Nutrient Transformations in LED Tubular Photobioreactors Used for a UASB Effluent System Followed by a Percolator Biological Filter

Abstract

This study investigated nutrient removal in tubular photobioreactors (PBR) illuminated by Light-Emitting Diodes (LEDs), treating the effluent from an Up-Flow Anaerobic Sludge Blanket (UASB) system followed by a Biological Percolator Filter (BPF). Designed as a tertiary treatment step, the PBRs aimed to minimize eutrophication while promoting microalgal growth through CO₂ assimilation released by bacterial activity—resulting in biomass with potential for value-added applications. The objective of this study was to elucidate the mechanisms responsible for nitrogen and phosphorus removal under a controlled environment. A key novelty of this work lies in the comparative evaluation of red and blue LED illumination in the pilot-scale PBRs used for wastewater treatment. All systems achieved >99% ammoniacal nitrogen removal, while soluble phosphorus removal achieved up to 95%. The highest nitrogen removal rate was observed under red light (10.60 mg L⁻¹ d⁻¹), although there was no difference for blue light, while blue light was more effective for phosphorus removal (3.28 mg L⁻¹ d⁻¹). Assimilation was the primary removal mechanism, supported by microalgae–bacteria interactions and artificial CO₂ injection. The research highlights distinct advantages of each light illumination: the blue-light photobioreactor was more suitable for tertiary treatment, whereas the red-light system showed potential for microalgal biomass-based co-product generation.

1. Introduction

The Up-Flow Anaerobic Sludge Blanket (UASB) process, followed by Percolating Biological Filtration (PBF), produces effluent that meets discharge standards in developing countries such as Brazil and has been widely studied for use in wastewater treatment plants [1]. However, this method does not have an effective removal rate of nutrients [2]. Therefore, implementing UASB-PBR post-treatment is crucial to mitigate the impact of the effluent from this type of wastewater treatment plant (WWTP) design [2,3]. The effluent from this type of WWTP contains significant residual loads of pollutants, particularly the nutrients nitrogen and phosphorus, which can lead to eutrophication. This is particularly problematic in lentic environments [4].

Only a few wastewater treatment plants are designed to remove nutrients, and it is common to remove one nutrient at a time (nitrogen or phosphorus). In general, activated sludge is the configuration used to remove these pollutants. However, it consumes a lot of electrical energy during operation, with energy-related costs accounting for 25% to 60% of total operating costs in conventional systems [5]. Moreover, some configurations do not completely remove nitrogen and phosphorus, such as UASB reactor treatments followed by PBF, in which aerobic conditions prevent the nitrification/denitrification process in the PBF, leaving the inorganic forms of these nutrients in the effluent [2].

Therefore, photobioreactors (PBR) could be a promising technological alternative to current forms of nutrient removal. Photobioreactors are able to remove nitrogen and phosphorus simultaneously. The microalgae present in this treatment assimilate inorganic nitrogen mainly in the form of nitrite (NO₂⁻), nitrate (NO₃⁻), and ammonium ions (NH₄⁺). The transformation of the different forms of inorganic nitrogen occurs across the plasma membrane, where NO₂⁻ and NO₃⁻ forms are reduced to NH₄⁺ and finally incorporated into amino acids [6]. It has also been observed that microalgae prefer NH₄⁺ to other forms of inorganic nitrogen due to lower energy consumption [7].

Similar to nitrogen removal, phosphorus removal occurs through assimilation by the microalgae’s metabolism. Phosphorus is available for direct biological metabolism in the form of orthophosphates (PO₄³⁻, HPO₄²⁻, H₂PO₄⁻, and H₃PO₄⁻). It is preferentially assimilated in the inorganic forms of H₂PO₄⁻ and HPO₄²⁻, and incorporated into organic compounds by phosphorylation after transport across the plasma membrane of the microalgae cell [8].

Thus, in line with the new management model of wastewater treatment plants, the use of wastewater as a resource for microalgae cultivation is considered a favorable approach for the production of microalgae biomass and co-products. Therefore, photobioreactors represent a promising alternative for the removal and reuse of nitrogen and phosphorus from wastewater, thereby contributing to the improvement of water quality [9,10].

Lighting is essential for the physiological responses of microalgae in photobioreactors, which can be artificially illuminated to enhance their performance. Light-emitting diode (LED) lighting has a longer lifetime, is more cost-effective, and is environmentally and human-friendly due to its lower energy consumption. In addition, LED lighting provides superior lighting quality with a range of specific wavelengths, making it an excellent choice for promoting microalgal biomass production and accumulation of specific compounds [11,12,13].

In addition, this treatment contributes to the formation of by-products from the production of algal biomass, which further enhances CO₂ sequestration, which is in line with the concept of sustainable wastewater treatment plants by reducing the operating costs of the plant and developing a technological alternative adapted to the feasibility of wastewater treatment [14].

Although several studies have been carried out using photobioreactors, few have focused on the effects of UASB-PBR effluent as a growth medium, particularly in systems combining microalgae and bacteria for nutrient removal and by-product generation. This study contributes uniquely to the field by comparatively assessing red and blue LED wavelengths to evaluate their specific effects on nutrient removal and biomass production. This research aims to investigate the transformation of nitrogen and phosphorus forms to better understand the main factors influencing their removal in a photobioreactor artificially illuminated by LEDs. To improve tertiary wastewater treatment, it is crucial to understand the effect of LED illumination at different wavelengths on the performance of microalgae photobioreactors.

2. Materials and Methods

2.1. Natural Consortium of Microorganisms and Preparation and Cultivation of the Inoculum

The consortium of microorganisms used to grow the inoculum was originally sampled in 2018 from a eutrophic pond used for tilapia farming at the Botanical Garden of the Federal University of Ouro Preto (UFOP), to obtain a variety of naturally occurring microorganisms.

A polyethylene container containing three liters of a consortium of natural microorganisms, with an average concentration of 139 ± 19 mg L⁻¹ of algal biomass, was filled with 20 L of synthetic wastewater. The synthetic wastewater was prepared according to the standard method of the Organization for Economic Cooperation and Development [15], with the addition of meat extract to the wastewater, containing the following chemicals: meat extract (650 mg L⁻¹), tryptone (160 mg L⁻¹), CH₄N₂O (30 mg L⁻¹), KH₂PO₄ (28 mg L⁻¹), NaCl (7 mg L⁻¹), CaCl₂. 2H₂O (4 mg L⁻¹), and MgSO₄.7H₂O (2 mg L⁻¹).

2.2. Photobioreactor Construction and Effluent Collection

The PBR was constructed on a pilot scale using an acrylic tube with an internal diameter of 76 mm, a height of 740 mm, and an acrylic thickness of 3 mm, with a useful volume of 3.35 L. A submersible aquarium pump was installed and operated continuously to prevent temperature-induced stratification of the photobioreactor fluid and sedimentation of the culture. A mixture of inoculum and effluent in volume proportions of 10 and 90%, respectively, was added to each photobioreactor. The photobioreactor had a tap at the bottom to collect effluent samples for analysis, and the top was left open to allow gas exchange with the external environment.

In order to achieve the required luminous flux, LED strips were arranged helically on the wall of a larger acrylic tube with an internal diameter of 145 mm and a wall thickness of 4 mm. To prevent excessive heat transfer from the LED strips into the growing medium, the LED strips were placed on another acrylic tube. However, five coolers and a portable mini fan were installed to help cool the LED strips.

The effluent used as a culture medium was collected from the Marzagão Wastewater Treatment Plant in Itabirito—MG, after it had passed through all the treatment stages of the plant—pre-treatment, UASB, BPF, and secondary decanter—at the Parshall flume at the outlet of the wastewater treatment plant. This type of effluent was chosen because it had a low organic content, low turbidity, and sufficient nitrogen and phosphorus concentrations for microalgae growth. The wastewater was collected in August, September, October, December 2018, and January 2019.

2.3. Experiment Design and Photobioreactor Operating Conditions

The photobioreactors were placed indoors to avoid interference from other light sources and operated on a 24:00 photoperiod. This photoperiod was chosen due to the need to maximize growth and algal biomass production. Chen et al. [16] found that the best specific growth rate was achieved with constant light. Furthermore, Ratledge and Cohen [17] highlighted that in cycles with periods of darkness, microalgae could lose up to 25% of the biomass produced through respiration.

The photobioreactors were operated in batches due to equipment limitations. Luminous flux in the form of Photosynthetically Active Radiation (PAR) was measured with a Delta OHM HD21012 photo-radiometer (Delta OHM, Italy) at various points on the inner wall of the photobioreactors when they were empty, accounting for the interference of the acrylic and to measure the luminous flux reaching the photobioreactor. The average of the flux values was used as a measure of distribution. A digital UV light meter (Instrutherm, model RS-232, Taiwan) was used to specifically measure 254 nm UV-C radiation to provide real evidence of the absence of UV radiation.

The wavelength of red light was chosen because it induces cell division in microalgae, while the wavelength of blue light induces an increase in cell size. A light flux of 200 µmol m⁻²s⁻¹ of photons was used because it did not cause photoinhibition and provided sufficient energy for photosynthesis. These wavelengths, together with the dark system as a control, were used to study the isolated effects of each, avoiding interactions arising from the combination of the different spectra. This allowed a better understanding of the microalgae’s responses to specific stimuli and provided more detailed information on the influence of each wavelength.

All photobioreactors, except the control (DARK), were equipped with a mesh of LED strips distributed over the entire lateral surface of the larger diameter acrylic to adjust the wavelength and luminous flux. The red photobioreactors were equipped with eight 5050 IP65 LED strips, and the blue photobioreactors were equipped with four 5730 IP65 LED strips and four 5050 IP65 LED strips to ensure that the luminous flux was close to 200 µmol m⁻²s⁻¹ photons. To evaluate the effect of light, the control reactors were set up with a kraft paper screen to minimize the influence of external light sources and to ensure a dark condition.

Microalgae were inoculated into all photobioreactors, including those kept in the dark. The photobioreactors were operated until nutrient limitation occurred, which was observed after approximately 84 h of operation. They were operated with CO₂ injections twice a day (every 12 h), except for the controls (

Table 1. Description of photobioreactor operating conditions.

Photobioreactor Identification				Irradiated Light	CO2 Injection Frequency
	Photobioreactor Blue	PBR B1	PBRB	Blue	Twice a day
		PBR B2
		PBR B3
	Photobioreactor Red	PBR R1	PBRR	Red	Twice a day
		PBR R2
		PBR R3
	Control	DARK 1	DARK	Dark	No injection
		DARK 2
		DARK 3

). The purpose of the CO₂ injection was to control the pH and prevent microalgae growth from being impaired by nitrogen volatilization, phosphorus precipitation, lack of inorganic carbon in the culture medium, or the combined effect of these factors [18,19,20,21,22]. The decision not to inject CO₂ into the DARK photobioreactor was made based on two main reasons: (i) in the absence of light, photosynthetic activity is inhibited, and therefore microalgae are unable to assimilate nutrients through photosynthesis [23], making CO₂ supplementation ineffective under dark conditions; and (ii) excluding CO₂ in the DARK group ensured a more accurate evaluation of the specific effect of light on microalgal growth, eliminating potential confounding variables. This approach allowed for a clearer comparison between illuminated and non-illuminated conditions regarding nutrient removal and biomass production.

The CO₂ was injected via a tube with a porous stone attached to a 99% concentration CO₂ cylinder to promote the smallest possible bubble size and facilitate gas solubility. The gas was manually injected at the same time that the pH was measured using a portable HACH HQ40d (Germany) multiparameter with a pHC 101 multiprobe (Germany). The injection was continued until pH < 7.0.

2.4. Microalgae Genera Identification

The microalgae were identified using an Olympus CX 31 microscope (Japan) at the Laboratory of Aquatic Ecology, Evolution, and Conservation (UFOP), based on the Bicudo et al. [24] database and www.algaebase.org.

2.5. Analytical Procedures

During the experiments, ammoniacal nitrogen (N_amon) and nitrate (NO₃⁻) were measured by liquid chromatography. According to Apha [25], spectrometry analysis was performed to analyze the ammoniacal nitrogen (4500 NH₃ C), total Kjeldahl nitrogen (NTK) and organic nitrogen (N_org) (4500 N_org B), and the soluble phosphorus (P_s) and total phosphorus (P_t) (4500 P D).

2.6. Chlorophyll a, Biomass Production, and Cell Growth

The analysis of chlorophyll a followed the ethanol extraction technique described in the Dutch norm NEN 6520 [26]. A defined sample volume was filtered through a membrane with a pore size of 0.7 µm, and this membrane was placed in a 16 × 100 mm glass tube, to which 10 mL of 80% ethanol was added. The cells were then subjected to a series of thermal shocks. The chlorophyll a was released into the ethanol, and the glass tube was placed in the fridge. Between six and 24 h later, the sample was read in a spectrophotometer at wavelengths of 665 nm and 750 nm. The sample was acidified with 0.4N HCl to a pH of approximately 2.6 to 2.8. After 2 min—to eliminate interference from pheophytin a, a phaeopigment that absorbs light in the same range as chlorophyll a—a new reading was performed on the spectrophotometer at the same wavelengths as before acidification.

Finally, the concentration of chlorophyll a was estimated using Equation (1), modified according to the Dutch standard [26].

Chlorophyll a = 29.6 × {(Eu_665 − Eu_750) − (Ea_665 − Ea_750)} × 10/(V × S)

(1)

where: Chlorophyll a = Chlorophyll a concentration (mg L⁻¹); 29.6 = specific absorption coefficient of chlorophyll a; Eu = absorbance of the unacidified sample; Ea = absorbance of the acidified sample; V = volume of the filtered sample (mL); 10 = volume of ethanol used (mL); and S = optical path (cm).

Biomass productivity was calculated using Equation (2), as proposed by Yao et al. [18], using SSV as a direct measure of biomass.

P = (SSV₁ − SSV₀)/(t₁ − t₀)

(2)

where: P = biomass productivity (mg L⁻¹ d⁻¹); SSV₁ = volatile suspended solids at time t₁ (mg L⁻¹); VSS₀ = volatile suspended solids at time t₀ (mg L⁻¹); and t₁ and t₀ = time (days).

3. Results and Discussion

3.1. Effluent Characterization

According to

Table 2. UASB-PBR effluent characteristics from Marzagão WWTP.

Parameter	Minimum and Maximum	Mean ± SD	CV (%)	n
Namon (mg L−1) 1	20.9–81.1	53.4 ± 24.5	46	9
NO3− (mg L−1) 2	0.8–8.5	3.8 ± 2.5	67	9
NTK (mg L−1) 3	23.6–74.6	58.7 ± 16.6	28	27
Ps (mg L−1) 4	2.2–11.6	8.7 ± 2.3	26	27
Pt (mg L−1) 5	5.0–13.3	10.4 ± 2.5	24	26

Note: ¹ N_amon = ammoniacal nitrogen; ² NO₃⁻ = nitrate; ³ NTK = total Kjeldahl nitrogen; ⁴ P_s = soluble phosphorus; ⁵ P_t = total phosphorus.

, the UASB-PBR effluent had different characteristics that affected the microalgae cultivation. The PBR B2 campaigns had the most significant differences in ammonia nitrogen measurements, which could be related to the treatment of the Marzagão Wastewater Treatment Plant or to the effluent’s characteristics. On the other hand, the DARK 2 had varying values in the ammonia nitrogen and phosphorus due to dilution caused by precipitation before sampling.

3.2. Inoculum Characterization

The microorganisms were identified at two different moments (in the inoculum and the photobioreactors). The identified microorganisms are shown in

Table 3. Identification of microorganisms in the photobioreactors and inoculum observations.

Phylum	Microorganisms	Inoculum	PBRR	PBRB
Chlorophytas	Chlorella sp.	Present	Present	Present
	Sphaerocystis spp.	Present	Present	Present
	Scenedesmus spp.	-	-	Present
Bacillariophyta	Diatomáceas	-	-	Present
-	Rotíferos	Present	-	-
-	Protozoários	-	-	Present

.

The presence of rotifers and protozoa altered the population balance due to predation on microalgae and bacteria, especially smaller ones such as Chlorella sp., which negatively affected the photobioreactor [27,28]. Tan et al. [22] stated that the high concentration of free ammonia and pH > 9.25 could be toxic to most microorganisms, especially protozoa. However, even in PBR B2, which reached a pH of 10.59, this was not sufficient to remove the protozoa.

3.3. Nitrogen and Phosphorus

Ammonium nitrogen (N-NH₄⁺) was practically removed from the culture medium in the photobioreactors, as shown in Figure 1a. It reached minimum values of 0.44 mg L⁻¹ in the blue light photobioreactors (PBRBs) and 0.32 mg L⁻¹ in the red light photobioreactors (PBRRs) at the end of the batch. This corresponds to a removal rate of 99%. The removal concentrations were 8.82 mg L⁻¹ d⁻¹ and 10.60 mg L⁻¹ d⁻¹ in the PBRBs and PBRRs, respectively. The dark photobioreactor (DARK) remained constant, with a removal rate of only 4%, and a lower removal rate of 0.40 mg L⁻¹ d⁻¹ was observed.

The ammoniacal nitrogen removal rates obtained in this study (8.82–10.60 mg L⁻¹ d⁻¹) are notably higher than those reported by Rada-Ariza et al. [29], who achieved a maximum volumetric ammonium removal rate of 50.9 mg L⁻¹ d⁻¹ under optimized conditions in a sequencing batch photobioreactor. While their system operated with synthetic wastewater and varying solids retention times (SRTs), the current study used real UASB-BPF effluent and LED lighting, showing competitive and robust nitrogen removal performance. These results reinforce the potential of LED-driven microalgal–bacterial PBRs in achieving efficient nitrogen removal in tertiary treatment applications.

Nitrogen in the form of nitrate (N-NO₃⁻) was completely removed in the DARK PBRs. The removal rate in the PBRBs was 32% (Figure 1b). In the PBRRs, the nitrate concentration increased by 5%. The complete removal of nitrate in the DARKs was due to the anaerobic conditions, where the depletion of dissolved oxygen in the batch reactors led to the consumption of nitrate by facultative aerobic microorganisms for respiration mechanisms [30].

Figure 2 shows the behavior of dissolved phosphorus in the photobioreactors. The results showed that 97% and 95% of the dissolved phosphorus was removed in the PBRB and PBRR photobioreactors. The removal rates observed for both treatments were 2.44 mg L⁻¹ d⁻¹ and 2.62 mg L⁻¹ d⁻¹, respectively. Similarly, the DARKs did not show a high removal, with only 5% of the dissolved phosphorus being removed, corresponding to a removal rate of 0.10 mg L⁻¹ d⁻¹.

According to the results, the PBRRs achieved a higher removal rate of dissolved phosphorus (2.62 mg L⁻¹ d⁻¹). However, considering the batch period, the PBRBs achieved a higher removal rate after 72 h of operation, with no significant removal rate after this time. Thus, our results showed that the PBRBs achieved a removal rate of 3.28 mg L⁻¹ d⁻¹ for dissolved phosphorus, based on the first three days of operation. Regarding the DARK photobioreactors, no phosphorus removal was observed (Figure 2).

This study found evidence that the rate of nitrogen and phosphorus removal was a time-dependent parameter. However, these data were not available in the literature, so calculations were required. Based on this, studies by Li et al. [31] reported total phosphorus removal rates ranging from 12.13 to 12.26 mg L⁻¹ d⁻¹ and ammonia nitrogen removal rates between 5.07 and 5.54 mg L⁻¹ d⁻¹. Data from Su et al. [32] were also calculated and showed total phosphorus removal rates of 0.33 to 0.40 mg L⁻¹ d⁻¹ and total nitrogen removal rates of 1.65 to 2.98 mg L⁻¹ d⁻¹. These comparisons demonstrate that the nitrogen and phosphorus removal rates achieved in both the red and blue LED photobioreactors are consistent with those reported in similar studies, reinforcing the efficiency and competitiveness of the system under controlled conditions.

The results showed that the nitrogen and phosphorus removal rates achieved in the blue and red photobioreactors were similar to the previous removal rates. Although the percentages of nutrient removal values were important, the rate is also crucial for understanding removal performance and designing an efficient wastewater treatment system.

In this study, the transformation of nitrogen and phosphorus during photobioreactor treatment was carefully investigated. According to the results obtained, three factors were essential to achieve high nitrogen and phosphorus removal simultaneously: (I) minimizing nitrogen volatilization, (II) minimizing nitrification and nitritation, and (III) adjusting the nitrogen and phosphorus ratio in the culture medium. The artificial injection of CO₂ every 12 h reduced nitrogen loss by volatilization due to better pH control. As a result, a high concentration of nitrogen was available for the assimilation and growth of the microalgae, thereby increasing phosphorus assimilation and improving the treatment. As suggested by Yan et al. [33], a sufficiently high nitrogen concentration was a prerequisite for the effective removal of phosphorus from wastewater by assimilation. The operation of photobioreactors under controlled conditions allowed the adjustment of the factors described above.

3.3.1. Nitrogen Transformations in the Photobioreactors Evaluated

Nitrogen transformations in the photobioreactors are shown in Figure 3. While the photobioreactors showed significant nitrogen transformations, the DARKs did not. Throughout the batch time, the levels of organic nitrogen, ammoniacal nitrogen, and NTK remained stable in the DARKs. In the absence of oxygen gas (DARKs), nitrate was removed by anoxic respiration, which occurs under anaerobic conditions.

Another possible transformation process in the photobioreactors was volatilization. It is important to note that there was no significant growth of algal biomass in the DARKs, and the pH did not exceed 8.5. As a result, there was no loss of volatilized nitrogen to the atmosphere (

Table 4. Nitrogen transformation in the photobioreactors.

Photobioreactor	Initial Nitrogen (mg L−1)				Final Nitrogen (mg L−1)				Assimilated Nitrogen		Nitrificated Nitrogen		Volatilized Nitrogen(a)		Nnt
	Norg	N-NH4+	N-NO3−	NTI	Norg	N-NH4+	N-NO3−	NTI	(mg L−1)	%	(mg L−1)	%	(mg L−1)	%	(mg L−1)	%
PBR B1	23.7	47.9	1.3	72.9	52.6	0.0	0.0	52.6	28.9	40%	−1.3	−2	20.3	28	23.7	33
PBR B2	20.8	22.8	1.3	44.9	31.9	1.3	5.0	38.3	11.1	25%	3.7	8	6.7	15	27.2	60
PBR B3	26.7	36.4	4.8	68.0	36.0	0.0	0.0	36.0	9.3	14%	−4.8	−7	32.0	47	26.7	39
PBR R1	17.2	57.0	0.9	75.1	64.3	0.9	0.0	65.1	47.0	63%	−0.9	−1	10.0	13	18.1	24
PBR R2	23.2	46.2	3.0	72.4	56.0	0.0	0.0	56.0	32.8	45%	−3.0	−4	16.4	23	23.2	32
PBR R3	38.5	25.0	2.1	65.6	66.9	0.1	6.4	73.3	28.4	43%	4.2	6	−7.7	−12	44.9	68
DARK 1	14.3	51.5	1.1	66.9	27.4	47.8	0.0	75.3	13.2	20%	−1.1	−2	−8.4	−12	62.1	93
DARK 2	17.4	15.7	0.8	34.0	18.3	14.2	0.0	32.5	0.9	3%	−0.8	−2	1.5	4	31.6	93
DARK 3	13.1	52.0	7.3	72.4	18.0	52.4	0.0	70.4	4.8	7%	−7.3	−10	2.0	3	65.6	91

Note: N_TI = initial total nitrogen = (N_org)initial + (N-NH₄⁺)initial + (N-NO₃⁻)initial; N_TF = final total nitrogen = (N_org)final + (N-NH₄⁺)final + (N-NO₃⁻)final; Assimilated nitrogen = (N_org)final − (N-org)initial; Nitrified nitrogen = (N-NO₃⁻)final − (N-NO₃⁻)initial; Volatilized nitrogen = N_TF − N_TI; N_nt = Untransformed Nitrogen = (N_org)initial + (N-NH₄⁺)final + (N-NO₃⁻)initial + Nitrified Nitrogen; Total = Assimilated nitrogen + Volatilized nitrogen + N_nt; Percentage values are given as a function of N_TI; (a) = used to close the balance sheet.

). On the other hand, in PBR B3, the volatilization of the nitrogen fraction was the main mechanism. The higher percentage of volatilized nitrogen in PBR B3 was related to the higher pH observed (9.0) during the first 12 h of cultivation. This can be explained by the physicochemical process of volatilization, which corresponds to the transformation of ammonium ions into ammonia, which is then lost to the atmosphere in the gas phase. Volatilization generally occurs at higher temperatures and pH [6,34].

In PBRBs and PBRRs (Figure 3), ammonia nitrogen (N-NH₄⁺) generally decreased due to nitrification (I), assimilation by microalgae (II) and volatilization (III). The nitrification processes were observed in all LED PBRs, as the nitrate concentration increased up to 72 h in both the blue and red light. It is well known that nitrification occurs in the presence of oxygen, where ammonia is oxidized to nitrite and nitrite is oxidized to nitrate (N-NH₄⁺ → NO₂⁻ → NO₃⁻) by autotrophic bacterial species [2,35].

These results showed that in PBR B1, PBR B3, PBR R1, and PBR R2, almost all of the ammonium was converted within 84 h of operation (Figure 3). It was also observed that the absence of ammonium resulted in nitrate transformation. Equally important, after 15 h of operation, there were high levels of dissolved oxygen in the LED photobioreactors, making denitrification unlikely. Therefore, it was emphasized that the microalgae were responsible for the assimilation of nitrate in the last 12-h batch. Thus, these results indicated that microalgae preferred to assimilate ammonium rather than nitrite or nitrate, as supported by Rashid et al. [36].

Further evidence of the microalgae’s preference for ammonium assimilation was demonstrated in PBR B2 and PBR R3 (Figure 3). These photobioreactors had adequate ammonium concentrations for microalgae assimilation throughout the batch time. Thus, ammonium assimilation by microalgae was predominant, and consequently, nitrate was not assimilated but accumulated. In PBR B1, PBR B3, PBR R1, and PBR R2, nitrate was removed 12 h after ammonium assimilation was complete, regardless of the concentration. It was therefore evident that the addition of 12 h would ensure nitrate removal from PBR B2 and PBR R3. As shown in the study by Su et al. [32], when ammonia nitrogen was not limiting in the experiment, nitrate was not removed, as observed in the results for PBR B2 and PBR R3.

In addition, the nitrification process was observed in PBR B2 and PBR R3. The rate of ammoniacal nitrogen oxidized to nitrate was 8% and 6% in PBR B2 and PBR R3, respectively (Table 4). As a result, this process did not remove the nutrient, so inorganic nitrogen remained a problem in the photobioreactors. These results showed that microalgal growth was affected because the ammonium was preferred for intracellular uptake by microalgae. Therefore, microalgae use more energy to convert nitrate to ammonium [6]. Based on the preference of microalgae to assimilate ammonia over other forms of nitrogen, the paper by Krustok et al. [37] showed that inhibition of nitrification in a photobioreactor resulted in higher microalgal growth rates. As a result, the dominant microalgal genera changed compared to the control experiment where nitrification occurred.

Considering the previous results, PBR B2 and PBR R3 showed that 60% and 68% of nitrogen was not converted (Table 4). These results were similar to those in photobioreactors, where nitrate assimilation by microalgae did not occur due to the limiting ammonia nitrogen concentration during the batch period [32]. It was observed that the PBR B2 and PBR R3 had lower concentrations of ammonia nitrogen, which does not contribute to an efficient assimilation process.

Further evidence of the preference for assimilation processes was the increase in organic nitrogen concentration, which is the rate at which nitrogen is converted into microalgal biomass. In the LED photobioreactors shown in Figure 3, the organic nitrogen concentration increased while the ammoniacal nitrogen concentration decreased. These processes occurred through nitrification and assimilation. In addition, after the consumption of ammoniacal nitrogen, nitrate was completely assimilated as an inorganic nitrogen source in PBR B1, PBR B3, PBR R1 and PBR R2.

From these results, it was clear that assimilation was the predominant mechanism in the artificially lit reactors. From the results, the percentage of removal by each process was calculated, and Table 4 shows the assessment of the forms of nitrogen after transformation. Nitrite could not be quantified. In the DARKs, 91% and 93% of the nitrogen was not transformed due to the anaerobic conditions in these reactors, resulting in low nutrient removal (Table 4).

The research conducted by González-Fernández et al. [38] highlights the importance of nitrogen assimilation by algal biomass rather than nitrification or denitrification processes in four ponds fed with pig slurry. In the anaerobically digested slurry, nitrogen was found to be converted to nitrite (36%) and nitrate (58%), followed by 12–27% assimilation and 9–16% denitrification. For fresh slurry analysis, the major nitrogen transformation was denitrification (40–57%) under natural lightning conditions and 20–23% denitrification under artificial lightning. The contribution of nitrogen transformation by assimilation was about 34% to 41% in the latter pond.

The current evidence on nitrogen transformations by microalgal and bacterial consortium is supported by the findings of Wang et al. [39]. It shows that nitrogen removal in wastewater can occur through different pathways: (I) nitrification and denitrification by bacteria, (II) nitrogen assimilation by microalgae and bacteria, and (III) nitrification processes due to oxygen production by microalgae and organic matter consumption by bacteria, resulting in energy benefits.

In this context, nitrifying and denitrifying bacteria play a fundamental role in complementing the direct ammonium assimilation performed by microalgae. These bacteria convert nitrogen into less toxic forms of dinitrogen gas (N₂), effectively removing it from the system and enhancing overall nitrogen removal efficiency. Meanwhile, microalgae contribute by regulating environmental conditions, producing oxygen via photosynthesis and releasing organic exudates, which support bacterial activity. This mutualistic interaction establishes a synergistic system where both groups benefit and optimize nutrient removal performance. Such interactions have also been documented by González-Camejo et al. [1], who demonstrated that the coexistence of nitrifying bacteria and microalgae in photobioreactors improved nitrogen removal while enhancing process stability [29,40,41].

3.3.2. Phosphorus Transformations in the Photobioreactors Evaluated

There was no phosphorus removal in the control reactors (Figure 4). As these reactors operate as anaerobic systems, there is practically no phosphorus removal, which is common in anaerobic biological treatment processes. This is probably because anaerobic treatment is less efficient at removing recalcitrant materials than aerobic treatment [2].

In contrast, within PBRB and PBRR, bacteria play an important role in phosphorus removal. Certain heterotrophic bacteria, known as phosphorus-accumulating organisms (PAOs), are capable of taking up large amounts of orthophosphate and storing it intracellularly as polyphosphate under aerobic conditions, thereby effectively removing phosphorus from the aqueous phase. Additionally, bacterial communities contribute to the mineralization of organic phosphorus compounds, converting them into bioavailable orthophosphate that can be readily assimilated by microalgae. These interactions are further enhanced by the oxygen released through microalgal photosynthesis, which supports aerobic bacterial activity, while bacteria in turn supply CO₂ and enzymes that facilitate nutrient cycling and promote algal growth. This synergistic relationship significantly improves the overall efficiency of phosphorus removal in photobioreactor systems [41,42,43].

The mechanism of phosphorus removal was investigated by analyzing dissolved phosphorus and quantifying orthophosphates and polyphosphates. The results revealed a decrease in dissolved phosphorus in all photobioreactors, accompanied by an increase in organic and particulate phosphorus (Figure 4). This suggests that biomass assimilation by the algae was the primary mechanism of phosphorus removal. This process is crucial as phosphorus plays a vital role in providing energy for microalgal metabolism and in the production of ATP and ADP [6].

Our results showed that during the first 12 h of the batch, the PBRBs started to remove phosphorus, in contrast to the PBRRs (Figure 4). This difference can be attributed to the phenomenon of “luxury uptake,” where microalgae absorb excess phosphorus under stress conditions. Such stress could have been induced by environmental factors affecting the process. Powell et al. [44] showed that “luxury uptake” can be optimized by exposing microalgae to controlled stress conditions.

Furthermore, the presence of Scenedesmus spp. in the photobioreactors under blue light wavelengths was associated with the removal of dissolved phosphorus. A similar pattern was observed by Beuckels et al. [45], who found that Scenedesmus accumulated more phosphorus in its biomass compared to Chlorella. These findings are also consistent with those of Kim et al. [46], where Scenedesmus grown under blue light showed a higher efficiency in removing dissolved phosphorus. This consistency between studies highlights the enhanced ability of Scenedesmus to remove phosphorus more efficiently under specific light conditions.

According to the results, the phosphorus content in the biomass on the last day of cultivation varied from 1.0% to 2.0% (

Table 5. Phosphorus concentration in the biomass.

	PBR B1	PBR B2	PBR B3	PBR R1	PBR R2	PBR R3
SSV (mg L−1)	683	455	872	875	1051	889
Organic phosphorus (mg L−1)	6.62	7.00	14.92	11.65	15.62	18.10
Biomass phosphorus (%)	1.0	1.5	1.7	1.3	1.5	2.0

). In line with previous studies, it is important to point out that a higher phosphorus content in the biomass was achieved compared to the results of Tan et al. [22], where the phosphorus content in the algal biomass was approximately 0.5% and 0.7%. A similar conclusion was reached by Beuckels et al. [45], who obtained phosphorus content between 0.5% and 1.3% in Chlorella cultures and 0.5% to 1.7% in Scenedesmus cultures. The phosphorus content of algal biomass can vary depending on the culture used. In open pond systems, the phosphorus content in the biomass is around 1%, while in more controlled cultures, it could reach 3.3%. It is known that nutrient recovery and the potential reuse as biofertilizer increases with increasing phosphorus content in the biomass [47,48].

In this study, the phosphorus content in the biomass on the final day of cultivation ranged from 1.0% to 2.0% (Table 5). Higher phosphorus content in the biomass is associated with greater nutrient recovery and potential reuse as biofertilizers. These results align with previous studies, such as Tan et al. [22], who reported phosphorus contents in algal biomass ranging from 0.52% to 0.69%. Similarly, Beuckels et al. [45] observed phosphorus contents ranging from 0.5% to 1.3% in Chlorella cultures and 0.5% to 1.7% in Scenedesmus cultures, concluding that Scenedesmus accumulates more phosphorus in its biomass than Chlorella.

Although complete removal of dissolved phosphorus was not observed in PBR B3 and PBR R3, the levels of phosphorus observed in the biomass were similar to the highest levels of phosphorus found in this study, confirming the robustness of the system (Table 5). Specifically, in PBR B3, it was shown that all dissolved phosphorus was removed after 72 h of batch time (Figure 4). On the other hand, the increase in phosphorus concentration after this batch period was probably due to “luxury uptake”, a mechanism that allows phosphorus to return to the culture medium, possibly intensified in PBR B3 due to the greater stress caused by the experimental conditions and the effluent, such as pH, which reached higher values in PBR B3 than in the others, as shown by Powell et al. [44].

To better understand the phosphorus transformations in the photobioreactors, the dissolved phosphorus fraction was analyzed as reactive and non-reactive phosphorus (Figure 5). The reactive dissolved phosphorus corresponded to orthophosphate forms, while the non-reactive dissolved phosphorus included acid-hydrolyzed forms (polyphosphates) that were filtered on the 0.45 µm pore membrane. Organic phosphorus is now referred to as particulate phosphorus for purposes of literature comparison [2].

According to the previous results, reactive dissolved phosphorus was the preferred form of phosphorus assimilation by algal biomass and was rapidly consumed in the initial period of PBRB operation. In contrast, PBR R1 and PBR R3 required more time to effectively remove phosphorus, as shown in detail in Figure 5. The PBR R2 increased the reactive dissolved phosphorus in the first 12 h, in contrast to the other photobioreactors. This could be explained by the higher concentration of non-reactive dissolved phosphorus during the initial period of the photobioreactor. At this point, the process of hydrolysis takes place rather than the assimilation of the reactive dissolved form. This was consistent with a more pronounced reduction of non-reactive dissolved phosphorus in the first 12 h of PBR R2, which was not observed in the other photobioreactors [6].

In addition, the results presented in Table 5 showed that reactive dissolved phosphorus was completely removed in PBRBs, PBR R1, and PBR R2 after 84 h of cultivation, whereas PBR R3 required 96 h. This may be related to the high concentrations of non-reactive forms of phosphorus in PBR R3, which were slowly converted to orthophosphates [2].

Another evidence supporting assimilation as the main mechanism is the increase in particulate phosphorus (Figure 5). It can be seen that the amount of particulate phosphorus was directly related to the amount of phosphorus assimilated by the algal biomass. This preferred assimilation mechanism is reported in

Table 6. Phosphorus transformations in the reactors.

Photobioreactor	Initial Phosphorus (mg L−1)				Final Phosphorus (mg L−1)				Assimilated Phosphorus		Mineralized Phosphorus (a)		Non-Transformed Phosphorus
	Particulate	Reactive Dissolved	Non-Reactive Dissolved	Total	Particulate	Reactive Dissolved	Non-Reactive Dissolved	Total	(mg L−1)	%	(mg L−1)	%	(mg L−1)	%
PBR B1	2.6	6.3	4.6	13.5	6.6	0.0	0.1	6.8	4.1	30%	6.8	50%	2.7	20%
PBR B2	2.1	5.3	2.9	10.3	7.0	0.0	0.0	7.0	5.0	48%	3.3	32%	2.1	20%
PBR B3	4.0	5.9	4.4	14.3	14.9	0.0	0.7	15.6	10.9	76%	−1.3	−9%	4.7	33%
PBR R1	2.5	8.1	2.4	13.0	11.7	0.0	0.1	11.8	9.2	71%	1.2	9%	2.6	20%
PBR R2	1.4	5.6	6.8	13.8	15.6	0.0	0.1	15.8	14.3	103%	−1.9	−14%	1.5	11%
PBR R3	1.9	5.5	4.7	12.1	18.1	0.0	1.4	19.5	16.2	133%	−7.4	−61%	3.3	27%
DARK 1	4.1	5.8	2.4	12.3	3.6	5.8	3.7	13.1	−0.5	−4%	−0.7	−6%	13.6	110%
DARK 2	1.7	2.2	2.4	6.3	5.4	0.6	1.8	7.9	3.7	58%	−1.5	−24%	4.2	66%
DARK 3	4.8	7.4	4.3	16.5	4.0	7.9	5.2	17.1	−0.8	−5%	−0.6	−4%	17.9	109%

Note: Non-reactive dissolved phosphorus = dissolved phosphorus − reactive dissolved phosphorus; Total phosphorus = particulate phosphorus + reactive dissolved phosphorus + non-reactive dissolved phosphorus; Assimilated phosphorus = (particulate phosphorus)final − (particulate phosphorus)initial; Mineralized phosphorus = final total phosphorus − initial total phosphorus; Unprocessed phosphorus = (particulate phosphorus)initial + (dissolved reactive phosphorus)final + (dissolved non-reactive phosphorus)final; The percentage values are given as a function of the initial total phosphorus.; (a) = used to close the balance sheet.

, where the PBR B2, PBR B3, PBR R1, PBR R2, and PBR R3 showed concentrations of organic phosphorus ranging from 48% to 133%. Therefore, the potential use of the produced algal biomass as a biofertilizer could be considered due to its high nutrient concentrations.

Although the reactors showed variability between replicates over time, some patterns could still be identified. This variability is likely to be due to the batch treatment, where, despite maintaining consistent conditions, slight variations in effluent characteristics may have influenced the process. In particular, PBR B produced less biomass than PBR R; however, it achieved higher phosphorus removal, as shown in Figure 5 and Table 6.

3.4. Production and Specific Growth Rate of the Biomass

Algal biomass in the photobioreactors grew, reaching SSV concentrations of 938 ± 102 mg L⁻¹ and 670 ± 187 mg L⁻¹ on day four in the red and blue PBRs, respectively (Figure 6). There was no significant growth in the control reactor, with concentrations of 182 ± 46 mg L⁻¹ SSV, consistent with the reduced anaerobic biomass production. Algal biomass productivity (Equation (2)) is a parameter that takes into account initial biomass concentration and time, and is more appropriate for assessing productivity. It was 198 mg L⁻¹ d⁻¹, 129 mg L⁻¹ d⁻¹, and 16 mg L⁻¹ d⁻¹ in the PBR R, PBR B, and PBR D, respectively.

The PBR R had a higher SSV concentration and productivity than the PBR B in every case. This was confirmed by Shu et al. [49], who obtained the best biomass production from Chlorella sp. under red light wavelength, while also testing green and blue wavelengths.

In order to clarify the growth of algal biomass and confirm that the growth of SSV was caused by microalgae, the behavior of chlorophyll a in the reactors was observed (Figure 7). In the red and blue photobioreactors, chlorophyll a increased in the same proportions as SSV, and more significantly in PBR R, with 13.16 ± 0.68 mg L⁻¹, compared to PBR B, with 9.34 ± 3.04 mg L⁻¹, both measured at the end of the experiment. The control did not present the same behavior as the other photobioreactors; the concentration of photosynthetic pigment was 0.90 ± 0.94 mg L⁻¹ from the initial moment and 0.63 ± 0.15 mg L⁻¹ of chlorophyll-a at the end of 96 h of cultivation.

Chlorophyll a/SSV ratios (%), a simple measure of cultivation status, was 1.4% in PBRs R, 1.4% in PBRs B, and 0.4% in the control. For photobioreactors, values between 1 and 1.5% of these ratios indicate a healthy population of microalgae, as observed in the blue and red PBRs, and values less than 1% indicate unhealthy algae, as in the control [50].

There have been few studies on the assessment of chlorophyll in microalgae cultivation systems. Santiago et al. [28] evaluated the production of algal biomass in two open ponds using UASB reactor effluent as a cultivation medium and obtained chlorophyll a/SSV ratios between 1.0% and 1.6%, with average SSV values of 124 mg L⁻¹ and 152 mg L⁻¹. Assemany et al. [51] obtained chlorophyll a/SSV ratios between 1.0% and 1.8%, with average SSV values between 95.5 mg L⁻¹ and 148 mg L⁻¹, using the same UASB reactor effluent to operate six open ponds for the cultivation of algal biomass. It should be noted that the data obtained by Santiago et al. [28] and Assemany et al. [51] were both from open pond systems (with natural sunlight source) operated in continuous flow systems, and with a microalgae–bacteria consortium. These studies reinforced the advantage of photobioreactor cultivation systems, which proved to be more efficient in algal biomass production, as the blue and red photobioreactors produced 4.4 to 9.8 times more concentrated SSV than the open ponds in question.

4. Conclusions

The present results confirmed that ammonia nitrogen removal was greater than 99% in all photobioreactors. Ammonia nitrogen removal was 8.82 mg L⁻¹ d⁻¹ and 10.60 mg L⁻¹ d⁻¹ in photobioreactors illuminated with blue wavelengths (PBRB) and photobioreactors illuminated with red wavelengths (PBRR), respectively. The higher rate observed in PBRR may be related to a higher initial concentration of the nutrient during the initial period.

Regarding nitrogen transformations in the photobioreactors, microalgae showed a preference for ammoniacal nitrogen assimilation. In the absence of ammoniacal nitrogen, nitrate was the second preferred substance. Assimilation was the main nitrogen transformation mechanism in PBR B1, PBR B2, PBR R1, PBR R2, and PBR R3, varying between 25% and 63%. In PBR B3, volatilized nitrogen was the main transformation mechanism, responsible for 47%, reaching a pH peak close to 9 in the first 12 h of the batch.

Analysis of dissolved phosphorus showed that 97% was removed by the PBRB and 95% by the PBRR. Although the PBRR removed high concentrations of phosphorus, the PBRB required less time and was, therefore, more efficient. As a result, the removal rate of dissolved phosphorus was 2.62 mg L⁻¹ d⁻¹ in the PBRR and 3.28 L⁻¹ d⁻¹ in the PBRB, mainly due to the presence of Scenedesmus spp. and blue light.

Regarding the phosphorus transformations in the photobioreactors, phosphorus was removed in the PBRBs during the first 12-h batch, but not in the PBRRs. Presumably, the luminous flux of the PBRB was sufficient to induce a state of stress in the microalgae biomass. This high luminous flux induces luxurious absorption in these photoreactors. The phosphorus content of the biomass varied between 1.0% and 2.0%. These results showed an increase in organic phosphorus, and it was possible to assume that assimilation was the predominant phosphorus removal mechanism in PBR B2, PBR B3, PBR R1, PBR R2, and PBR R3.

In summary, the present study confirmed that there were no differences in nitrogen removal between blue and red photobioreactors. PBRR produced more biomass and was preferred for by-product utilization. However, PBRB was more suitable for tertiary treatment of UASB-PBR effluent due to better phosphorus removal.

These findings are highly relevant for assessing the applicability of this technology in wastewater treatment plants, demonstrating the feasibility of achieving high nitrogen and phosphorus removal efficiencies, along with the potential valorization of the produced biomass. However, despite the promising results, it is essential to acknowledge the practical limitations of this study. The experiments were conducted in batch mode using a pilot-scale photobioreactor, whereas full-scale wastewater treatment plants typically operate under continuous flow conditions, which involve variable influent characteristics and flow rates depending on the municipality. Therefore, further research is needed to bridge this gap, including continuous pilot-scale experiments that more accurately simulate real operational scenarios and real-scale experiments. Additionally, future studies should investigate the combined use of red and blue LED illumination to determine whether this approach can synergistically optimize both biomass production and nutrient removal and quantify CO₂ uptake rates and energy efficiency compared to conventional systems. Exploring strategies to enhance the recovery and reuse of microalgal biomass as a by-product could further support the development of cost-effective and sustainable wastewater treatment solutions.