Optimization of Recovery of Nutrients from Pig Manure Slurry through Combined Microbial Fuel Cell and Microalgae Treatment

Abstract

Microbial fuel cells (MFCs) and microalgae–bacteria consortia represent two renewable and promising technologies of growing interest that enable wastewater treatment while obtaining high-value-added products. This study integrates MFCs and microalgae production systems to treat animal slurry, aiming to remove and recover organic and inorganic components while generating energy and producing biomass. The MFCs effectively eliminated Chemical Oxygen Demand (COD), organic nitrogen, and a portion of the suspended solids, achieving a maximum voltage of 195 mV and a power density of 87.03 mW·m⁻². After pre-treatment with MFCs, the slurry was diluted to concentrations of 10%, 50%, and 100% and treated with microalgae–bacteria consortia. The results showed a biomass production of 0.51 g·L⁻¹ and a productivity of 0.04 g·L⁻¹·day⁻¹ in the culture fed with 10% slurry, with significant removal efficiencies: 40.71% for COD, 97.76% for N-NH₄₊, 39.66% for N-NO₂⁻, 47.37% for N-NO₃⁻, and 94.37% for P-PO₄⁻³. The combination of both technologies allowed for obtaining a properly purified slurry and the recovery of nutrients in the form of bioelectricity and high-value biomass. Increasing the concentration of animal slurry to be treated is essential to optimize and scale both technologies.

1. Introduction

In the past century, fossil fuels have powered global economic and technological growth, but they have also caused major environmental issues like air and water pollution. Population growth and industrialization have worsened these problems, increasing wastewater production. Humanity now faces sustainability challenges, including the need for renewable energy, climate change impacts, and a lack of clean water [1,2,3]. This situation is exacerbated by the increased global demand for food, which has led to the intensification of livestock production in recent decades. Consequently, there is an increase in animal manure production, primarily slurry (liquid manure), making new strategies for manure management necessary to minimize its environmental impact and enhance its value [4]. In fact, to address these and additional problems, the United Nations proposed the 17 Sustainable Development Goals (SDGs), among which are “Affordable and Clean Energy”, “Climate Action”, and “Clean Water and Sanitation” [5]. In the European Union, and specifically in Spain, the bioeconomy emerges as an approach to promote the sustainable use of biological resources to generate economically valuable services [6]. Within this framework, the potential of micro-organisms to purify wastewater while producing bioenergy and bioproducts has been explored as a complement or alternative to regular wastewater treatment approaches that incorporate a combination of physical, chemical, and biological systems. Apart from micro-organism-based technologies, numerous methods have been demonstrated to be effective in treating pollutants from wastewater including adsorption, chemical precipitation, conventional coagulation, ion-exchange, electrolysis, and membrane technology [7].

On one hand, since the 20th century, research has been conducted on the ability of bacteria to use organic matter present in wastewater as a substrate to transform the energy in chemical bonds into electrical energy using Microbial Fuel Cells (MFCs). MFCs are bioelectrochemical devices that consist of a cathode and an anode. The operation of MFCs is based on the ability of micro-organisms to oxidize the organic matter in wastewater in the anode chamber to generate electrons, protons, and carbon dioxide (CO₂) under anaerobic conditions. On one side, the electrons produced are transmitted by the bacteria to the anode surface and travel through an external circuit to the cathode, producing electrical energy. Simultaneously, the protons travel internally and pass through a proton exchange membrane to reach the cathode. In the cathode chamber, the electrons and protons react with a final electron acceptor, which undergoes a reduction reaction [8]. The success of this treatment relies on the presence of electrochemically active bacterial species capable of directly transferring electrons to the anode [9,10]. Since its development, the structure and mechanisms of MFCs have been continuously optimized, resulting in ongoing advancements in wastewater treatment and direct electricity recovery, which are low in emissions and reduced in cost [11]. However, this technology faces numerous challenges that prevent it from providing applications in real environments such as the electron transfer mechanism in the anodic chamber, temperature, terminal electron acceptors, substrate, and the membrane and the electrode material, all of which could influence the performance and cost of MFCs [12]. Furthermore, although it allows reducing Chemical Oxygen Demand (COD) levels and turbidity in wastewater, it hardly reduces or recovers nitrogen (N) and phosphorus (P) present in wastewater or animal slurry.

On the other hand, there are microalgae and cyanobacteria, microscopic photosynthetic organisms that inhabit both marine and freshwater environments. Both microalgae and cyanobacteria can be cultivated in wastewater, offering two simultaneous advantages: wastewater purification and nutrient recovery in the form of biomass [13]. In the 1950s, Oswald determined that, in wastewater treatment with microalgae, there exists a key symbiotic mechanism between microalgae and bacteria [14]. Both communities mutually benefit as O₂ released during photosynthesis is utilized by heterotrophic bacteria to generate CO₂, which in turn is beneficial for the microalgal carbon fixation. Additionally, bacteria contribute phytohormones and essential macro- and micronutrients, significantly enhancing microalgae growth [15]. Despite the success of microalgae–bacteria systems and their large-scale application, they still present some limitations, especially when using wastewater such as animal slurry. This type of wastewater is characterized by high turbidity, which hinders light availability for photosynthesis, and high concentrations of N in the form of ammonium (N-NH₄⁺), which affects the electron transfer of photosystem II, making it unsuitable for microalgae growth [16].

Therefore, the objective of this study is to combine and optimize both technologies for wastewater treatment, specifically starting with wastewater from the Veterinary Teaching Farm—University of Murcia (Spain). The aim is to evaluate the purification capability of MFCs and their corresponding electricity production. Additionally, the study will assess the wastewater treatment capacity of microalgae cultures and their biomass production under different percentages of wastewater pretreated with MFCs.

2. Materials and Methods

2.1. Micro-Organisms and Culture Media

The microalgae used was Scenedesmus almeriensis, obtained from the culture collection of the Desalination and Photosynthesis Research Group at the University of Almería. This strain is suitable for wastewater treatment as it exhibits rapid growth and a great ability to adapt to stress conditions and various pH and temperature ranges [17]. For the preparation of the inoculum, it was necessary to prepare a mineral medium. This medium was prepared using chemical fertilizers, specifically 0.9 g·L⁻¹ NaNO₃, 0.14 g·L⁻¹ KH₂PO₄, 0.18 g·L⁻¹ MgSO₄ · 7H₂O, and 0.02 g·L⁻¹ Karentol. Slurry from the Veterinary Teaching Farm—University of Murcia (Spain) was used as fuel for the MFCs. Pig slurry also acted as the inoculum for the formation of an anaerobic bacterial community. The composition of the slurry is shown in

Table 1. Composition of the animal slurry from the Veterinary Teaching Farm—University of Murcia (Spain).

	Slurry
COD (mg·L−1)	2259.3 ± 73.1
BOD5 (mg·L−1)	888.6 ± 31.4
TN (mg·L−1)	603.3 ± 28.9
N-NH4+ (mg·L−1)	538 ± 36.6
N-NO3− (mg·L−1)	0.0
N-NO2− (mg·L−1)	0.04 ± 0.00
N-organic (mg·L−1)	65.3 ± 5.1
P-PO4−3 (mg·L−1)	14.1 ± 0.6
SS (g·L−1)	0.83 ± 0.0

Data are represented as mean of three independent determinations ± SD.

.

Once the slurry was pretreated with the MFCs, the resulting medium was used to prepare the culture media for S. almeriensis. For this, the pretreated slurry was diluted to 10% (10% slurry), 50% (50% slurry), and 100% (100% slurry) with distilled water. Additionally, a control treatment using the previously described mineral medium was added (fertilizers). A schematic representation of the methodology is shown in Figure 1.

2.2. Preparation of Proton Exchange Membranes with Catalytic Activity Based on Ionic Liquids

The proton exchange membrane used to separate the anode and cathode compartments is crucial because it must be impermeable to chemicals. In this case, it is essential that the membrane is not permeable to either the organic matter used as a substrate or to O₂. In this study, a Polymer Inclusion Membrane based on Ionic Liquids (PILIM) was used due to its cost-effectiveness, ionic selectivity, stability, and resistance to fouling, instead of traditionally employed membranes, which are more expensive and may present a series of drawbacks. In summary, the PILIM preparation procedure is based on the immobilization of the Ionic Liquid (IL) (70%) in an organic polymer (30%) by occlusion using the casting technique. After this, a platinum catalytic ink (0.5 mg Pt·cm⁻²) is sprayed onto the PILIM, prepared by dissolving platinum dispersed in Vulcan in a 50%/50% solution of H₂O and isopropanol, to which 20 µL of PTFE was added as a binder. The complete procedure is described in [18]. These types of membranes showed good results in previous studies due to their stability and efficiency in generating electrical energy and removing organic matter. Specifically, for their preparation, an IL called methyltrioctylammonium chloride, with the abbreviation [N_8,8,8,+1⁺] [Cl⁻], was used. The IL used was based on an ammonium cation and a chloride anion and was supplied by Sigma-Aldrich-Fluka Chemical Co. (Darmstadt, Germany) and was of the highest purity available. This IL is characterized by being liquid at room temperature, a typical property of ILs, and by being insoluble in water.

2.3. MFCs Test

The experimental setup involved reactors made from modified 250 mL glass bottles with cylindrical flanges, where the temperature was kept at 25 °C. The cathode was linked to the anode via a 1 kΩ resistor. The anode consisted of 100 g of graphite granules with a diameter of 3–5 mm and a graphite rod with a diameter of 3.18 mm. Anode chambers held 200 mL of feed and were sealed with a lid that had a sampling port, ensuring anaerobic conditions throughout the experiment. The membrane–cathode system was securely attached to the reactor flange using a round joint clip. All tests were performed in batch mode, using wastewater as the sole source of micro-organisms and fuel.

2.4. Electrochemical Analysis

To analyze the fuel’s performance in terms of electricity generation, polarization and internal resistance were calculated. The polarization test is a crucial tool in the analysis and optimization of the electrical performance of an MFC, providing detailed information on aspects such as the characterization of electrical performance and the evaluation of internal losses.

To analyze the electrical performance of each MFC, a polarization test was used. This method involves varying the external resistance using a resistor box to change the electrical current passing through the circuit and measure the voltage produced at each point. The voltage was continuously monitored every minute using a PCI 6010 data acquisition system (National Instruments, Austin, TX, USA). Additionally, the voltage was periodically checked offline with a DVM891 digital multimeter (HQ Power, Berlin, Germany). After 240 h of operation, polarization was measured using a variable resistor box (5.77 MΩ, 953 kΩ, 486 kΩ, 96.5 kΩ, 50 kΩ, 11 kΩ, 6 kΩ, 1.1 kΩ, 561 Ω, 94.5 Ω, and 1.5 Ω). Voltage measurements were taken once the cell had reached a pseudo steady state under a specific resistor value, which typically took about 1 min. To ensure accuracy, each measurement was performed three times, and the average value was reported. The relative standard deviation of the measurements was less than 10%, indicating high repeatability.

The internal resistance R_int(Ω) of each MFC was determined using Equation (1):

$$R_{int}(\mathsf{\Omega })={\displaystyle \frac{OCV\left(V\right)}{I\left(A\right)}}-R_{ext}(\mathsf{\Omega })$$

(1)

where OCV is the open circuit voltage, I is the current density at maximum power, and R_ext(Ω) is the external resistance at maximum power.

2.5. Lab-Scale Photobioreactors

Once the MFC tests were completed, the pretreated slurry was used for a second experimental plan. This test aimed to evaluate the growth of microalgae and the recovery of nutrients using the wastewater previously treated with the MFCs. Four treatments were tested: (i) control: fertilizer medium; (ii) 10% slurry: medium with 10% pig slurry and 90% distillated water; (iii) 50% slurry: medium with 50% pig slurry and 50% distillated water; (iv) 100% slurry: medium with 100% pig slurry. Three replicates of each treatment were prepared, resulting in 12 Erlenmeyer flasks (200 mL) with a final volume of 150 mL each by adding 30 mL of inoculum and 120 mL of the corresponding medium.

The flasks were placed in an orbital shaker incubator to promote mixing and prevent sedimentation. Artificial lighting was provided by twelve LED fluorescent lamps, with a photoperiod of 16 h of light and 8 h of darkness. The average light intensity in each flask was 200 µmol photons·m⁻²·s⁻¹. The experiment was conducted in batch mode for twelve days at a temperature of 26 ± 2 °C.

2.6. Growth Monitoring

For culture monitoring, a UV-VIS spectrophotometer (T80 PG Instruments Ltd., Woodway Lane, Alma Park, UK) was used, with the absorbance at 680 nm and 750 nm. Also, cell counts under an optical microscope using a Neubauer chamber at the beginning, middle, and end of the assay to monitor cell growth and determine cell density were conducted. Overall, monitoring the absorbance of microalgae cultures allows for tracking their growth, while cell counting of microalgae cells enables the determination of cell density in the different treatments, providing more accurate data on the growth of microalgae in the selected media. Cell density (C) was calculated using Equation (2), where N indicates the average number of cells present in the 5 small squares of the central square, 4 × 10⁻⁶ refers to the sample volume expressed in cm³ (mL) over the area of the small squares (0.004 mm³), and D is the dilution factor.

$$C={\displaystyle \frac{N}{4\times {10}^{-6}}}\times D$$

(2)

2.7. Chemical Analysis and Biomass Production

The initial and final assessments of the wastewater were conducted to evaluate the effectiveness of pollutant removal of each technology. The microalgal experiment additionally aimed to study biomass generation and overall biomass productivity.

Essential water quality parameters such as pH, Oxidation–Reduction Potential (ORP), and electrical conductivity (EC) were monitored using a portable multimeter, specifically the sensION + MM 150 from Hach (Loveland, CO, USA). Chemical Oxygen Demand (COD) and Total Nitrogen (TN) concentrations were determined using standardized photometric methods with a SpectroQuant Prove 300 spectrophotometer (Merck Millipore). Sample preparation for COD and TN involved the use of a TR 420 Thermoreactor (Merck Millipore) to ensure accurate results. Biological Oxygen Demand (BOD₅) was determined using a Velp Scientifica system for manometric BOD determination. Dicyandiamide was employed as a nitrification inhibitor and sodium hydroxide as an alkali to capture CO₂. Ammonium (NH₄⁺) levels were quantified using specific cuvettes and methods provided by Sigma-Aldrich^®, following ISO 23695 standards [19]. For Nitrites (NO₂⁻) and Nitrates (NO₃⁻), colorimetric assays were employed. NO₂⁻ were analyzed using the Griess reagent method, where the reaction with Griess reagent (consisting of sulfanilic acid and N-(1-naphthyl)-ethylenediamine) produced a red/pink compound measurable at 540 nm. NO₃⁻ were measured using High-Performance Liquid Chromatography (HPLC) on an Agilent Technologies system, employing an aqueous mobile phase with 20% methanol and 0.01 M octylamine adjusted to pH 6.5. The concentration of organic nitrogen (N-organic) was obtained as the difference between the TN concentration and the concentration of N-NH₄⁺, N-NO₃⁻, and N-NO₂⁻.

The biomass generation along with the productivity was determined through the Suspended solids (SS), which were measured by filtering samples through GF-F Whatman glass fiber filters (0.7 µm pore size), drying at 105 °C, and weighing after desiccation.

2.8. Statistical Analysis

Results are presented as mean ± standard deviation based on three independent replicates for each analysis. The data were processed using PASW Statistics 28 for Windows (SPSS Inc., Chicago, IL, USA). Student’s t-test was conducted with a significance level of p < 0.05 to assess differences among the treatments studied.

3. Results

The results of this study include an initial stage of treating slurry from the Veterinary Teaching Farm—University of Murcia (Spain) using MFCs, with the aim of purifying the wastewater and producing electricity. Following this pre-treatment, a second phase was carried out. The pretreated wastewater was diluted to 10%, 50%, and 100% to be used as a culture medium for S. almeriensis, allowing for the recovery of nutrients in the form of biomass and obtaining treated water that meets discharge or reuse regulations.

3.1. Slurry Pre-Treatment Using MFCs

3.1.1. Nutrient Removal

Within the 8-day MFCs trial employing pig slurry, the dynamics of COD were monitored. The acquired data, as shown in Figure 2A, revealed a progressive decline in COD levels, ranging from an initial value of 2259.3 mg·L⁻¹ on day 0 to a final value of 2003.3 mg·L⁻¹ on day 8. Figure 2B illustrates the percentage removal of COD alongside the elimination of various nutrients, TN, N-NH₄⁺, N-NO₃⁻, N-NO₂⁻, N-organic, and P-PO₄⁻³. Additionally, the removal of suspended solids is presented. The results showed an 11% removal of COD, a 1% removal of P-PO₄⁻³, and a 3.7% removal of TN present in the slurry. Among the different forms of N, no removal of N-NH₄⁺ was observed. However, a production of 42 mg·L⁻¹ of this nutrient was noted due to the mineralization of N-organic. Similarly, no removal of N-NO₃⁻ was detected, as it was absent in the slurry both at the beginning and the end. A 60.4% removal of N-NO₂⁻ and a 98.1% removal of N-organic were measured. Regarding the suspended solids, 28.4% were removed.

3.1.2. Electricity Generation

In this study, multiparametric data were measured following the characterization of the initial and final wastewater after the MFCs trial. The data showed a stable pH, ranging between 8.2 and 8.4. The conductivity varied from 7.5 μS·cm⁻¹ at the beginning of the trial to 6.8 μS·cm⁻¹ at the end. The ORP was stable at 28.40 ± 6.2 mV. These values are relevant to bacterial activity related to electricity production.

Regarding the voltage profile of the MFCs, Figure 3A shows that the voltage increased over time, reaching a maximum of 195 ± 4.2 mV at 85 h. In the polarization test, Figure 3B highlights two of the three types of losses that can be observed: activation losses, more prominent at low current densities, where a clearly defined exponential curve is observed, and ohmic losses, which are more significant at intermediate current densities and characterized by their linear behavior. Additionally, no mass transfer losses were observed, an advantage offered by single-chamber MFCs with an aerated cathode, where O₂ from the surrounding air is always available. This same graph provides information about the open circuit voltage (OCV), which in this case was 455 ± 1.3 mV. Furthermore, in the polarization test, by modifying the external resistance, it was possible to determine the resistance at which the power is maximized. In this case, the power reached its maximum at an approximate resistance of 1113 Ω.

On the other hand, Figure 3C shows the power curve obtained on the seventh day of the trial, and it can be seen that the maximum power generated was 87.03 ± 12.7 mW·m⁻². Additionally, regarding internal resistance R_int, the average R_int obtained in the system was 1805 ± 205.1 Ω.

3.2. Slurry Treatment Using Microalgae–Bacteria Consortia

3.2.1. Culture Monitoring, Cell Growth and Biomass Productivity

Once a pre-treatment with the MFCs was carried out, the pretreated slurry was diluted to 10%, 50%, and 100% with distilled water and used as a microalgae culture medium. The cultures were monitored over 12 days through absorbance and microalgae cell counting using an optical microscope.

Regarding the monitoring of culture growth by absorbance at 680 and 750 nm, the results are shown in Figure 4. It was observed that the cultures with fertilizers and 10% slurry as growth media increased absorbance at 680 nm from day 0 to day 12 of cultivation, with the highest values in the cultures with 10% slurry (Figure 4A). However, the cultures with 50% slurry and 100% slurry showed a slight growth during the first two days, followed by a decrease in absorbance at 680 nm to values lower than the initial ones (Figure 4B). The growth was also monitored with absorbance at 750 nm in the cultures with fertilizer medium and 10% slurry (Figure 4C), showing growth in both cases. The monitoring of absorbance at 750 nm in the cultures with 50% and 100% slurry showed the same trend as at 680 nm, with absorbance values decreasing (Figure 4D). During the test and at the end of it, significant differences were observed between the 10% slurry and fertilizer treatments (p < 0.05), with the highest absorbance values on day 12 in the culture with 10% slurry.

The counting results are shown in Figure 5A. It is observed that, over time, cell density increases in the fertilizer and 10% slurry treatments, while it decreases in the 50% and 100% slurry treatments. Additionally, the statistical analysis demonstrated that at the beginning of the trial, there were no significant differences between the data. However, at the end of the test, significant changes were observed between the treatments (p < 0.05), with cell density in the 10% slurry treatment higher than the fertilizer treatment. Also, low values of cell density were obtained in the 50% slurry and 100% slurry treatments.

Since the microalgae only grew in the samples with the fertilizer medium and 10% slurry, it was only possible to determine biomass growth and productivity in these samples, assigning a value of 0 to the productivity of the 50% slurry and 100% slurry treatments. The biomass produced was 0.35 and 0.51 g·L⁻¹, while the productivity obtained was 0.03 and 0.04 g·L⁻¹·day⁻¹, respectively. Statistically significant differences were observed between treatments (p < 0.05). Figure 5B shows the biomass produced and the productivity obtained.

3.2.2. Nutrient Recovery

The results indicated that cultures with 50% and 100% slurry did not exhibit growth. Consequently, nutrient recovery analysis was conducted on cultures utilizing 10% slurry as the growth medium and on cultures grown with chemical fertilizers. In this context, the medium’s composition was analyzed at the conclusion of the 12-day treatment period, following biomass separation via centrifugation. By comparing the initial and final concentrations of each nutrient (TN, N-NH₄⁺, N-NO₃⁻, N-NO₂⁻, P-PO₄⁻³, and COD), the nutrient consumption and the corresponding percentage of nutrients removal/recovery were determined.

Regarding the different forms of N measured in this study, the initial and final concentration, along with the consumption and removal percentage, are shown in Figure 6. The use of slurry as a culture medium implies that the predominant form of nitrogen is N-NH₄⁺. In the treatments with 10% slurry, the initial concentration of N-NH₄⁺ was 52 ± 1.0 mg·L⁻¹, decreasing to 6.7 ± 1.1 mg·L⁻¹ at the output, which implies an N-NH₄⁺ consumption of 4.62 ± 0.09 mg·L⁻¹·day⁻¹ (Figure 6A). This consumption represents a removal efficiency at the end of the assay of 97.8% of the N-NH₄⁺ present in the medium with 10% slurry. Regarding N-NO₃⁻, it is present at a concentration of 97.76 ± 0.11 mg·L⁻¹ in the medium with fertilizers and 15.84 ± 0.62 mg·L⁻¹ in the medium with 10% slurry (Figure 6B). Statistically significant differences were observed between both treatments related to N-NH₄⁺ consumption and removal (p < 0.05). Despite the slurry not containing N in the form of N-NO₃⁻, it originates from the inoculum used, as it still had N and P when the assay was initiated. After the 12-day assay, the measured values in the output water were 57.07 ± 2.13 mg·L⁻¹ and 8.33 ± 2.08 mg·L⁻¹ using the medium with fertilizers and 10% slurry, respectively. Therefore, a consumption of 3.7 ± 0.2 mg·L⁻¹·day⁻¹ was observed in the cultures with fertilizers and 0.68 ± 0.11 mg·L⁻¹·day⁻¹ in the cultures with 10% slurry (Figure 6C). The removal percentages were 41.63% in the cultures with fertilizers and 47.37% in the cultures with 10% slurry (Figure 6D). Statistically significant differences were observed between both treatments related to N-NO₃⁻ consumption (p < 0.05) but not for N-NO₃⁻ removal.

The initial and final N-NO₂⁻ values and consumption were also measured (Figure 6E). The removal was 0% in cultures with fertilizers and 39.7% in cultures with 10% slurry (Figure 6F). Fertilizers and 10% slurry treatments showed statistically significant differences related to N-NO₂⁻ consumption and removal percentage (p < 0.05). Finally, the initial and final TN of both treatments were determined. The initial TN was 105.1 ± 7.1 mg·L⁻¹ and 85.1 ± 5.2 mg·L⁻¹ using fertilizers and 10% slurry, respectively. The final TN in the output water was 70.2 ± 4.5 mg·L⁻¹ and 10 ± 0.6 mg·L⁻¹, respectively. These values imply a consumption of 3.2 mg·L⁻¹ and 5.5 mg·L⁻¹, respectively (Figure 6G). The TN removal percentages were 33.3% and 85.7% using chemical fertilizers and 10% slurry as the culture medium, respectively (Figure 6H). Statistically significant differences were observed between both treatments related to TN consumption and removal (p < 0.05).

In this study, it is important to note the removal of organic matter in samples with 10% slurry as the culture medium, where the COD concentration decreased from 179.92 ± 0.5 mg·L⁻¹ to 40.71 ± 3.6 mg·L⁻¹, resulting in a consumption of 0.66 mg·L⁻¹·day⁻¹ and a removal efficiency of 40.71%, as shown in Figure 7A,B. Statistically significant differences were observed between treatments with fertilizers and 10% slurry related to COD consumption and removal (p < 0.05).

Regarding P-PO₄⁻³, the initial concentrations were 34.5 ± 0.8 mg·L⁻¹ and 6.5 ± 0.1 mg·L⁻¹ using fertilizers and 10% slurry, respectively. After biomass separation, the P-PO₄⁻³ concentration in the output water was 20 ± 1.1 mg·L⁻¹ in the cultures with fertilizers as the culture medium and 0.36 ± 0.01 mg·L⁻¹ in the cultures with 10% slurry as the culture medium. Thus, the consumption was 1.3 ± 0.1 mg·L⁻¹·day⁻¹ and 0.56 ± 0.03 mg·L⁻¹·day⁻¹ in the cultures with fertilizers and 10% slurry, respectively (Figure 7C). In turn, the removal percentage was 42% in the cultures with fertilizers and 95% in the cultures with 10% slurry (Figure 7D). Statistically significant differences were observed between treatments with fertilizers and 10% slurry related to P-PO₄⁻³ consumption and removal (p < 0.05).

4. Discussion

In recent years, manure management has become a significant problem due to its environmental impact, especially because of greenhouse gas (GHG) emissions, notably CH₄, CO₂, H₂S, NH₃, and N₂O emissions. The magnitude of GHG emissions from livestock production depends basically on the type of animal, rearing method/system, manure management, and indoor/outdoor climate conditions [20]. NH₃ emissions from barns and slurry stores represent up to 80% of the total NH₃ emissions from agricultural activities. Currently, in many countries, mitigation solutions are now mandatory, such as diet manipulation, covering storage tanks, or slurry injection for soil application [4]. At the same time, alternative technologies have emerged that not only aim to solve the problems derived from manure management but also to recover nutrients (N, P) in the form of value-added products, such as composting and anaerobic digestion [21]. Although these technologies are already widely developed, in recent years, the use of MFCs [22,23] and microalgae–bacteria consortia [24,25,26,27] have emerged as promising alternatives. Therefore, in this study, animal slurry was treated using MFCs initially, and the pretreated slurry was used as a culture medium for the growth of S. almeriensis, diluted to 10%, 50%, and 100%.

First, the slurry was pretreated for 8 days using single-chamber MFCs with an aerated cathode. To evaluate the treatment capacity of the MFCs, the nutrients present in the slurry were measured at the beginning and end of the MFC treatment. The results showed a low COD removal rate over the 8 days of the trial, with an 11% COD removal at the end. This value is low compared to the yields obtained with microbial fuel cell systems in which others fuel at lower concentration are used [18,28,29]. In this trial, it is possible that the collected water was either stored for extended periods or contained antibiotics or chemicals used in cleaning livestock facilities, which could have reduced microbial activity and made the organic matter less biodegradable. [30]. Additionally, the high COD of the wastewater used in the experiments might also explain the results. In fact, other studies have observed lower COD reduction and coulombic efficiency when using slurry at high concentrations compared to MFCs fed with lower COD urban wastewater. [21]. Moreover, the literature suggests that high concentrations of N-NH₄⁺ can negatively impact COD removal in MFCs, as this inorganic component can disrupt bacterial enzymatic activity, alter intracellular pH, and induce osmotic stress [31]. In this trial, the initial concentration of N-NH₄⁺ was 538 ± 36.6 mg·L⁻¹, a considerably high value compared to the initial concentration observed in previous studies [29] (N-NH₄⁺, 198 ± 1 mg·L⁻¹) in which a higher COD reduction was reached was recorded.

The results showed the low efficacy of MFCs in eliminating TN, with an average removal efficiency of 3.7%. In the case of N-NH₄⁺, no removal efficiency was observed; instead, production was noted which could be due to mineralization of organic N as it is commented before. Typically, double-chamber MFCs with oxygenated aqueous cathodes are used to remove N-NH₄⁺ using this technology, where autotrophic bacteria can perform the nitrification process, allowing the oxidation of N-NH₄⁺ to N-NO₂⁻ and/or N-NO₃⁻. In this trial, as single-chamber anaerobic MFCs were used, nitrifying bacteria were unable to carry out their metabolic activity. Regarding N-organic, an average removal efficiency of 80.5% was achieved. This may be related to the ammonification process carried out by heterotrophic bacteria. Ammonification refers to chemical reactions in which amino groups (NH₂) associated with whereby N-organic is mineralized to NH₄⁺ [32]. During ammonification, N-organic is degraded, releasing N-NH₄⁺. This phenomenon, therefore, would explain the near-complete removal of N-organic and the increase in N-NH₄⁺. Concerning N-NO₂⁻ and N-NO₃⁻, low amounts were detected at both the beginning and the end of the trial. N-NO₂⁻ could be oxidized to N-NO₃⁻, but the absence of O₂ prevents this, so the initial amount of N-NO₂⁻ may have been transformed into gaseous N through a process known as heterotrophic denitrification where organic matter is oxidized under anaerobic condition using N-NO₂⁻ as an electron acceptor [33]. Also, N-NO₂⁻ could be eliminated by anaerobic ammonium oxidation (anammox), where, as the name suggests, N-NH₄⁺ is oxidized using N-NO₂⁻ as an electron acceptor, and, as a consequence, gaseous N is produced [34].

Presently, P is seen as a useful resource in wastewater due to its potential as a fertilizer, as P plays a crucial role in modern farming. Yet, prolonged application of P-rich animal waste to land results in P buildup in soil, which may result in movement through erosion, surface runoff, and underground leaching. This could lead to eutrophication, excessive algae growth, and O₂ depletion in surface waters [35]. The influence of MFCs on the P present in wastewater is of significant interest. This study showed a 1% removal with MFCs. Some studies suggest P recovery in MFCs through the precipitate that forms in single-chamber MFCs with an aerated cathode. The hypothesis regarding the precipitation mechanism is that, through the oxygen reduction reaction, the pH near the cathode increases locally, and the solubility of phosphate, ammonium, and magnesium reaches supersaturation, causing precipitation [36]. In this study, since P is usually at a low concentration in slurry compared to N, it is beneficial that it is not removed in the MFCs. This way, P remains available for subsequent use in microalgae cultures, as it is a fundamental nutrient for their growth.

In addition to analyzing the nutrient removal percentages, the pH and electrical conductivity were also evaluated at the beginning and end of the trial with the MFCs. Although most bacteria tend to show higher activity in a neutral pH environment, it has been demonstrated that the optimal pH for electricity production in single-chamber MFCs with an aerated cathode is between 8 and 10 [37]. In this trial, the pH was around 8, which likely contributed positively to electricity generation. The voltage increased over time, reaching a maximum of 195 ± 4.2 mV at 85 h. In a previous study that examined the behavior of different ILs, including the one used in this trial, both in electricity production and in the treatment of pig wastewater, it was shown that methyltrioctylammonium chloride can produce a maximum voltage of 159 mV, a value similar to that obtained in this trial [33].

Related to the voltage losses, there are three factors that can lead to losses and alterations in electrical performance in MFCs: electrode activation losses, ohmic losses, and mass transport losses [9]. The polarization test allows for a proper study of these three aspects. The first factor is electrode activation losses, which refer to the electricity losses caused by the activation barrier that must be overcome for oxidation at the anode or reduction at the cathode to occur. This is visualized as an exponential voltage loss curve, especially notable at low current densities, and can be corrected by (i) increasing the system temperature, (ii) increasing the surface area of the anode, and (iii) raising the concentration of redox mediators. Another factor is ohmic losses, related to the internal resistance of the MFCs. When current flows through the conductive materials during the transport of electrons and protons, some resistance to charge flow can be found, resulting in voltage losses. These losses typically occur at intermediate current densities, where electricity production is usually optimal. The last factor is mass transport losses, closely related to the supply of the oxidizing agent to the cathode (commonly O₂) and the removal of products [38,39]. If the O₂ supply to the cathode is insufficient or the reaction products accumulate because they cannot be removed, the efficiency of the MFCs will decrease, and voltage losses will occur, which will be visualized at high current densities [37]. The results highlight two of the three types of mentioned losses: activation losses, more prominent at low current densities, where a clearly defined exponential curve is observed, and ohmic losses, which are more significant at intermediate current densities and are characterized by their linear behavior. Additionally, no mass transport losses were observed, an advantage offered by single-chamber MFCs with an aerated cathode where oxygen from the surrounding air is always available. Furthermore, the anode was fed with wastewater that had a high concentration of organic matter. The concentration was related to the OCV; it was 455 ± 1.3 mV, with both the OCV and maximum power results being similar to those of [33], where an OCV of 424 mV and a maximum power density of 93 mW·m⁻² were obtained.

Following an initial pre-treatment of slurry using MFCs, the slurry was diluted to 10%, 50%, and 100% using distilled water. These treatments served as culture media for the growth of S. almeriensis. In addition, a treatment with chemical fertilizers was added, the composition of which is described in Section 2. Once the photobioreactors were inoculated with the corresponding culture media, the production was carried out in batch mode for 12 days. The growth of S. almeriensis was monitored in the four treatments using absorbance (680 and 750 nm) and cell counting with an optical microscope. The growth was observed in the cultures with fertilizer medium and 10% slurry, while the cultures with 50% and 100% slurry showed no growth. The absence of growth when using pig slurry at 50% and 100% concentrations can be explained by several factors such as (i) the high concentration of N-NH₄⁺. Previous studies indicate that values above 190–200 mg·L⁻¹ can be inhibitory to microalgal growth [27]. In this case, while the initial N-NH₄⁺ concentrations for fertilizers medium and 10% slurry medium were 5.5 and 52 mg·L⁻¹, for the 50% slurry and 100% slurry treatments, they reached 215.5 and 440 mg·L⁻¹, respectively. (ii) Another factor is the high turbidity of the pig slurry, which prevents light from penetrating the culture. Turbidity directly impacts light availability, as suspended particles in a turbid solution can scatter light, hindering the microalgae’s ability to utilize it for photosynthesis. In addition to these two reasons, the concentration of the initial inoculum in the assay could also be considered; a more concentrated inoculum or a greater amount of inoculum could be an alternative to address these two limitations. It may also be necessary to previously adapt the inoculum to the slurry by progressively increasing the concentration of slurry in the medium.

Additionally, higher values of absorbance and cell density were observed in cultures with 10% slurry compared to cultures produced with chemical fertilizers. In the latter, N and P are in excess, so they should not limit growth. However, no source of C was added, and they were not cultivated with aeration, so there could be a limitation of C, which is necessary for the proper growth of S. almeriensis. Furthermore, mixotrophic growth of S. almeriensis is possible in cultures with 10% slurry, since several microalgae, including species of the Scenedesmus genus, are mixotrophic and can simultaneously assimilate CO₂ and organic carbon [40].

In turn, biomass production was determined using dry weight and productivity, both being zero for the 50% and 100% slurry treatments and reaching productivity values 0.03 and 0.04 g·L⁻¹·day⁻¹ using the fertilizer medium and 10% slurry, respectively. The biomass productivity obtained with the 10% slurry medium is greater than that obtained with the medium with chemical fertilizers. The primary nutrients required for microalgal growth are N and P (along with C). In this study, the molar N/P ratio of the 10% diluted slurry was 26. The N/P ratio of algae and cyanobacteria is highly variable in nutrient-limited cells, ranging from approximately 5 mol N/mol P when P is abundant relative to N, to as high as 100 mol N/mol P when N is much more available than P. Under optimal nutrient-rich conditions, the cellular N/P ratio is somewhat more consistent, ranging from 5 to 19 mol N/mol P, with most observations below the Redfield ratio of 16 [41]. Therefore, a value of 26 can be considered high for an adequate growth of microalgal cultures and influence the biomass composition and their industrial applications. Environmental and operational conditions influence the composition of microalgal biomass, especially when wastewater is used as a culture medium. In previous studies, it has been observed that when diluted pig slurry is used as a growth medium for microalgae and inoculated with the genus Scenedesmus, the macromolecular composition shows approximately 40% proteins, 50% carbohydrates, and less than 10% lipids. The main applications of this biomass would be to produce biofertilizers or animal feed; it could also be used for biogas production [42].

The growth of microalgae using wastewater is not only influenced by the N/P ratio but also by the N and P sources, which induce specific conditions that can affect the community interactions and function of microalgae–bacteria consortia in wastewater treatment [43]. The culture medium with 10% slurry mainly presents N-NH₄⁺ as a N source, and the results showed a removal of more than 97% of it in the treated water. N-NH₄⁺ is eliminated in the microalgae–bacteria consortium through three main mechanisms: the oxidation to nitrite and nitrate (nitrification), assimilation into biomass (microalgae or heterotrophic bacteria), and volatilization as NH₃, which occurs at high pH and under intense aeration [15]. Since no increase in N-NO₂⁻ and N-NO₃⁻ concentration were observed in the effluent water, this mechanism is less likely, and the recovery of N in the form of biomass might have been favored.

These latter forms of N, N-NO₂⁻, and N-NO₃⁻ are present in very low concentrations, and in the outlet water, they are below 1 mg·L⁻¹. Overall, the TN measured at the outlet of the cultures produced with 10% slurry is 10 mg·L⁻¹. Therefore, the wastewater or slurry from the Veterinary Farm, in terms of TN, would comply with Directive 91/271/EEC for discharge, which establishes a maximum concentration of TN of 15 mg·L⁻¹ or 10 mg·L⁻¹, depending on whether the wastewater comes from facilities in which the pollution load is between 10,000 and 100,000 population equivalent (p.e.) or if it exceeds 100,000 p.e., respectively. The Directive defines 1 p.e. as “the biodegradable organic load with a five-day biochemical oxygen demand (BOD₅) of 60 g of oxygen per day.” The treatment plant at the Veterinary Farm of the University of Murcia has a treatment capacity of 500 m³·day⁻¹. Since an initial characterization of the wastewater was performed and the initial BOD₅ concentration (888.6 mg·L⁻¹) was determined, the pollution load of the water in terms of population equivalent was calculated, yielding a value of 7404.75 p.e. In this context, attention will be paid to the limits established for facilities with pollution loads between 10,000 and 100,000 p.e. Therefore, considering the TN concentration of the treated outlet water, it would comply with the discharge regulations.

This regulation also takes P concentrations into account. The results highlight the high percentage of consumption and removal (more than 94%) of P-PO₄⁻³ in cultures fed with 10% slurry. This removal efficiency could be related to two aspects: 1) P-PO₄⁻³ assimilation by microalgae and bacteria and 2) precipitation. The latter process can occur because, when microalgae absorb CO₂ for photosynthesis, an increase in pH can result, leading to P-PO₄⁻³ precipitation. This precipitation typically occurs at a pH between 9 and 11 [44]. Regarding assimilation, although it can be carried out by both microalgae and bacteria, [45], microalgae assimilation is the primary mechanism for phosphate removal. Normally, in a mixed environment, polyphosphate-accumulating organisms (PAOs) tend to release phosphate under aerobic conditions, while microalgae can use this released phosphate or the phosphate present in the medium to store it and incorporate it into biomass as polyphosphate, a process known as luxury phosphorus uptake [46,47]. In the effluent water, phosphorus concentration is observed at 1.10 mg·L⁻¹, with a removal efficiency of 94.37%, values that comply with the established limits since the maximum allowable concentration is 2 mg·L⁻¹, with a minimum removal efficiency of 80%.

The other relevant parameter in wastewater treatment is the concentration of COD in the effluent water. The outlet concentration is 106.7 mg·L⁻¹ in cultures with 10% slurry as the culture medium, complying with the regulation that sets a maximum value of 125 mg·L⁻¹ in treated water. Overall, the consumption of organic matter is carried out by heterotrophic bacteria thanks to the O₂ provided by the photosynthetic activity of the microalgae [48]. However, as mentioned previously it has also been found that some strains of the genus Scenedesmus can exhibit mixotrophic activity, consuming both organic and inorganic carbon.

The results obtained in this study are promising, as they demonstrate the potential to recover nutrients present in pig slurry in the form of bioelectricity and commercially valuable biomass for biofertilizers or animal feed, while simultaneously treating wastewater with high N-NH₄⁺ and COD content. However, this approach also presents several limitations such as (i) the need for the pre-treatment of pig slurry with MFCs. In this study, the use of a pre-treatment of pig slurry with MFCs to reduce turbidity has been assumed, but the direct use of pig slurry as a microalgal culture medium could be evaluated. (ii) Another limitation is scale limitations. The research has been conducted at a small scale, necessitating the upscaling of this technology. This is particularly crucial for MFCs, which have not yet been implemented at pilot or industrial scales. (iii) Finally, the dilution requirements is a limitation, as the necessary dilution of pig slurry to a 10% concentration for optimal microalgal growth presents a practical challenge. These limitations require optimization to render these technologies feasible under real-world conditions.

Further research and development are needed to address these challenges and enhance the applicability of this nutrient recovery and wastewater treatment approach in practical settings.

5. Conclusions

Single-chamber MFCs with aerated cathodes produce electrical energy effectively. However, challenges include difficulty in removing N compounds anaerobically, the inhibition of bacterial activity by high contaminant concentrations, and the necessity for fresh slurry for effective COD removal. Microalgae-based wastewater treatment is promising with 10% slurry but faces issues at higher concentrations (50% and 100%) due to limited light availability and high N-NH₄⁺ concentrations, which hinder the growth of microalgae. Using 10% slurry nearly eliminates N-NH₄⁺, and TN levels remain under regulatory limits (Directive 91/271/EEC). P-PO₄⁻³ removal is nearly 100%, meeting established limits, but pH control is needed for P recovery as a biomass. Microalgae–bacteria consortia effectively reduce COD to compliant levels. In the future, optimal pollutant reduction and improved MFC results require dual-chamber cells with oxygenated cathodes or alternative membranes like Nafion, despite higher costs and scalability issues. Diluting wastewater is necessary for microalgae experiments but raises sustainability concerns due to clean water usage. Recirculating wastewater could be an alternative, supporting future cultures, though turbidity may affect light availability. Promising methodologies need further research to address existing challenges.