Bioaugmentation with Electroactive Microbes—A Promising Strategy for Improving Process Performances of Microbial Electrochemical Technologies

Abstract

Microbial electrochemical technologies (METs), such as microbial fuel cells (MFCs) and microbial electrolysis cells (MECs), show promise for sustainable energy generation from biomass waste and wastewater. However, further work is necessary for their practical use. In particular, it has been argued that process performances, such as those for organics removal and energy generation, should be substantially improved to catch up with those of existing processes, such as anaerobic digesters. Recent work has reported that bioaugmentation (BA) with electroactive microbes (EAMs) can significantly improve the performance of MFCs and MECs, while previous reports have also documented BA cases with limited impacts. In this article, after summarizing EAMs that have been isolated and characterized as possible BA agents, we comparatively analyze past BA trials for MET processes. Based on the information thus obtained, key factors that should be considered for successful BA are suggested.

1. Introduction

Fossil fuels, particularly petroleum, are the major primary energy sources for human society. However, they are finite, and it is now evident that a significant portion of the planet’s fossil fuel reserves has already been extracted [1]. Moreover, the use of fossil fuels has led to environmental issues, such as global warming and plastic waste disposal. Global warming, in particular, is driven by carbon dioxide emissions from fossil fuel combustion [2]. Therefore, it is essential to explore alternative energy sources that are sustainable and environmentally friendly. Among these alternatives, biomass resources—especially biomass waste generated by our society—represent an attractive option [3].

Substantial amounts of biomass waste, such as food and agricultural waste, are discharged by human society [4]. It is projected that about 10 to 50% of the world’s energy consumption can be produced from biomass until the year 2050 [5], with biomass waste playing a significant role. Currently, some biomass waste is treated in anaerobic digesters, in which biogas—primarily methane gas—is produced and used as fuel to generate electricity [6]. In addition, after the discovery of electroactive microbes (EAMs) that can exchange electrons with extracellular electrodes, researchers have proposed the application of microbial electrochemical technologies (METs) to the treatment of biomass waste [7]. Among these technologies are microbial fuel cells (MFCs) and microbial electrolysis cells (MECs). MFCs generate electric power from biomass waste, while MECs primarily produce hydrogen gas [8,9]. Despite extensive laboratory studies, however, these technologies have yet to be implemented in practical applications, mainly due to insufficient process performances, including those for organics removal and power output. In many cases, reported values for laboratory MET devices are substantially lower than those achieved by anaerobic digesters [10].

A number of studies have been conducted to develop technologies for improving the process performances of MET devices, and these trials have been summarized in several review articles. For instance, numerous studies have examined configurations of MET devices, as reviewed by Javed et al. [11]. In addition, articles have documented advances in the development of electrode materials for improving electron transfer between microbes and electrodes [12,13,14,15]. Another crucial factor influencing MET-process performances would be microbes, in particular EAMs, in these devices [16,17,18]. MET devices for energy recovery from biomass waste and wastewater have been operated following inoculation with natural microbial mixtures, such as sludge, soil, sediment, and wastewater, in which naturally occurring EAMs contribute to current generation [19,20,21]. Despite that the performances of these devices are largely influenced by EAMs occurring in these devices, technologies for controlling their occurrence remain limited.

Recent studies have highlighted the potential of bioaugmentation (BA) with EAMs to enhance the performances of MET devices [22,23]. BA was originally developed as a technology for bioremediation of contaminated sites. In BA, specific microorganisms are introduced into contaminated sites to improve the ability to break down contaminants and/or to facilitate other biological processes [24,25]. Additionally, BA trials have also been exploited in bioprocesses for the treatment of wastewater [26] and biomass waste [27,28]. Although several studies have exploited BA trials to enhance the performances of MET processes, no comprehensive review has analyzed past BA trials for MET processes, and it has not been sufficiently understood how BA improves MET processes. We expect that comparative analyses of these results will provide us with valuable insights into the factors that should be considered for successful BA in MET processes.

In the present work, a particular focus is placed on BA with EAMs for improving the performance of MET processes. We begin by providing an overview of currently known EAMs as possible BA agents, followed by a discussion on the performance of MET processes and a comparative analysis of previous BA applications in MFCs and MECs. Based on the insights gained, we propose key factors that should be considered for successful BA in MET processes treating biomass waste and wastewater.

2. EAMs as Possible BA Agents

EAMs are capable of transporting electrons across cell membranes and cell walls, facilitating the use of extracellular inert electrodes as electron donors or acceptors [16,17,18]. This ability of microbes was first discovered in a study that observed anaerobic growth of a ferric iron-reducing bacterium, Shewanella putrefaciens IR-1 (taxonomy may have been changed), in a three-electrode electrochemical cell (EC) using lactate as the electron donor and an inert carbon electrode as the electron acceptor, resulting in anodic current generation (electrode reduction) coupled to lactate oxidation [29]. Since this bacterium is incapable of fermentative growth on lactate, the authors suggest that its anaerobic growth results from the ability of this bacterium to utilize the carbon electrode as an electron acceptor [29]. Following this observation, some other bacteria, such as Geobacter sulfurreducens [30], Pseudomonas aeruginosa [31], Shewanella oneidensis [32], and Thermincola ferriacetica [33], have also been found to be EAMs. Since then, a number of EAMs have been isolated from natural (e.g., soil and sediment) and engineered (e.g., wastewater and sewage sludge) habitats, and their electrochemical activities were characterized in pure cultures. Some of the EAMs isolated in the past 10 years are listed in Table 1, which we will discuss later.

Most of the EAMs isolated in previous studies are affiliated with the domain Bacteria, and many are affiliated with either the phyla Pseudomonadota, Thermodesulfobacteriota, or Bacillota. In addition, some EAMs affiliated with the domains Archaea have also been characterized, such as iron-reducing hyperthermophilic archaea, Ferroglobus placidus, and Geoglobus ahangari [34]. As per the Eukarya, some fungi [35,36] have been reported to be electrochemically active. Generally, electrochemical activities of EAMs affiliated with the domains Archaea and Eukarya are substantially lower than those of well-characterized bacterial EAMs, such as those affiliated with the genera Geobacter and Shewanella.

Studies have also been conducted to elucidate the molecular mechanisms underlying the electrochemical activities of EAMs [37]. Since cell membranes are made of lipid bilayers that act as barriers against charged molecules, ions, and electrons, EAMs need to equip their outer membranes and/or cell walls with special conduits for electrons, termed extracellular electron transfer (EET) pathways [38]. Different EAMs have different EET pathways (Figure 1), and they are categorized into two major types; one is a cytochrome-mediated pathway that is mainly present in Gram-negative bacteria, such as those affiliated with the genera Geobacter and Shewanella [38]. The other is a flavin-mediated pathway that is present in Gram-positive bacteria [39]. An exception would be EAMs in the genus Thermincola, since these Gram-positive bacteria use cytochrome-dependent pathways for EET [40]. In addition, some bacteria identified as EAMs, including P. aeruginosa [31], are known to use electron shuttles—relatively hydrophobic, charged compounds capable of transferring across cell membranes—for EET. Several EAMs, such as S. oneidensis MR-1 [41], G. sulfurreducens PCA [30], Listeria monocytogenes 10403S [42], and Thermincola potens JR [43], have been used as model organisms to study molecular mechanisms behind microbial electrochemical activities, and our knowledge on EET pathways comes from the results of these studies. Figure 1 presents schematic diagrams for different types of EET pathways.

S. oneidensis strain MR-1 is a Gram-negative bacterium affiliated with the phylum Pseudomonadota. This bacterium is one of the most intensively studied EAMs [41,44,45,46,47]. It was isolated from lake sediment in the USA [48] and is a facultative anaerobe utilizing a variety of electron acceptors, including oxygen, nitrate, nitrite, thiosulfate, elemental sulfur, trimethylamine N-oxide, dimethyl sulfoxide, fumarate, and anthraquinone-2,6-disulfonate, as well as both soluble and solid metals, such as iron, manganese, uranium, chromium, cobalt, technetium, and vanadium [44]. In contrast, it can utilize limited types of electron donors, including lactate, succinate, fumarate, N-Acetyl-glucosamine, and hydrogen [48], suggesting that it tends to thrive in nature under the syntrophic interaction with fermentative microbes that supply EAMs with electron donors. It is likely that the ecological niches of EAMs, such as MR-1, in their original ecosystems would be the final steps of organic matter decomposition, in which solid electron acceptors are used as electron sinks. Strain MR-1 has cytochrome-mediated EET pathways [41,45]. The major route of electrons is illustrated in Figure 1A, while more complete views can be found in other articles [45,46]. It is noteworthy that MR-1 has porin/cytochrome complexes (PCC) at the outer membrane, from which electrons are mainly transported to electrodes via water-soluble electron shuttles (ESs), such as riboflavin [49] and quinones [50]. PCC is comprised of multi-heme cytochromes, including outward deca-heme cytochromes that have flavin-binding sites [51]. It is conceivable that this organism is able to use ESs more efficiently than bacteria that do not have PCC since ESs do not need to transfer across cell membranes. Accordingly, studies have recorded relatively high current densities, such as over 0.2 mA cm⁻² (per electrode area), in ECs inoculated with MR-1 [52] or other Shewanella strains [53].

Table 1. A list of EAMs isolated and/or characterized in the past 10 years.

Strain	Taxon	Habitat	Electrochemical Activity 1	Description	Reference
ZH1	Pseudomonas aeruginosa	Palm oil mill sludge	451 mW m−2, 65 μA cm−2	Power generation from oil sludge	[54]
39E	Thermoanaerobacter pseudethanolicus	Hotspring	0.6 mA cm−2	Current generation from sugars at 60 °C	[55]
Z7	Citrobacter freundii	Aerobic sewage sludge	205 mW m−2, 0.1 mA cm−2	Current generation from sugars using mediators	[56]
D-8	Geobacter sulfurreducens	Rice paddy soil	1.1 mA cm−2	Grow on ethanol, glycerol and sugars	[57]
MK2	Stenotrophomonas maltophilia	Groundwater/sludge	27 μA cm−2	Petrotrophic exoelectrogen	[58]
WE1-13	Delftia	Deep aquifer	0.4 μA cm−2	Continental subsurface environments	[59]
WE2-4	Azonexus	Deep aquifer	350 nA cm−2	Continental subsurface environments	[59]
SCS5	Aeromonas jandaei	Wastewater sludge	7 μA cm−2	Isolated from acetate-fed MFC	[60]
WTL	Desulfuromonas soudanensis	Deep subsurface brine	58 μA cm−2	Halophilic iron-reducing bacterium	[61]
6	Clostridium amylolyticum	Soil	29 mW m−2, 20 μA cm−2	Fourteen other EAB were also isolated.	[62]
LAR-1	Citrobacter braakii	River sediment	610 mW/m−2, 0.2 mA cm−2	Current generation in acetate-fed MFC	[63]
LZ-1	Citrobacter freundii	Domestic sewage	865 μA cm−2	Facultative anaerobe	[64]
COM1	Pyrococcus furiosus	Volcanic marine sediment	0.2 mA cm−2	Current generation at 90°C	[65]
JhA	Anaerosinus	Coastal sediment	4 μA cm−2	Coastal gold mining site	[66]
R6	Clostridium sporogenes	Cu-contaminated soil	25 mW m−2, 20 μA cm−2	Resistant to 10 mg L−1 Cu2+	[67]
RNV-4	Dietzia	Intertidal zone sediment	30 μA cm−2	Aerobic growth in nutrient-rich media	[68]
KVM11	Citrobacter	Groundwater/sludge	36 μA cm−2	Petrotrophic exoelectrogen	[69]
EB-1	Mycobacterium fortuitum	Polluted river sediment	0.84 W m−2, 0.2 mA cm−2	Denitrifying bacterium	[70]
H	Bacillus cereus	Anaerobic digester	32 mW m−2, 25 μA cm−2	Isolated from Cr(VI)-reducing MFC	[71]
CL-1	Geobacter sulfurreducens	Paddy soil	1.0 mA cm−2	Current generation from acetate and ethanol	[72]
ND-2	Citrobacters	Rice paddy soil	20 μA cm−2	Isolated by electrode-plate cultivation	[73]
RPFA-12G	Geobacter sulfurreducens	Rice paddy soil	0.2 mA cm−2	Isolated by electrode-plate cultivation	[73]
AEDII12DO	Ferroglobus placidus	Hydrothermal system	68 μA cm−2	Archaeon, current generation at 85°C	[34]
234	Geoglobus ahangari	Hydrothermal system	57 μA cm−2	Archaeon, current generation at 80°C	[34]
10403S	Listeria monocytogenes	Rabbit	20 μA cm−2	Use of flavin for EET	[42]
LLD-1	Bacillus megaterium	Activated sludge	40 μA cm−2	Use of flavin for EET	[74]
S05	Klebsiella quasipneumoniae	MBR treating wastewater	0.07 mA cm−2	Causative agent of membrane fouling	[75]
DIF1	Bacillus cereus	MFC	10 μA cm−2	Use of flavin for EET	[76]
DIF2	Rhodococcus ruber	MFC	15 μA cm−2	Use of flavin for EET	[76]
Gut-S1	Enterococcus avium	Human feces	120 nA cm−2	Current generation from glucose	[77]
MCC 3673	Kluyvera georgiana	Lake sediment	39 μW cm−2	Power generation from oil cakes	[78]
E8	Pseudomonas protegens	Activated sludge	70 mW m−2, 80 μA cm−2	Potential for acid mine-drainage treatment	[79]
G7K4R3	Clostridium cochlearium	Mouse gut	0.53 mA cm−2	Current generation from glucose	[80]
JSUX1	Cystobasidium slooffiae	Activated sludge	21 μW cm−2	Eukaryote, current generation from Xylose	[35]
OR-1	Shewanella algae	Costal sediment	0.45 mA cm−2	This strain generates current from acetate.	[53]
MA-72	Paenibacillus dendritiformis	Freshwater sediment	0.5 μA cm−2	Current generation in peptone meat broth	[81]
EG	Alicyclobacillus hesperidum	Sewage treatment plant	188 mW m−2, 45 μA cm−2	Current generation under acidic conditions	[82]
YM18	Geobacter sulfurreducens	River sediment	1.1 mA cm−2	YM18 generates more current than KN400	[83]
NIT-T3	Desulfuromonas versatilis	Coastal sand	0.2 mA cm−2	Isolated from graphene-reducing enrichment	[84]
GY3	Lysinibacillus varians	Freshwater sediment	7.5 μA cm−2	Filamentous Gram-positive EAB	[85]
Isolate 1	Klebsiella pneumoniae	Tropical lake sediment	129 mW m−2, 0.07 mA cm−2	Isolated using MnO2 plates	[86]
Isolate 2	Agrobacterium salinitolerans	Tropical lake sediment	51 mW m−2, 0.03 mA cm−2	Isolated using MnO2 plates	[86]
Isolate 3	Serratia nematodiphila	Tropical lake sediment	23 mW m−2, 0.02 mA cm−2	Isolated using MnO2 plates	[86]
Isolate 4	Kosakonia oryzendophytica	Tropical lake sediment	80 mW m−2, 0.05 mA cm−2	Isolated using MnO2 plates	[86]
Isolate 5	Enterobacter cloacae	Tropical lake sediment	81 mW m−2, 0.04 mA cm−2	Isolated using MnO2 plates	[86]
S116	Pseudomonas stutzeri	Marine sludge	765 mW m−2	Marine sulfur-oxidizing bacterium	[87]
SAP-1	Geoalkalibacter halelectricus	Lake sediment	0.5 mA cm−2	Current generation under haloalkaline conditions	[88]
CS-1	Dechloromonas agitata	River sediment	−30 μA cm−2	Preferentially generate cathodic current	[89]
CS-2	Clostridium magenotii	River sediment	−10 μA cm−2	Preferentially generate cathodic current	[89]
SHE10	Shinella zoogloeoides	Sweet potato root	78 mW m−2, 22 µA cm−2	Plant endophytic EAB	[90]
EB1	Paenibacillus lautus	Activated sludge	1.6 μA cm−2	Current generation from starch	[91]
DWW1	Enterococcus faecalis	Dairy wastewater	144 mW m−2, 26 µA cm−2	Current generation from dairy wastewater	[92]
NTE-D12	Cellulomonas	Soil	0.9 μA cm−2	Possibly represent a novel species	[93]
PBH03	Pseudomonas aeruginosa	Anaerobic sludge	9 µA cm−2	Isolated using WO3 nanorod probes	[94]
NCIMB8826	Lactiplantibacillus plantarum	Saliva	20 μA cm−2	Current generation in the presence of flavin	[95]
YoMME	Paenibacillus profundus	Freshwater sediment	20 μA cm−2	Current generation from peptone	[96]
A1	Lysinibacillus sphaericus	Poultry dropping	4 μA cm−2	Current generation from poultry wastewater	[97]
AKS46	Paraclostridium	Solid waste disposal site	63 μA cm−2	May have multiple electron-transfer routes	[98]
NIT-SL11	Geotalea uranireducens	Municipal sewage	0.7 mA cm−2	Graphene oxide-reducing bacterium	[99]
620C	Pseudomonas citronellolis	Drilling waste	1 mW m−2, 3 μA cm−2	Use of pyocyanin as an electron shuttle	[100]
SQ-1	Klebsiella variicola	Activated sludge	0.6 mA cm−2	Facultative anaerobe	[101]
ADMFC1	Eubacteriales	Anaerobic digester	0.6 mA cm−2	Possibly represent a novel family	[102]
ADMFC2	Sulfurospirillaceae	Anaerobic digester	0.2 mA cm−2	Possibly represent a novel genus	[102]
ADMFC3	Geovibrio	Anaerobic digester	0.4 mA cm−2	Possibly represent a novel species	[102]
2Se	Brevundimonas diminuta	Activated sludge	102 mW m−2, 52 µA cm−2	Selenite reduction	[103]
AMB-1	Magnetospirillum magneticum	Water pond	27 µW m−2, 0.16 µA cm−2	Magnetotactic bacterium	[104]
MSR-1	Magnetospirillum gryphiswaldense	Wetland	11 µW m−2, 0.11 µA cm−2	Magnetotactic bacterium	[104]
KCf2	Bacillus altitudinis	Aquaculture sediment	67 mW m−2, 33 µA cm−2	Current generation from glucose	[105]
YH-1	Micrococcus	Metal-contaminated soil	8.3 µA cm−2	Halotolerant bacterium	[106]
RH-1	Gordonia	Metal-contaminated soil	2.2 µA cm−2	Halotolerant bacterium	[106]
CH-1	Stutzerimonas	Metal-contaminated soil	1.7 µA cm−2	Halotolerant bacterium	[106]
60473	Geobacter sulfurreducens	Lakeshore mud	1.5 mA cm−2	Useful in bioaugmentation	[23]

¹ Current and/or power densities per anode areas are indicated. Attention should be paid to direct comparison of these values, since they were obtained under different experimental settings.

Table 1. A list of EAMs isolated and/or characterized in the past 10 years.

Strain	Taxon	Habitat	Electrochemical Activity 1	Description	Reference
ZH1	Pseudomonas aeruginosa	Palm oil mill sludge	451 mW m−2, 65 μA cm−2	Power generation from oil sludge	[54]
39E	Thermoanaerobacter pseudethanolicus	Hotspring	0.6 mA cm−2	Current generation from sugars at 60 °C	[55]
Z7	Citrobacter freundii	Aerobic sewage sludge	205 mW m−2, 0.1 mA cm−2	Current generation from sugars using mediators	[56]
D-8	Geobacter sulfurreducens	Rice paddy soil	1.1 mA cm−2	Grow on ethanol, glycerol and sugars	[57]
MK2	Stenotrophomonas maltophilia	Groundwater/sludge	27 μA cm−2	Petrotrophic exoelectrogen	[58]
WE1-13	Delftia	Deep aquifer	0.4 μA cm−2	Continental subsurface environments	[59]
WE2-4	Azonexus	Deep aquifer	350 nA cm−2	Continental subsurface environments	[59]
SCS5	Aeromonas jandaei	Wastewater sludge	7 μA cm−2	Isolated from acetate-fed MFC	[60]
WTL	Desulfuromonas soudanensis	Deep subsurface brine	58 μA cm−2	Halophilic iron-reducing bacterium	[61]
6	Clostridium amylolyticum	Soil	29 mW m−2, 20 μA cm−2	Fourteen other EAB were also isolated.	[62]
LAR-1	Citrobacter braakii	River sediment	610 mW/m−2, 0.2 mA cm−2	Current generation in acetate-fed MFC	[63]
LZ-1	Citrobacter freundii	Domestic sewage	865 μA cm−2	Facultative anaerobe	[64]
COM1	Pyrococcus furiosus	Volcanic marine sediment	0.2 mA cm−2	Current generation at 90°C	[65]
JhA	Anaerosinus	Coastal sediment	4 μA cm−2	Coastal gold mining site	[66]
R6	Clostridium sporogenes	Cu-contaminated soil	25 mW m−2, 20 μA cm−2	Resistant to 10 mg L−1 Cu2+	[67]
RNV-4	Dietzia	Intertidal zone sediment	30 μA cm−2	Aerobic growth in nutrient-rich media	[68]
KVM11	Citrobacter	Groundwater/sludge	36 μA cm−2	Petrotrophic exoelectrogen	[69]
EB-1	Mycobacterium fortuitum	Polluted river sediment	0.84 W m−2, 0.2 mA cm−2	Denitrifying bacterium	[70]
H	Bacillus cereus	Anaerobic digester	32 mW m−2, 25 μA cm−2	Isolated from Cr(VI)-reducing MFC	[71]
CL-1	Geobacter sulfurreducens	Paddy soil	1.0 mA cm−2	Current generation from acetate and ethanol	[72]
ND-2	Citrobacters	Rice paddy soil	20 μA cm−2	Isolated by electrode-plate cultivation	[73]
RPFA-12G	Geobacter sulfurreducens	Rice paddy soil	0.2 mA cm−2	Isolated by electrode-plate cultivation	[73]
AEDII12DO	Ferroglobus placidus	Hydrothermal system	68 μA cm−2	Archaeon, current generation at 85°C	[34]
234	Geoglobus ahangari	Hydrothermal system	57 μA cm−2	Archaeon, current generation at 80°C	[34]
10403S	Listeria monocytogenes	Rabbit	20 μA cm−2	Use of flavin for EET	[42]
LLD-1	Bacillus megaterium	Activated sludge	40 μA cm−2	Use of flavin for EET	[74]
S05	Klebsiella quasipneumoniae	MBR treating wastewater	0.07 mA cm−2	Causative agent of membrane fouling	[75]
DIF1	Bacillus cereus	MFC	10 μA cm−2	Use of flavin for EET	[76]
DIF2	Rhodococcus ruber	MFC	15 μA cm−2	Use of flavin for EET	[76]
Gut-S1	Enterococcus avium	Human feces	120 nA cm−2	Current generation from glucose	[77]
MCC 3673	Kluyvera georgiana	Lake sediment	39 μW cm−2	Power generation from oil cakes	[78]
E8	Pseudomonas protegens	Activated sludge	70 mW m−2, 80 μA cm−2	Potential for acid mine-drainage treatment	[79]
G7K4R3	Clostridium cochlearium	Mouse gut	0.53 mA cm−2	Current generation from glucose	[80]
JSUX1	Cystobasidium slooffiae	Activated sludge	21 μW cm−2	Eukaryote, current generation from Xylose	[35]
OR-1	Shewanella algae	Costal sediment	0.45 mA cm−2	This strain generates current from acetate.	[53]
MA-72	Paenibacillus dendritiformis	Freshwater sediment	0.5 μA cm−2	Current generation in peptone meat broth	[81]
EG	Alicyclobacillus hesperidum	Sewage treatment plant	188 mW m−2, 45 μA cm−2	Current generation under acidic conditions	[82]
YM18	Geobacter sulfurreducens	River sediment	1.1 mA cm−2	YM18 generates more current than KN400	[83]
NIT-T3	Desulfuromonas versatilis	Coastal sand	0.2 mA cm−2	Isolated from graphene-reducing enrichment	[84]
GY3	Lysinibacillus varians	Freshwater sediment	7.5 μA cm−2	Filamentous Gram-positive EAB	[85]
Isolate 1	Klebsiella pneumoniae	Tropical lake sediment	129 mW m−2, 0.07 mA cm−2	Isolated using MnO2 plates	[86]
Isolate 2	Agrobacterium salinitolerans	Tropical lake sediment	51 mW m−2, 0.03 mA cm−2	Isolated using MnO2 plates	[86]
Isolate 3	Serratia nematodiphila	Tropical lake sediment	23 mW m−2, 0.02 mA cm−2	Isolated using MnO2 plates	[86]
Isolate 4	Kosakonia oryzendophytica	Tropical lake sediment	80 mW m−2, 0.05 mA cm−2	Isolated using MnO2 plates	[86]
Isolate 5	Enterobacter cloacae	Tropical lake sediment	81 mW m−2, 0.04 mA cm−2	Isolated using MnO2 plates	[86]
S116	Pseudomonas stutzeri	Marine sludge	765 mW m−2	Marine sulfur-oxidizing bacterium	[87]
SAP-1	Geoalkalibacter halelectricus	Lake sediment	0.5 mA cm−2	Current generation under haloalkaline conditions	[88]
CS-1	Dechloromonas agitata	River sediment	−30 μA cm−2	Preferentially generate cathodic current	[89]
CS-2	Clostridium magenotii	River sediment	−10 μA cm−2	Preferentially generate cathodic current	[89]
SHE10	Shinella zoogloeoides	Sweet potato root	78 mW m−2, 22 µA cm−2	Plant endophytic EAB	[90]
EB1	Paenibacillus lautus	Activated sludge	1.6 μA cm−2	Current generation from starch	[91]
DWW1	Enterococcus faecalis	Dairy wastewater	144 mW m−2, 26 µA cm−2	Current generation from dairy wastewater	[92]
NTE-D12	Cellulomonas	Soil	0.9 μA cm−2	Possibly represent a novel species	[93]
PBH03	Pseudomonas aeruginosa	Anaerobic sludge	9 µA cm−2	Isolated using WO3 nanorod probes	[94]
NCIMB8826	Lactiplantibacillus plantarum	Saliva	20 μA cm−2	Current generation in the presence of flavin	[95]
YoMME	Paenibacillus profundus	Freshwater sediment	20 μA cm−2	Current generation from peptone	[96]
A1	Lysinibacillus sphaericus	Poultry dropping	4 μA cm−2	Current generation from poultry wastewater	[97]
AKS46	Paraclostridium	Solid waste disposal site	63 μA cm−2	May have multiple electron-transfer routes	[98]
NIT-SL11	Geotalea uranireducens	Municipal sewage	0.7 mA cm−2	Graphene oxide-reducing bacterium	[99]
620C	Pseudomonas citronellolis	Drilling waste	1 mW m−2, 3 μA cm−2	Use of pyocyanin as an electron shuttle	[100]
SQ-1	Klebsiella variicola	Activated sludge	0.6 mA cm−2	Facultative anaerobe	[101]
ADMFC1	Eubacteriales	Anaerobic digester	0.6 mA cm−2	Possibly represent a novel family	[102]
ADMFC2	Sulfurospirillaceae	Anaerobic digester	0.2 mA cm−2	Possibly represent a novel genus	[102]
ADMFC3	Geovibrio	Anaerobic digester	0.4 mA cm−2	Possibly represent a novel species	[102]
2Se	Brevundimonas diminuta	Activated sludge	102 mW m−2, 52 µA cm−2	Selenite reduction	[103]
AMB-1	Magnetospirillum magneticum	Water pond	27 µW m−2, 0.16 µA cm−2	Magnetotactic bacterium	[104]
MSR-1	Magnetospirillum gryphiswaldense	Wetland	11 µW m−2, 0.11 µA cm−2	Magnetotactic bacterium	[104]
KCf2	Bacillus altitudinis	Aquaculture sediment	67 mW m−2, 33 µA cm−2	Current generation from glucose	[105]
YH-1	Micrococcus	Metal-contaminated soil	8.3 µA cm−2	Halotolerant bacterium	[106]
RH-1	Gordonia	Metal-contaminated soil	2.2 µA cm−2	Halotolerant bacterium	[106]
CH-1	Stutzerimonas	Metal-contaminated soil	1.7 µA cm−2	Halotolerant bacterium	[106]
60473	Geobacter sulfurreducens	Lakeshore mud	1.5 mA cm−2	Useful in bioaugmentation	[23]

¹ Current and/or power densities per anode areas are indicated. Attention should be paid to direct comparison of these values, since they were obtained under different experimental settings.

G. sulfurreducens PCA is another intensively studied EAM that was isolated from the surface sediments of a hydrocarbon-contaminated ditch in the USA [107]. It is a Gram-negative bacterium affiliated with the phylum Thermodesulfobacteriota. Different from S. oneidensis MR-1, this strain is an obligate anaerobe able to utilize relatively narrow ranges of electron acceptors, such as metal ions, metal oxides, nitrate and fumarate, and limited electron donors, such as acetate and hydrogen [108]. The ecological niches of this bacterium are considered to be similar to those of Shewanella described above. Figure 1B shows a simplified EET pathway of strain PCA. Similar to that of Shewanella (Figure 1A), it has a cytochrome-mediated pathway that includes PCC at the outer membrane [109]. However, a difference exists in the way to transfer electrons from cell surfaces to electrodes; PCA exploits conductive pili and/or nanowires for the transfer of electrons (Figure 1B) [110,111]. It is likely that this would be a reason why strains of G. sulfurreducens are able to attain high current densities compared to other EAMs, including Shewanella strains. In a previous study, current densities attained by the type strains of different species of Geobacter were compared in the same EC systems, showing that G. sulfurreducens attained higher current densities (e.g., 0.25 mA cm⁻²) than others, including G. metallireducens, G. daltonii, G. bemidjensis, G. chapellei, and G. pelophilus [112]. Furthermore, studies have isolated G. sulfurreducens strains that exhibit high electrochemical activities compared to strain PCA (Table 1); these include strains KN400 [113], D-8 [57], YM18 [83], and 60473 [23]. For instance, strain 60473 attained 1.5 mA cm⁻² in an EC system, while PCA attained 0.3 mA cm⁻² in the same system [23]. It would therefore be of interest to examine strain-level variations in the EET pathway that functions in G. sulfurreducens.

Some Gram-positive bacteria, such as L. monocytogenes [42] and Lactiplantibacillus plantarum [95], are also identified as EAMs. L. monocytogenes is affiliated with the phylum Bacillota and is a potential pathogen that can cause listeriosis, one of the most severe foodborne diseases [114]. On the other hand, L. plantarum, which is also affiliated with the same phylum, is one of the most widely studied lactobacilli and is extensively used in the food industry as a probiotic microorganism and/or a microbial starter [115]. Since their cell-surface structures are largely different from those of Gram-negative bacteria, their EET pathways are also largely different (Figure 1). The EET pathways of L. monocytogenes and L. plantarum are known to be flavin-mediated (Figure 1C), in which flavin molecules are reduced on flavin-carrier proteins in their cell walls and released into the extracellular space. Similar flavin-mediated EET pathways have been widely found in Gram-positive bacteria, including Bacillus cereus in the Bacillota and Rhodococcus ruber in the Actinomycetota [76]. Although this would be an effective EET strategy for bacteria with thick cell walls, the electrochemical activities of these bacteria are substantially lower than those of EAMs having cytochrome-mediated pathways (Table 1). For instance, L. plantarum attained ~20 μA cm⁻² in ECs containing nutrient-rich medium, while current densities increased to approximately 0.1 mA cm⁻² when the medium was supplemented with an artificial electron shuttle, such as 1,4-dihydroxy-2-naphthoic acid [95].

Exceptional Gram-positive EAMs would be those affiliated with the genus Thermincola in the phylum Bacillota. A study on T. ferriacetica has shown that this bacterium is able to attain ~1 mA cm⁻² in ECs with acetate as the electron donor at 60 °C, a density much higher than those of other Gram-positive EAMs [116]. In addition, studies have analyzed EET pathways of Thermincola EAMs [40,43,117], showing that they have cytochrome-mediated pathways that facilitate electron transfer across thick cell walls (Figure 1D). Recent work has also suggested that another Gram-positive EAM, Thermoanaerobacterium thermosaccharolyticum, also has a cytochrome-mediated EET pathway [118]. Comparative analyses suggest that EAMs having cytochrome-mediated EET pathways are able to generate more current in ECs than those having flavin-mediated EET pathways.

3. MET Processes and Their Performances

MET processes have been extensively studied in the laboratory for their application in generating energy—such as electric power and hydrogen—from biomass waste and wastewater [8,9]. For this purpose, two types of MET processes, namely MFC and MEC, have been examined. MFCs generate electric power from organic matter, while MECs generate hydrogen and/or methane gas. Schematic diagrams for MFC and MEC reactors frequently used in recent laboratory studies are shown in Figure 2.

In recent studies, single-chamber reactors equipped with air cathodes are commonly used for MFCs (Figure 2A), although double-chamber reactors are also available. This is because liquid-type cathodes tend to become rate-limiting due to the low solubility of oxygen in water. This problem can be overcome by using air cathodes that are membrane-type oxygen-permeable electrodes, thereby enabling single-chamber reactors [119]. In air cathodes frequently used in many studies, one side of a conductive basement (frequently made of carbon cloth) is overlaid with a polytetrafluoroethylene (PTFE) gas-diffusion layer, while the other side features a catalyst layer that holds oxygen-reduction catalysts such as platinum/carbon catalysts (PtC) (Figure 2A). Comparative analyses of double- and single-chamber MFCs operated using the same substrates demonstrate that power outputs from single-chamber MFCs are much higher (e.g., fourfold) than those of double-chamber MFCs [120], resulting in the frequent use of single-chamber rectors in recent MFC studies. In addition, sediment MFCs, also referred to as benthic MFCs or constructed-wetland MFCs, are a type of single-chamber MFC that have been examined for wastewater treatment and/or soil remediation [9] (Figure 2B).

In contrast, double-chamber reactors are commonly used for hydrogen production in MECs (Figure 2C), since hydrogen can only be produced when hydrogen-consuming microbes, such as methanogens, are not present around cathodes [121]. In double-chamber reactors, either cation- or anion-exchange membranes (collectively ion-exchange membranes, IEM) are used to separate anode and cathode chambers to alleviate pH changes in anode- and cathode chambers [122]. However, even in initially sterilized cathode chambers of double-chamber MECs, microbes tend to occur naturally and consume hydrogen several weeks after initiating the operation [123], suggesting the need to develop technologies that inhibit microbial growth and hydrogen consumption in MEC cathodes [124]. On the other hand, since stable production of hydrogen in MEC is difficult and hydrogen is in many cases converted to methane by methanogens occurring around cathodes, researchers have also attempted to apply MEC technology to enhance methane production in anaerobic digesters (termed AD/MEC) [125]. In such cases, single-chamber reactors can be used (Figure 2D).

In MECs, the efficient reduction of protons to form hydrogen molecules at neutral or alkaline pH needs relatively low potentials (e.g., −0.4 V or lower vs. the standard hydrogen electrode). Since the pH of the solution near the cathode tends to increase during hydrogen production, electric energy must be supplied through an external circuit to lower the cathode potential [126]. Cathode potentials are therefore set at 0.8 to 1.0 V lower than anode potentials. The cell voltages (0.8 to 1.0 V), however, are much lower than those of water-electrolysis cells (typically 1.8 V to 3.5 V) since oxidation of water at anodes needs substantially higher electrode potentials than microbial oxidation of organic matter [126]. Although this is a noticeable merit of MECs over water-electrolysis cells, increases in cell voltages in association with the formation of pH biases between anodes and cathodes, in particular those occurring on both sides of IEMs, should be suppressed for energy-efficient hydrogen production. Technologies to solve this problem would be necessary for the practical application of MEC technologies.

Studies on MFCs and MECs have shown that substrates for the anode reaction—generally any kind of organic matter—substantially influence current generation, thereby affecting power and hydrogen production [127,128,129]. It has been known that acetate is the best substrate for MFC and MEC performance among pure organic compounds and complex organic matter, such as food waste, domestic wastewater, and industrial wastewater. To cite an instance, a study analyzed electricity generation and anode microbial communities in MFCs fed with different substrates, including acetate, xylose, acetate/xylose mixture, and bioethanol effluent, showing that MFCs fed with acetate exhibit higher cell voltages and coulombic efficiencies than those fed with the other substrates [127]. It was also found that the anode microbial community in acetate-fed MFCs was less diverse than the other MFCs, and that genera known to include EAMs, such as Geobacter and Desulfuromonas, were abundantly present [127]. Similar results were also obtained in another study that examined MFCs operated with three different substrates (acetate, synthetic wastewater, and real domestic wastewater) and three different inocula (activated sludge, river sediment, and effluent from an MFC) [129]. They also analyzed the effects of substrate switches on MFC performance. The results showed that, regardless of inocula, acetate-fed MFCs generated higher cell voltages and power outputs than MFCs using the other substrates. In addition, it was found that cell voltages relatively dropped rapidly after the substrate was switched from acetate to complex organics. Analyses of microbial communities indicated that Geobacter was abundantly present in acetate-fed MFCs but decreased after the substrate switch [129]. In addition, anodes taken from paddy-field MFCs were used in laboratory MFCs fed with either acetate or glucose, and the anode microbial communities were subjected to metagenomic analyses [130]. It was found that acetate-fed MFCs generated more power than glucose-fed MFCs. In relation to this, metagenomics showed that G. sulfurreducens occurred only in anodes of acetate-fed MFCs, while other Geobacter, such as G. lovleyi and G. uraniireducens, occurred in anodes of glucose-fed MFCs. Given the results of a comparative study on current generation in pure-culture ECs inoculated with different species of Geobacter [112], it is suggested that the occurrence of G. sulfurreducens facilitates power generation in MFCs, for which acetate must be supplied as the anode substrate.

Results of past studies on MECs have been comparatively analyzed to uncover the effects of substrates on MEC performance, such as current generation and hydrogen production [128,131]. It is shown that MEC performance is largely dependent on substrates; namely, MECs fed with acetate generally show high performance compared to those of other MECs, while differences in performance are not apparent in MECs fed with other substrates, including fermentable organics, non-fermentable organics proteins, fermentation effluents, and domestic wastewater [128]. In one example, MECs supplied with a synthetic medium containing acetate as the major organic component generated more current than MECs supplied with a real-wastewater medium [132]. In both MECs, current generation was correlated with the enrichment of Geobacteraceae in anode biofilms; within the family, G. sulfurreducens was abundant in the acetate-medium MEC, while another Geobacter sp. occurred in the wastewater-medium MEC.

Collectively, based on the results of MFC and MEC studies, high electrochemical performance in MET processes is associated with the occurrence of G. sulfurreducens in anode biofilms, for which acetate must be supplied to anodes as the major substrate. This idea aligns with the results of pure-culture studies, in which G. sulfurreducens strains generated higher current densities compared to other EAMs (Table 1). However, this situation is not favorable for MET processes aimed at waste-to-energy conversion, since acetate is seldom the major constituent in biomass waste and wastewater.

4. BA for Improving MET Processes

It is widely accepted that the performance of MET processes treating complex organic matter, including biomass waste and/or wastewater, must be substantially increased for their practical application. This issue must be addressed before MET processes can be put into practice. To this end, studies have been conducted to develop methodologies to boost current generation in MET processes treating complex organic matter. One idea would be “pre-enrichment”; in a previous study, researchers conducted pre-enrichment of EAMs, including G. sulfurreducens, in MFCs either from activated sludge, river sediment, or MFC effluent by feeding acetate as the substrate, followed by trials of electricity generation from domestic wastewater [129]. However, it was shown that after the substrate was changed from acetate to wastewater, voltage and power outputs from these MFCs largely dropped, and G. sulfurreducens in anode biofilms decreased. It is likely that G. sulfurreducens occurs in anode biofilms from a variety of microbial sources only under strong selection pressures of acetate (the electron donor) and electrodes (the electron acceptors) [133]. Additionally, many studies have used EAM (G. sulfurreducens)-enriched microbiomes from pre-operated MET processes as inocula for the start-up of new MET processes. However, process performance did not increase sufficiently after biomass waste and/or wastewater were supplied as substrates [128].

BA with an EAM is another possible strategy for improving the performance of MET processes. In this trial, an EAM isolated in the laboratory is grown in pure culture, followed by its inoculation at an appropriate time point into a MET process. A merit of this trial is that engineers are able to steadily prepare inocula with expected catabolic and electrochemical activities; this would be difficult when naturally occurring microbiomes are used as inocula. In BA trials, one must carefully select EAMs to be used as inocula for BA of MET processes. Below, we review past trials of BA for MET processes and discuss their efficacy.

Table 2 summarizes past BA trials for MET processes, in which electrochemical performances were quantitatively compared between non-bioaugmented and bioaugmented reactors. Several BA trials that used mixed cultures as inocula are also included. Among these, one study comparatively examined electricity generation in MFCs augmented with an EAM (P. aeruginosa) and with a non-EAM (Escherichia coli) [134]. It was shown that MFCs with P. aeruginosa generated higher electric outputs than those of E. coli-augmented MFCs. In addition, higher redox currents and lower Tafel-slopes were observed in MFCs augmented with P. aeruginosa compared to the other group, indicating that bioaugmentation minimized electron-transfer losses. The authors suggested that electron transfer was enhanced by the synergistic interaction of the mixed consortia with the augmented EAM. In another study [135], MFCs were inoculated with an EAM, P. aeruginosa IIT BT SS1, which had been isolated from an anode of an MFC fed with dark fermentation spent media, and the effects of bioaugmentation were investigated. The study suggested that bioaugmentation with the Pseudomonas isolate effectively decreased the start-up time of MFC; however, after start-up, power output was not largely enhanced. It is assumed that ecological factors, such as microbial competition and environmental adaptability, may have caused the extinction of the BA agent. BA has also been attempted in constructed wetland MFCs, with positive effects reported [136].

BA trials have also been conducted for MECs (Table 2). For instance, G. sulfurreducens strain YM18 was introduced into the anode chambers of double-chamber MECs inoculated with paddy soil and fed with starch, and the effects of the BA agent on current generation and hydrogen production from starch were examined [22]. Strain YM18 is an EAM that was isolated from river sediment and reported to exhibit high electrochemical activity [83]. The results showed that MECs augmented with YM18 generated threefold greater current during a one-month operation and produced sixfold greater amounts of hydrogen than non-bioaugmented controls. Quantitative PCR and metabarcoding analyses confirmed the successful colonization of anode surfaces with YM18; these analyses detected that Geobacter shared 1 to 4% of the total bacteria. Although these results suggest the utility of bioaugmentation with YM18 for enhancing the performance of starch-fed MECs, the performance of YM18-inoculated MECs gradually decreased during a one-month operation [22]. The study therefore suggested the need to develop methods for maintaining BA agents stably in MET processes.

Table 2. BA trials for MET processes, in which electrochemical performances were quantitatively compared between non-bioaugmented and bioaugmented reactors.

Reactor Type 1	Substrate	BA Agent	Electrochemical Performance 2		Description	Reference
			Non-Bioaugmented	Bioaugmented
MFC
DC	LB medium	Pseudomonas chlororaphis PCL1391	49 μA	83 μA	Positive effects were observed only when the BA agent was contained in a slow-release tube. No power measuremet.	[137]
SC	Glucose	P. aeruginosa (P), Escherichia coli (E)	45 mW m−2	P: 230 mW m−2 E: 58 mW m−2	BAs with EAB (P) and non-EAB (E) were compared in relatively short time periods (~60 h).	[134]
SC	Fermented molasses	Pseudomonas aeruginosa IIT BT SS1	255 mW m−2	281 mW m−2	BA shortened start-up time of MFC.	[135]
CW	Textile dye wastewater	Electroactive bacterial community DC5	177 mW m−2	198 mW m−2	BA improved dye degradation and COD removal.	[138]
SC	Wastewater	Rumen microbes	634 mW·m−2	825 mW·m−2	The BA agent improved cathode performance.	[139]
CW	Root exudates	G. sulfurreducens DL-1	2 mW·m−2	Day 5: 91 mW·m−2 Day 30: 18 mW·m−2	The BA agent was injected to carbon-fibers anodes in sediment using sterile syringes.	[136]
CW	Haloxyfop-P-methyl (HM) and soil organics	Myrothecium verrucaria(fungus)	5.7 mW·m−2	11.7 mW·m−2	BA slightly improved HM degradation.	[140]
SC	Food waste	Geobacter sulfurreducens 60473	168 mW m−2	1760 mW m−2	Positive effects were observed even one month after BA, when Geobacter shared 40% of anode bacteria.	[141]
MEC
CW	Sediment organics	Acidimicrobiaceae sp. A6	3 mA m−2	7 mA m−2	Current increased after constructed wetland microcosm MECs were augmented with A6.	[142]
SC	Acetate	G. sulfurreducens PCA	0.012 mA cm−2	0.064 mA cm−2	Positive effects were observed only when the BA agent was encapsulated around anodes.	[143]
DC	Starch and yeast extract	G. sulfurreducens YM18	3.6 mA	13.3 mA	BA MEC generated sixfold more hydrogen than non-bioaugmented MEC.	[22]
SC	Acetate	G. sulfurreducens PCA	0.03 mA cm−2	0.27 mA cm−2	An anode was put in a dialysis bag, in which the BA agent was introduced along with graphite.	[144]
SC	Starch and yeast extract	G. sulfurreducens PCA, YM18, 60473	0.020 mA cm−2	PCA: 0.014 mA cm−2 YM18: 1.0 mA cm−2 60473: 1.6 mA cm−2	BA agents largely affect the effectiveness of BA. Hydrophobic cells of 60473 facilitate the colonization on anodes.	[23]

¹ Abbreviations are; DC, double chamber; SC, single chamber; CW, constructed wetland. ² Attention should be paid to direct comparisons of data in different studies, since these values were obtained under different experimental settings.

Table 2. BA trials for MET processes, in which electrochemical performances were quantitatively compared between non-bioaugmented and bioaugmented reactors.

Reactor Type 1	Substrate	BA Agent	Electrochemical Performance 2		Description	Reference
			Non-Bioaugmented	Bioaugmented
MFC
DC	LB medium	Pseudomonas chlororaphis PCL1391	49 μA	83 μA	Positive effects were observed only when the BA agent was contained in a slow-release tube. No power measuremet.	[137]
SC	Glucose	P. aeruginosa (P), Escherichia coli (E)	45 mW m−2	P: 230 mW m−2 E: 58 mW m−2	BAs with EAB (P) and non-EAB (E) were compared in relatively short time periods (~60 h).	[134]
SC	Fermented molasses	Pseudomonas aeruginosa IIT BT SS1	255 mW m−2	281 mW m−2	BA shortened start-up time of MFC.	[135]
CW	Textile dye wastewater	Electroactive bacterial community DC5	177 mW m−2	198 mW m−2	BA improved dye degradation and COD removal.	[138]
SC	Wastewater	Rumen microbes	634 mW·m−2	825 mW·m−2	The BA agent improved cathode performance.	[139]
CW	Root exudates	G. sulfurreducens DL-1	2 mW·m−2	Day 5: 91 mW·m−2 Day 30: 18 mW·m−2	The BA agent was injected to carbon-fibers anodes in sediment using sterile syringes.	[136]
CW	Haloxyfop-P-methyl (HM) and soil organics	Myrothecium verrucaria(fungus)	5.7 mW·m−2	11.7 mW·m−2	BA slightly improved HM degradation.	[140]
SC	Food waste	Geobacter sulfurreducens 60473	168 mW m−2	1760 mW m−2	Positive effects were observed even one month after BA, when Geobacter shared 40% of anode bacteria.	[141]
MEC
CW	Sediment organics	Acidimicrobiaceae sp. A6	3 mA m−2	7 mA m−2	Current increased after constructed wetland microcosm MECs were augmented with A6.	[142]
SC	Acetate	G. sulfurreducens PCA	0.012 mA cm−2	0.064 mA cm−2	Positive effects were observed only when the BA agent was encapsulated around anodes.	[143]
DC	Starch and yeast extract	G. sulfurreducens YM18	3.6 mA	13.3 mA	BA MEC generated sixfold more hydrogen than non-bioaugmented MEC.	[22]
SC	Acetate	G. sulfurreducens PCA	0.03 mA cm−2	0.27 mA cm−2	An anode was put in a dialysis bag, in which the BA agent was introduced along with graphite.	[144]
SC	Starch and yeast extract	G. sulfurreducens PCA, YM18, 60473	0.020 mA cm−2	PCA: 0.014 mA cm−2 YM18: 1.0 mA cm−2 60473: 1.6 mA cm−2	BA agents largely affect the effectiveness of BA. Hydrophobic cells of 60473 facilitate the colonization on anodes.	[23]

¹ Abbreviations are; DC, double chamber; SC, single chamber; CW, constructed wetland. ² Attention should be paid to direct comparisons of data in different studies, since these values were obtained under different experimental settings.

In order for BA agents to stably work in MECs, researchers have developed methods to contain them in electrode-associated three-dimensional (3D) capsules [143,144]. For instance, electrode-associated 3D capsules containing G. sulfurreducens strain PCA were used for current generation in MECs [143]. When MECs were fed with artificial wastewater, MECs with the 3D capsules generated twice as much current and three times as much hydrogen as those without the capsules. The authors suggested that encapsulated bacterial anode could help overcome the problem of contamination by non-EAMs, as well as shear and friction forces in wastewater plants. A similar idea has also been examined previously [137], where bioaugmentation of mixed-culture MFCs with slow-release tubes containing P. aeruginosa not only doubled the current but also maintained the effect for longer periods.

Harada et al. [23] also attempted BA to improve the performance of MECs. Unlike other studies on BA trials for MET processes, in which researchers used existing isolates of EAMs as BA agents (Table 2), Harada et al. isolated novel EAMs for the purpose of applying them to BA trials [23]. In that work, researchers hypothesized that EAMs that are able to stiffly adhere to electrodes would be effective BA agents that could substantially and stably improve MEC performance. To address this idea, the authors isolated a novel EAM, G. sulfurreducens strain 60473, from microbes adhering to an anode of a sediment MFC. The anode had been intensely washed under a flow of sterile water to remove microbes that had loosely adhered to the anode. Physiological characterizations using hydrocarbon-fractionation assays showed that the cell surface of strain 60473 is more hydrophobic than those of other G. sulfurreducens strains, including PCA and YM18, resulting in high affinity to graphite. The utility of strain 60473 as a BA agent was demonstrated in experiments where starch-fed MECs were augmented with either 60473, YM18, or PCA. It was shown that bioaugmentation with 60473 substantially and stably (over a month) improved current and hydrogen production compared to non-BA controls, while no significant effects were observed with PCA. BA with YM18 also exhibited positive effects, although its effects were smaller than those of 60473. It was also found that Geobacter accounted for a large portion (more than 40%) of the total bacteria in anode biofilms in 60473-augmented MECs one month after initiating the operation, while the relative abundance of Geobacter was much lower in YM18-augmented (approx. 10%) and PCA-augmented (less than 1%) MECs. Based on these results, the authors suggest that the ability of an EAM to tightly adhere to electrodes is important for successful BA in improving MEC performance.

The utility of strain 60473 as a BA agent has also been demonstrated in food waste-fed MFCs inoculated with digester effluent [141]. MFC performance, including power output, COD removal, and Coulombic efficiency, was largely increased in 60473-augmented MFCs compared to non-BA control MFCs. In these 60473-augmented MFCs, maximum power densities per anode area and per reactor volume reached 1760 mW m⁻² and 32 W m⁻³, respectively. These values are much higher than those reported previously for MFCs treating food waste [145,146] and are equivalent to or higher than those reported for MFCs fed with acetate as the sole substrate [133]. Analyses of microbes in anode biofilms indicate that after one-month of operation, Geobacter comprised approximately 40% of the total bacteria in 60473-augmented MFCs, whereas its abundance was approximately 0.2% in non-BA control MFCs. It is therefore suggested that MFCs treating biomass waste, including food waste, can generate similar levels of power to those of acetate-fed MFCs, provided that sufficient amounts of G. sulfurreducens are present in the anode biofilms. In addition, the findings that BA with G. sulfurreducens 60473 improves COD removal in starch-fed MECs [23] and food waste-fed MFCs [141] suggest that current generation from fermentation products, such as acetate, is the rate-limiting step in the conversion of fermentable substrates into electricity in MET processes. This step should therefore be the target of BA for improving the performance of entire MET systems.

5. Factors That Need to Be Considered for Successful BA

Based on comparative analyses of the results of past BA trials for MET processes (Table 2), factors that should be considered for successful BA are discussed here. We aim to provide researchers and engineers with information on effective BA strategies that facilitate the practical application of MET processes for the treatment of biomass waste and/or wastewater.

First, the electrochemical activities of BA agents, such as those expressed as current densities per anode area (Table 1), must be greater than those attainable by naturally occurring anode microbiomes (Table 2). Among the EAMs isolated and characterized so far, the activities of EAMs affiliated with G. sulfurreducens are substantially higher than those of other EAMs (Table 1). It is therefore reasonable to consider that G. sulfurreducens strains should be used as BA agents, even though they are able to utilize limited organics, such as acetate, as electron donors.

Second, in order for G. sulfurreducens strains to efficiently and stably generate currents, they must colonize anodes and form biofilms in the presence of other microbes [147,148,149]. It has been widely known that G. sulfurreducens is able to form conductive biofilms on electrodes in pure-culture ECs [148]. On the other hand, it has also been shown that structures of biofilms formed by different G. sulfurreducens strains, in particular those in the initial phase of current generation, are substantially different [23]. The study also showed that different abilities to form biofilms are attributable to differences in cell-surface hydrophobicity among these strains [23], thereby suggesting that G. sulfurreducens strains with hydrophobic cells may be potent BA agents capable of forming stable biofilms on electrodes. It is noteworthy that a methodology for isolating G. sulfurreducens with hydrophobic cell surfaces has been described [23].

Third, since G. sulfurreducens strains can utilize only limited organics—such as acetate—as electron donors, they must have abilities to interact with other microbes, in particular fermentative microbes that degrade complex organic matter, such as starch and proteins, and excrete fermentation products, such as acetate, for successful BA of MET processes treating biomass waste and/or wastewater. Organic substrates must be efficiently transferred from fermentative microbes to EAMs for efficient current generation. Since studies have shown that substrates for mixed-culture MFCs affect the species of Geobacter present in anode biofilms [130], it is conceivable that different Geobacter strains may have different abilities to interact with fermentative microbes. However, studies are scarce on this subject. Further studies are therefore necessary for understanding the ecological interactions of Geobacter strains with fermentative microbes in mixed-culture MET processes.

6. Perspectives

As described above, massive work has been performed to gain knowledge on EAMs and develop MET processes that exploit them. In addition, efforts have been made to use EAMs through BA to improve MET processes (Table 2). Although several studies have shown that BA with EAMs can substantially improve MET processes [23,141], the performance of MET processes reported in these studies are still insufficient for their practical applications. To cite an instance, a study on BA reported 32 W m⁻³ for MFCs fed with food waste and augmented with G. sulfurreducens strain 60473 [141]. Based on the reactor structures and performance data reported in the work, the energy-production rate of this MFC is calculated to be equivalent to a methane-production rate of ~0.1 L L⁻¹ D⁻¹, a value substantially lower (1/10 or less) than those reported for ADs treating food waste [150]. Although the reactor structure and electrode material were not optimized in that work [141], the data suggest that more effective BA schemes should be developed for constructing MET processes whose energy-production rates are comparable to or more than those of anaerobic digesters. To this end, EAMs that are more active than those known to date would be necessary. More studies should therefore be done to isolate novel and active EAMs from natural environments and/or to breed EAMs that have been isolated in the laboratory and identified to be electrochemically active. Although special care should be taken in the use of genetically engineered EAMs for their containment, genetic engineering would be a possible strategy for breeding EAMs (41). In addition, studies should be conducted to combine the latest methods for improving the performance of MET devices—including optimized reactor configurations, electrochemically active electrodes, and effective BA—and to evaluate the process performances.

For the practical application of BA with EAMs, in addition to its effectiveness in improving the performance of MET processes, we also need to consider if it is economically feasible. It is reasonable to predict that BA needs considerable costs; however, it would be possible to develop cost-effective BA by optimizing operational schemes, such as the interval of BA and the amounts of EAM cells to be inoculated for maintaining MET performance. The costs of BA may be dependent on what BA agents are used; it is recommended that an EAM that is able to actively grow after introduction into MET processes and stably form a major population in the electrode biofilm should be used, thereby reducing the amountsof cells needed. We expect that, in combination with the introduction of advanced electrodes and reactors, BA will contribute to the development of MET processes with sufficient performances for practical application to energy generation from biomass waste and/or wastewater.