Microorganisms and Microbial Communities in Bioelectrochemical Systems for Wastewater Bioremediation and Energy Generation

Abstract

Water resource sustainability is a critical global concern, leading to extensive scientific research. Proposed alternatives for wastewater effluent use include the promising Bioelectrochemical Systems (BES) that not only treat wastewater effectively but also generate electricity, produce biofuels, and synthesize valuable compounds through integrated microbial and electrochemical processes. BES research aims to enhance device design and develop superior electrochemical materials for optimal performance. The efficiency of treatment and energy co-generation depends on the metabolic characteristics of microbial communities responsible for oxidation-reduction processes in wastewater. The diversity of these communities, along with electron transport mechanisms and metabolic pathways, significantly impacts BES functionality and effectiveness. This study focuses on microorganisms in various BES setups, presenting their electrochemical performance. It compiles data on microbial ecology, emphasizing controlled communities and model microorganisms from wastewater treatment systems. The study highlights the scarce research on native microbial communities for agroindustrial wastewater. Its main goal is to consolidate information on microorganisms with electrogenic capacity, demonstrating their potential in different bioelectrochemical systems. These applications can transform wastewater bioremediation and enable the production of green energy, biofuels, and high-value compounds.

1. Introduction

The development of humanity has led to an exponential increase in the consumption of natural resources and the generation of waste derived from anthropogenic activities. One of the most consumed resources is water. It is estimated that in the world the consumption for domestic, agricultural, and industrial activities is greater than 4000 km³ of water per year [1]. As a consequence, a large amount of wastewater with a high environmental impact has been generated. According to UNESCO: “in low-income nations only 8% of domestic and industrial wastewater is treated” [2]. Wastewater treatment systems are classified into: (I) mechanical or primary processes: settling, screening, sedimentation; (II) biological or secondary processes: aerobic and anaerobic microbial metabolism of organic compounds; and (III) physicochemical or tertiary processes: advanced oxidations, filtrations with ionic charges, chlorination, ozonation, among others [3]. One of the most widely applied strategies for the purification of organic matter in wastewater is carried out through Wastewater Treatment Plants (WWTP), which combine primary and secondary methods [4]. The implementation of WWTPs is costly and low-income countries, whose productive activities correspond mainly to the exploitation of raw materials, and do not incorporate WWTPs into their agricultural or industrial production processes. These treatments are carried out on domestic and industrial wastewater, mainly in urban areas, and according to recent data, only an average of 20% of the wastewater produced are treated in the world before being returned to the ecosystems [5]. In recent years, research has been carried out for the non-conventional biological treatment of wastewater using bioelectrochemical systems (BES) [6, 7, 8, 9].

The BES can be wastewater treatment systems adapted and designed based on a microbial metabolism that, through reactions of oxidation and reduction of organic matter, carry out the purification of contaminated water, producing at the same time electrical energy, biofuels, or other chemical compounds of interest [6, 8, 10]. There is a general classification of BES based on their application; (a) microbial fuel cells (MFCs): developed mainly for electricity production, but have recently been used for wastewater treatment and as biosensors for the detection of toxic chemicals; (b) microbial electrolysis cells (MEC) developed for the electrochemical production of biofuels such as hydrogen or methane, and finally, (c) microbial electrochemical technologies (MET): used to desalinate water or produce chemical compounds such as hydrogen peroxide [11]. The configuration of the BES and the origin and differentiation of microbial communities to be used in these systems depend on the composition of the substrate and the compounds of interest that can be generated from a specific cellular metabolism. The different types of BES are designed and implemented according to the objective of wastewater treatment, and some are coupled to conventional secondary treatments, such is the case of wetlands connected to MFC, in which symbiotic microorganism-plant relationships are used as a strategy of optimization in the efficiency of reduction of organic matter content [12, 13]. BES have also been implemented for the treatment of residual water or a fixed effluent, with cascade feeding or simultaneous feeding under continuous operation, in which different microbial communities have been used in each stage or system to increase the production rate energy and decontamination levels [14, 15]. Moreover, various conventional wastewater treatment technologies have been effectively combined with BES. For instance, MFCs have been coupled with stabilization ponds, while BES have been integrated into aerobic or anaerobic treatment systems specifically designed for nutrient (nitrites/nitrates, sulfates) or heavy metal (Fe, Cu, Cd, etc.) removal purposes [7, 16, 17, 18, 19, 20, 21, 22, 23].

For the design and adaptation of BES technologies in the unconventional treatment of wastewater, it is necessary to know individually the metabolic responses and the enzymatic machinery of the microorganisms present in the system. Currently, microorganisms of different genera with the ability to degrade organic matter and with potential for application in bioelectrochemical systems have been described. Mainly bacteria and archaea have been studied, although there are reports of electrogenic activity in eukaryotes, such as yeasts [10, 24, 25]. On the other hand, in a significant amount of research, detailed descriptions of the mechanisms of mass and energy transport in the cell have been made; and in recent years, some studies have been registered that seek to adapt diverse microbial communities to BES [12, 23, 25, 26, 27, 28, 29, 30]. However, in the case of microbial communities, due to the complexity of the interactions, it is difficult to establish the metabolic processes and mass and energy transport mechanisms involved in bioelectrochemical processes. This review presents a consolidation of the information on the main microorganisms used in BES, the metabolic processes they carry out for the degradation of organic matter and the use of substrates for the cogeneration of energy and the production of other metabolites of interest, and the possible interactions between the microorganisms of the microbial communities in these bioelectrochemical systems.

2. Microorganisms in bioelectrochemical systems

Microorganisms in bioelectrochemical systems are responsible for the transformations of the substances or compounds present in the organic matter or substrate, since through their metabolic processes they carry out oxidation and reduction reactions, which in turn constitute the mechanisms of energy generation and high value compounds. Its implementation for wastewater treatment and cogeneration of energy or compounds of interest depend on the interaction between the physicochemical characteristics of the substrates and the metabolic capacity associated with microbial diversity [7, 14, 19, 23, 31, 32, 33, 34, 35, 36]. In the case of MFCs, the main metabolism described corresponds to the redox (electrogenic) degradation of organic matter. In MEC systems, microorganisms with a metabolism of methanogenesis and hydrogenogenesis related to the production of biofuels have been recorded [28, 37, 38, 39].

The biotechnological capacity of cells to produce energy called “electrogenic potential” was first described in 1911 by Potter, who evaluated the electrogenic potential of Escherichia coli and Saccharomyces cerevisiae [40]. Advances in molecular biology and microscopic characterization techniques have allowed the identification of multiple microorganisms and their metabolism, as well as their ability to generate energy. Some main microbial groups have been identified, with species known as model electrogenic microorganisms, which present adaptive characteristics of great interest in BES, such as the presence of some cellular structures (pili and nanotubes) and the excretion of mediating substances that act as direct and indirect electron transport mechanisms in the different bioelectrochemical systems [41, 42, 43, 44]. The predominant microorganisms found in these systems belong to the Bacteria domain, mainly the phyla Proteobacteria, Chloroflexi and Firmicutes [13, 26, 33, 45, 46, 47]; and the genera Geobacter sp., Shewanella sp., and Pseudomonas sp.

3. Model electrogenic microorganisms

The Electrogenic model microorganisms have a remarkable electron transfer capacity, exhibiting physical and metabolic characteristics that allow the production of usable energy from the consumption of organic matter [10, 11, 48]. The bacterial genus Geobacter sp. it has been reported as the one with the highest electricity production rates in bioelectrochemical systems [32, 43, 49, 50]. Several studies have reported this electrogenic capacity; J Chen and his collaborators established that in a double-chamber MFC fed with synthetic wastewater, in which power density values of 71.6 mW/m² and 65.4 mW/m² were achieved at the anode and the cathode, respectively; the predominant genus in the microbial community developed from activated sludge from a WWTP was Geobacter sp. [51]. Similarly, Shen et al. reported that in an MFC designed for the co-degradation of phenolic compounds, a production of 267.2 mW/m² was reached with the predominance of Geobacter sp. within the identified bacterial genera [52]. Also, Paitier et al., in a bottle-type open cathode MFC, characterized the microbial community and identified the predominance of Geobacter in the formation of the electrogenic biofilm on the anode during one month of operation, reaching an energy performance of approximately 50 mW/m². The authors reported a predominant exponential growth of Geobacter during the first 5 days of biofilm formation [50]. In the report by McAnulty et al., an MFC that converts methane directly into an electric current, the capacity of Geobacter sulfurreducens to produce electrons from acetate was evaluated. A maximum power density of 160 mW/m² was obtained [53]. On the other hand, considering different types of ESP, in a MEC dedicated to methane production, Zhao et al. evaluated the performance and interaction between Geobacter sp. and Methanosaeta sp. in the reduction of CO₂ to CH₄, reaching 3017.6 mL of CH₄ in 51 days of operation; the authors suggest from their results that Geobacter develops a key metabolism as a precursor to methanogenesis in bioelectrochemical systems [41]. However, although Geobacter sp. is a microorganism with a great electrogenic potential, several studies indicate that it is not the only relevant microorganism in BES [11, 19, 42, 45, 52, 54, 55, 56, 57]. For example, for the development of electrochemical technologies with unconventional materials, such as ceramic biocathodes as those made of terracotta, the predominance of fermentative bacteria has been identified, which exhibits metabolic capacities different from Geobacter, proving that there is an influence on the interaction between the microorganisms and materials used in BES. It has been suggested that Geobacter interacts more efficiently with conductive materials such as carbon felt [58].

For the bacterial genus Pseudomonas sp. that it is present in almost all ecosystems, the electrogenic potential associated with its metabolism has also been identified; and it has been established that it plays an important role in electron transport in BES [33, 42, 52, 59]. P. aeruginosa stands out as the model microorganism. However, other species within this same genus with high electrogenic capacity have also been reported. In the study by Ilamathi et al. of a single-chamber MFC, the power densities determined for a P. aeruginosa strain were 601 μW/m² and 323 μW/m² for a P. fluorescens strain [60]. Arkatkar et al. obtained a current of 0.036 mA in a double-chamber MFC inoculated with P. aeruginosa [42]. In turn, non-conventional MFC designs have been tested using this microorganism. For example, Zhang et al. reported a power density of 3322 mW/m² for an MFC with a cylindrical reactor design [61]. Similarly, in the comparative study by Bagchi and Behera, this microorganism was used to evaluate the influence of the manufacturing material on an MFC, reaching power density values of 0.96 W/m³ for an MFC made of plastic and 0.69 W/m³ for the MFC built with ceramic materials [52]. Also, alternative strategies for the operation of bioelectrochemical systems using P. aeruginosa have been evaluated. For example, Qiao et al. evaluated the effect of the implementation of inhibitors such as sodium hyaluronate, on the formation of biofilm on the anode in an MFC inoculated with P. aeruginosa, indicating a current density of 4.8 μA/cm² [62]; Yong et al. evaluated the mixed aerobic-anaerobic operation of an MFC inoculated with P. aeruginosa, obtaining a current density of 99.8 mA/cm² [16].

Similarly, Shewanella sp., recognized as a facultative bacterium, has been reported as an exoelectrogenic microorganism [32, 57, 60, 63]. The presence of these microorganisms in nutrient-limited cultures has been described, a characteristic that may favor their implementation in BES [26]. This bacterial genus has been widely identified in studies with bioelectrochemical systems in which various microbial communities have been used as inoculum. In a comparative study carried out by Hassan et al., in a double-chamber MFC, they compared microbial communities in petrochemical and domestic wastewater, reaching current densities of 156 mA/m² and 123 mA/m², respectively. The authors reported the active presence of Shewanella sp. in the cells in which wastewater of domestic origin was used [64]. In particular, the Shewanella oneidensis MR-1 strain has been identified as an electrogenic reference, as reported by Wang et al., in their study of a non-conventional laminar flow MFC fed with synthetic wastewater and glycerol, in which a potential for 16.05 mW [65]. In another work by Wang et al., the mutualistic interaction between Shewanella oneidensis and Escherichia coli in a double-chamber MFC fed with a synthetic substrate, a current density of 2.0 μA/cm² was achieved [44]. Likewise, Hirose et al. reported a power density of approximately 250 mW/m² in a single-chamber electrolysis cell fed with a synthetic substrate, electrogenic activity that was attributed to the metabolic response of S. oneidensis MR-1. As stated by the authors, this microorganism possesses molecular mechanism that enables it to detect electrode potentials and effectively regulate its catabolic pathways, so this capability allows the microorganism to adapt and optimize its metabolic activities in response to the electrical conditions presented by the electrodes [66]. This molecular mechanism provides valuable insights into the intricate processes underlying the bioelectrochemical interactions and further enhances our understanding of the microbial behavior within the context of electrochemical systems.

On the other hand, another variety of electrogenic bacteria has been described in BES, and some genera are distributed depending on the origin of the wastewater or even depending on the differentiation of the microbial communities through the operation time of the system, within the frequently described bacterial genera, Clostridium sp. [4, 28, 49, 67, 68]. As an illustration, the Clostridium beijerinckii strain was subjected to evaluation in both a H₂ fermentation reactor and an open cathode MFC, with the feedstock being port drainage sediment, in this study yielded impressive results, including the production of 104 mmol/L of H₂, 5 mmol/L of acetate, 33 mmol/L of butyrate, 3 mmol/L of lactate, and 1 mmol/L of ethanol; additionally, the power density achieved was recorded at 1.2 W/m² [28]. These findings demonstrate the potential of this strain and highlight the feasibility of harnessing its capabilities for efficient and sustainable bioenergy production. Another bacterial genus studied has been Desulfovibrio sp.; that in a double-chamber MFC it reached an electrical production of 185 mW/m² and in a MEC, where it was the predominant bacterial genus, a hydrogen production of 0.28 m³-H₂/m³-d was reached [26]. Also, reports have been made of the electrogenic activity of Klebsiella sp. in a double-chamber MFC, and from finding in a symbiotic relationship of Klebsiella variicola and P. aeruginosa, a power density of 14.78 W/m³ was reached [69]. Additionally, the presence of Acinetobacter sp., Bacillus sp., Thiobacillus sp., Desulfomonas sp., among other genera, has been reported. Some of the archaea studied according to their electrogenic potential are P. furiosus with an electrical production, in terms of power density, of 225 mW/m². The current density produced by Ferroglobus placidus and Geoglobus ahangari has been reported to be 680 mA/m² and 570 mA/m², respectively. The study of some archaea in BES has also been carried out in mixed communities including bacteria, where volume power density values between 2138 mW/m³ and 6924 mW/m³ have been recorded [4, 11, 53, 62]. Recently, it has been reported that some genera of archaea, such as Methanosaeta and Methanosarcina, have symbiotic interactions with other electrogenic microorganisms, such as Geobacter sp. [4] which generates a particular interest in its presence and functionality in the microbial interactions of ecosystems in BES.

The microbial community present in bioelectrochemical systems is not exclusively constituted by electrogenic microorganisms, they coexist with a great variety of microorganisms that through their metabolic processes and enzymatic machinery carry out the degradation of complex substrates such as polymers, carbohydrates, proteins, hydrocarbons, fats and oils, among others; contributing to obtaining of precursor substances for electrogenic metabolism, which significantly influence energy productivity in BES [15, 22, 49, 64, 70, 71, 72, 73, 74, 75]. The presence of different microorganisms in bioelectrochemical systems indicates the important ecological role they play in nature, as well as suggesting their participation in bioanode formation and electron transport in bioelectrochemical systems [47, 68, 76, 77]. However, a considerable amount of BES studies focusses primarily on the design and configuration of devices developed as MFCs or MECs, and research on microorganisms and their interactions is relatively minor. The study of microbial communities can be considered more complex due to the interactions that occur between microorganisms when they are active in BES. In these systems, ecological successions or interdependent mixed metabolisms could occur between electrogenic microorganisms and those that do not present electrogenic activity. Knowledge of the symbiotic relationships of microbial communities in BES would allow the adaptation of this technology for the treatment of a wide variety of substrates and environments, including wastewater from multiple industries. Table 1 presents a list of electrogenic microorganisms reported by various authors, which were evaluated in the different BES using multiple substrates.

Microorganisms	Type of BES	Substrate	Electrochemical response	References
Geobacter sp.	MFC	Activated sludge	71.6 mW/m^2	[51]
Geobacter sp.	MFC	Phenolic waste water	267.2 mW/m^2	[75]
Geobacter sp.	Bottle-type air cathode single chamber MFC	Waste water + acetate	50 mW/m^2	[50]
Geobacter sulfurreducens	MFC-MEC (CH4)	Synthetic wastewater (sodium acetate)	160 mW/m^2	[53]
Geobacter sp. and Methanosaeta sp.	MEC (CH4)	Activated sludge + glucose	3017.6 mL CH4	[41]
Pseudomonas aeruginosa	Single-chamber MFC.	Modified King's B médium + azo dye: Orange 16 (RO-16) and black 5 (RB-5)	601 μW/m^2	[78]
Pseudomonas fluorescens			323 μW/m^2
Pseudomonas aeruginosa	Two-chamber MFC.	Synthetic wastewater (glucose)	0.036 mA	[42]
Pseudomonas aeruginosa	Non-conventional MFC (cylinder).	Anaerobic activated sludge + glucose	3322 mW/m^2	[9]
Shewanella oneidensis MR-1	MFC.	Synthetic wastewater (glycerol)	16.05 mW	[79]
Shewanella sp.	Two-chamber MFC.	Industrial (petrochemical) and domestic waste water	156 mA/m^2; 123 mA/m^2	[64]
Shewanella oneidensis MR-1	Single-chamber MFC.	LMM + NaCl	250 mW/m^2	[66]
Shewanella sp. and E. coli	Two-chamber MFC (U shaped).	Saline solution + glucose	2,0 μA/cm^2	[44]
Clostridium beijerinckii	MEC	Port drainage sediment	104 mmol/L H2; 1.2 W/m^2	[28]
Desulfovibrio sp. (Comunidad mixta)	Two-chamber MFC.	Activated sludge + sodium acetate	185 mW/m^2	[26]
	MEC.		0.28 m^3-H2/m^3-d
Klebsiella variicola and P. aeruginosa	Two-chamber MFC.	Domestic waste water + POME	14.78 W/m^3	[69]
Geobacter sp.; Petrimonas sp.; Erysipelotrichia sp.; Escherichia sp.; Shigella sp.	Two-chamber MFC	Food waste sludge	422 mW/m^2	[56]
Clostridium sp.; Chthonomonas sp.	Double-chamber BES	Active sludge	99 mW/m^2	[54]
Chloroflexi sp.; Thiobacillus sp.; Desulfovibrio sp.; Desulfomicrobium sp.; Desulfobulbus sp.; Desulfuromonas sp.	GW-MFC	Active sludge	24.7 mW/m^2	[19]
Native bacteria	Compost soil MFC (CSMFC)	Compost (soil)	3.2 mW/m^2	[21]
Clostridium sp.; Paracoccus sp.; Pseudomonas sp.; Arcobacter sp.	Pilot scale MFC (1-year operation)	Domestic sewage	2 mA	[80]
Desulfovibrio sp.	Two-chamber MFC	Simulated textile wastewater	258 mW/m^2	[30]
Citrobacter sp.; Thiobacillus sp.; Thioclava sp.; Corynebacterium sp.; Arcobacter sp.; Desulfovibrio sp.; Geobacter sp.	Two-chamber MFC	Domestic anaerobic and aerobic sludges and simulated wastewater (with RB19 dye)	85.2 mW/m^2	[18]
Methylobacterium extorquens	MEC	Synthetic wastewater (16 g/L succinate)	2.53 mM/g-wet cell/h (formate production)	[39]
Rummeliibacillus sp.; Burkholderia sp.; Enterococcus sp.; Clostridium sp.	Cylinder-type air cathode single chamber MFC.	Food waste hydrolysate.	0.97 kWh/kg COD	[81]
Azospirillum sp.; Petrimonas sp.; Pseudomonas sp.; Geobacter sp.	Three-electrode single chamber BES	Synthetic wastewater (1 g/L acetate)	2.81 mW/cm^2	[74]
Native bacteria	Two-chamber MFC	Domestic waste water	1856 mW/m^2	[82]
Native bacteria	Single-chamber air-cathode Ceramic MFC	Activated sewage sludge	189 mW/m^2	[83]
Thauera sp.; Pseudomonas sp.; Candidatus Methylomirabilis oxyfera	Two-chamber MFC	Anaerobic granular sludge	98.7 mW/m^2	[47]
A. woodii; M. maripaludis	Two-chamber MEC	Synthetic medium	Electromethanogenesis 0.1–0.14 μmol/cm^2 h^1 (–400 mV); Acetate 0.21–0.23 μmol/cm^2 h^1 (–400 mV)	[84]
Parabacteroides sp.; Proteiniphilum sp.; Catonella sp.; Clostridium sp.	Air-cathode MFC	Cow manure	44 mW/m^2	[85]
Cloacibacillus sp.; Proteiniphilum sp.; Clostridium sp.; Sphaerochaeta sp.	Two-chamber MFC	Biogas slurry (corn stover)	296 mW/m^2	[70]
Native bacteria	Fixed-bed air-cathode MFC	Synthetic medium + activated sludge	1.149 mA	[7]
Azoarcus sp.; Nitratireductor sp.; Ochrobactrum sp.; Serratia sp.	Single-chamber air-cathode MFC	Granular anaerobic sludge	22.8–112.7 mA/m^3	[86]
Native bacteria (rhizobacteria)	Integrated drip hydroponics MFC	Domestic waste water	31.88 mW/m^2	[21]
Anammox Planctomyces; Bacillus sp.; Desulfovibrio sp.; Geobacter sp.; Nitrospira sp.; Thauera sp.	CW-MFC	Domestic waste water (sludge)	3714.08 mW/m^2	[12]
Geobacter sp.; Pseudomonas sp.	1000 L modularized MFC system	Municipal waste water	7.58 W/m^2	[87]

Table 1.

Summary of electrogenic microorganisms reported.

4. Electron transport mechanisms in electrogenic microorganisms

The energy production in the BES is directly related to the chemical transformations of the organic matter present in the substrates fed in these systems [19, 75, 88, 89]. In the chemical reactions of oxidation and reduction, the mechanisms of electron transport (ET) are essential to ensure that the substances that are oxidized give up or transfer electrons to the substances that are reduced, that is, substances that gain electrons and process that is used in BES to achieve power generation [10, 35, 41, 46]. Figure 1 shows a representation of the flow of electrons and protons resulting from cellular metabolism present in a bioelectrochemical system. Direct and indirect ET mechanisms have been identified and used for the general characterization and classification of electrogenic microorganisms [6, 11, 18]. Direct electron transport (DET) mechanisms refer to the physical contact between electrogenic microorganisms and the electrode, and direct contact between the outer membrane of the bacteria and the anode surface [24]. The indirect electron transport (IET) mechanism, also known as mediated electron transport (MET), occurs due to the presence of mediating substances that facilitate the flow of electrons toward the surface of the anode electrode [33, 35, 57]. These substances are produced by some microorganisms in their cellular metabolism, as occurs in the oxidation of compounds and the generation of fermentative products, such as hydrogen [22, 90, 91]; or they can be added to the systems to facilitate the electron transport process [25]. The development of direct or indirect mechanisms for electron transport in BES depends on the enzymatic machinery of the electrogenic microorganisms used and on the experimental conditions such as the composition and concentration of substances of the substrate used in the operation of the bioelectrochemical system [42]. The indirect electron transport mechanism presents a lower efficiency in the production of electricity in the BES [6].

Figure 1.

Representation of the flow of electrons and protons resulting from cellular metabolism present in a BES (MFC double chamber).

In electrogenic microorganisms such as Geobacter sp. and Shewanella sp., mainly DET mechanisms have been identified, associated with the formation of membrane-extending structures known as pili [26, 42]. The pili are short membrane extensions that establish direct contact with the outside of the cell or other microorganisms for the exchange, mainly, of ions, nutrients, and genetic material [10, 41, 52, 72, 75, 92]. Figure 2 shows a schematic representation of the direct mechanism of electron transfer through a pili of Geobacter sp. The formation of these membrane extensions has been described as a mechanism of ecological adaptation of cells to the environment. Different studies have established that the genetic machinery for the appearance of pili is based on the expression of cytochrome C (OmcS) [85, 86]. Similar to Geobacter sp., the bacterial genera Shewanella sp. and Rhodoferax sp. possess protein complexes of type C cytochromes [8, 93, 94, 95].

Figure 2.

Schematic representation of DET mechanism of Geobacter sp. (pili and cytC).

On the other hand, for Shewanella sp. the formation of nanotubes that allow the direct transport of electrons between the cell and its environment has been reported [96, 97]; and also, indirect transport mechanisms have been described with the presence of the group of quinones and quinol in the cytoplasmic membrane, which transfer electrons to extracellular acceptors such as minerals containing Fe (III), mediating substances that finally carry out the transport of electrons toward the electrodes of the BES [60]. Figure 3 shows a schematic representation of both electron transport mechanisms for Shewanella sp.

Figure 3.

Schematic representation of (a) DET (nanotubes) and (b) IET from Shewanella sp.

Bacteria such as Pseudomonas sp. are included in microorganisms with indirect electron exchange mechanisms, as they excrete biomolecules to facilitate the transfer or transport of electrons [32, 33, 92]. For the bacteria Pseudomonas sp., and Lactococcus sp. the presence of phenazine-type pigments (cyclic hydrocarbons with the presence of two N groups), riboflavins and quinones, produced by secondary metabolic pathways, has been identified. Some examples of these pigments are pyocyanin and 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ), mediating substances that carry out the electron transport process in BES systems [35, 52]. The metabolic performance of P. aeruginosa depends, to a large extent, on the presence of phenazines, since these substances have been reported to be involved in virulence, signaling, iron metabolism, and electron transport [35]. The phenazine of greatest interest in the study of bioelectrochemical systems is pyocyanin (PYO) due to its participation in the transfer of electrons at high catalytic reaction rates and low overpotential [61, 96, 98]. Figure 4 shows a schematic representation of the indirect electron transfer mechanism for Pseudomonas sp.

Figure 4.

Schematic representation of IET through phenazines (PYO) in Pseudomonas sp.

5. Microbial metabolism in bioelectrochemical systems

Some main metabolic routes that electrogenic microorganisms carry out to achieve the degradation of compounds present in the substrates in BES have been identified [66, 92, 99]. In general terms, it can be considered that the oxidation of organic matter is carried out by microorganisms for cell growth and maintenance. In this process, ions associated with the energy transfer mechanisms are generated, as described in Figure 5, and correspond respectively to the anode and cathode reactions that take place in the BES [48, 89]. The metabolism and electrogenic activity of microorganisms on controlled substrates, especially synthetic substrates with acetate (CH₃COO▬) or simple sugars as carbon source, has been extensively investigated [6, 9, 18, 20, 49, 85]. Different authors have evaluated the electrogenic capacity of the so-called model microorganisms from these substrates to establish efficiencies in electron transfer processes and to identify the reaction stoichiometry present in the metabolism of microbial consortia [4, 64, 84, 100, 101]. However, the diversity in the origin of the wastewater or substrates used in the BES generate significant variations in the electrogenic potential of the system due to the composition of the substances present in these substrates; for example, it has been established that in domestic wastewater predominates oxidizable organic matter [9, 55, 80, 102]. In industrial wastewater, the composition depends on the productive activity from which it comes; and components such as fats, dyes, petroleum derivatives and a large number of recalcitrant contaminants have been identified [14, 17, 30, 56, 73, 75, 89, 103]. In substrates of agricultural origin, a variety of complex carbon compounds and nitrogen compounds derived from pesticides and fertilizers have been found [47, 89].

Figure 5.

Schematic representation of IET through phenazines (PYO) in Pseudomonas sp.

$${\mathit{CH}}_{\mathit{3}}{\mathit{COO}}^{-}+{\mathit{\text{2O}}}_{\mathit{2}}\to {{\mathit{\text{2HCO}}}_{\mathit{3}}}^{-}+H^{+}\left(\mathit{\text{∆G}}=-\mathit{847,6}\mathit{kJ}/\mathit{mol}\right)\left(E°=\mathit{0,805}V\mathit{vs}\mathit{SHE}\right)$$E1

The usable energy in bioelectrochemical systems depends on organic compounds that can be biochemically transformed through redox reactions, giving rise to different substances and metabolic intermediates. Eq. (1) presents a simplified reaction of the transformation of a simple substrate such as acetate, which is achieved through the electrogenic metabolism of microbial communities in a BES [47, 93]. However, in a complex substrate such as domestic or industrial wastewater, in which the presence of multiple compounds has been identified, there are many more intermediate reactions, as a product of the metabolic activity of the microorganisms involved in the oxidation and reduction processes. For example, for MEC-type systems used for the production of methane or hydrogen, a series of complementary reactions known as methanogenesis and hydrogenogenesis occur [28, 37, 39, 41, 53, 70]. The reactions present some variations, since what is intended in these BES is the production of biofuels, so the oxidation of organic matter is carried out until obtaining methane gas (CH₄) or until obtaining biohydrogen (H₂) [7, 11]. In this type of system, there is generally a symbiotic relationship of anaerobic microorganisms, especially bacteria and archaea, which degrade organic matter and produce biofuels with an interdependent metabolism; that is, the microorganisms in the present community mutually benefit from the metabolic intermediates and by-products produced by the successive substrate transformation reactions [11, 25, 53, 84]. Figure 6 presents the stoichiometry of redox and methanogenesis of organic compounds in a BES using CO₂ as carbon source [47].

Figure 6.

Metabolic reactions of methanogenesis in BES.

Nutrients derived from nitrogen and sulfur from wastewater represent a particular interest for bioremediation processes due to their high impact on ecosystems. These compounds have been described as the main cause of the effects of eutrophication and pollution of natural water sources [19, 22, 71, 104]. The use of BES for the purification of nitrites, nitrates, and sulfates in wastewater has been shown to be a promising strategy to reduce the degree of pollution and co-generation of energy. An example is the study carried out by Mahmoudi and collaborators in which the performance of a bioelectrochemical reactor with two chambers (anaerobic and aerobic) was evaluated, in which a current generation of 0.841 mA and an ammonium and COD removal of 54.6% and 87.2% were observed. The authors concluded that the COD/N ratio is one of the parameters with the greatest influence on the nitrification process to control the growth of autotrophic and heterotrophic bacteria [7]. In some cases, the wastewater may contain other nutrients derived from sulfur, so a series of stoichiometric adaptations of electrogenic metabolism for sulfate reduction have been proposed [89]. Simplified metabolic reactions for nitrates and sulfates in BES are presented in Figure 7.

Figure 7.

Metabolic redox reactions of nitrites/nitrates (N) and sulfates (S) in BES.

In nature, there are ecological successions and symbiotic relationships between microorganisms in which different metabolisms are expressed for the assimilation of complex nutrients and pollutants [23, 46, 84]. The expression of specific metabolic pathways could represent a strategy for the optimization of bioelectrochemical systems, since the electrogenic metabolism of a diverse microbial community would allow an increase in the efficiency and scope of these systems for the treatment of different contaminated environmental matrices, which are constituted by recalcitrant organic and inorganic compounds [17, 48, 64, 72, 74, 75, 105].

6. Microbial communities in bioelectrochemical systems

Numerous studies report BES in which a great diversity of consortiums and microbial communities are used for the treatment of domestic and industrial wastewater for the degradation or reduction of polluting compounds such as organic waste, dyes, pesticides, phenolic compounds and petrochemicals; as well as for the production of metabolites with high added value such as volatile fatty acids, organic acids, biofuels, among others [8, 41, 50, 64, 80, 89, 106, 107, 108]. In some cases, the presence of “model” microorganisms is indicative of electrogenic activity. As reported by Liang et al., there is a presence of Geobacter sp. and Pseudomonas sp. in a 1000-L reactor, in which a production of 7.68 W/m² was obtained with synthetic wastewater and 3.64 W/m² with domestic wastewater [87]. However, the diversity of the microbial communities used in bioelectrochemical systems depends fundamentally on the origin or nature of the inoculum or the substrate used.

In the treatment of domestic wastewater in these systems, the presence of Paracoccus denitrificans and Clostridium sp., denitrifying bacteria with the capacity to metabolize sucrose, glucose, starch, lactic acid, and other sugars, has been reported [80]. Also, the genera Enterobacter sp. involved in the electrochemical reduction of oxygen and Escherichia sp. involved in the reduction of extracellular iron [36]. Urine has been evaluated as domestic waste for its treatment in BES, achieving a degradation of antibiotics such as ampicillin greater than 99%. In these investigations, the most abundant bacterial phyla identified were Proteobacteria, Firmicutes, Bacteroidetes, Chloroflexi, Actinobacteria. The most abundant bacterial genera were Pseudomonas sp., Anaerolineae sp. known for their cellulolytic capacity and their cellular adhesiveness; and the genera Nitrospira sp. and Methyloversatilis sp. associated with the metabolism of nitrogenous compounds (NH4/NO3) [76]. Xiao et al. reported the presence of the bacterial genera Desulfovibrio, Lysinibacillus, Clostridium and Lachnoclostridium in an MFC directly inoculated with wastewater, achieving greater efficiency in energy production of 543.75 mW/m² and a removal of nutrients such as total nitrogen with 76.15%, ammoniacal nitrogen with 83.23%, and a COD reduction of 75.59%, compared to that achieved with a defined inoculum or consortium. The authors indicate a higher microbial diversity in the MFC operated with wastewater [103].

In the case of industrial wastewater, bioelectrochemical treatments of wastewater from the food industry have been reported in the presence of Geobacter sp. and Petrimonas sp. known for their action to oxidize or ferment substrates such as brewery waste [56], or the metabolic activity of Pseudomonas sp. to degrade azo-type dyes derived from the textile industry or even for the production of pigments [78]. Some archaea of the order Methanomicrobiales and Methanosarcinales have also been identified in wastewater from the meat industry that could establish syntrophic interactions with acetate-oxidizing microorganisms and also develop exoelectrogenic activity [4]. In an investigation published by Marshal et al., in a BES with an inoculum from brewery wastewater, there were described the genera Acetobacterium sp., Sulfurospirillum sp. and Desulfovibrio sp. participating in functions such as fixation of carbon in the electrode, indication of a microaerophilic ecosystem and in the possible expression of protein hydrogenases, formate dehydrogenase and proton-to-hydrogen reducing cytochromes [109]. In a study describing the electrogenic behavior of controlled microbial consortia, strains of Methanococcus maripaludis and Acetobacterium woodii were used as model microorganisms for the hydrogenotrophic synthesis of methane and acetate; electromethanogenesis rates of 0.1–0.14 μmol cm⁻² h⁻¹ at −400 mV (vs SHE) and 0.6–0.9 μmol cm⁻² h⁻¹ at −500 mV were observed and acetate formation was observed at rates of 0.21–0.23 μmol cm⁻² h⁻¹ at −400 mV and 0.57–0.74 μmol cm⁻² h⁻¹ at −500 mV, respectively [84]. Some microorganisms such as Desulfovibrio sp. and Thiobacillus sp., Azospirillum sp. and Mycobacterium sp. have the ability to metabolize nutrients such as SO₄, PO₄, and NO₃/NO₂, and transform them into simple molecules and gases with less impact on the environment [19, 30, 74, 97]. Some cyanobacteria, photosynthetic microorganisms, have also been related to the production of intermediate compounds such as acetate and lactate, and it is suggested that they could be used for the denitrification of organic matter and co-generation of energy through exoelectrogens in BES [13].

On the other hand, the characterization of microbial communities in bioelectrochemical systems has been subject to studies evaluating the performance of BES that are subjected to different operational conditions. For example, Koók et al. reported that the predominance of the genera Geobacter sp., Azospirillum sp., Castellaniella sp., Pandoraea sp., Treponema sp., Clostridium sp. varies in relation to the external resistance used in an MFC. The authors report a higher yield, but a lower stability of the system when a dynamic system of external resistances is used, which is attributed to the acidification of the biofilm. These results suggest the adaptive evolution of the microbial community in response to the operational conditions of the BES [110]. In a mixed system known as MFC-CW that has been described as an MFC coupled to a constructed wetland, Tao et al. reported that the most abundant phyla were Proteobacteria, Cyanobacteria, Bacteroidetes, Acidobacteria, Chloroflexi, and Nitrospirae, in the case of the predominant genera Geobacter sp., Desulfobulbus sp., Bacillus sp., and Geothrix sp. In this mixed system, a nitrate reduction greater than 90% and a power density of 6.09 mW/m² were achieved when xylose was used as carbon source; in comparison, only a 10% nitrate reduction and a power density of 2.91 mW/m² were achieved when cellulose was used [13]. In another study by Ge et al., using an MFC-CW, reduction values of COD, NO³⁻-N, total inorganic nitrogen, and total phosphorus of 71.9, 70.1, 63.2, and 91.2%, respectively, were reported; and current and power densities of 47.77 mA/m² and 6.74 mW/m², respectively [19]. The predominant electrogenic microorganisms found were the Proteobacteria, Actinobacteria, Acidobacteria, Bacteroidetes, and Chloroflexi phyla. The genera Geobacter sp. and Dechloromonas sp. associated with autotrophic denitrification and phosphate accumulation; the genus Thiobacillus sp. related to pyrite-driven autotrophic denitrification and the genera Desulfovibrio sp., Desulfomicrobium sp., Desulfobulbus sp., and Desulfuromonas sp. identified as sulfate-reducing bacteria for the oxidation of organic compounds such as biofilm-detached biomass and residual organic pollutants [19]. For the treatment of organosulfur compounds, Elzinga et al. used a type H MFC, and determined an abundance of the families Halomonadaceae, Clostridiaceae, Eubacteriaceae, and Clostridiaceae. The coulombic efficiency associated with this system in relation to different compounds was 1.7% for methanethiol, 3.4% for ethanethiol, and 5.0% for propanethiol and dimethyl disulfide [22]. Research in microbial ecology for BES has increased in the recent years. However, there are few reports focused directly on the study of native or autochthonous microbial communities in BES. Examples include the work of Revelo et al., who evaluated the effect of the inoculum in an MFC with anodic and cathodic chambers separated by a salt bridge. Anaerobic sludge from a solid waste treatment plant and sulfidogenic sludge and wastewater from artisanal tanneries were evaluated. The researchers reported reductions of 97.9% in organic matter content and 86.3% in chromium content, as well as a coulombic efficiency of 0.92% [111]. In a recent study, Agudelo-Escobar et al. reported the electrogenic capacity of native microorganisms in complex communities originating from wastewater from the coffee agroindustry, in an open cathode MFC with an unconventional design, achieving a reduction in organic matter of up to 70% and a power density of 21.16 W/m³. The authors reported the presence of the genera Clostridium sp., Cohnella sp., Aneurinibacillus sp., Candida sp., Gluconacetobacter sp., Clostridium sp., Acetobacter sp., Bacillus sp., Weissella sp., Leucononostoc sp., and Lactobacillus sp., microorganisms known mainly in anaerobic or microaerophilic ecosystems, associated with the reduction of organic compounds and that they suggest are related to the electrochemical performance of the system [112]. Do et al. indicate that the bacterial community at the anode is mainly affected by the type of substrates used and ultimately this influences current generation [113]. It has been shown that current may be sensitive not only to COD concentrations, but also to the presence of aerobic and electrogenic bacteria, as well as nutrient ratios in water samples [114]. The success of the implementation of a BES system for the treatment of wastewater, the co-generation of energy and the generation of high value-added products, will be conditioned by the type and origin of the substrate used and by the choice of microorganism or microbial community, which will be responsible for carrying out the metabolic transformation and use of organic matter in the system. Figure 8 proposes a consolidated scheme that groups the main components identified in the wastewater with the intermediate metabolites and final products that can be obtained through the main metabolic activities identified in the electrogenic microorganisms in the BES.

Figure 8.

Unified scheme unveiling wastewater transformation by electrogenic microorganisms.

What is proposed is a route to identify the best bioelectrochemical alternative. To do this, the purpose of the implementation must be established, that is, to identify whether the main objective is bioremediation or treatment of a specific wastewater or the obtaining of biofuels, energy generation, or obtaining high value-added metabolites. The origin and composition of the wastewater must be identified, to establish what type of substances will be present in the substrate and thus choose the adapted microbial community with the metabolic capacity to transform that type of substrate. For example, for the treatment of domestic wastewater, the use of an MFC or a combined system such as the MFC-CW could be implemented. For industrial wastewater, depending on their origin, MECs or METs could be implemented coupled to gas capture systems, in which the use of microbial communities with metabolic capacity for the successive transformation of organic substrates, until reaching the production of biofuels such as H₂ and CH₄.

7. Perspectives of electroactive microorganisms

Microorganisms and different microbial communities play a fundamental role in oxidation-reduction processes for the transformation of organic matter, energy production, and obtaining compounds of interest in bioelectrochemical systems. Some main metabolic pathways have been identified that are directly associated with the degradation of organic matter for the recovery of electrical energy or the obtaining of biofuels for some specific microorganisms. However, in diverse biological systems, such as microbial communities, in which there are ecological successions and different metabolic reactions that are essential to achieve the transformation of complex organic and inorganic compounds, as well as the transformation of highly polluting substances such as heavy metals, pesticides, and other contaminants; the combination of metabolic pathways that; they do not necessarily obey bioelectrochemical processes; have not been clearly established. Although knowledge of the microbial communities responsible for the biochemical functioning of BES has been progressively deepening in relation to technological advances in molecular biology and genetic engineering, the population and ecological study of microbial communities is constantly updated [45, 54, 59, 113], and research directly focused on the ecological study of complex microbial communities in bioelectrochemical systems is scarce.

Scientific advances have made it possible to identify which microorganisms are present in these systems and the possible metabolic mechanisms that favor their ability to transform organic matter and electrochemical performance. However, research focuses mainly on the design, configuration, and electrochemical performance of these systems. The detailed study of the changes in the diversity and abundance of microbial communities in BES provide fundamental information to determine the role that each microorganism plays in the microbial community and establish their participation in the oxidation-reduction processes of organic matter, the energy production, the electron transport mechanism, and the formation of biofilms and their interaction with the substrate and other microorganisms in BES systems; there is fundamental information for the implementation of bioelectrochemical systems as biotechnological alternatives for wastewater treatment and co-generation of clean energy. As a perspective, it is necessary to deepen the investigation of the biological interactions of microbial communities to analyze the electrochemical potential with application in the optimization of bioelectrochemical systems. For example, for the implementation of BES as biotechnological tools such as biosensors based on microbial fuel cells known as economic in situ sensing technologies of wastewater quality [113, 114].

There is evidence of microorganisms' capability to generate electricity and biofuels from complex organic matters such as cellulose, pectin, pesticides, dyes. While many studies delve into the treatment of domestic wastewater, industrial wastewater, particularly of agricultural origin, could be considered as unconventional substrates for biotechnological processes like BES. Agro-industrial wastewater consists mainly of organic matter and nutrients derived from both plant sources (biomass) and the addition of manure and fertilizers to crops. This composition makes them substrates with high potential for microbial metabolism.

Access to wastewater treatment systems in rural and agricultural areas is primarily limited by the difficulty of reaching these locations. Moreover, the extensive cultivation areas and their geographical separation make it challenging to aggregate the waste. The use of bioelectrochemical systems as an alternative for treating agro-industrial waste shows promise as a sustainable strategy that allows for the implementation of local treatment systems in agricultural zones. Considering the properties and composition of agro-industrial wastewater, as well as the adaptability of electrogenic microorganisms to different environmental conditions and substrates, supports the applicability of these systems in rural areas.

It is necessary to delve deeper into the study and characterization of native microbial communities in agro-industrial wastewater responsible for energy production through the oxidation-reduction of organic matter in bioelectrochemical systems. These communities offer several advantages, such as their adaptation to the composition, their development under ambient conditions, and their stable ecological relationships in these substrates. Compared to the introduction of exogenous microorganisms and microbial consortia into the treatment matrix, native microbial communities do not require adaptation phases to achieve electrogenic metabolism.

In-depth exploration of biostimulus and bioaugmentation techniques holds great promise in the optimization of wastewater bioelectrochemical treatment processes. These techniques involve adapting and enhancing the microorganisms present in the system, leading to a range of benefits in terms of electrogenic potential and overall efficiency of BES [9]. Through biostimulus techniques, the electrogenic activity and performance of the microorganisms can be stimulated and optimized providing suitable growth factors, adjusting environmental parameters, or employing specific additives that enhance microbial activity and promote electron transfer processes. Considering bioaugmentation, it is proposed to evaluate the introduction of selected microorganisms or microbial consortia into the existing native microbial community of the BES. These added microorganisms could improve specific capabilities, such as enhanced electron transfer abilities or the ability to degrade specific complex organic or remove inorganic compounds. By introducing these specialized microorganisms, the electrogenic potential of the system can be enhanced, leading to improved performance in terms of electricity generation or pollutant removal.

Both biostimulus and bioaugmentation techniques offer exciting opportunities for optimizing wastewater bioelectrochemical treatment processes. Through the adaptation and enhancement of microorganisms, these strategies can unlock the full potential of BES, improving their electrogenic performance and overall efficiency. However, further research is needed to understand the specific mechanisms and conditions that lead to optimal results, as well as to ensure the long-term stability and sustainability of these approaches in real-world applications.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Notes/thanks/other declarations

All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.