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. 2022 Aug 3;16(3):534–545. doi: 10.1111/1751-7915.14128

Enhancing electrical outputs of the fuel cells with Geobacter sulferreducens by overexpressing nanowire proteins

Zhigao Wang 1, Yidan Hu 1,2, Yiran Dong 1,2,3,4,5, Liang Shi 1,3,4,5,✉, Yongguang Jiang 1,2,✉
PMCID: PMC9948223  PMID: 36815664

Abstract

Protein nanowires are critical electroactive components for electron transfer of Geobacter sulfurreducens biofilm. To determine the applicability of the nanowire proteins in improving bioelectricity production, their genes including pilA, omcZ, omcS and omcT were overexpressed in G. sulfurreducens. The voltage outputs of the constructed strains were higher than that of the control strain with the empty vector (0.470–0.578 vs. 0.355 V) in microbial fuel cells (MFCs). As a result, the power density of the constructed strains (i.e. 1.39–1.58 W m−2) also increased by 2.62‐ to 2.97‐fold as compared to that of the control strain. Overexpression of nanowire proteins also improved biofilm formation on electrodes with increased protein amount and thickness of biofilms. The normalized power outputs of the constructed strains were 0.18–0.20 W g−1 that increased by 74% to 93% from that of the control strain. Bioelectrochemical analyses further revealed that the biofilms and MFCs with the constructed strains had stronger electroactivity and smaller internal resistance, respectively. Collectively, these results demonstrate for the first time that overexpression of nanowire proteins increases the biomass and electroactivity of anode‐attached microbial biofilms. Moreover, this study provides a new way for enhancing the electrical outputs of MFCs.

Geobacter sulfurreducens strains with overexpressed nanowire proteins were constructed and used as electricigens in microbial fuel cells. Enhanced electrical outputs based on promoting biofilm formation on anode demonstrated that nanowire proteins are promising genetic tools to improve electrochemical performance of microbes for bioelectricity production.

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INTRODUCTION

Microbial extracellular electron transfer (EET) is an important metabolic process, in which microorganisms exchange electrons with various extracellular acceptors such as metal oxides, organic compounds and electrodes (Jiang et al., 2019; Logan et al., 2019; Shi et al., 2016). The microorganisms with the capability to exchange electrons with electrodes are called as electroactive microbes that have shown great promise as biocatalysts in bioelectrochemical systems (BESs) for various biotechnological applications (Logan et al., 2019; Shi et al., 2019). One of extensively studied BESs is microbial fuel cells (MFCs) in which electroactive microbes convert chemical energy to electrical energy through transferring electrons, generated by the oxidation of organic substrates, to electrodes via EET (Santoro et al., 2017; Verma et al., 2021). Therefore, MFC is an attractive bioelectrochemical technology for coupling wastewater treatment and energy recovery from organic matter (Santoro et al., 2017, Verma et al., 2021). However, the applications of MFCs have practically stagnated due to low output of power density (Mateo et al., 2018).

Electroactivity of microbes is a primary bottleneck for improving electricity production of MFCs (Zhao et al., 2020). Geobacter spp. are promising for bioelectrochemical applications because of a strong capacity for EET (Reguera & Kashefi, 2019). For example, G. sulfurreducens generates the highest current density of wild‐type microbes available in pure culture (Nevin et al., 2008; Rotaru et al., 2015; Yi et al., 2009). The electrical output of MFCs is significantly impacted by the thickness and conductivity of G. sulfurreducens biofilm grown on the surfaces of electrodes (Hu et al., 2021; Malvankar et al., 2012; Reguera et al., 2006; Steidl et al., 2016). Generally, thick biofilms with high conductivity are expected to generate high current density. On the other hand, biofilm conductivity is a decisive variable for biofilm thickness (Steidl et al., 2016). The conductivity of G. sulfurreducens biofilm is conferred by membrane‐bound and matrix‐associated electroactive c‐type cytochromes and protein nanowires (Jiménez Otero et al., 2021; Liu et al., 2021; Malvankar et al., 2012; Richter et al., 2009, 2012; Yalcin et al., 2020).

Geobacter sulfurreducens nanowires are conductive filaments with the base anchored in cell membrane and the distal end extending through the biofilm (Liu et al., 2021; Wang et al., 2019; Yalcin et al., 2020). The nanowires could transfer electrons from bacterial membrane to extracellular electrodes by direct contact (Liu et al., 2020; Reguera et al., 2005; Wang et al., 2019; Yalcin et al., 2020). Previous study revealed that EET across membrane and through the biofilm are two limiting steps for catalytic current generation (Strycharz et al., 2011). Therefore, protein nanowires are ideal targets for genetic engineering of G. sulfurreducens to improve electrical output of MFCs (Dantas et al., 2015; Leang et al., 2013).

Geobacter sulfurreducens has been proposed to generate two types of nanowires, namely, conductive pili (e‐pili) comprising PilA protein (Liu et al., 2021) and c‐type cytochrome filaments comprising OmcZ or OmcS proteins (Wang et al., 2019; Yalcin et al., 2020). E‐pili and OmcZ are required for thick electroactive biofilm and optimal current production of G. sulfurreducens (Nevin et al., 2009; Reguera et al., 2005, 2006; Steidl et al., 2016; Vargas et al., 2013). However, deletion of OmcS and its homologue OmcT did not impair the current production of G. sulfurreducens (Nevin et al., 2009). Previous study suggested that the reciprocal relationship between expressions of omcS and omcZ genes may render the role of OmcS in current production underappreciated (Wang et al., 2019, Yalcin et al., 2020). In addition, OmcZ is highly concentrated at the biofilm–electrode interface and may serve as an electrochemical gate facilitating electron transfer from biofilm to the electrode (Inoue et al., 2011). The EET efficiency of G. sulfurreducens could not be improved by replacing the endogenous e‐pili with the more conductive one from G. metallireducens (Tan et al., 2017). On the contrary, the G. sulfurreducens variants with greater abundance of e‐pili and c‐type cytochromes have stronger capacity for current production in MFCs than the wild type (Hernández‐Eligio et al., 2022; Leang et al., 2013; Yi et al., 2009). These findings indicated that the abundance of nanowires may be critical for enhancing EET efficiency of G. sulfurreducens. To determine whether nanowire proteins are applicable for further improving the electrical output of MFCs, the genes encoding PilA, OmcZ, OmcS and OmcT proteins were overexpressed in G. sulfurreducens via a high‐copy‐number vector in this study. The results show for the first time the enhanced electricity production of G. sulfurreducens MFCs via overexpression of bacterial nanowire proteins.

EXPERIMENTAL PROCEDURE

Bacterial strains and growth conditions

Wild‐type G. sulfurreducens PCA (ATCC 51573) was obtained from the American Type Culture Collection. G. sulfurreducens strains were routinely grown anaerobically in NBFA medium (Coppi et al., 2001). The gas phase was 80% N2 and 20% CO2. All the procedures referring to Geobacter cells were performed in an anaerobic chamber (Coy Laboratory Products) that was filled with 5% H2, 20% CO2 and 75% N2. Escherichia coli DH5α was cultured in Luria‐Bertani (LB) medium and used for gene cloning experiments. Kanamycin was added to the bacterial cultures with a final concentration of 200 μg ml−1 when necessary. The cultivation temperature of Geobacter and E. coli strains was 30 and 37°C, respectively. The bacterial growth curves were obtained by measuring the absorbance of cultures at 600 nm (OD600). Table 1 lists all the bacterial strains used in this study.

TABLE 1.

Bacterial strains and plasmids used in this study

Strains or plasmids Relevant genotype or uses Source or reference
Bacterial strains
G. sulferreducens
PCA Wild‐type G. sulferreducens Liu et al. (2015)
Control strain G. sulferreducens PCA containing empty pAWP78 vector This study
GNW1 G. sulferreducens PCA containing pOEpilA This study
GNW2 G. sulferreducens PCA containing pOEomcZ This study
GNW3 G. sulferreducens PCA containing pOEomcS This study
GNW4 G. sulferreducens PCA containing pOEomcT This study
E. coli DH5α Host for vector cloning Takara Co. Inc., Japan
Plasmids
pAWP78 IncP‐based broad host range vector Puri et al. (2015)
pOEpilA Expression cassette of pilA in pAWP78 This study
pOEomcZ Expression cassette of omcZ in pAWP78 This study
pOEomcS Expression cassette of omcS in pAWP78 This study
pOEomcT Expression cassette of omcT in pAWP78 This study
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DNA manipulation and reagents

Genomic DNA of G. sulfurreducens was isolated using the MiniBEST Bacteria Genomic DNA Extraction Kit (TaKaRa) as the template for PCR cloning of pilA, omcZ, omcS and omcT (Table S1). The start codon of pilA gene was changed from TTG (Richter et al., 2012) into ATG via site‐directed mutagenesis by PCR. As omcT and omcS share the same promoter (Mahadevan et al., 2008; Mehta et al., 2005), the promoter and omcT coding region were amplified individually and then ligated. The plasmids were prepared with the QIAprep Spin Miniprep Kit (Qiagen). PCR amplification of the backbone of pAWP78 vector (Addgene No. 61263) was performed as described previously (Puri et al., 2015). All the PCR reactions were performed using the PrimeSTAR HS (Premix) Kit (TaKaRa, Japan) with high‐fidelity DNA polymerase. The PCR products were purified using the GeneJET Gel Extraction Kit (Thermo Fisher Scientific).

Gene fragments and pAWP78 vector were ligated to produce the following expression constructs, pOEpilA, pOEomcZ, pOEomcS and pOEomcT. Ligation was performed using Gibson Assembly Cloning Kit (NEB) based on the overlapping sequences added at the 5′‐end of primers (Table S1). The ligation products were transformed into E. coli DH5α, and bacterial cells were incubated on LB plates with kanamycin. Positive clones were screened by colony PCR followed by bidirectional sequencing. Sequencing‐confirmed constructs were electroporated into G. sulfurreducens PCA (Coppi et al., 2001). Empty pAWP78 vector was transformed into G. sulfurreducens PCA as a control. The sequences of all the expression cassettes are shown in the supplementary materials (Table S2).

If not mentioned, analytical grade or biochemistry grade chemicals were purchased from the Sinopharm Chemical Reagent Co. Ltd or Sigma‐Aldrich Co. Llc.

Western blot analysis

Polyclonal antibodies specific for PilA, OmcZ, OmcS and OmcT were prepared by Proteintech Group, Inc (Wuhan, China). Equal amounts of cells were lysed by protein‐loading butter for the constructed strains and the control. Ten microliters of cell lysates were used to perform sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE). Following SDS‐PAGE, the proteins were probed and visualized by Western blot with the antibodies of each nanowire protein, goat anti‐rabbit IgG‐HRP (TransGen Biotech) and super sensitive ECL luminescence reagent (Meilunbio).

Fe(III) reductions

All constructed strains were tested for their ability to reduce ferric citrate or ferrihydrite as described before (Liu et al., 2015). Ferrihydrite was synthesized and characterized as previously described (Liu et al., 2015). At predetermined time points, the produced Fe(II) was determined with the ferrozine assay (Stookey, 1970).

Biofilm assays

Geobacter sulfurreducens strains with OD600 of 0.1 were grown at 30°C with fumarate in 24‐well plates for biofilm assay (Hu et al., 2019). At predetermined time points, the plates were washed twice using phosphate buffer solution (PBS, pH = 7.0). The bacterial biofilms on plates were stained with 0.1% crystal violet solution for 15 min. After washing twice with PBS, the stained biofilm was dissolved and suspended in 1.0 ml ethanol solution (95%). OD570 was measured with a SpectraMax 190 microplate reader (Molecular Devices).

Construction of MFC

Dual‐chamber MFCs with 140 ml working volume were used to evaluate the current‐producing capacity of the bacterial strains. Anode and cathode chambers were separated by Nafion 117 membranes (DuPont Inc.) as previously described (Li et al., 2018). Carbon cloth was used as the electrodes. Dimensions of the anode and cathode were 1.0 × 1.0 cm (i.e. the geometric area is 1.0 cm2) and 2.5 × 3.0 cm, respectively. The anodic electrolyte was prepared by replacing fumarate in NBFA medium with equal molar concentration of sodium chloride. The cathodic electrolyte contained K3[Fe(CN)6], K2HPO4 and KH2PO4 (50 mM each). Mid‐log G. sulfurreducens culture suspension was inoculated into 50 ml fresh medium and incubated at 30°C until OD600 of the culture reached 0.5. The bacterial cells were harvested by centrifugation at 5000 g, and the supernatant were discarded. Then, the cell pellets were re‐suspended in 140 ml anodic electrolyte supplemented with kanamycin for maintaining the expression constructs. Subsequently, the cell suspension was injected into anode chamber. During growth of electroactive biofilm, the anode and cathode were connected by a 2 kΩ external resistor to measure the voltage generated. The potential of MFCs was recorded with a PS2024V multi‐channel data acquisition unit (SMACQ, China). The MFCs of each G. sulfurreducens strain were set up in triplicates and repeated at least three times.

Electrochemical analyses

Electrochemical, biochemical and microscopical analyses of MFCs were performed upon reaching the highest output voltage of each MFC, that is 160, 190, 180, 140 and 160 h for the control strain, GNW1, GNW2, GNW3 and GNW4, respectively. Electrochemical analysis was carried out on a CHI 1000C multichannel potentiostat (CH Instruments, China) as previously described (Li et al., 2018). EIS was measured at the open circuit potential (OCP). The sinusoidal excitation signal was set as 10 mV and the frequency varied linearly from 10,000 to 0.01 Hz. To generate polarization curves, LSV analysis was performed on a two‐electrode mode with a scan rate of 0.1 mV s−1 from OCP to −0.1 V. Power density (P) was calculated by P = VIS−1, where V is the output voltage, I is the current, and S is the projected area of the anode surfaces. Cyclic voltammetry (CV) curves were obtained in a three‐electrode system with an Ag/AgCl reference electrode by voltage scanning at a rate of 5 mV s−1 from −0.7 to 0.1 V.

Biofilm imaging

Biofilms on the anodic carbon cloth were stained using the LIVE/DEAD BacLight Viability Kit (Thermo Fisher Scientific) as suggested from the manufacturer's instructions. The structure of electroactive biofilms was characterized via three‐dimensional fluorescent images taken using an TCS SP8 confocal microscope (Leica). To display the biomass of live cells, only green fluorescence at 500 nm was detected in the confocal imaging. Image processing was implemented in the LAS AF Lite‐version 2.3.0 software.

Measurement of the bacterial proteins on electrodes

To measure the amounts of proteins on the anodes, the anodic carbon cloths were cut into pieces and placed in 5 ml of 0.2 M NaOH solution. After vertexing for 1 min, bacterial cells were lysed by incubating the tube at 96°C for 20 min. The lysate was cooled to room temperature and total protein concentrations in the extracts were measured with the bicinchoninic acid protein assay kit (Beyotime). According to the manufacture's protocol, colour reactions were developed in 96‐well plates and the absorbance of product solution was measured at 562 nm using a SpectraMax 190 microplate reader (Molecular Devices). The standard curve of absorbance against protein concentrations was created using bovine serum albumin solutions with defined concentrations.

Statistical analysis

The differences between the constructed strains and the control strain in different measurements were examined by independent‐samples t‐test with SPSS 20.0 for Windows, and differences were considered to be significant at p < 0.05.

RESULTS AND DISCUSSION

Growths and Fe(III) reductions

In this study, we constructed four G. sulfurreducens strains with the expression vectors of nanowire proteins, namely, GNW1 (pOEpilA), GNW2 (pOEomcZ), GNW3 (pOEomcS) and GNW4 (pOEomcT) (Table 1). Western blot analyses confirmed that PilA, OmcZ, OmcS and OmcT proteins were overexpressed in GNW1, GNW2, GNW3 and GNW4, respectively (Figure S1). All the constructed strains grew slower than the control strain (i.e. G. sulfurreducens PCA with empty pAWP78 vector) with fumarate as the electron acceptor (Figure S2). This finding suggested that overexpression of the nanowires maybe a metabolic burden and thus decreased cell growth. However, the constructed strains reduced soluble ferric citrate at comparable rates (Figure 1A). It was also shown that ferrihydrite reduction by GNW1 and GNW3 was slightly faster than the remaining constructed and control strains (Figure 1B). These results are consistent with previous reports that nanowires were not needed for reducing soluble Fe(III) and that PilA and OmcS have critical roles in reducing solid‐phase Fe(III) (Liu et al., 2014, 2018; Mehta et al., 2005; Nevin et al., 2009; Reguera et al., 2005).

FIGURE 1.

FIGURE 1

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Reduction in ferric citrate (A) and ferrihydrite (B) by the control and the constructed G. sulfurreducens strains. The expression constructs are displayed in parentheses. The values reported are the means and standard deviations of triplicate measurements.

Biofilm formation

After inoculation in the 24‐well plates, all the G. sulfurreducens strains formed cohesive biofilms on the bottom of the plate wells. The biomass of the biofilms reached their peaks between the 4th and 5th day and decreased subsequently (Figure 2). Compared with the control strain, biomass of the biofilms from the constructed strains increased 16% to 146% (p < 0.05), which decreased in the order of GNW2 > GNW1 > GNW3 > GNW4 > control strain. To evaluate the effects of nanowire proteins on the biofilm‐forming capability of individual cells, the ratios of biofilm biomass to cell growth at the 4th day were calculated (Figure S3). The ratios for the constructed strains were significantly higher than that for the control strain (p < 0.05). These results revealed that overexpression of PilA, OmcZ, OmcS and OmcT can promote the formation of G. sulfurreducens biofilm. Previous study also found that PilA deficiency impaired the biofilm formation of G. sulfurreducens on solid surface when fumarate was used as electron acceptor (Reguera et al., 2007; Richter et al., 2012). These findings suggested that pili plays an important structural role in cell–cell aggregation during biofilm differentiation (Reguera et al., 2007, Richter et al., 2012). On the other hand, Geobacter pili may mediate the secretion of OmcS and OmcZ (Gu et al., 2021). In addition to their electron transfer roles, cytochromes may act as important structural components in biofilm matrix (Hu et al., 2021). Similarly, the results of this investigation also indicated the structural roles of OmcZ, OmcS and OmcT in biofilm formation. Comparison between the constructed strains also revealed that overexpression of OmcZ had the most significant promotion for biofilm formation in well plate, which provided further evidence for an important role of OmcZ in maintaining biofilm structure.

FIGURE 2.

FIGURE 2

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Biofilm formation of the control and the constructed G. sulfurreducens strains. OD570, optical density at 570 nm. The expression constructs are displayed in parentheses. The values reported are the means and standard deviations of triplicate measurements. Asterisks represent significant differences between the constructed G. sulfurreducens strains and the control strain at the same day (p < 0.05).

Bioelectrochemical characterizations

During the incubation period in the MFCs, the peak voltage outputs of the constructed G. sulfurreducens strains were 0.470–0.578 V in comparison with a peak value of 0.355 V for the control strain (p < 0.05) (Figure S4). The maximum values of electrochemical curves were used for the following analyses. As shown in Figure 3A, the calculated current output of GNW3 and GNW4 increased much faster than the control strain, indicating that overexpression of OmcS and OmcT enhanced the attachment of G. sulfurreducens to electrode. Compared with the control strain, all the constructed strains generated much higher electrical outputs that increased by 32% to 62% (p < 0.05) (Figure 3A). The maximum current density of the constructed strains was 2.35 ± 0.06 A m−2 (n = 3) to 2.89 ± 0.06 A m−2 (n = 3), while the control strain only produced 1.78 ± 0.05 A m−2 (n = 3) (Figure 3A).

FIGURE 3.

FIGURE 3

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Bioelectrochemical characterization of the control and the constructed G. sulfurreducens strains. (A) Current output curves, the values reported are the means and standard deviations of triplicate measurements; (B) polarization curves (output voltage vs. current density) generated by LSV at the scan rate of 0.1 mV s−1; (C) power density output curves (power density vs. current density); (D) Nyquist plots of MFCs. The expression constructs are displayed in parentheses. Curves in (B), (C) and (D) are representative (n = 3).

The polarization curves of the constructed strains were very similar and increased much faster than that of the control strain under linear sweep voltammetry (LSV) conditions (Figure 3B). As shown on the power output curves (Figure 3C), the power density of the constructed strains was elevated by 2.62‐ to 2.97‐fold in comparison with that of the control strain (p < 0.05). The constructed strains produced the maximum power density of 1.39 ± 0.08 W m−2 (n = 3) to 1.58 ± 0.08 W m−2 (n = 3), while the control strain only produced 0.53 ± 0.62 W m−2 (n = 3). Considering that the polarization curves reflect the increase of current density with the reduction in external potential, the reducing slope of polarization curves is thus positively correlated with the internal resistance of the MFCs. Compared with the control MFC, the MFCs of the constructed strains had very small reducing slope at the linear portion of the polarization curves (Figure 3B), and therefore their internal resistance was smaller. This finding was also evidenced by the data of electrochemical impedance spectroscopy (EIS) (Figure 3D). The diameter of the semicircle appearing in the Nyquist plots is positively correlated with charge transfer resistance (Malvankar et al., 2012). The MFCs of the constructed strains had shorter diameters in the Nyquist plots than that of the control strain (p < 0.05) (Figure 3D), indicating that overexpressed nanowires effectively reduced the charge transfer resistance of MFCs. Similarly, previous electrochemical impedance analysis revealed that the biofilm of an OmcZ‐deficient mutant was more electrically resistant than that of the wild‐type G. sulfurreducens (Richter et al., 2009).

Results in this study demonstrate for the first time that overexpression of nanowire proteins can enhance current production through decreasing the internal resistance of MFCs. These findings are also consistent with the fact that the conductivity of protein nanowires is the critical factor for current production of electrode‐attached biofilms (Liu et al., 2021; Lovley & Walker, 2019; Wang et al., 2019; Yalcin et al., 2020). However, given that the known nanowire proteins have distinct electrochemical properties (Gu et al., 2021; Wang et al., 2019; Yalcin et al., 2020; Yalcin & Malvankar, 2020), the similar power density of the constructed strains is unexpected. According to the shape of polarization curves, the electrical output efficiency of MFCs probably reached the limitation of substrate turnover rate, which might be caused by the limited metabolic rate of G. sulfurreducens, or the substrate diffusion limitation between biofilm and bulk solution.

The kinetics of redox reactions at cell‐electrode interfaces were also determined by cyclic voltammetry (CV) analysis through measuring current response at a low scan rate of voltage (Figure 4). The current density at the redox peaks of all the constructed strains showed significant increase compared with that of the control strain, indicating improved electroactivity of biofilms derived from the constructed strains. Previous investigation demonstrated that the peak current of CV curves is positively correlated with the cell density at the electrode surface and the number of membrane‐bound electron transfer proteins in the individual cells (Fricke et al., 2008). Therefore, the results of CV analysis were consistent with the increased biomass of electrode‐attached biofilms observed in the following section and overexpression of nanowire proteins in G. sulfurreducens.

FIGURE 4.

FIGURE 4

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CV curves obtained at the scan rate of 5 mV s−1. Shown are the CV data of the control strain and the constructed G. sulfurreducens strains overexpressing PilA (A), OmcZ (B), OmcS (C) and OmcT (D). The expression constructs are displayed in parentheses. Curves are representative (n = 3).

The first derivatives of CV data were used to calculate the midpoint potentials of electrode‐attached biofilms (Figure S5). GNW3 and the control strain had the same midpoint potential of −0.39 V (vs. Ag/AgCl). The midpoint potential of GNW1, GNW2 and GNW4 is between −0.37 V (vs. Ag/AgCl) and − 0.36 V (vs. Ag/AgCl), higher than that of the control strain. Given that the midpoint potentials of OmcZ and OmcS are −0.42 V (vs. Ag/AgCl) and − 0.41 V (vs. Ag/AgCl) respectively (Fricke et al., 2008; Inoue et al., 2010; Qian et al., 2011), GNW2 and GNW3 were expected to have midpoint potentials lower than that of the control strain, which was inconsistent with the present results. Therefore, these findings indicated a complex regulation of redox components in G. sulfurreducens as suggested in previous studies (Hernández‐Eligio et al., 2022; Park & Kim, 2011). In addition, reversible peaks near the top of the CV curves at approximately −0.30 V (vs. Ag/AgCl) were resolved in all the biofilms of the constructed strains. First derivate analysis also revealed a current wave near −0.30 V (vs. Ag/AgCl) for the control strain (Figure S5). Therefore, this reversible peak reflects an intrinsic electrochemical characteristic of G. sulfurreducens biofilm.

Biomass and images of electrode‐attached biofilms

The protein amounts of the electrode‐attached biofilms were measured to evaluate the abundance of electroactive cells on the anode (Figure 5). The protein amounts of the constructed strains increased by 39% to 62%, as compared with that of the control strain (p < 0.05). The amounts of proteins from the biofilms of the constructed strains ranged from 7.23 ± 0.36 g m−2 (n = 3) to 8.45 ± 0.35 g m−2 (n = 3), while those for the biofilms of the control strain was 5.21 ± 0.28 g m−2 (n = 3). In addition, comparison between the constructed strains revealed that overexpression of OmcS had the most significant enhancement for biofilm formation on electrode. However, overexpression of OmcZ had relatively weaker promotion for electroactive biofilm. This result may be ascribed to the inhibition of omcZ gene on the expression of omcS gene (Park & Kim, 2011). After normalization with measured proteins, the maximum power density of the constructed strains was 0.18 ± 0.01 W g−1 (n = 3) to 0.20 ± 0.01 W g−1 (n = 3), which were 1.74‐ to 1.93‐fold higher than that of the control strain with 0.10 ± 0.01 W g−1 (n = 3) (p < 0.05) (Figure 5).

FIGURE 5.

FIGURE 5

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Protein amounts of electrode‐attached biofilms and biofilm biomass‐normalized maximum power density. Shown are the amounts of proteins normalized with the areas of anodes. The maximum power density was normalized with the biofilm‐associated protein amounts. The values reported are the means and standard deviations of triplicate measurements. The expression constructs are displayed in parentheses. Asterisks represent significant differences between the constructed G. sulfurreducens strains and the control strain (p < 0.05).

As shown in Figure 6, Geobacter cells formed active biofilms on the surface of the carbon filaments of anodes. Three‐dimensional side‐view image revealed that the thickness of biofilms from the constructed strains was 110–130 μm, which were significantly larger than the thickness of biofilms from the control strain with only 75 ± 13 μm (n = 3) (p < 0.05). These results further evidenced the measured larger protein amounts of the constructed strains.

FIGURE 6.

FIGURE 6

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Confocal scanning laser microscopy images of the electrode‐attached G. sulfurreducens biofilms. (A) control strain; (B) GNW1 (pOEpilA); (C) GNW2 (pOEomcZ); (D) GNW3 (pOEomcS); (E) GNW4 (pOEomcT). The images are three‐dimensional top views (middle panels), lateral side views (right panels) and horizontal side views (bottom panels) of biofilms. Side‐view images show denser biofilms for the constructed strains with overexpression of nanowire proteins than that for the control strain. Images are representative (n = 5). Scale bar, 100 μm.

Enhancement of biofilm‐formation capability was thought to be beneficial for increasing the abundance of active current‐producing cells on electrode. In this study, the biomass of electrode‐attached biofilm was increased for all constructed strains, and the current production was substantially improved. Moreover, increase in average power output normalized with protein amounts suggested that the EET efficiency of individual cells was also improved. These results were consistent with previous reports that PilA and OmcZ were critical components of conductive biofilms (Inoue et al., 2011; Nevin et al., 2009; Steidl et al., 2016). In previous study, the abundance of OmcS was positively correlated with the current productivity of electrode‐attached biofilm (Holmes et al., 2006). Consistent with this finding, the results from this study showed that overexpressed OmcS and OmcT also participated in electron transfer to electrodes. In addition, the present results revealed that the constructed strain with overexpressed OmcZ had lower current production than that with overexpressed OmcS. Given that omcZ can downregulate omcS expression (Park & Kim, 2011), the total abundance of extracellular cytochromes of the biofilms with overexpression of omcZ may be lower than that with overexpression of omcS. Thus, the results from this investigation suggested that all the Geobacter nanowire components play important roles in the development of conductive biofilm.

Up to date, several genetic strategies have been developed to engineer G. sulfurreducens for improving its EET efficiency and current production (Dantas et al., 2015; Hernández‐Eligio et al., 2022; Leang et al., 2013; Tan et al., 2017). All these studies indicated the importance of protein nanowires for the formation of thick conductive biofilms as emphasized in this study. Therefore, bacterial nanowire proteins are promising synthetic biology parts for improving electrochemical performance of microbes by genetic engineering. Moreover, protein nanowires are interesting conductive bionanomaterials which can be constructed in G. sulfurreducens at low cost (Liu et al., 2020).

CONCLUSIONS

Overexpression of nanowire proteins (e.g. PilA, OmcZ, OmcS and OmcT) can substantially increase electrical outputs of the MFCs with G. sulfurreducens. The increased bioelectricity production was attributed to the decreased internal resistance as well as improved biofilm formation on the anodes. These results demonstrate a new approach for enhancing the performance of MFCs. It should be noted that overexpression of nanowire proteins may cause metabolic burden for constructed cells and the performance of MFCs is close to the upper limit in this study. Rational engineering of the metabolic flux of G. sulfurreducens may further improve the electricity production of MFCs with this species.

FUNDING INFORMATION

This work was supported by the National Key Research and Development Program of China (No: 2018YFA0901303); National Natural Science Foundation of China (No: NSFC91851211, 41772363); and the Fundamental Research Funds for the Central Universities, China University of Geosciences (Wuhan).

CONFLICT OF INTEREST

The authors declare no competing interests.

Supporting information

Figure S1

Figure S2

Figure S3

Figure S4

Figure S5

Table S1

Table S2

Click here for additional data file. (660.7KB, docx)

ACKNOWLEDGEMENTS

This work was supported by the National Key Research and Development Program of China (No: 2018YFA0901303); National Natural Science Foundation of China (No: NSFC91851211, 41772363); and the Fundamental Research Funds for the Central Universities, China University of Geosciences (Wuhan).

Wang, Z. , Hu, Y. , Dong, Y. , Shi, L. & Jiang, Y. (2023) Enhancing electrical outputs of the fuel cells with Geobacter sulferreducens by overexpressing nanowire proteins. Microbial Biotechnology, 16, 534–545. Available from: 10.1111/1751-7915.14128

Contributor Information

Liang Shi, Email: liang.shi@cug.edu.cn.

Yongguang Jiang, Email: jiangyg@cug.edu.cn.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author.

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Associated Data

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Supplementary Materials

Figure S1

Figure S2

Figure S3

Figure S4

Figure S5

Table S1

Table S2

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Data Availability Statement

The data that support the findings of this study are available from the corresponding author.

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