Bio-Power Generation in Microbial Fuel Cell with Vermicompost Using Eisenia foetida

Abstract

This research emphasizes the effect of using Eisenia foetida in vermicompost for power generation in microbial fuel cells (MFCs). By accelerating the organic decomposition, the bioenergy generation is improved. A vermicompost-microbial fuel cell employing electrogenic microorganisms was used to convert chemical energy into electrical energy. In this work, substrates of black soil, tree bark, leaves, eggshells, and ground tomatoes were used. The vermicompost MFC has a copper cathode and a stainless steel anode. In this study, the performance of MFCs was evaluated using different numbers of Eisenia foetida specimens, with three specimens (MFC_W3), five specimens (MFC_W5), and seven specimens (MFC_W7). Our key findings show that by increasing the number of Eisenia foetida specimens does not bring higher power densities; as a result, the best power density was observed in MFC_W3 and MFC_W5 at the end of the fourth week, both presenting a total of five Eisenia foetida specimens with a power density of 192 mW m⁻². Therefore, optimal results were found when 330 g of substrate and five Eisenia foetida specimens were used to achieve a maximum current density of 900 mW m⁻² and a maximum power density of 192 mW m⁻². This type of microbial fuel cell can be considered as an alternative for power generation with a significantly reduced environmental impact, considering the use of organic waste. It can be considered a game-changer in waste management and bioenergy projects.

1. Introduction

The organic residues created from food waste, animal breeding, food industry, agro-industry, etc., are currently considered a high pollution source due to the greenhouse emissions generated when decomposed and the waste of water and energy used to produce them. In 2020, it was reported that 100 kg per person per year of food is wasted at the consumption stage in developed countries, and the food systems contribute between 21 and 37% of the world’s greenhouse gas emissions [1,2]. Although there are some strategies to reduce organic wastes, essential actions must be taken to recycle them, such as biofuel production, biodegradation of naphthalene with mixed microbial consortia [3], microbial biohydrogen production [4], extractive production of microbial oil using hydrophobic adsorbents [5], animal feed (when applicable), composting, and some other techniques, less common, such as algae production, fish production, etc. Also, there have been efforts to produce compostable polymers, but the results have not yet been practical [6].

Composting is one of the most practiced techniques for organic waste management. This process allows the recovery of nutrients from organic waste and restores them to the environment [7]. It is based on the decomposition of organic compounds by biochemical reactions that produce carbon dioxide, water, minerals, and stabilized organic matter [6]. Vermicomposting is a non-thermophilic, bio-oxidative process where worms and associated microbes transform matter [7]. According to the description of the vermicomposting process, it is possible to infer that bioenergy can be obtained simultaneously by using microbial fuel cells [8].

The benefits of using vermicompost can be covered in bioprocesses as a sustainable alternative for the management of poultry waste, not only in terms of nutrient recycling but also by providing a clean source of energy [9]; composting has been promoted as an important alternative for the treatment of organic waste [10]; the use of vermicomposting reduces methane emissions compared to natural composting [11]; the use of fertilizers derived from vermicompost, which is created from organic waste of animal or plant origin, has been proven to be an extremely effective method of enhancing soil quality. In [12], promoting sustainable development, composting is an important alternative. In [10], composting represents an effective and environmentally friendly method of recycling organic waste, whereby it is converted into stable and mature fertilizers. In [10], finally, the nutritional content can be improved by combining thermophilic biodegradation in rotating drum containers and vermicomposting based on Eisenia foetida [13].

Microbial fuel cells (MFCs) are electrochemical devices in which microorganisms catalyze organic substrate oxidation by metabolic processes to produce electrical energy [14,15,16,17,18,19,20]. Different microorganisms have been used to manage different waste sources and produce energy. Using microalgae in fuel cells is an alternative for biomass production and water treatment [21,22,23,24,25,26]. Besides microalgae microorganisms, several studies with aerobic and anaerobic microorganisms as electro-generators have been developed to produce bioenergy; for example, Segundo, R.-F. et al. [27] obtained energy from pepper wastes in a single-chamber microbial fuel cell using Rossellomorea marisflavi strain, obtaining a maximum power density of 154.142 mW cm⁻² and a maximum current density of 4.834 A cm⁻²; Kuo-Ti Chen (2020) [28] developed thermophilic microbial fuel cells (thermo-MFCs) equipped with a hydrophobic membrane electrode to remove and recover ammonia and water from leachate. Chen et al. [29] used a three-dimensional graphene (3D GR) as the anode of a miniaturized microbial fuel cell (mini-MFC) and used Shewanella bacterial culture to promote highly efficient extracellular electron transfer. This mini-MFC’s maximum output voltage and power density were 820 mV and 23.8 mW m⁻². Considering the microbial fuel cell applications, most cases contemplate room temperature operation, where mesophilic microorganisms would be the main electro-generators to produce bioenergy. The use of mesophilic microorganisms has been widely analyzed [30,31,32,33,34,35,36]; notably, some research highlights the use of Escherichia coli in microbial fuel cells. Lalitha Priya R. et al. [37] show the use of the Escherichia coli biofilm electrode in a dual-chamber fuel cell, which, in optimized operating conditions, provided 778 mV open-circuit potential, 3.47 mA m⁻² of current density, and 1660 mW m⁻² power density. Meanwhile, Othman et al. [38] evaluated a photo-stimulated bacterial consortium that showed promising results using electrogenic bacterial strains E. coli and E. cloacae as consortium at optimum conditions.

Using biological components promises moderate environmental impact [14,39]. However, implementing microbial fuel cells for power generation presents several challenges, such as improving performance, reducing cost, achieving automation, and offering an easy way to scale up [39,40]. An advantage of microbial fuel cells is greener energy, considering that the energy could come from waste; therefore, the power is obtained and achieves organic waste management. Most studies have focused on using wastewater as fuel [41,42,43,44]. However, as mentioned above, solid organic waste is increasing, and the soil’s microbial fuel cells have also attracted attention [45,46,47,48,49,50]. A good alternative is the vermicompost microbial fuel cells, though few works exist on the subject. In 2011, Karim et al. used three different wastes: spent tea, sugarcane, and coconut meat for the vermicompost; after the compost was obtained, they took out the worms and proved the electricity generation using metallic electrodes and a multimeter [51]. Nandy et al. [7] showed the use of membrane-MFCs with soil obtained from vermicomposting; they compared the results with soil obtained without using earthworms, and their results showed a maximum power density of 4 mW m⁻² and a maximum of 66% chemical oxygen demand (COD) removal when the vermicompost soil was used. Youn et al. [52] presented a system of several continuous chambers that allow obtaining energy from food waste. They put food waste in two chambers and made the vermicompost, then the organic matter passed to another chamber and fed the membrane-MFCs the results show an open circuit voltage of 0.75 V and a maximum power of 0.41 mW. Yukimoto et al. [53] used worms from the Megascolecidae family and compared the results of using and not using worms; the maximum power density obtained was 7.2 mWm⁻². Edwards and Arancon [54] and Hajam et al. [55] employed banana and watermelon peels, used Eudrilus eugeniae, and evaluated the use of Nafion ${\mathrm{MFC}}^{\circledR }$, clay, and cotton membranes. Sandy L. Calderon et al. [56] developed a double-compartment microbial fuel cell for electrical energy generation using Shewanella spp. strains and gold nanoparticles as an anode biocatalyst. The results demonstrated the formation of Shewanella spp. colonies on the electrode surface and the occurrence of electrochemical activity under aerobic and anaerobic conditions. A stabilized average power density of 281 mW cm⁻² was recorded as well as a maximum current density of 0.11 mA cm⁻² after 72 h of operation, and a coulombic efficiency of 65% under anaerobic conditions was obtained. The previous works of vermicompost microbial fuel cells have established a background for its optimization; in this work, we present vermicompost as an energy source in a membraneless-MFCs and compare the use of different numbers of Eisenia foetida specimens. Also, in this case, the soil with the Eisenia foetida specimens is used directly in the fuel cell, meaning the separation is unnecessary. The study shows the composition of the organic matter and the energy generated after four weeks of decomposing using Eisenia foetida, known as the Californian red worm. Therefore, the design without membrane and an exposed cathode, along with using Eisenia foetida, presents an alternative for improving the vermicomposting fuel cells.

2. Materials and Methods

2.1. Substrate Preparation

The substrate (330 g) was prepared with natural black soil, tree bark, and ground leaf (Nutrigarden). It was enriched with ground eggshell (1 g) and tomato (1 g) to feed the Eisenia foetida specimens. We used lombricompost gdl^® (Zeigen, Mexico City, Mexico) Californian red worm (Eisenia foetida) of 4 cm and 1 g. The feeding was constant each week, and the polarization curves (Section 2.4) were obtained after the feeding.

2.2. MFC Assemble

The research is based on the quantification of the effect of the number of Eisenia foetida species and its relationship with the electrical power density, for which a stainless steel electrode is chosen for the anode due to its low corrosion in atmospheres containing oxygen and copper is selected as the cathode for its electrical conductivity.

The exposed cathode was made from half of a type M copper pipe (1.9 cm). The metal sheet (7 × 6.5 cm) was polished with Fandeli sandpaper (grades 1500 and 2000) to achieve adequate micro finishing. Meanwhile, the anode was made of Scrmushuzi SS304 stainless steel mesh (7 × 6.5 cm), number 20. The structure was observed with an 8× magnified image obtained with a Zeigen stereoscope model DECO-TV (Zahner-Elektric Gmbh & Co. KG, Kronach, Germany; work distance 112 mm). The connection between both electrodes was made using Plusivo PVC-coated 22-gauge copper wire.

The camera for the cell consists of a plastic container (7 × 10 × 5.5 cm). The Eisenia foetida specimens were placed at the bottom, and a substrate layer (2.5 cm) was put over them. The anode was implemented and covered with another layer of substrate (1.5 cm), and finally, the cathode remained exposed to the air. After the cell assembly, potable water (15 mL) was added.

We assembled six fuel cells to evaluate the dependence between the number of Eisenia foetida specimens in the microbial fuel cells and the energy generated. Two fuel cells without any Eisenia foetida specimens were used as control ( ${\mathrm{MFC}}_{CT}$), two fuel cells with three Eisenia foetida specimens ( ${\mathrm{MFC}}_{W3}$), another two using five Eisenia foetida specimens ( ${\mathrm{MFC}}_{W5}$), and finally, another two fuel cells using seven Eisenia foetida specimens ( ${\mathrm{MFC}}_{W7}$).

The statistical behavior of the microbial cells was constructed; with the experimental results of the current density during the four weeks, the average was calculated; then, two deviations were added to this average, one to the right and one to the left for the average point.

2.3. Microbial Fuel Cell Performance

The performance of the MFCs was evaluated using polarization and power density curves. The polarization curves were obtained using a potentiostat/galvanostat Zennium XC and Thales XT software (2.09.24.2024). The voltage range was from open circuit potential to 0 V (scan rate = 1 mV/s). The performance was evaluated at the end of each week for four weeks. After that period, a stack of two ${\mathrm{MFC}}_{W3}$ connected in a series arrangement was used to power a light emission diode.

2.4. Substrate Analysis

2.4.1. Physicochemical Analysis

The substrate composition analysis was conducted weekly for four weeks. The sample was taken after the electrochemical measurements. The Walkley and Black method determined the percentage of organic matter and carbon. The content of nitrogen was determined by the Kjeldahl method. We used an Orion potentiometer (Thermo Fisher, Waltham, MA, USA) for the pH measurements; the dried sample (1 g) was ground in deionized water (25 mL) and stirred (10 min). The Eisenia foetida specimen was observed with a Zeigen stereoscope model DECO-TV (Zeigen, Mexico City, Mexico; work distance 112 mm) .

2.4.2. Microbiological Analysis

A microbiological analysis of each MFC was made after four weeks. The microbiological sample was obtained using a swab procedure in the anode. The study presents the count of mesophilic aerobes and the total coliforms. The isolation and count of mesophilic aerobes colonies were made according to the NOM-092-SSA1-1994 [57] procedure using the plate method. The total coliforms were determined according to the NOM-112-SSA1-1994 procedure using the most probable number (MPN) method [58]. The microbiological analysis was performed by a certified Laboratory (Laboratorio de Referencia Lab-Tec S. A. de C. V.). Also, a composite sample of the substrate was analyzed after four weeks for a count of total coliforms, fecal coliforms, and Escherichia coli colonies; the analysis procedure was in accordance with the NOM-113-SSA1-199 [59] method; a selective Violet Red Bile Agar was used for the count of Escherichia coli colonies. This analysis was performed in a certified Laboratory (Laboratorio de Referencia Lab-Tec S. A. de C. V.). The morphology of the Escherichia coli was analyzed using a Hitachi SU3500 scanning electron microscope (Hitachi, Tokyo, Japan; 5 kV) .

3. Results

3.1. Microbial Fuel Cell (MFC) Operation and Performance

Figure 1a shows the scheme of the MFCs used in this work; as can be seen, the anode (Figure 1b) is immersed in the soil with the electrogenic microorganism and the Eisenia foetida specimens. In these vermicompost fuel cells (solid-phase microbial fuel cells), the organic matter is oxidized and transformed into simple molecules, producing ${\mathrm{CO}}_2$. The microorganisms act as biocatalysts [7,14,15,60], and the electrons generated in the anode move to the exposed cathode reduces the oxygen from the air, and the subproduct of the complete oxidation–reduction reaction is water.

The performance of MFCs was evaluated each week for four weeks. Figure 2, Figure 3, Figure 4 and Figure 5 show the polarization and power density curves for the four different MFCs in the first, second, third, and fourth weeks, respectively. It is observed that on the first week (Figure 2), the polarization curves for ${\mathrm{MFC}}_{CT}$, ${\mathrm{MFC}}_{W3}$, and ${\mathrm{MFC}}_{W7}$ do not show the three characteristic regions; these curves present high resistance, particularly on the diffusion zone; this effect could be due to the lack of bacteria colony growth to the moment, especially when compared to the polarization curve of the fourth week, shown in Figure 5, where curves are better defined, except for ${\mathrm{MFC}}_{W7}$, which, as described further, presents anomalies due to the excess of Eisenia foetida specimens. It is observed that ${\mathrm{MFC}}_{W3}$ presents a notable change in the polarization curve, improving each week, while ${\mathrm{MFC}}_{W5}$ presents a well-defined polarization curve since Week 1. By the end of the experiment, its behavior is quite similar to that of ${\mathrm{MFC}}_{W3}$.

Table 1. Performance parameters of MFCs for four weeks.

MFC	${\mathrm{MFC}}_{\mathit{C}\mathit{T}}$			${\mathrm{MFC}}_{\mathit{W}3}$
Week	OVCmax/V	jmax/mAm−2	Wmax/mWm−2	OVCmax/V	jmax/mAm−2	Wmax/mWm−2
Week 1	0.84	140	14	0.84	70	8
Week 2	0.81	140	28	0.94	480	134
Week 3	0.81	140	32	0.98	840	156
Week 4	0.82	560	96	1.08	840	192
σ	0.014	210	36	0.099	367	80

presents the performance parameters: open-circuit voltage (OCV) and maximum current and power densities. It is shown that the four MFCs present a similar OCV between 0.81 and 0.84 V in the first week, but the MFCs with Eisenia foetida specimens ( ${\mathrm{MFC}}_{W3}$, ${\mathrm{MFC}}_{W5}$, ${\mathrm{MFC}}_{W7}$) present voltage increase each week, while the OCV in ${\mathrm{MFC}}_{CT}$ remains constant; this could be related to a difference reaction mechanism when the Eisenia foetida specimens are present. As the voltage, the current in ${\mathrm{MFC}}_{CT}$ stays constant for three weeks, and on the fourth week, a considerable increase in the current density is observed; this could be due to the natural degradation of organic matter by the microorganisms in the soil. Table 1 and

Table 2. Performance parameters of MFCs for four weeks (part two).

MFC	${\mathrm{MFC}}_{\mathit{W}5}$			${\mathrm{MFC}}_{\mathit{W}7}$
Week	OVCmax/V	jmax/mAm−2	Wmax/mWm−2	OVCmax/V	jmax/mAm−2	Wmax/mWm−2
Week 1	0.95	680	106	0.87	280	36
Week 2	0.95	510	156	0.95	200	36
Week 3	0.98	640	160	0.98	840	66
Week 4	1.05	900	192	1.05	290	52
$\sigma$	0.047	162	36	0.075	58	14

show that the minimum values of standard deviation ( $\sigma$) correspond to the open-circuit potentials ( ${\mathrm{OVC}}_{max}$), with an average variation of 0.059 ± 0.4. However, the variation in standard deviation related to current densities presents values ranging from 36 to 367; thus, the main challenges are focused on this variable, which is related to the quantity of electrogenic bacteria and the conductivity in the microbial cell.

As mentioned, ${\mathrm{MFC}}_{W3}$ presents an increase in OCV from 0.84 V in the first week to 1.08 V in the fourth week; also, an increase is observed in the current density from 70 mA m⁻² to 840 mA m⁻²; therefore, the power density shows the same behavior, increasing from 8 to 192 mW m⁻² (Figure 2 and Table 1). This behavior could be due to Eisenia foetida’s conditioning to the soil and its reproduction rate, considering that there are three specimens in the first week. In contrast, at the end of the fourth week, there are five Eisenia foetida specimens. The OCV in ${\mathrm{MFC}}_{W5}$ also presents a gradual increase from 0.95 to 1.05 V (Figure 2 and Table 1), and the current and the power densities show an increment from 680 to 900 mA m⁻², which is higher than other membraneless vermicompost fuel cells reported. As can be seen in Figure 2, the current in the first week is higher than in ${\mathrm{MFC}}_{W3}$, but in the fourth week, both values are similar. It is essential to highlight that in ${\mathrm{MFC}}_{W3}$, by the end of the tests (fourth week), there is evidence of Eisenia foetida specimen reproduction, with a total of five specimens, while at ${\mathrm{MFC}}_{W5}$, there are the same number of initial specimens (five Eisenia foetida specimens). Therefore, this could explain the similar performance in the fourth week for $MF{C}_{W3}$ and $MF{C}_{W5}$. In the case of ${\mathrm{MFC}}_{W7}$, a similar increase in OCV is observed as in ${\mathrm{MFC}}_{W3}$.

In the Microbial fuel cell control ( ${\mathrm{MFC}}_{CT}$) shown in Figure 6a, the central line formed by green triangles corresponds to the average current density during the four weeks. At average current densities of 1.6 mA ${\mathrm{m}}^{-2}$, it shows a standard deviation (σ) of 0.19; however, at average current densities of 224 mA m⁻², the standard deviation is 191, meaning that the maximum contributions are made in the concentration region, which is related to the diffusion of the species. In the microbial fuel cell ( ${\mathrm{MFC}}_{W3}$) shown in Figure 6b, the standard deviation is 19 for average current densities of 11 mA m ${\mathrm{MFC}}^{-2}$. For average current densities of 488 mA m⁻², the standard deviation is 335, presenting high values of standard deviation in the activation and concentration regions, as a function of time during the four weeks this microbial fuel cell is the one that increases its power density the most. In the Microbial fuel cell ( ${\mathrm{MFC}}_{W5}$) shown in Figure 6c, the minimum standard deviation of 0.98, obtained in the activation region, for average current density values of 1.1 mA m⁻², the maximum standard deviation of 105 when 617 mA m⁻² is obtained. It is important to note that in the ohmic region, there is a decrease in the standard deviation, understanding that a better conductivity is presented in the microbial cell. In the microbial fuel cell ( ${\mathrm{MFC}}_{W7}$) shown in Figure 6d, the minimum standard deviation variations are presented compared to ${\mathrm{MFC}}_{CT}$, ${\mathrm{MFC}}_{W3}$, and ${\mathrm{MFC}}_{W5}$. The minimum standard deviation value of 0.65 is obtained at 0.005 mA m⁻², and the maximum standard deviation variation of 52 is related to the average current density of 229 mA m⁻².

However, it presents a weird behavior on the current response (Figure 2 and Table 2), showing a decrease in the second and fourth weeks and a higher value in the third week. According to those values, the power density increases in the third week and decreases in the fourth week. However, compared to ${\mathrm{MFC}}_{W3}$ and ${\mathrm{MFC}}_{W5}$, the ${\mathrm{MFC}}_{W7}$ presents a lower performance and, by the end of the last week, is even lower than the power generated in ${\mathrm{MFC}}_{CT}$, meaning that it is counterproductive to have an excessive number of Eisenia foetida specimens.

The performance of ${\mathrm{MFC}}_{W3}$ and ${\mathrm{MFC}}_{W5}$ overcame those previously reported for membrane-less vermicompost MFCs (

Table 3. Performance of MFCs with different substrates and electrodes.

Substrate	Electrodes	Power Density/mWm−2	Reference
Vermicompost (watermelon peels) using Eudrilus eugeniae	Carbon fiber sheet electrodes; Separator: clay and cotton cloth	0.056	Budihardjo et al. (2021) [60]
Vermicompost with 10–12 matured worms	Anode: aluminum mesh and carbon cloth; Cathode: carbon cloth Membrane: Nafion 117	4.0	Nandy et al. (2015) [7]
Vermicompost (banana peels) using Eudrilus eugeniae	Graphite electrodes; Membrane: Nafion 212	5.6	Hajam et al. (2023) [55]
Vermicompost using Megascolecidae specimens	Anode: Carbon felt; Cathode: Carbon felt; Membraneless	7.2	Edwards (2021) [61]
Vermicompost	Anode: Carbon cloth; Cathode: Pt on Carbon cloth; Membrane: Nafion 117	52.3	Yukimoto et al. (2017) [53]
Shewanella spp. strains isolated from Odontesthes regia	Anode: Carbon felt impregnated with multi-walled carbon nanotubes; Cathode: Carbon felt; Membrane: CMI-7000	281	Sandy L. Calderon et al. (2020) [56]
Vermicompost (natural black earth, tree bark, leaves) using Eisenia foetida;	Anode: Stainless steel; Cathode: Copper; Membraneless	192	This Work

); however, some two-chamber (membrane) fuel cells present higher power density [56], which led to continuing the optimization of soil MFC as an alternative power source and environmental technique.

According to the previously described performance results, the best performance is observed in ${\mathrm{MFC}}_{W3}$. Therefore, three ${\mathrm{MFC}}_{W3}$ were connected in series to energize a light emitter diode as an experimental test of the MFC operation and possible application. The arrangement and the schematic with the light on is shown in Figure 7.

3.2. Substrate Characterization

Each MFC was connected to evaluate its performance, and after four weeks, the soil compositions and the Eisenia foetida specimens’ state were analyzed. The same substrate was used for the four MFCs ( ${\mathrm{MFC}}_{CT}$, ${\mathrm{MFC}}_{W3}$, ${\mathrm{MFC}}_{W5}$, and ${\mathrm{MFC}}_{W7}$). Therefore, the composition (% organic matter, %C, and %N) of the soil in each MFC in the first week was the same (Table 3), and in all of them, the pH $=8.0$. In the second week, there was a decrease of 2% in the organic matter (O. M.) in the MFCs with Eisenia foetida specimens; it could be assumed that Eisenia foetida precisely metabolized this organic matter. This decrease in the O. M. was observed during Weeks 3 and 4. The composition in ${\mathrm{MFC}}_{CT}$ also presents a reduction in organic matter; however, in the first two weeks, it only reduced by 0.3%, and for the fourth week, the percentage was approximately halved. These results correspond to its electrochemical behavior, supporting the evidence of degradation by the microorganisms present in the soil, even without Eisenia foetida specimens.

The carbon percentage (

Table 4. Physicochemical composition of the substrate.

Week	Fuel Cell	O.M./wt%	C/wt%	N/ wt%	pH
Week 1	${\mathrm{MFC}}_{CT}$	45.01	26.11	1.90	8.0
	${\mathrm{MFC}}_{W3}$	45.01	26.11	1.90	8.0
	${\mathrm{MFC}}_{W5}$	45.01	26.11	1.90	8.0
	${\mathrm{MFC}}_{W7}$	45.01	26.11	1.90	8.0
Week 2	${\mathrm{MFC}}_{CT}$	44.87	26.02	2.00	8.0
	${\mathrm{MFC}}_{W3}$	42.54	24.94	1.90	8.0
	${\mathrm{MFC}}_{W5}$	42.14	24.73	2.02	8.0
	${\mathrm{MFC}}_{W7}$	42.10	24.85	1.80	8.0
Week 3	${\mathrm{MFC}}_{CT}$	44.70	25.93	1.90	8.0
	${\mathrm{MFC}}_{W3}$	43.00	24.67	1.90	8.0
	${\mathrm{MFC}}_{W5}$	42.63	24.44	1.90	8.0
	${\mathrm{MFC}}_{W7}$	42.84	24.42	1.90	8.0
Week 4	${\mathrm{MFC}}_{CT}$	22.40	13.00	2.00	7.5
	${\mathrm{MFC}}_{W3}$	20.01	11.60	2.10	7.5
	${\mathrm{MFC}}_{W5}$	17.14	10.00	2.10	6.8
	${\mathrm{MFC}}_{W7}$	16.00	9.00	2.11	8.0

) in the four MFCs decreased; however, only a 0.18% decrease was observed in ${\mathrm{MFC}}_{CT}$, related to the characteristic biological activity in composting. The reduction in ${\mathrm{MFC}}_{W3}$, ${\mathrm{MFC}}_{W5}$, and ${\mathrm{MFC}}_{W7}$ corresponded to 1.69% (average), according to the decomposition in vermicomposting with the Eisenia foetida.

In all the MFCs, the O. M. and the %C decreased considerably; as described above, this reflects the increment in the energy generated from the combined biochemical reactions of the microorganisms and the Eisenia foetida specimens.

Concerning the nitrogen percentage, a light increment of 0.12% was observed in the second and fourth weeks in all the MFCs, possibly due to the composting process’s thermophile phase. At the end of the tests, the amount of nitrogen was approximately 2.1%, and according to NMX-FF-109-SCFI-2008, the C/N ratio must be under 20 for composting systems [61]; therefore, as is observed in Table 3 in all cases, this parameter meets the Mexican regulations. Energy generator microorganisms such as E. coli, which belong to the aerobic mesophilic microorganisms [62], need a slightly alkaline medium for development. Table 4 shows that the pH for the four MFCs during the first three weeks was 8, indicating that this substrate is adequate for the vermicomposting process and Eisenia foetida reproduction. In the fourth week, it was approximately 7.5, which is optimal for vermicompost [61,62]. The microbiological analysis of the substrate is shown in

Table 5. Microbiological analysis of the MFC substrate at the end of the fourth week.

Analyzed Parameters	Aerobic Mesophilic/CFU cm−2	Total Coliforms/CFU g−1	Fecal Coliforms/MPN g−1	Escherichia coli/MPNg−2
Substrate	200,630	164	77	110

; it was observed that the number of aerobic mesophiles was high compared to the total coliforms, indicating that several types of mesophiles could contribute to biopower generation. Also, E. coli has been proven as an electro-generator in MFC [37,38]. Figure 8a,b show the SEM micrographs of the E. coli at 2000× and 5000×, respectively.

3.3. Eisenia foetida Development

The specimens’ adequate survival and growth led to the development of the electron generators’ microorganisms. In all cases, the Eisenia foetida specimens remained alive and showed a length growth of approximately 4 cm; their length increased from 5 cm to 9 cm (Figure 8c). The number of Eisenia foetida present in each MFC was verified at the end of the fourth week; in ${\mathrm{MFC}}_{CT}$, ${\mathrm{MFC}}_{W5}$ and ${\mathrm{MFC}}_{W7}$, there was the same number of Eisenia foetida specimens as at the beginning. Figure 8d shows a magnified image of the Eisenia foetida, which shows its digestive tract. No clitellum was observed during the reproduction season [63]. Meanwhile, in ${\mathrm{MFC}}_{W3}$, there was an increase in the specimens, resulting by the end of the test in five Eisenia foetida specimens. Therefore, the number of Eisenia foetida specimens in ${\mathrm{MFC}}_{W3}$ and ${\mathrm{MFC}}_{W5}$ was the same in the fourth week, which explains the similar performance of these two MFCs in the last week.

3.4. Microbiological Analysis

Table 6. Microbiological analysis of the MFC substrate at the end of the fourth week.

Analyzed Parameters	${\mathrm{MFC}}_{\mathbf{CT}}$	${\mathrm{MFC}}_{\mathit{W}3}$	${\mathrm{MFC}}_{\mathit{W}5}$	${\mathrm{MFC}}_{\mathit{W}7}$
Aerobic mesophilic/CFU cm−2	8820	9950	9740	7310
Total coliforms/CFU cm−2	4	8	2	2

shows the microbiological analysis of the aerobic mesophilic and coliform microorganisms present in the anode of each MFC. It is observed that ${\mathrm{MFC}}_{W3}$, which presents the best electrochemical performance (192 mW m⁻²), shows the highest account of aerobic mesophiles (9950 CFU cm⁻²), the ${\mathrm{MFC}}_{W5}$ which also offers a maximum power density of 192 mW m⁻² showing a value of 9740 CFU cm⁻² for the aerobic mesophiles. In contrast, the ${\mathrm{MFC}}_{CT}$ and ${\mathrm{MFC}}_{W7}$ present lower values, 8820 CFU cm⁻² and 7310 CFU cm⁻², respectively; this microorganism account also agrees with power results, 96 mW m⁻² and 52 mW m⁻².

The coliforms correspond to the aerobic mesophile microorganism classification; therefore, a similar relation between the coliforms and power generated was expected, as observed in Table 6; this relation is in agreement with ${\mathrm{MFC}}_{W3}$, which presents a high performance and the higher number of total coliforms. The same behavior is observed for ${\mathrm{MFC}}_{CT}$ and ${\mathrm{MFC}}_{W7}$; however, in this case, the ${\mathrm{MFC}}_{W5}$ presents a high power density, but the number of total coliforms is the lowest. This could indicate the presence of different types of mesophilic microorganisms, or support the contribution of the Eisenia foetida for the power generation.

4. Conclusions

The studied bio-electrochemical prototype (MFCs) has been proven capable of transforming chemical energy from organic waste into electrical energy thanks to the action of present microorganisms, especially the families of aerobic mesophiles and coliforms, which are called electrogenic. This work presents the advantages of using Eisenia foetida specimens for energy generation. The energy obtained in the vermicompost MFC offers a greener alternative for energy production, as evidenced by powering an LED with an array of three MFCs connected in series. The best results were obtained after four weeks of substrate degradation. The number of microorganisms present in the soil is an indicator of the power generated. At the same time, the growth conditions in bacteria are favored by Eisenia foetida species. It can be assumed that the bacterial colonies may originate from the digestion of Eisenia foetida. The research suggests the optimal number of species is 3–5 ( ${\mathrm{MFC}}_{W3}$ and ${\mathrm{MFC}}_{W5}$) for the added substrate volume, considering the power density results and the number of Eisenia foetida in the MFC. ${\mathrm{MFC}}_{W3}$ showed an increase in the number of species from three to five after the fourth week, and the power density increased from 8 to 192 mW m⁻², while in ${\mathrm{MFC}}_{W5}$, the power increased from 106 mW m⁻² to 192 mW m⁻². The results also indicate that more Eisenia foetida specimens do not translate into better performance in power density development. For example, ${\mathrm{MFC}}_{W7}$ shows values of 36 mW m⁻² (first week) to 52 mW m⁻² (fourth week) compared to ${\mathrm{MFC}}_{W3}$ or ${\mathrm{MFC}}_{W5}$, which, as mentioned before, both achieved power density values of 192 mW m⁻².

The investigated substrate is rich in organic matter, transforming into carbon and nitrogen, essential for soil conditioning. In this substrate, Eisenia foetida specimens play a significant role, promoting the growth of bacterial colonies that release electrons and protons to be harnessed by the electrochemical system through the anode and cathode. The ideal pH for the development of total coliforms is approximately 7.5, and the pH obtained during the four weeks of experimentation is 8, indicating a favorable pH for bacterial life. Therefore, vermicomposting MFCs are an excellent alternative for obtaining energy, and they also use organic solid wastes, decreasing contamination.

In the present investigation, when there are high standard deviation variations (MFCCT), the lowest power density values are related to Figure 5a and Figure 6a, respectively. Conversely, when the standard deviations are minimal, the highest power densities are observed in the ohmic region (Figure 5c and Figure 6c), respectively. This hypothesis is proposed for future research.