge decrement and electrical performance of sludge microbial fuel cell. Chem. Eng. J.; 2019; 372, pp. 572-580. [DOI: https://dx.doi.org/10.1016/j.cej.2019.04.164]

43. Zhao, J.; Wang, L.; Tang, L.; Ren, R.; You, W.; Farooq, R.; Wang, Z.; Zhang, Y. Changes in bacterial community structure and humic acid composition in response to enhanced extracellular electron transfer process in coastal sediment. Arch. Microbiol.; 2019; 201, pp. 897-906. [DOI: https://dx.doi.org/10.1007/s00203-019-01659-3] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30993372]

44. Chen, C.Y.; Chen, T.Y.; Chung, Y.C. A comparison of bioelectricity in microbial fuel cells with aerobic and anaerobic anodes. Environ. Technol.; 2014; 35, pp. 286-293. [DOI: https://dx.doi.org/10.1080/09593330.2013.826254]

45. Wu, M.S.; Xu, X.; Zhao, Q.; Wang, Z.Y. Simultaneous removal of heavy metals and biodegradation of organic matter with sediment microbial fuel cells. RSC Adv.; 2017; 7, pp. 53433-53438. [DOI: https://dx.doi.org/10.1039/C7RA11103G]

46. Wingfors, H.; Hägglund, L.; Magnusson, R. Characterization of the size-distribution of aerosols and particle-bound content of oxygenated PAHs, PAHs, and n-alkanes in urban environments in Afghanistan. Atmos. Environ.; 2011; 45, pp. 4360-4369. [DOI: https://dx.doi.org/10.1016/j.atmosenv.2011.05.049]