Contents
Introduction
Microbial reduction
Bacteria Shewanella Sulfate reducing bacteria Escherichia Other bacteria Mechanism of bacterial reduction of GO Exoelectrogens Non-exoelectrogens Fungi Algae Application of MrGO Wastewater treatment Biological application Other applications Issues and challenges in the application of microbial reduction of GO
Application of microbial-rGO in MFC and BPV
Anodes Cathodes
Conclusions and outlook
Disclosure of potential conflicts of interest
Acknowledgements
References
1 Introduction
Graphene is a single-atom-thick sheet of sp2 hybridized carbons densely organized in a hexagonal arrangement. It is the first two-dimensional (2D) substance that has been heralded as the cornerstone of materials science research [1–2]. In fact, it is the fundamental element of other carbon allotropes, namely fullerene, carbon nanotubes and graphite. Ever since its discovery in 2004, graphene has been widely researched due to its extraordinary characteristics such as superior electron mobility, great thermal and electrical conductivity and exceptional mechanical properties, which lead to its promising application in various sectors, such as energy storage [3], biosensing [4], nanoelectronics [5], and biomedical fields [6].
Despite the massive potential and great expectations, the commercialization of graphene has yet to be achieved. One of the biggest bottlenecks is their production, which has low degrees of reproducibility. Therefore, the main drive in graphene research is to achieve mass production in order to accommodate industrial needs. The synthesis of graphene can be categorized into top-down and bottom-up strategies. The top-down strategy is based on the basic concept of exfoliating layers of graphene from graphite. The first method proposed to produce graphene is the mechanical exfoliation of graphite by using adhesive tape, which produces high-quality graphene flakes, but is cumbersome, yield-limited, and hence impractical to scale up [2]. Other top-down strategies such as liquid phase exfoliation [7] and electrochemical exfoliation [8] have also been proposed to produce graphene in bulk. Nonetheless, these methods are energy-consuming and require expensive equipment even with shorter production times. Additionally, bottom-up strategies such as chemical vapor deposition (CVD) [9] and epitaxial growth on silicon carbide [10] build the graphene sheet from scratch but are often laborious and expensive, making them unfeasible for the industrial production of graphene.
Among the developed production strategies, the chemical oxidation of graphite to produce graphene oxide (GO), followed by the subsequent reduction of GO to produce reduced graphene oxide (rGO), is the most practical and economically viable route to produce graphene derivatives [11–12]. Graphite is a three-dimensional (3D) material comprised of millions of graphene layers. By oxidizing graphite with strong oxidizing agents, oxygen functional groups such as epoxide, carbonyl, carboxyl, and hydroxyl groups are introduced into the graphite structure to form graphite oxide. This not only increases their interlayer distances but also makes the material hydrophilic, thereby enabling them to be exfoliated in water using sonication and ultimately producing a single or few layers of the oxidized form of graphene sheets, known as GO [13]. The most established method for the synthesis of GO is the Hummers method, in which graphite is oxidized by concentrated sulfuric acid, potassium permanganate, and sodium nitrate followed by a brief sonication [14]. Due to its hydrophilic properties, GO could be easily processed in solution on a large scale at a low cost. Nonetheless, oxygen functional groups must be removed to restore the constitutional properties of pristine graphene, particularly the electrical conductivity [15].
rGO is a graphene-like sheet comprised of graphene domains scattered with residual oxygen functional groups resulting from the reduction of GO. Despite having more flaws than pristine graphene, which results in poorer conductivity and mechanical qualities, rGO’s excellent synthetic scalability has allowed the development of numerous innovative graphene-based materials with superior physical attributes [12]. To date, different GO reduction strategies have been proposed, such as chemical, biological, photo- and thermal-mediated reduction. Chemical reduction is one of the most extensively used methods due to the well-established reduction mechanism of common reducing agents that has been long proposed [16]. However, chemical reducing agents such as hydrazine, sodium borohydride, and hydroquinone are potentially hazardous to humans as well as the environment. Also, the chemical reduction of GO tends to remove the oxygen functional groups completely, which results in the irreversible agglomeration of rGO and disruption of the electron transfer [15]. Therefore, scientists have shifted their focus to eco-friendly biological reductants such as plant extracts, biomolecules, and microorganisms [17]. Moreover, numbers of reports suggest the application of biocompatible graphene for bio-related usages such as drug administration, biosensors, bioimaging, and tissue engineering [18]. The presence of trace concentrations of toxic chemicals upon chemical reduction of GO could be hazardous for biological usage. As a result, the use of green GO reduction to address the aforementioned issues by employing biological reducing agents such as microorganisms has become increasingly important [17]. Recently, interest in photoreduction and thermal reduction of GO has increased, but commercialization of these techniques is difficult to achieve as specialized instruments are required. In addition, energy-intensive procedures make these production methods unviable and unsustainable for large-scale applications [19]. Instead, the ability of microorganisms to reduce GO effectively under ambient conditions could be a more economically viable and environmentally friendly option for large-scale production of rGO [20].
A recent comprehensive review published by Agarwal and Zetterlund [19] discussed the existing GO reduction methods, including chemical, biological, photo- and thermal-mediated reduction. Meanwhile, detailed chemical and biological reduction of GO was also reviewed by Chua and Pumera [16] and Agharkar et al. [17], respectively. However, there has yet to be a recent review discussing the reported microbial reduction of GO in detail. In this review, the reaction conditions and the proposed reduction mechanism of GO by the microorganisms will be summarized and compared in terms of their reduction efficiency (Tab.1 [20–54]). Then, the application of microbially reduced GO (MrGO), particularly in microbial fuel cells (MFC) and biophotovoltaics (BPV) will be discussed (Fig.1).
Tab.1 List of publications on the microbial reduction of GO [20–54] (the reaction conditions and properties of the resulting rGO including ID/IG ratio obtained from Raman analysis and carbon-to-oxygen (C/O) ratio obtained from XPS analysis were compared) |
| Microorganism | Reaction conditions | C/O ratio | ID/IG ratio | Potential application | Ref. | ||||
|---|---|---|---|---|---|---|---|---|---|
| GO | Microorganisms | Duration | Temperature | Anaerobic/aerobic | |||||
| S. oneidensis MR-1, S. amazonensis SB2B, S. baltica 10735T, S. putrefaciens CN32 & S. putrefaciens W3-18-1 | 2 mg | 108 cells·mL−1 Overnight culture in 10 mL of Shewanella Federation-defined medium with lactate | 72 h | RT a) | Anaerobic | %C-C b): GO = 28; CrGO = 83; MR-1 = 56; SB2B = 75; 10735T ≥ 95; CN32 = 91; W3-18-1 = 54 | ‒ | ‒ | [21] |
| S. oneidensis MR-1, Shewanella sp. ANA-3 | 50 mL (0.3 mg·mL−1) | 100 mL of 12-h Culture in trypticase soy broth (TSB) medium | 24‒60 h | RT a) | Anaerobic and aerobic (shaken at 200 r·min−1) conditions separately | GO = ~1.4; rGO-60 h = ~3.1 | ‒ | ‒ | [22] |
| Shewanella oneidensis MR-1 | 0.5 mg·mL−1 | (OD600 = 0.1) of Overnight culture in TSB medium | 48 h | RT a) | Anaerobic and aerobic (shaken at 250 r·min−1) conditions separately | GO = 1.03; MrGO = 2.13; CrGO = 6.17 | GO = 0.85; MrGO = 1.0; CrGO = 1.0 | ‒ | [20] |
| Shewanella oneidensis MR-1 | 0.8 mg·mL−1 | 1:1000 Dilution of overnight bacteria culture + 15 mmol·L−1 lactate | 40 h | ‒ | Anaerobic | ‒ | ‒ | ‒ | [40] |
| Shewanella sp. CF8-6 | 20 mL (0.2 mg·mL−1) | 100 mL Bacteria culture + 0.2 mg·mL−1 sodium acetate | 12 h | ‒ | Anaerobic | GO = 0.86; rGO = 1.23 | GO = 1.11; rGO = 1.26 | Dye decolourization | [48] |
| Shewanella xiamenensis BC01, Shewanella putrefaciens CN32 | 0.17 mg·mL−1 | 1 mL Bacterial suspension + 10 mL sodium acetate medium | 8 d | 28 °C | Anaerobic | ‒ | ‒ | Dye decolourization | [23] |
| Shewanella oneidensis MR-1 | 0.01 mg·mL−1 | Bacterial suspension (OD600 = 0.5) in mineral medium | 10 h | 30 °C | Anaerobic | GO = 1.82; rGO = 2.79 | GO = 0.92; rGO = 1.02 | Oxygen evolution reaction | [50] |
| Sulfate-reducing bacteria | 100 mL (0.1 mg·mL−1) | 20 mL SRB + 30 mL fresh medium | 6 d | 37 °C | Anaerobic | GO = 2.12; rGO = 4.02 | GO = 1.02; rGO = 1.40 | Electrochemical sensing | [25] |
| Desulfovibrio desulfuricans | 5 mg | 10 mL Bacteria (0.5×109 cells per mL) in M9 medium | 24 h | 25 °C | Anaerobic | ‒ | GO = 0.92; rGO = 1.13 | Anti-biocorrosion | [24] |
| Enterococcus avium BY7 | 0.4 mL | 0.5 mL Bacteria (OD600 = 0.5) + 50 mL medium | ‒ | 30 °C | Anaerobic | GO = 1.39; rGO = 2.54 | ‒ | Heavy metal removal | [26] |
| Geobactersulfurreducens | 0.4 mg·mL−1 | 100 mL G. sulfurreducens suspension (OD600 = 0.8) in growth media with 20 mmol·L−1 acetate | 48 h | 30 °C | Anaerobic | GO = 2.78; rGO = 5.5 | GO = 0.93; rGO = 1.18 | Oxygen evolution reaction | [27] |
| Geobactersulfurreducens | 0.6 mg·mL−1 | 5% Inoculum of bacteria | ‒ | 30 °C | ‒ | GO = 0.83; rGO = 2.04 | GO = 0.945; rGO = 1.324 | ‒ | [28] |
| Escherichia coli | GO film (drop casting 5 mg·mL−1 of GO suspension on SiO2/Si(100) substrate) | Bacterial suspension | 48 h | 37 °C | Anaerobic | ‒ | GO = 1.31; rGO = 0.97 | Antibacterial coating | [29] |
| Escherichia coli | 0.02 mg·mL−1 | 107‒108 cfu·mL−1 Cells in saline (0.5% NaCl) | 0.5‒2 h | 37 °C | Aerobic (continuous shaking) | GO = 2.68; rGO = 5.78 | GO = 0.95; rGO = 0.72 | ‒ | [30] |
| Escherichia coli | 0.5 mg·mL−1 | 200 mg Bacteria in 20 mL of water | 72 h | 37 °C | ‒ | ‒ | GO = 1.3; rGO = 2.6 | ‒ | [31] |
| Escherichia fergusoni | 0.5 mg·mL−1 | 200 mg Bacteria in 20 mL of water | 72 h | 37 °C | ‒ | ‒ | GO = 1.58; rGO = 1.96 | ‒ | [32] |
| Bacillus sp., E. coli, E. cloacae, S. baltica, and extremophile consortium | 30 mL (0.4 mg·mL−1) | 30 mg·mL−1 Bacterial biomass | 72 h | 20‒25 °C | Aerobic (shaken at 150 r·min−1) | ‒ | GO = 1.09; S. baltica-rGO = 0.99; Extremophile consortium-rGO = 1.05 | ‒ | [33] |
| Halomonas eurihalina and Halomonas maura | 1 mg·mL−1 | 1 mL Bacteria culture (OD520 = 2.5) | 5 d | 32 °C | Aerobic: agitation speed of 110 r·min−1; Anaerobic: without agitation in dark | ‒ | GO = 1.24; H. eurihalina-rGO = 1.04; H. maura-rGO = 1.05 | Biological applications | [51] |
| Fontibacillus aquaticus | 0.015 mg·mL−1 | 25 mL Bacterial culture grown in basal mineral medium | 30 d | ‒ | ‒ | ‒ | GO = 0.23; rGO = 0.11 | ‒ | [34] |
| Bacterial suspension from riverside | GO coated on silicon substrates | Microbial culture | 72 h | 28 °C | Anaerobic | GO = 3.24; MrGO = 8.1; UVrGO = 3.7; CrGO =15.9 | GO = 1.4; MrGO = 1.55; UVrGO = 1.6; CrGO = 1.9 | ‒ | [35] |
| Lactobacillus plantarum | 0.5 mg·mL−1 | 200 mg Bacteria biomass | 7 d | 30 °C | ‒ | GO = 1.7; rGO = 3.3 | GO = 0.94; rGO = 0.92 | ‒ | [36] |
| Mixed culture of microorganisms from anaerobic sludge | 0.1 wt.% of GO dispersion | 200 mg Microbial cells | 24 h | 30 °C | Anaerobic | ‒ | GO = ~ 0.9; MrGO = ~1.5; CrGO = ~ 1.8 | Biological applications | [37] |
| Azotobacter chroococcum | 100 mL (1 mg·mL−1) | 100 mg A. chroococcum | 72 h | RT a) | ‒ | GO = 2.23; rGO = 4.18 | ‒ | ‒ | [38] |
| Pseudoalteromonas sp. CF10-13 | 0.2 mg·mL−1 GO + sodium alginate | 50% Bacteria inoculum in LB medium | ‒ | 80 °C | Anaerobic | ‒ | GO = 1.03; rGO = 1.30 | Dye decolourization | [49] |
| Bacillus marisflavi | 0.5 mg·mL−1 | 200 mg B. marisflavi biomass | 72 h | 37 °C | ‒ | ‒ | GO = 1.4; rGO = 1.7 | Biological applications | [53] |
| Bacillus subtilis | 5 mg | 10 mLCell suspension (0.5×109 cells per mL) in M9 medium + 10 µg of VK3 | ‒ | 25 °C | Anaerobic | ‒ | GO = 0.92; rGO = 1.01 | Biological applications | [41] |
| Pseudomonas aeruginosa | 0.5 mg·mL−1 | 200 mg P. aeruginosa biomass | 48 h | 37 °C | ‒ | ‒ | GO = 1.4; rGO = 2.03 | Biological applications | [52] |
| Gluconobacter roseus | 0.5 g | 0.1 g (wet weight) G. roseus dispersed in phosphate buffer + 5 g/100 mL sorbitol | 24 h | 37 °C | ‒ | ‒ | GO = 1.12; rGO = 0.87 | MFC | [54] |
| Bacteriorhodopsin (bR) extracted from Halobacterium salinarum | GO film on SiO2 substrate | 1 mL (5 mg·mL−1) The purple membrane of H. salinarum | 3 h | 39 °C under irradiation of ~80 mW·cm−2 yellow light | ‒ | ‒ | bR-reduced GO = 0.08; CrGO = 0.15 | ‒ | [39] |
| Baker’s yeast | 200 mL (0.5 mg·mL−1) | 200 mg Baker’s yeast in deionized water | 72 h | 35‒40 °C | ‒ | GO = 2.2; rGO = 5.9 | GO = 0.80; rGO = 1.44 | ‒ | [42] |
| Yeast extract | 0.05‒0.5 mg·mL−1 | 30 mL Culture solution containing 0.5% yeast extracts | 15 min | Autoclaved at 121 °C | ‒ | GO = 1.8; rGO = 3.1 | GO = 0.98; rGO = 0.999 | Nanocomposites formation | [43] |
| Rhizopus oryzae | 1 mg·mL−1 | Small pieces of semi dried mycelia of R. oryzae | 24 h | 37 °C | ‒ | ‒ | GO = 0.96; rGO = 1.17 | Antibacterial coating | [44] |
| Ganoderma lucidum | 50 mL (0.1 mg·mL−1) | 50 mL G. lucidum extract (1 g mushroom powder in 100 mL Milli-Q water) | 16 h | 85 °C | ‒ | ‒ | GO = 0.94; rGO = 0.99 | Biological application | [45] |
| Ganoderma sp. | 1 mg·mL−1 | 20 mg Ganoderma extracts powder in 20 mL of deionized water | 24 h | 37 °C | ‒ | ‒ | GO = 1.8; rGO = 2.1 | Cancer therapy | [46] |
| Algal extracts of Scenedesmus vacuolatus, Chloroidium saccharophilum, Leptolyngbya JSC-1 | 100 mL (1 mg·mL−1) GO | 5 g Algae extracts in 100 mL water | 24 h | 95 °C | ‒ | ‒ | ‒ | Heavy metal removal | [47] |
a) RT means room temperature; b) Fraction of reduced carbon was shown as no C/O ratio was reported from the study. |
2 Microbial reduction
2.1 Bacteria
2.1.1 Shewanella
Shewanella, a group of heterotrophic and facultative anaerobes ubiquitous in diverse environments, is one of the first bacteria species tested for GO reduction [21]. Shewanella uses a variety of inorganic or organic molecules as terminal electron acceptors in their respiratory route. The external electron transfer (EET) ability of Shewanella has been widely researched for various applications such as bioremediation, toxic metal removal, and electricity generation in the MFC [22].
The ability of five strains of Shewanella from different habitats to reduce GO under strictly anaerobic conditions was investigated by Salas et al. [21]. X-ray photoelectron spectroscopy (XPS) analysis showed that rGO synthesized by two strains of S. putrefaciens (CN32 and W3-18-1) had the highest fraction of reduced carbon (%C-C) after the reduction process which was even higher than the chemically reduced GO (CrGO) by using hydrazine at room temperature (83%). Meanwhile, the %C-C of rGO synthesized by S. amazonesis SB2B, S. oneidensis MR-1, and S. baltica 10735 were 75%, 56%, and 54%, respectively. In a study by Wang et al. [22], Shewanella sp. ANA-3 exhibited better GO reduction than the MR-1 strain due to its faster growth. Interestingly, Shewanella achieved better GO reduction under aerobic conditions than anaerobic conditions due to its faster growth under aerobic conditions. As it was believed that oxygen would be a more energetically favourable electron acceptor than GO, better GO reduction under aerobic conditions could be due to the rapid expenditure of oxygen by densely populated Shewanella, which eventually created an anaerobic condition. Also, GO could attach physically to the bacterial surface, thus reducing the amount of oxygen reaching the bacteria. Therefore, Shewanella shuttled the electrons to GO instead of the oxygen molecules [22]. Meanwhile, Lehner et al. [20] reported that there was no significant difference in the amount of rGO produced by S. oneidensis under aerobic and anaerobic conditions. In addition, MrGO exhibited better storability and a larger surface-area-to-thickness ratio than CrGO. Because of the higher energy required to reduce C=O bonds, S. oneidensis is unable to reduce C=O bonds. As a result of the presence of C=O, π‒π stacking of MrGO is prevented [20].
Shen et al. [23] demonstrated the synthesis of 3D rGO hydrogel, which contains live bacteria, extracellular polymeric substances (EPS), and rGO. Out of the seven strains of bacteria, including Shewanella, Escherichia, and Bacillus, only Shewanella xiamenensis BC01 and Shewanella putrefaciens CN32 were capable of forming rGO hydrogel after incubating with GO dispersion for 8 d under anaerobic conditions. They proposed that the rGO hydrogel be self-assembled by the Shewanella through stacking, bridging, rolling, and cross-linking. After the oxygen functional groups on the GO were removed, hydrophobic rGO sheets were stacked together due to π–π interaction. The rGO sheets rolling occurred because a higher number of oxygen functional groups, such as carboxyl, were found on the edge of the GO sheets [55]. Thus, the edge of rGO sheets tends to be reduced first and become hydrophobic, while the centre of the GO sheets remains hydrophilic, thereby resulting in the rolling of the rGO sheets. Next, the motile bacteria may bridge the graphene fragments remaining in the GO solution and bind them to the hydrogel. EPS synthesized by the bacteria also functioned as a cross-linker to gel the bacteria/rGO complex together [23].
2.1.2 Sulfate reducing bacteria
Sulfate-reducing bacteria (SRB) are a varied group of bacteria capable of utilizing soluble sulfate as the terminal electron acceptor in the respiratory pathway [24]. They are commonly found in anoxic environments and play an important role in the carbon and sulphur cycles. SRB have been extensively studied in biotechnological applications to remove toxic metals. Guo et al. [25] reported that the SRB could reduce GO at 37 °C after 6 d (Tab.1). Interestingly, N and S atoms were also introduced onto the rGO during the reduction process, resulting in N and S-doped rGO. XPS analysis showed that the percentages of N and S atoms doped on the rGO were 6.11% and 1.1%, respectively. The resulting N, S-doped rGO displayed remarkably better electrochemical sensing of heavy metal ions Cd2+ and Pb2+ simultaneously than single-doped rGO, which could be attributed to the synergistic effects of N and S dopants [25]. Song et al. [24] also reported that N and S-containing functional groups were introduced into the rGO after being reduced by Desulfovibrio desulfuricans at 25 °C for 24 h (Tab.1).
Another SRB, Enterococcus avium BY7, was also used to synthesize rGO under anaerobic conditions [26]. Results showed that adding 0.4 and 0.6 mL of GO enhanced the growth of the bacteria, while 0.8 mL and above inhibited their growth. Interestingly, after the reduction process, rGO was able to act as a matrix to immobilize large numbers of bacteria to form BY-rGO particles. The resulting BY-rGO particles promoted the growth of the immobilized SRB and sulfate reducing ability. Moreover, the BY7 immobilized in the rGO particles can survive better under extreme conditions such as pH 2.0‒12.0 and temperature of 10‒45 °C. This could be due to rGO protecting the microbial activity of the bacteria after immobilization [26]. Similarly, after incubating Geobacter sulfurreducens with GO under anaerobic conditions, rGO hydrogel was formed. Scanning electron microscopy (SEM) showed that the bacteria were embedded inside the rGO sheet after the reduction process. Moreover, multiple heteroatoms doped-rGO (N, S, P, Fe, and Cu) were formed during the reduction process, which could be due to the abundance of these elements in the bacterial cells. The resulting rGO hydrogel was effective in the oxygen evolution reaction due to the synergistic effect of different heteroatoms presented on the rGO [27]. The doping of N and P on rGO reduced by G. sulfurreducens was also observed, which might be due to the interaction of GO with phospholipids or other macromolecules that are rich in the cell membrane of the bacteria [28].
2.1.3 Escherichia
Another species of bacteria tested in the reduction of GO was E. coli due to its industrial scalability and abundance. Akhavan & Ghaderi [29] incubated the GO film with E. coli in Luria‒Bertani (LB) medium under anaerobic conditions. After 48 h of incubation, XPS analysis showed that about 60% of the oxygen functional group in the GO film was removed. However, no reduction of GO was observed in the LB medium containing the bacteria without glucose content which indicated that the glycolysis process might be involved in the reduction of GO [29]. Similarly, Zhao et al. [30] reported that no reduction of GO was observed by incubating E. coli with GO in saline (0.5% NaCl) under anaerobic conditions. This could be due to no glucose being provided during the reduction process, so the glycolysis process could not be carried out. However, GO was reduced by E. coli incubated in saline with continuous shaking. The GO reduction was completed in 0.5‒2 h, much shorter than most microbial reductions that required 24‒72 h [30]. Meanwhile, E. coli [31] and E. fergusoni [32] reportedly reduced GO effectively in an aqueous medium after 72 h. Interestingly, all of the studies on GO reduction by Escherichia were conducted at 37 °C instead of room temperature (Tab.1).
2.1.4 Other bacteria
To expand the range of bacteria with potential GO reduction ability, Vargas et al. [33] incubated Enterobacter cloacae, Bacillus sp., E. coli, Shewanella baltica, and an extremophile consortium with GO aerobically for 72 h. Different degrees of GO reduction were observed, with S. baltica and the extremophile consortium exhibiting the best reduction ability based on thermogravimetric analysis (TGA) and Raman spectroscopy of the resulting rGO. Meanwhile, Chouhan et al. [34] developed a facile GO reduction method by utilizing nanomaterial-resistant Fontibacillus aquaticus isolated from the soil samples near a nanomaterial-contaminated pond. Fourier transform infrared spectroscopy (FTIR) and XPS analyses showed that the C=O functional group on GO was reduced after their interaction with the resistant bacteria. Bacterial suspensions extracted from riverbanks were also shown to reduce GO under aerobic conditions. Results showed that CrGO remains the most effective in reducing oxygen functional groups, followed by ultraviolet-photocatalysis-reduced GO (UVrGO) and MrGO [35–56]. Lactobacillus plantarum was also used to reduce GO at 30 °C for 7 d (Tab.1). A significant decrease in the intensity of epoxy and alkoxy functional groups was observed from FTIR after the reaction. Furthermore, it showed good stability in water, indicating that L. plantarum might play a role as a stabilizing agent [36].
Other than SRB, other microorganisms were also reported to synthesize heteroatom-doped rGO for various applications. For instance, an N-doped rGO with an N/C ratio of 8.4 was synthesized by incubating GO with microorganisms extracted from the anodic chamber in MFC, mainly consisting of Staphylococcus. The formation of the C−N bond could be due to the reaction between the oxygen functional group on the GO and amine, nitrite, or other nitrogen-containing substances [37]. Similarly, Chen et al. [38] have observed the formation of a C−N peak in the XPS spectra of rGO after being reduced by Azotobacter chroococcum, which could be due to the introduction of the hydrazine group during the reaction, hence resulting in N-doped rGO.
Akhavan [39] attempted to use bacteriorhodopsin (bR) isolated as a purple membrane from Halobacterium salinarum to reduce GO. The bR molecules and GO were separated by a membrane filter to prevent the attachment of molecules to the GO. Through the light-driven proton pumping process of bR molecules, the electrons were produced and adsorbed by the GO. A sufficient accumulation of electrons in GO can cleave the hydroxyl and open the epoxide group. Lastly, the reduction was completed by the reactions of opened epoxide and hydroxyl groups with protons to form hydroxyl and water, respectively. As a result, bR-reduced GO showed a similar deoxygenation level as CrGO. In addition, bR-reduced GO showed better electrical conductivity and lower sheet resistance than CrGO, which could be due to no formation of the C−N bond on bR-reduced GO during the reduction process [39].
2.2 Mechanism of bacterial reduction of GO
In general, the bacteria tested for the reduction of GO can be separated into two categories: bacteria with EET ability, known as exoelectrogens, and bacteria without EET ability. Using bacteria to reduce GO emerged as some bacteria, such as Shewanella and Geobacter, have been well-studied for their ability to transfer electrons externally to uncommon terminal electron acceptors other than oxygen in their respiratory pathway. Shewanella, for example, has been shown to use a variety of organic and inorganic compounds as terminal electron acceptors in their respiratory pathway, including iron oxides, arsenate, chromium oxides, and dimethyl sulfoxide [21]. Thus, the following sections will discuss the mechanism for GO reduction by exoelectrogens and non-exoelectrogens.
2.2.1 Exoelectrogens
In order to understand the direct electron-transfer mechanism of Shewanella, Salas et al. [21] incubated S. oneidensis MR-1 deficient in different membrane proteins commonly involved in anaerobic metal reduction with GO. Results showed that MtrA protein was required for the reduction of GO while CymA was not, even though CymA has been proven to be an essential protein in anaerobic respiration. However, Jiao et al. [40] found out that CymA was required for S. oneidensis MR-1 to reduce GO, while MtrA and MtrB were also equally important (Fig.2). This is because CymA mutant strains cannot restore any reduction of GO even with the electron shuttles, implying that it is a crucial protein in transporting the electrons to GO. Also, three membrane-bound cytochrome c in the outer membrane, MtrC, MtrF, and OmcA were tested for their involvement in GO reduction. Results suggested that MtrC and OmcA are the main terminal reductases for GO, and they can partially compensate for the absence of one another while MtrF is unessential [40]. Cytochrome c was also involved in the GO reduction by Geobacter sulfurreducens under anaerobic conditions, as the bacterial cells treated with bipyridine could not reduce GO [27].
Besides direct EET, indirect EET via electron mediator compounds was also involved in the Shewanella reduction of GO. Results showed that the addition of exogenous electron shuttle 9,10-anthraquinone-2,6-disulfonic acid (AQDS) (4.7-fold) had a more significant impact than the in-situ produced electron shuttle riboflavin (2.7-fold), indicating that the electron shuttle also played an essential role in the reduction of GO by Shewanella [40]. This could be supported by the observation from Wang et al. [22], who reported the reduction of GO by spent medium, indicating the redox-active compounds secreted by Shewanella could be involved in GO reduction. Similarly, Fan et al. [37] reported the complete reduction of GO by the spent medium from the mixed culture of microorganisms obtained from the anodic chamber of the MFC.
2.2.2 Non-exoelectrogens
Akhavan & Ghaderi [29] proposed that GO reduction by E. coli under anaerobic conditions was related to glycolysis, as no reduction occurred when glucose was not supplied. It was the hydrogen or electrons that were generated in the glycolysis process that reduced the GO. Meanwhile, Liu et al. [41] found out that under anaerobic conditions, Bacillus subtilis can only reduce GO in the presence of menadione, also known as vitamin K3 (VK3), with the help of transmembrane succinate:menaquinone oxidoreductase (SQR), an enzyme capable of transferring electrons from intracellular succinate to extracellular VK3. B. subtilis transfers electrons from succinate to VK3. Then, the reduced form of VK3 transfers electrons to GO, forming rGO. The oxidized VK3 returns to the SQR to receive electrons again. Similarly, Chen et al. [38] suggested that the reduction of GO by A. chroococcum was catalyzed by Mo-nitrogenase, an enzyme that is commonly found in many bacteria and archaea that catalyzes biological nitrogen fixation. GO was first attached to the MoFe protein of the nitrogenase. Then, C−O−C and C=O bonds were cleaved to form hydroxyl groups by the addition of electrons and protons. Lastly, the hydroxyl groups were removed through dehydration [38].
Under aerobic conditions, the reduction of GO by the bacteria was unrelated to the respiratory metabolism. Zhao et al. [30] observed a significant increase in the superoxide anion radical (O2•−) during the GO reduction by E. coli under aerobic conditions. They proposed that GO may act as an electron shuttle to transport electrons from cytochrome c of E. coli to extracellular oxygen, thereby resulting in the formation of O2•−. The GO was then reduced to rGO by the O2•− (Fig.3). This was demonstrated by using superoxide dismutase to prevent GO reduction or by conducting experiments under completely anaerobic conditions to prevent O2•− production. Also, direct contact between the bacterial cells and GO was required as no reduction occurred in GO coated with bovine serum albumin [30]. Similarly, Vargas et al. [33] proposed that bacteria could reduce GO by generating reactive oxygen species due to the interaction between GO and bacteria. Also, physical disruption of the bacterial cell membrane by GO will lead to the leakage of cellular compounds that contribute to the reduction of GO (Fig.3). The chemical oxidation mechanism was proved by experiment with aerobic conditions so that the use of oxygen instead of GO as the final electron acceptor could be achieved. Additionally, cell respiration was inhibited by not providing carbon sources and nutrients throughout the experiment to prevent GO reduction through the respiration pathway [33].
Fig.3 Possible GO reduction mechanism by bacteria: (1) Leakage of cytoplasmic compounds through physical disruption of bacterial cell membrane by GO [33]; (2) Direct EET [21,29,40]; (3) Indirect EET through exogenous or self-secreted electron shuttle [22,37,40–41]; (4) Generation of ROS such as O2•− [30,33]. |
2.3 Fungi
Besides bacteria, fungi such as baker’s yeast and Rhizopus oryzae have also been explored to reduce GO due to their low cost, wide availability, and scalability, which make them an ideal reducing agent for use in the industry. Khanra et al. [42] reported a novel strategy to reduce and functionalize GO simultaneously by using baker’s yeast as the reductant. The reaction was conducted at 35–40 °C and completed after 72 h (Tab.1). The amine functional groups from the nicotinamide adenine dinucleotide phosphate (NADPH) can efficiently react with the epoxy functional groups of GO, thereby resulting in the attachment of NADPH to the rGO surface. The resulting rGO exhibits superior water dispersibility due to the presence of amine functional groups, thus preventing the aggregation of rGO [42]. One-pot in-situ fabrication of bacterial cellulose (BC)/rGO composites was reported by Nandgaonkar et al. [43]. GO suspension was added to the mannitol culture medium containing Gluconacetobacter xylinus and subjected to autoclave at 121 °C for 15 min. Eventually the GO was reduced by the yeast extract and attached to the BC to produce BC/rGO composites in a single step. This research presented a cheap and facile method for fabricating biocompatible cellulose-based materials for various biomedical applications [43]. Similarly, the mycelia of a “generally recognized as safe (GRAS)” microorganism, Rhizopus oryzae, were incubated with GO at 37 °C for 24 h to synthesize rGO (Tab.1) [44].
The mushroom extract has also been tested to reduce GO due to its high polysaccharide content, which has been proven to exhibit excellent reducing potential [45]. Gurunathan et al. [46] reported the reduction of GO by using the mycelia extract of Ganoderma sp. at 37 °C for 24 h (Tab.1). Meanwhile, Muthoosamy et al. [45] reported the reduction of GO by using whole Ganoderma lucidum extract in 16 h, which is shorter than the process reported by Gurunathan et al. [46] but at a higher temperature (85 °C). This was because the heat was required to liberate the polysaccharides of G. lucidum, which is responsible for the reduction of GO. Furthermore, it was demonstrated that the extract could be reused at least three times to reduce GO with up to 75% conversion efficiency [45].
2.4 Algae
Nowadays, algae are increasingly being researched in the biosynthesis of nanoparticles due to their ubiquitous nature and environmentally friendly approach. Algal cells contain various bioactive compounds, such as pigments and antioxidants, which can act as reductants to synthesize metal nanoparticles such as Au and Ag [57]. To date, the only publication regarding the utilization of algae as a green reductant for GO was reported by Ahmad et al. [47]. Effective GO reduction was achieved by adding GO to three different algal extracts: Scenedesmus vacuolatus, Chloroidium saccharophilum, and Leptolyngbya JSC-1. Then, the mixture was kept at 95 °C for 24 h. As a result, S. vacuolatus was the most effective algal extract in removing oxygen functional groups on the GO due to the lowest zeta potential (−17.1 mV) achieved after the reduction process [47]. However, the actual mechanism of GO reduction by algal extracts was unknown.
2.5 Application of MrGO
2.5.1 Wastewater treatment
Several studies have reported that rGO synthesized by microorganisms has the potential to be a robust and environmentally friendly adsorbent for dye decolourization in wastewater treatment. Azo dyes such as Congo red and methylene blue are toxic and carcinogenic pollutants that are difficult to decompose due to their complex aromatic structures [23]. As a result, they have a detrimental impact on industrial wastewater treatment. In a study by Shen et al. [23], self-assembled 3D rGO biohydrogel synthesized by Shewanella xiamenensis BC01 achieved a 97% Congo red decolourization rate without the addition of sodium acetate medium, which is much higher than the suspended bacterial cells that can hardly work without the medium. In addition, rGO hydrogel was more manageable to remove from the liquid than the bacterial suspension after the treatment, making the separation step easier and more suitable for industrial applications. Han et al. [48] created a 3D poly(vinyl alcohol)/rGO aerogel (PVA-GA) by first reducing GO with Shewanella sp. CF8-6, then mix with PVA as a cross-linker to form an aerogel. As a result, a removal rate of 94.62% and 93.97% was achieved for methylene blue and Congo red, respectively. The superior pollutant removal performance of the PVA-GA was due to its 3D porous structure, which provided an abundance of adsorption sites for the pollutants [48]. Besides Shewanella, a marine bacterium, Pseudoalteromonas sp., was also used to reduce GO at 80 °C under anaerobic conditions to synthesize 3D rGO hydrogel with sodium alginate. The resulting rGO hydrogel was superior in the adsorption of methylene blue and Congo red with a removal rate of 87% and 92%, respectively, showing its potential to be applied in dye decolourization in sewage treatment [49].
MrGO has also been tested to remove heavy metal pollutants in wastewater treatment. rGO biohydrogel synthesized by Shewanella putrefaciens CN32 was used to treat wastewater containing hexavalent chromium (Cr(VI)). As a result, Cr(VI) was successfully reduced to less toxic Cr(III) after 20 h of incubation. The rGO hydrogel removed Cr(VI) at a rate of 96.31% in the presence of sodium lactate medium, compared to 47.38% for the cell suspension [23]. Yan et al. [26] reported that the tolerance of E. avium immobilized in the rGO particles towards various heavy metals such as Ni(II), Pb(II), Ti(I), Cu(II), Fe(III), and Cd(II) except Cr(III) was improved. The metal removal efficiency of the rGO particles was 40%‒50% higher than free bacterial suspension. This was because the rGO provided protection for the bacteria under adverse conditions while enhancing the EET efficiency from the bacteria to the heavy metal, thereby accelerating the biodegradation of the pollutants [26]. Meanwhile, Wang et al. [50] reported that rGO reduced by S. oneidensis MR-1 could recover Pd nanoparticles effectively. The amount of Pd recovered by the bacterial cells was only 37.8%, while 90.4% of Pd was recovered by the rGO. SEM and transmission electron microscopy (TEM) showed that after the reduction of GO, the bacteria adhered to the ultrathin rGO sheets. At the same time, Pd nanoparticles were uniformly dispersed on the surface of rGO and the bacterial cells. The resulting Pd-rGO has a high catalytic rate in the oxygen evolution reaction, which can be attributed to the high dispersion of Pd over the rGO sheets that supply an abundance of catalytically active sites for the reaction [50]. rGO synthesized from algal extracts achieved a 74%‒93% and 82%‒95% removal rate for Cu and Pb within 30 min of exposure. The effective heavy metal removal could be attributed to the homogenous and thin flat sheet of rGO providing an enormous surface for the adsorption of cationic heavy metals [47].
2.5.2 Biological application
One of the major advantages of reducing GO by using microorganisms is the ability to synthesize biocompatible graphene for biological applications. Raveendran et al. [51] attempted to synthesize biocompatible rGO using the extremophiles Halomonas eurihalina and Halomonas maura. Through the alamar blue assay, they found that MrGO enhanced the growth of mouse fibroblast cells which might be attributed to the bacterial EPS on the rGO. This indicates the safety of extremophilic reduced GO that could be used in biological applications [51]. Similarly, a green reduction method of GO was proposed using Pseudomonas aeruginosa as the reducing agent. The resulting rGO is highly biocompatible towards primary mouse embryonic fibroblasts, while chemically synthesized rGO showed significant toxicity [52]. Also, MrGO synthesized by Bacillus subtilis was tested for in-vivo toxicity using the zebrafish model. Results showed that MrGO had little or no effect on the zebrafish embryos’ hatching rate, heart rate, and body length. Meanwhile, GO and CrGO exhibited significant toxicity to the zebrafish embryos at high concentrations [41].
On the other hand, rGO synthesized by using Bacillus marisflavi biomass showed potential to be applied in the nanotherapy of cancer cells [53]. Cytotoxicity of the as-synthesized rGO to the MCF-7 breast cancer cell line was demonstrated by decreasing cell growth, the elevated release of ROS, and the generation of lactate dehydrogenase. Similarly, rGO synthesized from Ganoderma sp. extract exhibited cytotoxicity to the MDA-MB-231 breast cancer cell line [46].
The N-doped rGO synthesized by a mixed culture of microorganisms mainly consisting of Staphylococcus exhibited a haemolytic rate of 2.18%, slightly higher than CrGO (1.32%). This could be due to the small sheets and sharp edges of N-doped graphene in the aqueous solution that caused mechanical damage to the cells. However, it was still below the acceptable haemolytic rate (5%) for clinical use. Also, the resulting MrGO exhibited low thrombogenicity and did not cause platelet adhesion, making it more appropriate for biological applications than chemically synthesized N-doped graphene, even though it has a lower N/C ratio [37].
2.5.3 Other applications
Akhavan and Ghaderi [29] reported the antibacterial property of the rGO film after being reduced by E. coli. E. coli proliferated well on the GO film coated on a SiO2 substrate at the start of the incubation. However, after 24 h, the number of surviving bacteria decreased after the GO was reduced. The bacteria were detached from the surface of the rGO film. This could be due to the change in surface charge and the functional groups on the GO surface during the reduction process [29]. Similarly, rGO synthesized by Rhizopus oryzae was coated on an aluminium plate to test its antibacterial activity towards E. coli. The rGO-coated aluminium plate exhibited superior cytocompatibility toward fibroblast cell lines compared to the GO-coated aluminium plate. This is because of the hydrophobic nature of the rGO, which prevents the attachment of bacteria to its surface, making it a perfect material for nontoxic antibacterial coating in biomedical applications [44].
A copper coupon coated with GO was tested for its anti-biocorrosion performance by incubating the copper-GO coupon with D. desulfuricans for 7 d. As a result, GO on the copper coupon was successfully reduced to rGO by D. desulfuricans and avoided the corrosion of the copper. This was because that the resulting rGO blocked the direct interaction between the copper coupon and D. desulfuricans [24].
2.6 Issues and challenges in the application of microbial reduction of GO
XPS is commonly used in the characterization of rGO to indicate the degree of reduction in the carbon-to-oxygen (C/O) ratio before and after the reduction, allowing better quantification of the reduction reactions [20]. The highest C/O ratio is achieved by GO reduced by bacterial suspension (Fig.4) [35]. This might be due to different microorganisms extracted from the river, which are responsible for the removal of different oxygen functional groups, resulting in better oxygen removal efficiency and a higher C/O ratio [35]. Some microorganisms could not remove certain oxygen-functional groups that require higher reduction energy. For example, Lehner et al. [20] reported that S. oneidensis could not reduce the C=O bond as the peak area of the C=O bond was unchanged after the reduction process. Therefore, utilizing an assortment of mixed species of microorganisms may enhance the effectiveness of GO reduction.
As most of the microbial reduction of GO is carried out at room temperature, it is more environmentally friendly compared to other reduction methods. Also, microbial reduction of GO is more economically viable for large-scale production of graphene compared to hydrothermal reduction strategies, which are more energy-intensive and expensive. However, microbial reduction processes generally require a longer reaction time (24‒72 h) (Tab.1) which is longer than chemical and thermal reduction methods [19].
Besides, the efficacy of microbial reduction of GO could be lowered compared to chemical reduction methods such as hydrazine. XPS and Raman analysis showed that the chemical reduction of GO had better oxygen removal efficiency compared to the microbial reduction of GO [20,35,37]. However, CrGO tends to stack tightly together due to the π‒π stacking of the rGO sheets caused by strong van der Waals interactions [20,37]. This might increase the difficulty in applying CrGO in which extra steps are needed to separate the stacked rGO sheets or functionalize by surfactants, therefore incurring additional costs during production [58].
3 Application of microbial-rGO in MFC and BPV
As the adverse effects of greenhouse gas emissions and global warming have become more evident, alternative technologies to fossil fuels for sustainable and clean energy production have emerged, such as MFC and BPV. In these energy production systems, microorganisms such as bacteria and microalgae are utilized as biocatalysts in the anodic chamber to catalyze electricity generation by breaking down electron donors such as organic substrate (MFC) or water (BPV) in the presence of light. The electrons generated from the biocatalytic processes are then transferred to the anode and transported across an external circuit to the cathode [59–60]. At the cathode, the oxygen reduction reaction (ORR) completes the circuit. The electrons will combine with the protons that diffuse through the proton exchange membrane and the oxygen in the air to produce water. In fact, the cathodic ORR is achievable by receiving electrons from the cathode with the help of electrochemically active microorganisms. This could replace expensive chemical cathodic catalysts such as platinum, which are unsustainable for the wide-scale implementation of MFC and BPV technology [59–60].
MFC and BPV provide an attractive and economical solution for supplying sustainable energy for our expanding population while providing unparalleled advantages such as bioremediation of wastewater, removal of nutrients, synthesis of value-added products, and hydrogen and biogas production [61–62]. However, the commercialization of MFC and BPV has been hindered by their low power output due to the low EET efficiency from the microorganisms to the anode [63–64]. Thus, a cost-effective anode with superior electrical conductivity and long-term stability is required to meet the requirements for industrial applications. Apart from that, a poor ORR at the cathode also resulted in low power output from MFC and BPV. This is caused by several factors, such as slow oxygen mass transfer in the liquid phase, fouling that may impede the electron acceptor access to the cathode, and biofilm formation that may form anaerobic zones or inhibit the mass transfer of electron acceptor [59–60]. Thus, a biocompatible and high-conductivity cathode material could significantly improve the power generation of the MFC and BPV. Owing to their large surface areas, exceptional conductivity, lower thickness, high mechanical stability, and good biocompatibility, graphene-based materials have been considered an excellent material that could minimize the internal resistance of the electrode, act as a catalyst to mediate the transmission of electrons between microbes and the electrodes, as well as enhance the growth of microorganisms on the electrodes [65–66]. Moreover, the synthesis of rGO by using graphite as the precursor is an inexpensive route among other fabrication methods of graphene electrodes, which fulfils the idea of utilizing cheap electrode material for MFC and BPV devices [67].
3.1 Anodes
Yuan et al. [68] demonstrated a simple one-pot reduction of GO by adding GO-amended fresh acetate medium into the anodic chamber of MFC. As a result, the GO was reduced by the bacteria and automatically assembled onto the bacterial biofilm to form a biofilm/rGO network on the anode. Cyclic voltammetry measurement showed that the rGO bioanode had the highest redox peak current compared to the glassy carbon electrode (GCE) and GO/GCE, indicating its superior electrical conductivity. Through the formation of the self-assembled 3D-like biofilm/rGO network, the catalytic activity of the acetate oxidation by the bacteria has been enhanced due to the increase in the accessible active exoelectrogens and the acceleration of EET kinetics, thereby increasing the maximum power output to 1905 mW·m−2 (Tab.2 [68–76]). Additionally, the porous 3D structure of the biofilm/rGO network was favourable for the transport of substrate inside the biofilm, thereby increasing the interfacial area between the anolyte, biofilm, and anode [68]. Similarly, Zhu et al. [69] developed the in-situ fabrication of rGO anode with Shewanella putrefaciens as the inoculant in the MFC. Based on the SEM analysis, the 3D rGO structure resulted in better attachment of S. putrefaciens compared to the bare carbon felt (CF) anode. Electrochemical impedance spectroscopy (EIS) analysis was conducted to study the internal resistance of the MFC. Interestingly, the solution resistance and charge transfer resistance remained constant throughout the incubation period, except that the diffusion resistance decreased significantly after GO was reduced. This could be due to the formation of rGO increased the surface area of the anode, which increased the capacity of the electrode to absorb electrolyte. Therefore, the electrolyte could be more easily diffused to the surface of the anode, reducing the diffusion resistance. As a result, a maximum power output of 225.7 mW·m−2 was achieved by the rGO-CF anode compared to the bare CF anode (0.014 mW·m−2) (Tab.2) [69].
| Type of bioelectrochemical cell | Electrode material | Microbial reductant of GO | Maximum power density | Ref. |
|---|---|---|---|---|
| Single chamber MFC | Anode: MrGO-carbon cloth; Cathode: carbon cloth-Pt | Activated anaerobic sludge | 1905 mW·m−2 | [68] |
| H-shaped MFC | Anode: MrGO-carbon felt; Cathode: Pt sheet | Shewanella putrefaciens | 225.7 mW·m−2 | [69] |
| Soil MFC and plant MFC | Anode: MrGO-graphite felt; Cathode: graphite felt | Soil microbes | SMFC = 40 mW·m−2; PMFC = 49 mW·m−2 | [70] |
| Single chamber MFC | Anode: MrGO-zeolite carbon felt; Cathode: stainless steel wire mesh | Mixed anaerobic sludge | 280.56 mW·m−2 | [71] |
| Algal BPV | Anode: rGO-coated glass; Cathode: Pt-coated glass | Langmuir–Blodgett method | 0.148 mW·m−2 | [72] |
| Dual chamber MFC | Anode: carbon cloth; Cathode: MrGO-carbon cloth | Aerobic activated sludge | 323 mW·m−2 | [73] |
| Dual chamber MFC | Anode: carbon felt; Cathode: MrGO-carbon felt | Activated sludge | 65.4 mW·m−2 | [74] |
| Dual chamber MFC | Anode: MrGO-carbon felt; Cathode: MrGO-carbon felt | Activated sludge | 124.58 mW·m−2 | [75] |
| Dual chamber MFC | Anode: graphite felt; Cathode: MrGO-graphite felt | Anaerobic activated sludge | 163.8 mW·m−2 | [76] |
In addition, Goto et al. [70] demonstrated that GO added to the soil could be reduced by the soil microbes, thereby increasing the EET to the anode and enhancing the electricity generation in soil MFC and plant MFC. GO could also be used to enrich exoelectrogens in the environment. For instance, the number of Geobacter sp. has increased after incubating GO in paddy soil, river water, and water channel sediment [77], while the marine exoelectrogen Desulfomonas sp. was enriched from a coastal sample [78]. After the GO was reduced, rGO, along with the microbes, was self-aggregated into a 3D hydrogel complex due to the π‒π stacking of the rGO, which was also observed by Yuan et al. [68]. Since then, conductive hydrogel complexes have been developed by incubating GO with anaerobic sludge [79] and artificial dialysis wastewater (ADWW) anaerobically for 21‒30 d [80]. The resulting hydrogels have generated a higher and steadier current than that of a graphite felt (GF) anode. Rathinam et al. [54] reported an improvement in the performance of bioelectrodes due to Gluconobacter roseus wrapped with rGO after reducing by the bacteria. Chronoamperometry analysis showed that the rGO-modified bioelectrode increases current density by 10-fold compared to a bare CF anode. This could be due to the higher conductivity of rGO and decreased electron transfer resistance at the electrode–electrolyte interface. Therefore, the efficiency of EET to the anode was improved, and the loss of electrons from the microbes into the electrolyte was reduced [54].
Meanwhile, an MFC with a CF anode modified with GO–zeolite composite has increased the power density by 3.6 times compared to a bare CF anode [71]. This could be due to the reduction of GO on the anode surface by the bacteria in the anodic chamber. Enhanced biofilm formation was observed on the GO–zeolite modified anode (GZMA) due to the hydrophilic nature and porous structure of the composite. Hence, the conductivity of the anode is improved, and the internal resistance of the MFC is lowered. This was supported by EIS analysis which showed 18.9 times lower charge transfer resistance of the GZMA compared to the bare CF anode. As a result, a higher maximum power output of 280.5 mW·m−2 was obtained compared to 77.8 mW·m−2 obtained from the bare CF anode (Tab.2). In addition, the higher removal efficiency of chemical oxygen demand (COD) was also achieved in GZMA due to enhanced electrogenic activity of the microbes in the MFC [71].
To the best of our knowledge, there was no related publication on the application of microbially-reduced GO in BPV devices. However, it is worth mentioning that according to a study by Ng et al. [72], the incorporation of Langmuir–Blodgett method fabricated rGO anode in the algal BPV device has resulted in a 119% increase in maximum power output compared to indium tin oxide (ITO) anode. This could be due to the formation of porous three-dimensional structures of the rGO anode that promoted the adherence of microalgal cells to the anode, thereby enhancing the EET efficiency [72]. Ng et al. [81] also reported a similar result as the rGO anode BPV achieved 61.49% higher maximum power output compared to the ITO anode.
In short, the application of rGO to the anode of the MFC and BPV systems has improved the EET efficiency from the electrochemically active bacteria and microalgae to the anode due to the increased specific surface area of the anode after the coating of rGO and enhanced substrate transport and microbial attachment on the anode. Moreover, 3D biofilm/rGO scaffolds or hydrogel complexes can be formed if the reduction of GO is carried out in-situ by the microorganisms, which is a more facile, cost-effective, and environmentally friendly approach in the fabrication of rGO electrodes. Through the formation of 3D scaffolds, long-distance electron transfer from the microorganisms was made feasible as the rGO embedded in the biofilm serves as a conductive platform to transfer electrons from the top or intermediate layer of the biofilm to the electrode. Thus, the number of accessible active microorganisms at the anode could be significantly increased [68–69,82].
3.2 Cathodes
To date, lesser attention has been paid to the fabrication of nanostructured materials to improve the biocompatibility and conductivity of the biocathode compared to the anode [73,83]. A one-pot fabrication method for a 3D rGO/biofilm cathode has been reported by Zhuang et al. [73]. Similar to the preparation of rGO bioanode, GO was added to a fresh medium before being used to replace the cathode medium after the power output of the MFC was stable. After incubation, 3D rGO/biofilm composites were formed. As a result, a 103% increase in the maximum power output of MFC with rGO-biocathode (323.2 mW·m−2) compared to the carbon cloth (CC) biocathode (159 mW·m−2) was observed (Tab.2). An electrochemical study indicated that rGO-biocathode had faster EET kinetics and less charge transfer resistance, which eventually enhanced the ORR of the bacteria. This could be due to the increased specific surface area of the cathode, which can accommodate a higher amount of electrochemically active microorganisms. Even though the performance of rGO-biocathode MFC was still lower than the MFC with Pt/C cathode (464.1 mW·m−2), rGO-biocathode should still be considered for large-scale application from the economic practicability and environmental sustainability viewpoint [73].
Meanwhile, Chen et al. [74] fabricated an rGO-biocathode through polarity reversion of an rGO-bioanode. GO was first reduced by the microorganisms in the anodic chamber. After three cycles of MFC operation, polarity reversion was carried out by changing the anodic chamber into a cathodic chamber without changing the medium but the anaerobic conditions into aerobic conditions. EIS analysis showed that charge transfer, polarization, and ohmic resistance of the rGO-biocathode were all decreased, indicating the conductivity, electrochemical kinetics, and interaction between the microorganisms and the cathode were improved. Thus, the rGO-biocathode had a better catalytic effect on the ORR by the microorganisms. As a result, a 1.22 times improvement in the maximum power density (65.4 mW·m−2) was achieved in rGO-biocathode MFC. Meanwhile, microbial diversity analysis showed that the exoelectrogens in the biocathode were enriched, which may be due to the increase in catalytic activity [74]. Furthermore, Chen et al. [75] developed a dual rGO-modified bioelectrode MFC by in-situ microbial reduction of GO and polarity reversion. As a result, a maximum power output of 122.4 mW·m−2 was obtained [75].
In addition, the MFC with rGO-bioelectrode can enhance the removal of oxytetracycline, an antibiotic that is extensively used in the livestock industry. This could be due to the enhanced EET process caused by applying highly conductive rGO electrodes [84]. Similarly, Song et al. [76] reported improved Cr(VI) removal and higher maximum power output in rGO-biocathode MFC compared to GF biocathode. The higher Cr(VI) removal rate was due to the increased specific surface area of the rGO, enabling easy adsorption of Cr(VI) and its subsequent reduction to Cr(III). Also, the rGO biocathode improved the electrical conductivity of the electrodes and the EET between the electrode, bacteria, and Cr(VI) [76].
4 Conclusions and outlook
Due to the difficulties in producing pristine graphene, the synthesis of rGO via the reduction of GO is one of the most promising strategies for achieving large-scale graphene production. As a result, research has been conducted using various reduction strategies on GO, with chemical reduction being the most extensively researched. Although several chemical reductants are available today for synthesizing rGO, the hazardous properties of chemical reductants have prompted the discovery of rGO synthesis by microorganisms. The utilization of microorganisms in the reduction of GO has been summarized in this review. They are environmentally benign, even with a lower degree of oxygen functional group removal compared to CrGO. However, the resulting MrGO is both highly dispersible and biocompatible, making it beneficial in specific applications, particularly in the biomedical field. In order to further improve the efficiency of oxygen functional group removal, the exact functional group or biomolecules from the microorganisms responsible for GO reduction should be identified and isolated.
Because of its superior biocompatibility with microorganisms such as bacteria and microalgae, rGO material should be used and investigated as the electrode of MFC and BPV. This is because of the highly porous 3D structure of rGO and its increased surface area, which favours the microbial attachment to the electrode surface, thus promoting biofilm formation and the microbe–electrode–electrolyte interaction. Therefore, EET from the microorganism to the electrode is enhanced, and the internal resistance of the system is reduced, thereby contributing to the improved performance of the system. The GO-reducing ability of microorganisms enables the synthesis of self-assembled MrGO electrodes in the MFC, which further brings down the cost and simplifies the fabrication process of graphene or rGO electrodes.
The most difficult challenges to be addressed before the practical commercialization of graphene-based electrodes and their use in MFC and BPV are sustainable large-scale production, cost, and quality of the graphene. The quality of rGO strongly depends on the production method. However, some methods capable of producing high-quality rGO are costly, complicated, yield-limited, and require expertise, thus impractical for industrial application. Therefore, future research should focus on improving the affordability of 3D graphene materials due to their superb electrical conductivity, strong catalytic activity, and exceptional biocompatibility. The long-term stability, performance, scalability, and repeatability of the rGO bioelectrode in MFC and BPV should also be investigated. Meanwhile, developing low-cost, environmentally friendly, and facile fabrication methods for MrGO composite electrodes doped with conductive polymers, metal or metal-organic frameworks is a topic worth exploring. For instance, inorganic metal oxides such as manganese dioxide [85], titanium dioxide [86–87], nickel foam [88], and cobalt monoxide [89] were used for the functionalization of rGO due to their abundance and low cost. By anchoring metal oxide on rGO as a composite matrix, the stability and electrical conductivity of the metal oxide could be improved. However, research in the utilization of rGO composite in the electrodes of MFC and BPV remains underexplored compared to other electrode applications such as supercapacitors [90] and lithium-ion batteries [91]. After all, the electrode material must be cheap in order to lower the manufacturing cost of the MFC and BPV, thereby realizing the commercialization of these bioelectricity generation devices to provide sustainable energy for our ever-growing population.