Isolation and Electrochemical Analysis of a Facultative Anaerobic Electrogenic Strain  sp. SQ-1

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 Geobacter sp., Shewanella sp., Pseudomonas sp., Citrobacter sp., Klebsiella sp., Escherichia sp., Bacillus sp., and Saccharomyces cerevisiae have been employed as biocatalysts in MFC (Bond et al. 2003; Ieropoulos et al. 2005; Kim et al. 2005; Rabaey et al. 2005; Cao et al. 2019; Jimenez Pacheco et al. 2023). Studies on these isolated bacteria helped scientists better understand the specific mechanisms of extracellular respiration (Bishir et al. 2023; Meylani et al. 2023). More importantly, it is easier to manipulate these electricigens and optimize their power generation performance in MFC. Among the studied electricigens, Geobacter sp. and Shewanella sp. are the most representative anaerobic and facultative anaerobic strains, respectively. Geobacter sp. can use ambient ferric iron or solid electrodes as electron acceptors for extracellular electron transfer, but it cannot secret ETMs. As for Shewanella sp., the “nanowires” extended from the outer membrane give it an advantage in extracellular electron transfer, by which the ferric reduction is accelerated. However, it showed much lower power generation in MFC or other bio-electrochemical systems than strictly anaerobic Geobacter sp. under the same conditions (Marsili et al. 2008; Pirbadian et al. 2014).

To date, several facultative anaerobic electricigens such as Shewanella sp., Pseudomonas sp., Citrobacter sp., and Klebsiella sp. were isolated and able to achieve the metabolic pathway shift between electricigenic respiration and anaerobic fermentation in MFC, which showed prospective applications in bioremediation, wastewater treatment, heavy metal leaching, and the global carbon cycle (Chaudhuri et al. 2003; Rabaey et al. 2004; Deng et al. 2010). The wild-type Klebsiella variicola generates electricity by utilizing the palm oil mill effluent as a substrate, achieving the maximum power density of 4,426 mW m^–3 and a high coulombic efficiency of 63% in MFC (Islam et al. 2018). Various types of bacteria within the Klebsiella genus have been isolated from diverse habitats, including soil sediment (Zhang et al. 2008), palm oil mill effluent (Islam et al. 2018), activated sludge (Kim et al. 2006), and food industry wastewater (Ramu et al. 2020). Another facultative anaerobic electricigen named Shewanellaoneidensis MR-1 is a model dissimilatory iron-reducing bacterium which uses iron oxides as electron acceptors and adsorbs phosphate anions and metal ions during the bioreduction process (Long et al. 2021; Ge et al. 2022; Yu et al. 2022a).

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.

Experimental

Materials and Methods

Growth medium, inoculation, and culture

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 Geobacter sulfurreducens PCA was purchased from Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Germany) and cultured in the growth medium.

Isolation and identification of the electricigenic strain

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.

MFC construction and operation

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 G. sulfurreducens PCA, respectively. The anode and cathode compartments were separated by a proton exchange membrane (PEM; Nafion 117, DuPont Co., USA) with an effective area of 4π cm². In the anode chamber, a carbon rod with a working area of 9.6 cm² was used as an anode. The growth medium containing 20 mM sodium acetate was used as anolyte and 10% inoculum of the isolated strain or G. sulfurreducens PCA was added to the chamber. A small carbon brush (2.5 cm in diameter and 2.5 cm long) connected by a titanium wire served as an electrode in the cathodic chamber containing 50 mM potassium ferricyanide and 0.1 M PBS solution. The MFC was purged strictly with high-purity nitrogen for at least 20 min before running experiments. A 1,000 ohms resistance was connected in an external circuit except when plotting the polarization curve. Besides, the output voltages were simultaneously monitored by a Keithley instrument (Model 2400; Keithley Instruments, Inc., USA) at an interval of 24 h. The recorded voltage was converted to output power.

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.

Half-cell experiments

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 (E_i) to –0.6 V (E_f). All potentials in this study are versus SCE unless otherwise stated.

Electrochemical impedance spectroscopy

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 (R_ct) and ohmic resistance (R_Ω) were calculated by fitting the measured impedance data to an equivalent circuit (EC): R(Q[RW]) using ZView® software (Scribner, LLC, USA).

Morphological analysis

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).

Analysis of ferric reduction

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 o-phenanthroline (1,10-phenanthroline) method was used to measure the total Fe(II) concentration (Georgi et al. 2017; Islas et al. 2018). Aliquots were taken from the medium, and the same volume of 0.5 M HCl was added. The mixture was vigorously shaken and incubated in an anaerobic incubator for 24 hours to extract the adsorbed Fe(II). After centrifugation at 10,000 × g for 5 min, the supernatants were used for Fe(II) measurements. Experiments were performed in triplicate.

Results and Discussion

Enrichment, isolation, and identification of the strain SQ-1

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 Klebsiella variicola SB5531^T (Fig. 1C). Additionally, the isolated strain SQ-1 and the strictly anaerobic electricigen G. sulfurreducens PCA were cultured in the same growth medium under anaerobic conditions for 3 days. Their bacterial solutions showed a different color. (Fig. 1D).

Electricity generation performance of Klebsiella sp. SQ-1 in MFC

In this study, the electrochemical activity of the isolated strain SQ-1 was evaluated in MFC. The output voltage was recorded using the isolated Klebsiella sp. SQ-1 and G. sulfurreducens PCA as inoculums, respectively. The strain SQ-1 showed a rapid increase of output voltage from 110 to 180 h, which reached approximately 0.53 V (Fig. 2A). The output voltage of G. sulfurreducens PCA showed an identical pattern in fed-batch MFCs under similar conditions (Fig. 2B). The maximum output voltage (0.57 V) of the strain SQ-1 was higher than that of G. sulfurreducens PCA (0.52 V). More importantly, the strain SQ-1 was able to maintain a high output voltage for a longer time than that of G. sulfurreducens PCA in each fed-batch MFC, which suggested that the facultative strain SQ-1 had better electricity generation performance than either the strictly anaerobic G. sulfurreducens PCA or the mixed bacteria (Fig. 1A).

Output voltage and power density of Klebsiella sp. SQ-1 in MFC

A) The output voltage performance of the strain SQ-1 in a dual-chamber MFC using sodium acetate as a carbon source. Arrows show the supply of sodium acetate. B) The output voltage performance of a model electricigen Geobacter sulfurreducens PCA in a dual-chamber MFC using sodium acetate as a carbon source. Arrows show the supply of sodium acetate. C) The output voltage (circle) and power density (square) curve for the strain SQ-1. D) The output voltage (circle) and power density (square) curves for G. sulfurreducens PCA. E) Comparison of the maximum output voltage of mixed microflora, G. sulfurreducens PCA, and Klebsiella sp. SQ-1 in MFC. F) Comparison of maximum output power density of mixed microflora, G. sulfurreducens PCA, and Klebsiella sp. SQ-1 in MFC.

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 G. sulfurreducens PCA showed a maximum power density of 460 mW m^–2 when the output voltage was 0.47 V (Fig. 2D). To compare the electricity production performance of SQ-1 with other reported facultative anaerobic electrogenic bacteria, the electrical generation performance of some reported electricigens was summarized in Table I. The Table I shows that SQ-1 demonstrates superior electricity production capacity and higher output power.

Table I

Electrical generation performance of reported electricigens.

Strains	Substrates	Voltage (V)	Power density (mW m^–A)	Current density (μA cm^–2)	References
Klebsiella sp. SQ-1	sodium acetate	0.59	560	625	this work
Geobacter sulfurreducens PCA	sodium acetate	0.47	460	605	(Deng et al. 2015)
Pseudomonas aeruginosa	glucose	/	/	52.1	(Yong et al. 2017)
Klebsiella pneumonia	glucose	0.621	40.26	/	(Guo et al. 2020)
Shewanella oneidensis	lactate	/	41	/	(Watson and Logan 2010)
Citrobacter freundii	citrate	/	204.5	/	(Huang et al. 2015)
Escherichia coli	glucose	/	/	34	(Qiao et al. 2009)
Rhodoferax ferrireducens	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 Klebsiella sp. SQ-1, with high power generation capacity, might be one of the potential candidates in engineering biological catalysts for MFC.

Electrochemical performance of Klebsiella sp. SQ-1 in half-cell experiments

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.

Electrochemical performance of Klebsiella sp. SQ-1 in a three-electrode system

A) Chronoamperometrlc curves for the strain SQ-1 In a three-electrode system using sodium acetate as electron acceptors. Inset, cyclic voltammograms of the enriched strain SQ-1 biofilm on electrodes in dltferent stages; a) before blofllm formation; b) after blofllm formation with a current density below 2 μA cm^–2. B(1) The morphology of the strain SQ-1 blofllms under the scanning electron microscope (5,000x). Blofllms were attached to the graphite surface. B(2) The appearance of bacterial culture after 5 days of Incubation when the growth medium Is supplemented with lron(III) oxide. A magnet Is placed outside the tube. The arrow shows that the magnet attracts the black suspension. B(3) The Initial appearance of bacterial culture. C) Voltammograms of the strain SQ-1 blofllm. Scan rates: 1, 2, 5, 10, 20, 50 mV s^–1. Inset, the linear dependence of the cathodic peak current density at -0.37 V versus scan rates or square roots of scan rates. D) The Nyquist plot for blofllms attached to working electrodes.

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 (E₁) and –0.43 V (E₂), respectively (Fig. 3A).

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 Klebsiella genus (Kim et al. 2006; Zhang et al. 2008; Peng et al. 2013; Bajithun et al. 2022). Multilayered biofilms were found on the carbon electrode surface, which might show that it has enough capability to generate a highly stable current. Besides, the strain SQ-1 was inoculated into the growth medium containing iron(III) oxide as electron acceptor and acetate sodium as electron donor, and the color of the iron(III) oxide medium changed from brown to black after 5 days of incubation (Fig. 3B(2) and 3B(3)).

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 (v) or the square root of scan rates (v^1/2) was obtained at approximately –0.37 V when the basic medium (without electron donor or acceptor) was used. Their good linear relationship (R₁²=98.7 and R₂² = 96.3) suggested that the biofilms of SQ-1 showed surface-controlled and diffusion-controlled kinetics (Fig. 3C inset). A similar pattern of kinetics was found in G. sulfurreducens, Citrobacterfreundii, or mixed culture biofilms, which were formed on carbon electrodes, ITO (Indium Tin Oxide) electrodes, or gold surfaces under non-turnover conditions (Liu et al. 2010; Liu et al. 2015; Zhou et al. 2017).

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.

Ferric reduction of Klebsiella sp. SQ-1

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 Klebsiella sp. SQ-1, batch cultures were supplemented with different amounts of ferric citrate and incubated under aerobic or anaerobic conditions. An approximate OD₆₀₀ value of 3.51 was measured after five days of culture in aerobic conditions without the presence of hydrous ferric oxide (Fig. 4B). A lower OD₆₀₀ value (OD₆₀₀ = 2.24) was obtained when the medium was supplemented with 5 mM HFO, indicating the growth of strain SQ-1 was affected by the extracellular HFO. However, the bacterial growth maintained at a high and similar level even if the medium was supplemented with higher concentrations of ferric citrate (10–70 mM). Therefore, it was speculated that metabolic pathways were modified in these facultative bacteria when both oxygen and HFO were present in their living environment. Aerobic respiration was the principal pathway the strain SQ-1 utilized to grow and proliferate. However, some energy flow was diverted to the extracellular electricigenic respiration, producing less biomass. By contrast, a much lower OD₆₀₀ value ranging from 0.1 to 0.56 was measured in anaerobic conditions (Fig. 4B) when the growth medium was supplemented with 5 mM to 70 mM ferric citrate, which suggested that the HFO served as an extracellular electron acceptor and sustained the growth of Klebsiella sp. SQ-1.

To estimate the Fe(III)-reduction efficiency of strain SQ-1, the amount of reduced Fe(II) in the medium was measured with o-phenanthroline. The amount of Fe(II) at the stationary phase gradually increased when the concentration of Fe(III) increased from 5 mM to 40 mM under anaerobic conditions (Fig. 4C). The concentration of reduced Fe(II) increased from 1.25 mM to 6.00 mM (Fig. 4D). The amount of Fe(II) at the stationary phase started to fall when the concentration of Fe(III) was higher than 40 mM, which suggested that high concentration of Fe(III) might inhibit extracellular electricigenic respiration of the strain SQ-1. The highest reduction ratio of ~25% was calculated when 5 mM Fe(III) was supplied (Fig. 4D). Notably, the bacterial biomass was at a low level (OD₆₀₀ = 0.1, Fig. 4B) under this condition, which suggested that the strain SQ-1 has a strong ferric reduction ability. However, the reduction ratio decreased to approximately 15% when the concentration of Fe(III) increased to 40 mM and dropped to 4% when the concentration of Fe(III) was 70 mM (Fig. 4D).

The power generation performance of Klebsiella SQ-1 in MFC and half-cell experiments has confirmed its status as an electrochemically active bacterium capable of extracellular electron transfer, i.e., accomplishing electron exchange reactions between microbial cells and solid materials. In ferric reduction reactions, SQ-1 utilized HFO as an electron acceptor for extracellular electron transfer. The Fe(III) ions were reduced to Fe(II) or other iron ions in various valence states, which may undergo complicated chemical reactions to produce Fe₃O₄. This may explain why a magnet bar magnetically attracted the bacterial suspension (Fig. 3B).

Conclusions

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 Klebsiella variicola based on its 16S rDNA gene sequences. Using sodium citrate as a substrate, the maximum power density of 560 mW m^–2 was achieved in a dual-chamber MFC, which was higher than that of G. sulfurreducens PCA under similar conditions. Moreover, the strain SQ-1 showed high ferric reduction activity and achieved 15% of ferric reduction in the presence of 40 mM HFO under anaerobic conditions. Aerobic respiration was the optimal energy-generating way for Klebsiella sp. SQ-1 to grow and proliferate when cultured in aerobic conditions. When the medium was supplemented with HFO and the bacteria were cultured in anaerobic conditions, extracellular electricigenic respiration and anaerobic fermentation were employed to sustain its growth. Considering its high power generation and ferric reduction capacity, the facultative anaerobic strain Klebsiella sp. SQ-1 might be a promising biocatalyst in microbial electrochemistry. During our experimental procedures, the most difficult problem to solve is the selection of electrodes. Since some microorganisms cannot attach well to the electrode rods, we tried many kinds of electrodes so that the enriched microorganisms can attach well and have stable electricity production performance. This prompts us to search for and optimize electrode materials, one of our future research topics. It is also intriguing to investigate the strong ferric reduction ability of Klebsiella sp. SQ-1. The molecular mechanism of ferric reduction can also be studied using gene knockout and complementation techniques to identify genes related to electron transport and verify their functions in extracellular electron transport.

eISSN:: 2544-4646
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Isolation and Electrochemical Analysis of a Facultative Anaerobic Electrogenic Strain Klebsiella sp. SQ-1

Article Category: ORIGINAL PAPER

Published Online: Apr 27, 2024

Page range: -

Received: Dec 07, 2023

Accepted: Feb 26, 2024

DOI: https://doi.org/10.33073/pjm-2024-013

Keywords
microbial fuel cell, sp., electrochemical analysis, extracellular respiration, facultative electricigens

© 2024 Lei Zhou et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

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