Electrochemical oxidation of FOG with WAS
In this regard, pH was an important parameter to be controlled for a proper MEC performance and was highly effective for the electrochemical oxidation of FOG with WAS, resulting in significant energy harvest from the treatment of organics. Results showed pH 7.2 when WAS with FOG (100 + 11%) was used. Results also revealed that the MEC pH values further decreased to less than 6.5 more than 11% FOG. Microbial electrolysis cell (MEC) are a type of bioelectrochemical system that showed stability and was evaluated based on the bio-electrochemical characteristics, substrate contents, and microbial biomass that contributed to bio-electricity generation. The voltage produced was examined for current density, COD removal, and number of viable cell growth, which increased tremendously as shown in Table 1. Our findings showed that the majority of solids were organics, VS 15.7 ± 7.64 g/L TVS. The results showed that the VFA was enhance remarkabely with FOG added and 2.94 ± 0.19 g/L 11% FOG (Table 1). The results indicate that the redox potential with FOG concentrations can improve microbial growth, resulting in increased electron transfer capacity and conductivity [38]. The possibility of a good conductive environment and electroactive microorganisms had been rooted in higher quantities of electron donor organic at 11% FOG. The results also indicated that WAS + FOG could improve the electrochemical performance, metabolites 7.32 ± 0.21 moles/mL, viable of cells 5.96 ± 0.73 CFU/mL and 17.91 ± 7.23 mA electricity generation by supplementation of 11% WAS-FOG (Table 1).
Anode biofilm EPS characteristic
Displayed in Fig. 1 are the characteristics of extracellular polymeric substances (EPS) in anode Fig. 2. The dense EPS encases microbial biomass interacts with FOG, reducing nutrients and providing a source of carbon and energy for microbial metabolism [38]. In this respect, high concentrations of FOG or organics may lead to changes in microbial cell morphology, volume, and metabolic activity, whereas the availability of excessive substrate could result in cellular stress, alter growth kinetics, and reduce electrochemical performance [27, 30]. The composition and diversity of the microbial community in the biofilm may shift in response to changes in substrate concentration. Certain species may outcompete others under high substrate loads, leading to alterations in electrochemical activity [52]. Previous studies indicated that the WAS with FOG with optimum organic substrates may alter the composition and influencing its ability to support microbial activity and electron transfer [35, 22]. The characteristics of EPS having significant protein, polysaccharide, lipids, and iron in the anode biofilm of bio-electrochemical play a crucial role in modulating extracellular transfer to make current density and microbial structure, particularly in the presence of optimum concentrations of WAS-FOG.
EPS is composed of various organic molecules, including proteins (highest percentage 3.4 mg/gVS), polysaccharides (1.7 mg/gVS), lipids (1.02 mg/gVS), and iron (0.18 mg/gVS), secreted by microorganisms within the biofilm matrix (Fig. 3a). EPS forms a complex three-dimensional matrix helps for microbial cells growth, which could provide structural support, adhesion to surfaces, and create microenvironments for microbial growth and metabolism of organics [7]. EPS-specific compositions such as conductive protein and redox-active molecules electron accepting capacity (EAC) of 0.91 mmol e-1g-1, extracellular electron transfer (EET) of 3.5 mmol e-1g-1, electron exchange capacity (EEC) of 1.9 mmol e-1 g-1, can enhance electron exchange-transfer within the biofilm, and facilitate efficient electron transfer to the electrode surface, leading to increased current density in MEC [37, 35]. At 11% of WAS-FOG, EPS supports microbial adhesion to surfaces and facilitates the formation of multicellular aggregates within the biofilm, affecting microbial adhesion, biofilm architecture, and spatial distribution of microorganisms due to high WAS-FOG concentration (SM2). In conclusion, the role of EPS in modulating current density and microbial structure in MEC with WAS-FOG concentrations is essential for optimizing system performance and designing effective dosage strategies for FOG treatment and energy recovery. Additionally, electron transport across the anodic biofilm thickness of 69.5 µm in MEC is facilitated by the conductivity was 73.5 mS/cm matrix of EPS, cell volume number 19.93 and NADH/NAD+ ratio 28.6, specialized structures like pili, and periodic polarization reduction-oxidation of organics (Fig. 3b). These factors work together to create efficient pathways from microbial metabolism to channel electron transfer on the electrode surface, enabling the generation of bio-electricity. Optimizing these mechanisms can enhance the performance and scalability of MEC for various applications, including high organic wastewater treatment, and energy recovery [2, 7].
Bio electrochemical performance
The impact of waste-activated sludge (WAS) and fat, oil, and grease (FOG) on electrolysis for metabolite yields, including acetate, butyrate, ethanol, lactate, and total metabolites, can vary depending on various factors such as FOG dosage, microbial community composition, and anode charge capacity [35]. Metabolites produce 18.6 mmol mg-1 DW compared to the initial stage of 1.56 at the electrode area, influenced by the combined metabolic activities of microbial communities degrading FOG (Fig. 3a). Monitoring the total metabolite concentrations provides insights into the overall microbial activity and substrate utilization [35]. The performance of MEC was evaluated by characterizing electron transfer capacity (ETC) and current density was significantly changed after adding FOG. In this context, microbial electrolysis is associated with NADH/NAD+ ratio and the volume of cells is linked to redox-mediated [42]. Therefore, we confirmed redox homeostasis via electroactive microbes in 11% FOG under EET conditions estimating intercellular NADH/NAD + ratio, viable cells, and maximum current density (Fig. 3b). Supplemented with WAS-FOG, EET activity was confirmed in a bioelectrochemical reactor, the current density was observed with 11% FOG (215.7 µA/cm2), higher than that 13% FOG (117.3 µA/cm2), and 9% FOG (178.5 µA/cm2) (Fig. 3a). The microbial electrolysis was positively influenced by organic oxidation with the exoelectrogenic anode side and electrons released [48, 21]. The high concentrations of organic compounds like WAS-FOG can exert toxic effects on microorganisms, including electroactive bacteria, and could disrupt the cellular redox balance, affecting the NADH/NAD + ratio and impairing electron transfer processes [26, 14]. This toxicity may inhibit microbial growth and microbial metabolic activity, thereby reducing the overall electrochemical performance. While organic substrates like WAS-FOG can serve as electron donors for electroactive bacteria, excessively high concentrations of FOG 13% and low levels of 1.5% also may overwhelm the microbial community's capacity for substrate utilization. At good concentrations of WAS-FOG 11%, electroactive bacteria may have better access to the available WAS-FOG, leading to higher metabolic activity, and the EET 17.49 mM and, current production was 783 mV (Fig. 4a).
Bio-electrochemical current production
Results from this investigation indicate that using WAS-FOG for electroactive microorganism growth could improve the current production, similar to results obtained by other researchers [39, 11]. The optimum concentrations of FOG provide an abundance of organic substrates for microbial metabolism. This can lead to higher rates of substrate utilization by electroactive microbes at the anode. With ample organic substrates available, electroactive microbes can produce more electrons through metabolic processes like the oxidation of organic matter [3, 8]. This can result in higher electron transfer rates to the anode, and increased current generation at optimum substrate concentrations may promote the formation of dense and robust biofilms at the anode surface (Fig. 3b). These biofilms can support the growth and activity of electroactive microbes, enhancing electroactive electron transfer (EET) efficiency of 11% FOG present time variation trends (Fig. 4a), indicating good performance at long-term operation. The current density of 215.7 µA/cm2 in 11% FOG and 178.5 µA/cm2 was significantly higher than that of 13% FOG 117.3 µA/cm2 and 7% FOG 139.6 µA/cm2 (Fig. 4b). The good amount of organic as 11% FOG, may promote the formation of dense and robust biofilms at the anode surface and biofilms can support the growth and activity of electroactive microbes, enhancing electron transfer efficiency. However, excessively high concentrations of FOG such as 13% may lead to changes in pH and accumulation of toxic byproducts, which can inhibit metabolite production, and microbial activity and impair electron transfer processes, ultimately limiting current production [50, 24].
Different microbial species in the electrolysis system may metabolize WAS and FOG substrates differently, leading to variations in metabolite yields (Fig. 3a). In this respect, some microorganisms may preferentially produce certain metabolites over others based on their metabolic pathways and substrate preferences to improve the MEC performance [15, 28]. The electrolysis of FOG can be used to produce current and power density with very high efficiency at remarkable bioelctricity production (Fig. 4b, 5a). Cyclic voltammetry (CV) efficiency curves showed the redox capacity and electrochemical performance of MEC (Fig. 5b). As can be seen in Fig. 5b, performing significant long-term stable operation to achieve higher current densities and CV efficiencies may result in increased current production. While lower energy consumption could improve overall process efficiency, the WAS and FOG might be significant specific qualities. We expect that our research will enhance the bio-electricity consideration of energy conservation in electroactive biofilms by harnessing the EET capabilities of Geobacter bacteria. Therefore, it may be possible to electronically modulate the biochemical pathways involved in flavor and texture development through EET-mediated electron transfer for bio-energy processing [15, 22].
Results from the present study agree with those obtained from previous investigations, where FOG, as a bio-energetic component balance between energy intake and utilization for microbial electrolysis, will be key to generating electricity [29, 32, 41]. By controlling the electrochemical conditions such as potential reduction-oxidation, EET, current density, and substrate composition of WAS-FOG, it may be possible to influence the types and concentrations of flavor compounds and textural precursors generated during electrolysis [35]. Moreover, for optimum WAS-FOG, we found that an increase in intercellular electron acceptor causes an enhanced intercellular NADH/NAD + ratio, mean metabolites increased, reduction of chemical oxygen demands, and substitute protein helping for EET mechanism for energy conversion at the anode side [7, 34]. Similarly, by controlling the electrochemical conditions of redox potential, current density, and substrate composition, it may be possible to influence the types and concentrations of flavor compounds and textural precursors generated during electrolysis to alleviate extracellular redox balance at anode areas [2, 30].
Biofilm morphology analysis electron transportable
A biofilm formation, cell surface interactions between the electroactive biofilm with the electrode, and also play a positive role in microbial electron transfer to the anode surface [35]. Biofilm is often encased in a polymeric matrix that secretes itself, allowing it to cling to one another and attach to anode surfaces [16]. Within MECs, microorganisms initially adhere to the electrode in the form of a micro-colony, eventually developing into a fully formed biofilm [6]. In previous studies, the biofilm thickness of 50–100 µm and the spongy structure of the biofilm impact EET processes and bio-electricity production. Data from Fig. 2 show that increasing conductivity of 73.5 mScm2 at 11% FOG and developing thicker biofilms of 69.5 µm provide a larger surface area for microbial attachment and larger electron transfer to the electrode. However, decreasing biofilm thickness and conductivity may create diffusion limitations for substrates and electron acceptors, reducing overall metabolic activity and electrical output at higher and lower concentrations of FOG. Furthermore, increasing the surface of electrodes with biofilm thickness by controlling conditions, the supplement of nutrients, and microbial community composition is essential for maximizing EET efficiency and bio-electricity generation in MEC [36, 40]. The mechanism of electron transport over the anodic biofilm in MEC was observed with a matrix of biofilm including conductive pili nanowires and periodic polarization factors contributing to electron transport.
Figure 2, a well-developed 0.5-in-length pili electroactive biofilm produces a conductive matrix, which extends from the cell-cell membrane and facilitates electron transfer over longer range distances and time-efficient electron transfer. Electrically conductive pili provide an electronic network for electron transport between microbial cells and the electrode, bypassing the bulk resistance of the biofilm matrix and improving electron transfer efficiency. Periodic polarization techniques can be used to modulate the density and conductivity of pili in the biofilm, enhancing electron transport rates 17.49 mV, current density 215.7 mA/cm2 (Fig. 4a), thus increasing power density to 783 mV, more than 25-fold at 11% FOG (Fig. 4b). More than 200% conductivity increases were observed in WAS-FOG-modified pili strains compared with findings by other researchers [38, 12]. In previous studies, periodic polarization involves applying an intermittent electrical potential to the electrode surface, which can induce changes in microbial metabolism, biofilm thickness structure, and electron transfer mechanisms for energy recovery [31, 12]. By periodically switching between 5 to 11% of FOG, organic oxidizing and reducing conditions, periodic polarization can stimulate the current production increment from 218–783 mV and drastically decrease 457 mV at 13% FOG (Fig. 4b), due to oxidative stress generation byproduct [35] and the activity of electroactive microorganisms, promoting the formation of conductive structures like pili (Fig. 2) and enhancing electron transport for energy efficiency [53].
Modulating of electroactive biofilm, the density and distribution of pili by periodic polarization can enhance electron transport across the biofilm and improve the efficiency of bio-electricity generation in MEC [12, 47, 48]. The Geobacter strain, expressing the highest amount of growth of conductive pili with a high number of cell volume. Bacteria cell with conductive pili is capable of extracellular electron transfer (EET) in process. As for WAS-FOG-rich strains, pili-abundant regions were observed with dominant conductivity increases over that of the background substratum (Fig. 2). The displayed power density was increases of 3540 mW/cm− 2 respectively (Fig. 4a), which was 200% greater than the current density by the FOG at 5%. This study was supported by pre-existing literature, where pili moderations increase WAS-FOG concentration prompted conductivity increases of 200–700%. Strain pili with 10.0 nm cross-sectional height and in length several microns were in all specimens (Fig. 2). Furthermore, observations overview the experimental, we found to detect a significant change in pili height, diameter and length between WAS-FOG as a potential nutrient compared to the control experiment.
Mechanism of electron transfer for bio-electricity
An electrochemical technique using electrolysis of FOG can be used to produce current (Fig. 5a) and cyclic voltammetry can be used to clarify the anode's electron transfer mechanism and catalytic effectiveness in redox processes [32, 25]. However, the intense redox peak for MEC operation was present at a higher current (1.5 mA) when the voltage applied reached 0.0002 V as shown in Fig. 5b. With CV analysis, a similar tendency was also detected which unequivocally demonstrates that the type of substrate the anolyte uses affects its capacity to generate bioelectricity [32, 25]. During WAS-FOG degradation pathways, redox reactions occur within microbial cells, resulting in the co-enzyme NADH-NAD+ working as intercellular reduction-oxidation capacity and an electron carrier where it carries electrons released during metabolic reactions and transfers them to electron acceptors [13, 11]. The microbial cell-to-cell, electrostatic potential gradients are established due to the redox reactions and the presence of charged molecules. Electrostatic potential gradients across the cell membrane drive the movement of electrons through specific transport channels, facilitating electron transfer from electron carriers to external electron acceptors [18, 49]. The act of electrostatic interactions between the anode surface and biofilm pili facilitates electron transfer, thus enabling the conversion of microbial metabolic activity into electric current (Fig. 4a,b,5a). The microbial electrolysis extracellular electron transfer (EET) mechanisms that play a crucial role in bio-electricity generation were analyzed in the anode and cathode of MEC. The present study, explore the opportunity to enhance the current production at 11% FOG electrolysis. The incorporation of WAS and FOG concentration of between 5–11% FOG considerably improved electrolysis. The highest current production was observed at 11% FOG, the current production trend 427˃ 458 ˃ 546 ˃788˂635 at different FOG ratios. The concentrations of extracellular electron acceptors, the concentrations of co-enzymes NAD+/NADH ratio, types of nutrient substrates enhanced microbial electrolysis and biofilm thickness influence EET processes and overall electrical output at the anode; these factors interact in the context of yields of metabolites and current density generation (Table 1). The mechanism involved for generation current, special attention on microbial species, enzymes NAD+/NADH ratio, and nutrient substrate play crucial pars in cellular metabolism oxidize organic substrate, producing electrons and protons as metabolites (Table 1). Electroactive microbes utilize EET to transfer electrons from intracellular NADH to extracellular acceptor anode. Table 1 shows that the flow of electrons from the microbial metabolism to the anode creates a current that could be measured as current density, which was achieved 3540 mW m− 2 compared to previous studies [20, 13, 11, 19].
Published work showed that FOG is a good extracellular electron acceptor because under electrolysis conditions it has significant levels of heme contents for EAB, affects electron production and current generation, and accepts electrons from microbial metabolism [2]. Electroactive biofilm transfers electrons from intracellular redox reactions to extracellular electron acceptors, a process that can occur through direct contact between the microbial cell and the electron acceptor conductive pili in the extracellular environment (Fig. 5a). The NADH-NAD+ ratio was observed as a key indicator of the cellular redox state; a high NADH-NAD+ ratio promotes microbial cells increase, which might increase cell volume (Fig. 2) and extracellular electron transfer (Fig. 3b) from intracellular metabolic pathways to extracellular electron acceptors to cause more current density at anode side (Fig. 4a,b). This facilitates the release of electrons from microbial metabolism for bio-electricity via cellular metabolism to maintain or increase the NADH-NAD+ ratio, enhancing EET efficiency and electrical output (Table 2).
Microbial communities
From Fig. 6 shows the changes in microbial community size of relative abundance compared to lower and higher concentrations of WAS-FOG ratio. As shown in Fig. 6, bacterial communities at phylum stages genus comprised Geobacter S., Shewanella, E. coli, Lactococcus, and Geobacter biofilm. The notable result evident from Fig. 6, average segments of the dominant phyla were achieved Geobacter S., and Shewanella increased from 10.5% and 22.0–31.1%, 40.2%, and 73.4% after adding the FOG, consistently domination indicating a significant notable EAB effect of the FOG addition. At the surface of electrodes, microorganisms form biofilms, and biofilms have conducive pili which can transfer electrons from the microbial metabolism to the electrode. Our findings from this study showed that Geobacter sulfurriecens species' total microbial richness of 3.2 was influenced by the FOG addition of 11%, indicating their active role in FOG microbial electrolysis. Geobacter S. is highly efficient in metabolizing organic substrates, and producing electrons with oxidation-reduction catalyzed by acetate dehydrogenase NADH-dependent reactions. The Geobacter S. electrochemically active metabolism is optimized for efficiently producing reduced equivalents NADH that can be utilized for EET, enabling efficient electron flow to the electrode surface (Table 2) [19]. Additionally, Geobacter S. strains have been reported to produce power densities as high as 3,800 mW m− 2 (Fig. 4a,b,5a), which is comparable to power production by E. Coli or Shewanella strains [19, 2]