Microbial Fuel Cell (MFC) is a bio-electrochemical system that converts chemical energy to electricity by utilizing electricigens as biocatalysts, with practical applications in biomass degradation, sewage disposal, and clean energy generation (Elmaadawy et al. 2022; Ren et al. 2022; Thapa et al. 2022). Electricigens are microorganisms that transfer electrons generated during metabolic processes to the extracellular environment for respiration. When electricigens function as biocatalysts in MFCs, they play vital roles in substrate oxidation and the transfer of electrons (or hydrogen).
The electricity generation process consists of several steps: anodic oxidation, electron transport in an external circuit, proton migration, and cathodic reduction. Generally, two types of inocula are utilized in MFC: mixed culture and pure culture. In mixed culture, microbial interactions offer good adaptability to the environment because the microflora can degrade a wide variety of substrates and facilitate concurrent power generation. Additionally, some microbes secrete specific electron transfer mediators (ETMs), which may contribute to the performance boost of the MFC (Kiely et al. 2011; Islam et al. 2018). However, this synergy effect in microflora does not always happen. Antagonistic interactions and metabolic conflicts between different microbes give rise to a performance decrease in MFC.
In contrast, using simple substrates as carbon sources, such as acetate, formate, citrate, and pyruvate (Zhou et al. 2016; Ali et al. 2017; Zhao et al. 2017), pure culture achieved better performance than mixed culture. Different types of electricigens like
To date, several facultative anaerobic electricigens such as
Previous electrochemical studies have demonstrated that the low power output by electricigens poses a significant hurdle to the industrial applications of MFC (Bello et al. 2017). Therefore, the long-term challenge is to screen superior exoelectrogenic strains with high efficiency and strong environmental adaptability. In this study, a facultative anaerobic strain SQ-1 was isolated from sludge in a biotechnology factory. The electricity generation capacity and ferric reduction of this strain were investigated by using dual-chamber MFCs and an electrochemical workstation.
The activated sludge was collected from an anaerobic waste water tank in New Yangshao Biological Technology Factory, Henan, China. The sample was inoculated in 100 ml of growth medium containing 0.6 g KCl, 1.5 g NH4Cl, 0.3 g KH2PO4, 0.1 g MgCl2, 0.1 g CaCl2, 10 ml of trace element solution, and 10 ml of vitamin solution as previously described (Liu et al. 2014; Zhou et al. 2016). Meanwhile, 20 mM acetate sodium and 40 mM fumarate were supplemented as electron donors and acceptors. The initial pH of the medium was adjusted to 7.2 and autoclaved at 121°C for 15 min. The mixed microflora (or pure bacteria) was incubated at 30°C under aerobic or anaerobic static conditions. Anaerobic conditions were created in serum vials by purging high-purity nitrogen for 20 min and the vial was sealed with a rubber plug. The model electricigen
The enrichment of facultative anaerobes was achieved by injecting 10% of the sludge sample into 200 ml of growth medium in a 500 ml flask. The microbes were cultured at 30°C, 180 rpm for 7 days. Five percent of the selective inoculation was transferred into the growth medium under anaerobic conditions and cultured at 30°C for 7 days to deplete aerobic bacteria further. The finally as-obtained mixed bacteria were utilized as inoculum for screening facultative anaerobic electricigens using a dual-chamber MFC.
After enriching the electrochemically active biofilm on the anode, the electrode was transferred to a phosphate-buffered saline (PBS) solution and the biofilm was detached by vigorous shaking to prepare a bacterial suspension for use. Subsequently, the well-dispersed samples were serially diluted from 10–1 to 10–6 and plated on solid growth medium plates in an anaerobic incubator. After one week of culture at 30°C, single colonies were selected and inoculated into the liquid growth medium. Once the single colonies grew well in the serum vials, an MFC device was employed to assess their voltage-producing capabilities. The entire isolation procedure was iterated until single colonies of facultative anaerobic electricigens were isolated, showing similar output power density. Following the selection of the electrogenic microorganism, the isolated single colony was subcultured again, and the cultured bacterial solution served as an inoculum for subsequent MFC operations and half-cell experiments.
Discrete colonies were used as templates for PCR. The following universal primers were used to amplify the 16S rDNA: 27F (5’-AGAGTTTGATCCTGGCT CAG-3’) and 1492R (5’-GGTTACCTTGTTACGA CTT-3’). The 16S rDNA gene sequences were compared in the National Centre for Biotechnology Information (NCBI) databank, and the related neighborjoining phylogenetic tree was constructed by using the Molecular Evolutionary Genetics Analysis (MEGA, version 6.0) package (Tamura et al. 2013). A bootstrap analysis was based on 1,000 resamplings.
The isolated SQ-1 strain was inoculated into the growth medium at a 5% inoculum volume and cultured at 30°C under anaerobic conditions for 3 days. Subsequently, 10 ml of the bacterial solution was transferred into the MFC anode chamber to form a microbial membrane. A dualchamber MFC having a total volume of 150 ml and an effective volume of 100 ml in each section (anode or cathode) was used to test the electrogenic ability of the isolated electricigen SQ-1 or
All the electrodes were cleaned several times with 5.0 M NaOH and 75% ethanol to remove impurities and stored in deionized water before the next use. All measurements were performed at 30°C. The experiments were conducted in triplicate under the same operating conditions.
The isolated SQ-1 strain was inoculated into the growth medium at a 5% inoculum volume and cultured at 30°C under anaerobic conditions for 3 days. Subsequently, 5 ml of the bacterial solution was transferred into the quartz bioreactor to form a microbial membrane. Half-cell electrochemical experiments were performed by a 50 ml quartz bioreactor in a three-electrode system, consisting of working and counter electrodes as well as a saturated calomel reference electrode (SCE, Hg/Hg2Cl2 saturated KCl, + 0.244 V vs. hydrogen standard electrode (SHE)). The polished graphite with a geometric surface area of 2.6 cm2 served as the working and counter electrode, respectively. The anaerobic bioreactor was sealed with a rubber plug and purged with high-purity nitrogen for 20 min before running experiments. The growth of biofilm was monitored by an 8-channel potentiostat (CHI 1040C; CH Instruments Inc., USA) at a constant potential of 0.30 V with stirring at 30°C. Meanwhile, the cyclic voltammogram (CV) was tested at a scan rate of 5 mV s–1 between the potential ranges of +0.3 V (
Electrochemical impedance spectroscopy (EIS) analysis was performed in a three-electrode system to evaluate the contribution of internal resistances in biofilms, and this system was connected to an electrochemical station (CHI 760E; CH Instruments Inc., USA) (Islam et al. 2017). The EIS was acquired at OCV by applying AC potential in the frequency range of 100,000–5,000,000 Hz at 10 mV amplitude to prevent biofilm detachment and maintain systematic stability. The EIS data was plotted in a Nyquist curve. The charge transfer resistance (Rct) and ohmic resistance (RΩ) were calculated by fitting the measured impedance data to an equivalent circuit (EC): R(Q[RW]) using ZView® software (Scribner, LLC, USA).
The morphological characteristics of biofilm on carbon electrodes were analyzed with a scanning electron microscope (Hitachi S-3400N, Japan) at 15 kV, as mentioned before (Zhou et al. 2016).
Ferric citrate was used as an electron acceptor to verify the capability of ferric reduction under aerobic and anaerobic conditions. The microbial growth was estimated by measuring the optical density (OD) of the cell culture at a wavelength of 600 nm (OD600). The
Various organic compounds were utilized in the MFC for power generation, such as glucose, sodium citrate, sodium lactate, and glutamic acid (Sarmin et al. 2021; Qiu et al. 2022; Yu et al. 2022b). The output voltage of the MFC using mixed microflora as an inoculum and sodium acetate as a carbon source was measured. It showed a maximum output voltage of 0.55 V after 35 hours of running. When there was enough sodium acetate in the culture, a voltage of 0.50 V or above was maintained for at least 50 hours in three fed-batch MFCs (Fig. 1A). Moreover, the polarization curve was plotted when the MFC stably ran in the second batch (Fig. 1B). The maximum power density was 260 mW m–2 when the output voltage was 0.48 V. This MFC system showed average electricity generation performance. It may be due to the antagonism between specific microbial populations or the lack of substrate metabolite-based synergy, which inhibited the biofilm enrichment on the anode or the electron transfer between microbes and electrode surface (Hsieh et al. 2015; Feichtmayer et al. 2017).
In an MFC system catalyzed by mixed microflora, it is believed that both the biofilms enriched on the anode and free microorganisms in the solution contributed to electricity generation. Once electrochemically active biofilm was enriched from activated sludge in the MFC, the anode was used to isolate facultative anaerobic electricigens using the spread plate technique. A single colony was picked from the agar plate and passaged five times. The isolated electricigen, SQ-1, was identified through 16S rDNA sequencing, which was found to be 99.23% homologous to
In this study, the electrochemical activity of the isolated strain SQ-1 was evaluated in MFC. The output voltage was recorded using the isolated
The maximum power density of SQ-1 reached 560 mW m–2 when the corresponding output voltage was 0.59 V in MFC (Fig. 2C) and
Electrical generation performance of reported electricigens.
Strains | Substrates | Voltage (V) | Power density (mW m–A) | Current density (μA cm–2) | References |
---|---|---|---|---|---|
sodium acetate | 0.59 | 560 | 625 | this work | |
sodium acetate | 0.47 | 460 | 605 | (Deng et al. 2015) | |
glucose | / | / | 52.1 | (Yong et al. 2017) | |
glucose | 0.621 | 40.26 | / | (Guo et al. 2020) | |
lactate | / | 41 | / | (Watson and Logan 2010) | |
citrate | / | 204.5 | / | (Huang et al. 2015) | |
glucose | / | / | 34 | (Qiao et al. 2009) | |
glucose | / | / | 14.6 | (Liu et al. 2007) |
Facultative electricigens showed many advantages over anaerobic electricigens, such as less stringent growth conditions and faster microbial growth. However, low power generation in a bio-electrochemical system impeded their applications in the MFC (Vikromvarasiri et al. 2016; Sunarno et al. 2019). The facultative anaerobic electricigen
The chronoamperometric curve of SQ-1 showed that the current density increased from 7 μA cm–2 and drastically reached the maximum value of approximately 625 μA cm–2 after operating for 43 hours by utilizing sodium acetate as the sole carbon source (Fig. 3A). The current density gradually decreased after it reached the plateau due to the depletion of substrate and accumulation of metabolites.
The cyclic voltammogram (CV) method was performed to analyze its electrochemical characteristics, and a typical sigmoidal CV curve was obtained when the biofilm formed on a carbon sheet electrode with a maximum current density of 625 μA cm–2. The strong catalytic activity indicated that the biofilms and dissociated microbes effectively transfer electrons to the electrode surface under the turnover status. Meanwhile, two pairs of redox peaks under the non-turnover status were shown (Fig. 3A inset). Their midpoint potentials were located at –0.35 V (
The morphological characteristics of the strain SQ-1 were studied using a scanning electron microscope (Fig. 3B(1)). SQ-1 is a rod-shaped, non-flagellated bacterium, approximately 5 μm in length. These morphological characteristics are consistent with many other reported bacteria within the
The black suspension was magnetically attracted to the side of a magnet bar, indicating that the strain SQ-1 has a strong ferric reduction ability.
To investigate the electron transfer kinetics of the strain SQ-1, CVs were recorded at scan rates of 1, 2, 5, 10, 20, and 50 mV s–1 under the non-turnover condition (Fig. 3C). A linear relationship between cathodic peak values and scan rates (
Moreover, the Nyquist plot for the strain SQ-1 biofilms attached on working electrodes in a half-cell system was shown in Fig. 3D. The equivalent circuit EC: R (Q[RWJ) was used to fit the impedance spectra of biofilms where Rs is the solution resistance; Ret is the charge transfer resistance at the interface of electrodes; Qct refers to the constant phase element, representing the double-layer capacitance; Rbiofilm is the biofilm resistance; Qbiofilm represents the double-layer capacitance caused by biofilms on the surface of electrodes. The equivalent circuit fitted well with the model, and a high Rct (148 ohms) was achieved when effective electrochemical biofilms formed on the electrodes’ surfaces.
The strain SQ-1 showed a typical microbial growth curve in both aerobic and anaerobic conditions when the hydrous ferric oxide (HFO) was absent (Fig. 4A). The OD600 value measured after 30 hours of growth in aerobic condition (OD600 = 3.46) was much higher than that in anaerobic conditions (OD600 = 0.47), which suggested that aerobic respiration plays a dominant role in the growth of strain SQ-1. To further explore the effect of HFO on the growth of
To estimate the Fe(III)-reduction efficiency of strain SQ-1, the amount of reduced Fe(II) in the medium was measured with
The power generation performance of
A facultative anaerobic electricigen was isolated from a wastewater tank in a biotechnology factory after a series of aerobic and anaerobic enrichment procedures. The strain SQ-1 was identified as a relative of