Integration of Aquaculture Wastewater Treatment and Chlorella vulgaris Cultivation as a Sustainable Method for Biofuel Production

Abstract

The integration of microalgae cultivation in the treatment of aquaculture wastewater (AWW) offers a sustainable solution for the recovery of nutrients and the valorisation of biomass. In this study, the potential of Chlorella vulgaris for growth in raw AWW and its variants was investigated and the efficiency of nutrient removal, biochemical composition of biomass, biodiesel potential by FAME analysis, and biogas production were evaluated. C. vulgaris was cultivated in three media: raw AWW, microelement-enriched AWW, and a synthetic base medium. Raw AWW allowed for the highest biomass production (2.4 g VS/L) and nutrient removal efficiency (ammonia: 100%, phosphate: 93.7%, nitrate: 37.8%). The addition of microelements did not significantly improve growth or nutrient uptake. The biomass grown on AWW showed a favourable lipid profile for biodiesel, dominated by C16:0 and C18:1. The highest biogas and methane yields were recorded for biomass from raw AWW as 358 ± 11 L/kg VS and 216 ± 7 L/kg VS, respectively. The results confirm that AWW is a suitable medium for the cultivation of C. vulgaris, enabling efficient wastewater treatment and the production of high-quality biomass.

1. Introduction

Aquaculture and microalgae could form a promising integrated biorefinery, where wastewater from aquaculture can be used to produce algal biomass, while the biomass produced can be used for a range of applications, including the production of food, feed, biofuels, and biofertilisers [1,2,3]. The growing demand for sustainable and efficient systems to meet global food security, energy, and environmental goals is driving research into the cultivation of microalgae in aquaculture effluents [4,5].

Globally, aquaculture is one of the most rapidly developing sectors of food production [6]. According to the FAO 2024 report, global fisheries and aquaculture production will increase to 223.2 million tonnes in 2022, which corresponds to an increase of 4.4% compared to 2020 [7]. This will generate considerable quantities of wastewater that require sustainable management [8]. Aquaculture wastewater (AWW) contains significant amounts of organic matter (100–150 mg/L as COD), nitrogen (3–7 mg/L as ammonium and 2–110 mg/L as nitrates), and phosphorus (2–50 mg/L as phosphates), which, if left untreated, can contribute to pollution and eutrophication in aquatic ecosystems [1,3]. However, this wastewater can be effectively utilised in integrated systems with microalgae to ensure the recycling of nitrogen and phosphorus for the production of algal biomass [2,9,10,11].

The integration of wastewater treatment with microalgae cultivation is not only beneficial from an environmental point of view, but in terms of economic viability [12,13]. A review of recent advances in the utilisation of AWW for algae cultivation has shown that microalgae can produce biomass rich in proteins, lipids, carbohydrates, and vitamins, making them ideal candidates for animal feed and food ingredients [14]. Microalgae biomass can also be a food source for fish [15,16]. In addition, the lipids produced by microalgae can be further processed into biodiesel, carbohydrate-rich biomass is suitable for bioethanol production, while the anaerobic digestion of microalgae biomass leads to the production of biomethane, all renewable energy sources [17,18]. Microalgae have also been shown to contribute to soil health and fertility when processed into biofertilisers and are an acceptable alternative to chemical fertilisers [19,20].

There are many types of microalgae that could be supported by the nutrients available in AWW. According to the literature, microalgae, such as Nannochloropsis gaditana, Palmaria palmata, Ulva lactuca, Chlamydomonas, Haematococcus pluvialis, Selenastrum sp., Monoraphidium griffithii, Scenedesmus, Chlorella sorokiniana, and Chlorella vulgaris, have been shown to be the most suitable for the simultaneous treatment of AWW and biomass yield [3]. However, C. vulgaris, in particular, has attracted attention due to its rapid growth rate, high nutrient uptake, ability to thrive under a wide range of environmental conditions, and tolerance to high concentrations of nitrogen and phosphorus compounds in AWW [21,22]. In the study on the removal of toxic pollutants and nutrients from wastewater, the concentrations of nitrates, COD and BOD were reduced by up to 93%, 95% and 92%, respectively [23]. Ma et al. [24] found that the monoculture of C. vulgaris is the optimal strain for the removal of ammonium nitrogen, total nitrogen, and orthophosphate from artificial marine culture wastewater, with a removal rate of 97.15%, 95.90%, and 96.62%, respectively, in 72 h. The cultivation of Chlorella on undiluted agricultural wastewater with a high ammonium concentration of 221 mg/L/day showed the ability to synthesise proteins in up to 52% of the biomass [25]. In other experiments, an ammonium concentration of 4315 ± 834 mg N/L stimulated lipid accumulation of up to 50% of the biomass with a simultaneous ammonia and phosphorus removal of over 95% [26]. Chlorella has also been reported to be the most commonly consumed microalgae as a food source for humans and animals [27]. Chlorella extracts had a biostimulant effect on plants to promote their growth and increase the quality of fruits by improving plant metabolism and synthesis of antioxidants [28].

To summarise, the integration of microalgae cultivation with aquaculture offers the possibility of utilising aquaculture wastewater while producing valuable bio-based products. However, further research is needed to develop waste-free conversion technologies on a commercial scale [3]. In this study, we investigated the growth of C. vulgaris in raw wastewater without pH regulation compared to growth in a base medium and a microelement-enriched wastewater. The potential of an AWW treatment with nutrient removal was evaluated. The utilisation of the C. vulgaris biomass in terms of production efficiency, fatty acid methyl ester (FAME) composition, and biogas production was also determined.

2. Materials and Methods

2.1. Experimental Study Organisation

The experiment was divided into four sequential stages (S). In Stage 1 (S1), the aim was to evaluate the growth capacity of C. vulgaris microalgae in different types of culture media and to determine the efficiency of nutrient uptake from each medium tested. Stage 2 (S2), the biochemical composition and properties of the C. vulgaris biomass obtained were analysed. In Stage 3 (S3), the content of fatty acid methyl esters (FAMEs) in the biomass produced in all three experimental variants was determined, focussing on the suitability for biodiesel production. In Stage 4 (S4), the biogas and methane potential of the algae biomass cultivated in the tested media were evaluated. All test stages were carried out in three variants (V), which differ in the type of culture medium used to cultivate the microalgae biomass. In Variant 1 (V1:AWW), C. vulgaris was cultivated in raw aquaculture wastewater. In Variant 2 (V2:BM), the microalgae were cultivated in a basic medium that served as a control. Variant 3 (V3:AWW+M) consisted of aquaculture wastewater supplemented with a microelement solution.

2.2. Materials

2.2.1. Inoculum of the C. vulgaris Biomass

The C. vulgaris (BA-166) algae selected for cultivation on aquaculture effluents were obtained from the Culture Collection of Baltic Algae (CCBA) (University of Gdansk, Institute of Oceanography, Gdansk, Poland). The algae cells were pre-cultured in 1000 mL flasks with an 8 ppt F/2 culture medium [29] at 25 °C under continuous illumination with white fluorescent light (12,000 lux) and shaking at 100 rpm. The inoculum was collected in the logarithmic growth phase by centrifugation of the microalgae cells (800 rpm, 15 min) and then seeded into the photobioreactors used in the actual experiments at a concentration of 50 mg VS/L.

2.2.2. Characteristics of the Growth Media

Three different media were used to cultivate the biomass of C. vulgaris: aquaculture wastewater (AWW) (V1:AWW), a synthetic base medium (V2:BM) and AWW with the addition of microelements (V3:AWW+M).

The AWW was obtained from an aquaculture research facility of the Department of Ichthyology and Aquaculture, Faculty of Animal Bioengineering, University of Warmia and Mazury in Olsztyn, Poland (53.75392 N, 20.46189 E). The recirculating aquaculture system (RAS) was used for rearing Nile tilapia (Oreochromis niloticus), a freshwater species. The fish were reared in black tanks with a total volume of 1500 L, under controlled temperature conditions of 23 ± 1 °C and continuous aeration. The fish were fed commercial pelleted feed (Nutra, Skretting AS, Stavanger, Norway). The system included a settling tank and a dissolved waste removal system. A filtration system with 0.45 nm pore size philtres (Eheim Classic 600, EHEIM GmBH & Co. KG, Deizisau, Germany) and aerobic biological beds were used to remove unutilised nutrients, including biogenic compounds and organic loads. The AWW used in the present study was collected directly after mechanical filtration and before the biological treatment stage using a biofilm treatment. The chemical composition of the AWW used as the growth medium is shown in

Table 1. The characteristics of the microalgae growth media used in this study.

Parameter	Unit	V1:AWW	V2:BM	V3:AWW+M
Ammonia	mg N-NH4/L	1.03 ± 0.09	76.30 ± 2.80	4.85 ± 0.25
Nitrate	mg N-NO3/L	98.60 ± 4.10	56.70 ± 2.10	96.70 ± 3.60
Phosphate	mg P-PO4/L	10.50 ± 0.80	33.60 ± 1.40	6.69 ± 0.50
pH	–	6.74 ± 0.05	7.05 ± 0.04	6.70 ± 0.06
Iron	mg Fe/L	0.094 ± 0.008	3.270 ± 0.120	2.440 ± 0.095
Potassium	mg K/L	47.6 ± 2.3	190.0 ± 6.2	53.8 ± 2.1

.

The base medium (BM) was prepared by mixing tap water with a nitrogen–phosphorus fertiliser (Azofoska; Grupa INCO S.A., Góra Kalwaria, Poland) and a micro-nutrient solution (MicroPlus; Intermag, Olkusz, Poland). According to the manufacturer, Azofoska contains 13.3% total nitrogen (NTOT), of which 5.5% is nitrate nitrogen (N–NO₃) and 7.8% ammonium nitrogen (N–NH₄), 6.1% P₂O₅ soluble in neutral ammonium citrate solution (C₆H₁₇N₃O₇) and water, 4.0% water-soluble P₂O₅, 17.1% water-soluble K₂O, 4.5% MgO, and 21.0% SO₃. The MicroPlus solution contains 2.3 g/L boron (as acid), 1.2 g/L copper in chelate form with EDTA, 22.3 g/L iron in chelate form with EDTA, 9.5 g/L manganese in chelate form with EDTA, 0.6 g/L molybdenum (as ammonium salt), and 3.5 g/L zinc in chelate form with EDTA. The fertiliser and the micronutrient solution were added to the tap water at a concentration of 1.35 g/L and 0.1 mL/L, respectively (Table 1).

The V3:AWW+M medium consisted of AWW enriched with the MicroPlus solution (Intermag, Olkusz, Poland), as described above, at a final concentration of 0.1 mL/L (Table 1).

2.2.3. Anaerobic Sludge Inoculum

The anaerobic reactors used in S4 were inoculated with anaerobic sludge obtained from closed digesters at the Municipal Wastewater Treatment Plant in Olsztyn, Poland (53.81321 N, 20.44956 E). The facility operates based on an activated sludge process, incorporating the enhanced biological removal of organic compounds as well as nitrogen and phosphorus. The anaerobic digestion chambers have an active volume of 10,000 m³ and operate under mesophilic conditions at a temperature of 37 ± 1 °C. The organic loading rate (OLR) is maintained at approximately 2.2 kg VS/m³·d, with a hydraulic retention time (HRT) of approximately 20 days. The feedstock introduced into the digesters consists of pre-thickened sewage sludge with a moisture content of approximately 95 ± 1%. The characteristics of the anaerobic sludge are presented in

Table 2. The characteristics of the anaerobic sludge used in Stage 4 of the experiment.

Parameter	Unit	Value ± SD
Total solids (TS)	[% FM *]	3.9 ± 0.3
Volatile solids (VS)	[% TS]	60.2 ± 3.5
Mineral solids (MS)	[% TS]	39.8 ± 3.5
Total carbon (TC)	[mg/g TS]	372 ± 35
Total organic carbon (TOC)	[mg/g TS]	322 ± 17
Total nitrogen (TN)	[mg/g TS]	30.8 ± 2.5
C/N ratio	[–]	10.2 ± 0.4
Total phosphorus (TP)	[mg/g TS]	2.4 ± 0.2
pH	[–]	7.26 ± 0.10
Protein	[% TS]	20.5 ± 1.5
Lipids	[% TS]	2.3 ± 0.3
Carbohydrates	[% TS]	3.1 ± 0.8

* FM—fresh mass.

.

2.3. Experimental Stands and Procedures

2.3.1. Continuous Cultivation of C. vulgaris

C. vulgaris was grown in three vertical, cylindrical photobioreactors on a laboratory scale with an active volume of 2.4 L (inner diameter 70 mm and 660 mm working height) made of transparent plexiglass [30,31]. The photobioreactors were illuminated with fluorescent lamps (6500 lx, cold white light, Osram, Munich, Germany) in a 12:12 light–dark cycle. The temperature of the culture was maintained at 23.0 ± 2.0 °C using a TERMIO-1 (TER-MOPRODUKT, Bielawa, Poland) temperature recorder with a PT1000 temperature sensor. Compressed air was continuously supplied at 200 L/h (Mistral 200, Aqua Medic, Bissendorf, Germany) via a silicone tube (5 mm inner diameter) to a valve (4 mm inner diameter) at the bottom of the reactors to provide agitation and CO₂ supply and to ensure the homogeneity of conditions throughout the reactor volume.

After inoculation, the microalgae were cultivated continuously for 10 days in three parallel photobioreactors. The three experimental variants differed in the microalgae growth medium used (Table 1).

2.3.2. Biogas Potential of Microalgae Biomass

The methane yield (CH₄) from the biomass of C. vulgaris was determined by the volumetric measurement of gas production using the Automated Methane Potential Test System II (AMPTS II, BPC Instruments AB, Lund, Sweden). The fermentation experiments were carried out at a controlled temperature of 37 ± 1 °C. The system continuously monitored the partial pressure changes in the reactors. Each AMPTS reactor consisted of a 500 mL glass vessel equipped with an individual automatic stirrer operating at 100 rpm for 30 s every 10 min. A total of 12 reactors were used for triplicate experiments for the C. vulgaris biomass under three experimental variants and a control containing only anaerobic sludge inoculum. Approximately 200 g of anaerobic sludge inoculum was added to each reactor, followed by the addition of the microalgae biomass. The initial organic loading rate (OLR) was approximately 5 g VS/L. Anaerobic conditions were maintained by flushing the substrate and sludge mixture with pure nitrogen gas.

Gas production data was recorded daily using dedicated process control software that automatically normalised gas volumes to standard conditions (101.3 kPa, 0 °C, dry gas). Measurements were continued until complete degradation of the biodegradable organics was achieved. The test was terminated when ten consecutive gas volume measurements deviated by less than 1%. Endogenous biogas produced by the anaerobic sludge was not included in the methane production calculations.

The methane yield was expressed as the volume of CH₄ produced (normalised litres, NL) per kilogram of introduced volatile solids (VS) over a 20-day period. Methane and biogas production kinetics were modelled using nonlinear regression based on a first-order kinetic equation as described by Llabrés-Luengo and Mata-Alvarez [32]: Y(t) = −Y_max (e^−kt − 1), where Y represents methane or biogas yield at time t (NL/kg VS), Y_max is the maximum achievable yield (NL/kg VS), k is the kinetic constant (1/day), and t is the time (days). The CH₄/biogas production rate was calculated as the product of k and Y_max. Model performance and fit quality were assessed using the coefficient of determination (R²) calculated using Statistica software (version 13.1, StatSoft, Tulsa, OK, USA).

2.4. Analytical Methods

During the experiment, samples were taken from the photobioreactors every day. The media properties were determined during the cultivation process, after the microalgae were harvested from the medium by centrifugation at 110,000 rpm for 3 min (MPW-251, Donserv, Warsaw, Poland). The supernatant was analysed for orthophosphate (P-PO₄), nitrate–nitrogen (NO₃), ammonia–nitrogen (AN), potassium (K), and iron (Fe) using a DR 5000 spectrophotometer with an HT 200 s mineralizer (Hach-Lange, Düsseldorf, Germany). The pH value of the aqueous solutions was determined using a pH metre (1000 L, VWR, Darmstadt, Germany).

The algal cells were harvested by prior sedimentation and subsequent centrifugation (MPW-251, Donserv, 110,000 rpm, 3 min). The light intensity in the photobioreactors was measured with the luxmeter NL-100 (Hanna Instruments, Woonsocket, RI, USA). The total solids (TS), volatile solids (VS) and mineral solids (MS) were measured by the gravimetric method. The chlorophyll concentration in the mixture of 1 mL sample and 500 mL distilled water using by analyser bbe Moldaenke GmbH (Schwentinental, Germany). The dried samples (at 105 °C) were analysed for total carbon (TC), total organic carbon (TOC), and total nitrogen (TN) content using a Flash 2000 Elemental Analyzer (Thermo Scientific, Delft, The Netherlands). Total protein content was calculated by applying a conversion factor of 6.25 to the measured total nitrogen (TN) values. Carbohydrate concentrations were determined spectrophotometrically at 600 nm using a DR 2800 device (Hach-Lange GmbH, Düsseldorf, Germany), following the anthrone method, with glucose used as the standard. Lipid extraction was performed using the Soxhlet technique with a Büchi extraction unit (Flawil, Switzerland), and the lipid content was expressed as a percentage of total solids.

The lipid content in the dried biomass was assessed through solvent extraction using hexane. Before the extraction, the samples underwent a 30-s pretreatment with the ultrasonic processor UP400S (Hielscher Ultrasonics, Teltow, Germany) set at 100% amplitude. Following lipid extraction, transesterification was carried out in accordance with the protocol developed by Van Wychen et al. [33]. Subsequently, the composition of FAMEs was analysed using a Bruker 450 gas chromatograph (Billerica, MA, USA) equipped with a CP-Sil 88 Tailor Made FAME capillary column (50 m × 0.25 mm × 0.39 mm) and a flame ionization detector (FID). Details regarding the chromatographic parameters are available in the publication by Chakrabarty et al. [34].

The composition of biogas produced in the headspace of digesters was measured every 24 h using a gastight syringe (20 mL injection volume) and a gas chromatograph (GC, 7890A Agilent, Agilent, Santa Clara, CA, USA) equipped with a thermal conductivity detector (TCD). The GC was fitted with the two HayeSep Q columns (80/100 mesh), two molecular sieve columns (60/80 mesh), and a Porapak Q column (80/100) operating at a temperature of 70 °C. The temperature of the injection and detector ports were 150 °C and 250 °C, respectively. Helium and argon were used as the carrier gases at a flow of 15 mL/min.

2.5. Statistical Analysis

The experimental variants were performed in triplicate. The results were also analysed in triplicate and the data were shown as a mean ± standard deviation (SD). The results were processed statistically using the Statistica 13.3 PL package (StatSoft, Inc.). One-way analysis of variance (ANOVA) was conducted to determine the significance of differences between the variables. The homogeneity of variance in groups was tested with Levene’s test, whereas Tukey’s RIR test was used to determine the significance of differences between the analysed variables. In all statistical tests, a 95% confidence level was applied, corresponding to a significance level of p = 0.05.

3. Results and Discussion

3.1. Aquaculture Wastewater Treatment

This study investigated the effectiveness of nutrient removal from AWW by C. vulgaris (Figure 1). The content of ammonia and nitrate nitrogen in a raw AWW, in a basic growth medium, and in AWW with microelements is shown in Table 1. The efficiency of nitrogen removal is shown in Figure 1. The base medium in V2:BM showed the highest initial concentration of ammonium nitrogen of 76.3 ± 3.8 mg/L. In AWW and AWW+M it was initially 1.03 ± 0.9 g/L and 4.85 ± 1.2 mg/L, respectively. It should be noted that the AWW used in this study contained a lower concentration of ammonia and a higher concentration of nitrate, since in RAS with biofilters the nitrogen is produced in the form of nitrates [35]. During the experiment, the ammonia concentration in V1:AWW and V3:AWW+M was reduced to 0 within two days (Figure 1). In V2:BM, the final ammonia concentration was 6.6 ± 2.3 g/L, so that the reduction of ammonia nitrogen was 91.3% (Figure 1). The fluctuation of nitrate concentration during this study showed an increasing trend in V2:BM, with the final increase in nitrate concentration being 71.4%. On the other hand, nitrate removal was observed in V1:AWW and V3:AWW+M, which was 39.1% and 21.8%, respectively.

The results obtained also confirm the high efficiency of C. vulgaris in phosphate removal from aquaculture wastewater (Figure 1). For the variants V1:AWW and V3:AWW+M, where the initial phosphate concentrations were 10.50 ± 1.5 mg/L and 6.69 ± 1.9 mg/L, respectively, very high removal efficiencies of 93.7% and 94.9% were observed after only three days of cultivation. This indicates the strong potential of C. vulgaris for utilising the available nutrients. Importantly, the addition of microelements in V3:AWW+M did not significantly affect the biomass growth or phosphorus uptake rates, suggesting that the naturally occurring components in aquaculture wastewater are sufficient to support active algal growth. By contrast, the removal efficiency in V2:BM was 72.9%, which could indicate the release of phosphorus from unutilised components of the medium or a lower uptake efficiency under conditions of nutrient excess.

The efficiency of the wastewater treatment determined in this study corresponded to the values in the literature. Esteves et al. [9] showed an efficiency of 98% for nitrate–nitrogen removal and 92.7% for phosphate removal using C. vulgaris microalgae and AWW as a culture medium. In studies with Scenedesmus obliquus, Chlorella sorokiniana, and Ankistrodesmus falcatus, nutrient uptake from AWW was in the range of 86.45–98.21% for ammonia, 75.76–80.85% for nitrate, and 98.52–100% for phosphate [1]. The experiments conducted by Lugo et al. [36] showed that Chlorella sorokiniana achieved a lower percentage of nutrient removal from AWW, namely 70% of phosphorus and 78% of nitrogen. In the production of the Chlorella minutissima biomass using AWW as growth medium, Hawrot-Paw et al. [37] achieved 88.6% nitrate removal and 99% removal of dissolved orthophosphates. In the study by Viegas et al. [10], a removal of 100% of total nitrogen and total phosphorus from raw wastewater from the aquaculture of brown crabs was determined.

Nutrient uptake by microalgae cells depends on various environmental factors that influence algal growth, such as light, temperature, turbulence, and nutrient availability [38]. Nutrient concentration in the growth medium is one of the most important factors, and the Redfield ratio of 106:16:1 C/N/P is widely used as a guide to quantify potential nutrient limitation. According to the literature, the average N/P ratio suitable for cultivation of Chlorella sorokiniana is 15–26 [39]. Yu et al. [40], on the other hand, indicated that Chlorella sp. is able to efficiently remove nutrients from wastewater at N/P ratios between 5 and 15. Studies on the effects of the N/P ratio on nutrient removal from municipal wastewater by C. vulgaris showed that phosphorus uptake is strongly influenced by the N/P ratio [41]. In addition, a low phosphate concentration in the growth medium impairs the efficiency of nitrate removal by microalgae.

In this study, the growth media in V1:AWW and V3:AWW+M were characterised by a low ammonium concentration in which the nitrate form predominated; while, in V2:BM, an ammonium-rich initial medium was used. This had an effect on the nitrogen uptake pathways of Chlorella sp. It is known that the microalga Chlorella sp. prefers ammonium to synthesise amino acids via the glutamine synthetase–glutamate synthase pathway when both ammonium and nitrate are available in the growth medium [42]. Scherholz and Curtis [43] based this preference on the inhibitory effect of ammonium on the transport and reduction of nitrate, as ammonium assimilation is less energetically costly than nitrate assimilation. The process is also associated with the production of other forms of nitrogen, such as nitrate [35]. However, excess ammonium can be toxic to microalgae and can impair the photosynthetic machinery [44,45]. When the growth medium in V2:BM contained a high concentration of ammonium, it was utilised by C. vulgaris to produce amino acids, while the nitrate concentration increased. In V1:AWW and V3:AWW+M, the low amount of ammonia increased nitrate assimilation by the cells via nitrate reduction to nitrites by nitrate reductase and further reduction of nitrite to ammonium by nitrite reductase [42,46].

However, the assimilation of ammonia or nitrates is also related to the pH of the growth medium [35]. During this study, the pH decreased when ammonia was used as a nitrogen source in V2:BM; while, in V1:AWW and V3:AWW+M, the pH increased during nitrate assimilation as the sole nitrogen source for the microalgae cells (Figure 1). According to the literature, the assimilation of ammonium by microalgae generates H₂O and H⁺ as a result of peptide formation by the condensation polymerisation of amino acids, which leads to a pH drop [44]. On the other hand, chemical compounds, such as O₂ and OH⁻, are formed as a result of nitrate assimilation, which leads to an increase in pH [47]. In the experiment, the final pH in V2:BM decreased to 4.86 ± 0.3 as a result of ammonium assimilation; while, in V1:AWW and V3:AWW+M, it remained between 6.70 ± 0.2 and 8.76 ± 0.1 throughout the experimental period, which is within the optimal range for Chlorella sp. (Figure 1) [40,48]. Elevated pH can promote the release of free ammonia from ammonium, which can inhibit the growth of microalgae [49]. Therefore, in high pH environments, algae may prefer to use nitrate to avoid the toxicity of ammonium, whereas in neutral or slightly acidic environments they favour ammonium as it is more easily assimilated [36].

Phosphorus is essential for the growth of microalgae and many cellular processes, such as energy transfer and nucleic acid biosynthesis. The preferred form of phosphorus in which it is taken up by algal cells is orthophosphate [50]. When nutrients are abundant and light is the growth-limiting factor, most algal species have a remarkably constant phosphorus content of about 1% of dry weight [35]. The concentration of orthophosphate in the growth medium influences the content of lipids, proteins, and carbohydrates in the biomass [13,37]. Some of the symptoms of phosphorus depletion are similar to those observed in nitrogen-deficient crops. The chlorophyll a content and photosynthetic activity in the cells tend to decrease, while the lipid content increases [51]. During the experiment, the total chlorophyll concentration increased so that phosphorus limitation was not observed (Figure 2). The lack of significant effects of microelement addition in our study suggests that C. vulgaris can efficiently utilise the nutrients present in aquaculture wastewater without the need for supplementation, which increases its applicability in sustainable wastewater treatment strategies.

3.2. Growth and Characterisation of the C. vulgaris Biomass

At the beginning of the experiment, the biomass concentration in all variants was around 0.05 ± 0.001 gVS/L (Figure 2a). At the end of the cultivation period, the biomass concentration in the variant V1:AWW reached 2.4 ± 0.08 gVS/L and was statistically higher compared to the other experimental variants (p ≤ 0.05). In V2:BM, the final concentration was 2.21 ± 0.11 gVS/L; while, in V3:AWW+M, it reached 2.09 ± 0.09 gVS/L (Table 2, Figure 2a). The logarithmic growth phase was observed between days 2 and 8, after which the C. vulgaris population entered the stationary growth phase (Figure 2a). The strongest increase in the chlorophyll a concentration was recorded between the third and sixth day. In V1:AWW, the concentration reached 44.1 ± 2.1 mg/L; while, in V2:BM, it was 38.5 ± 3.4 mg/L. In the case of V3:AWW+M, the chlorophyll a concentration was lower and totalled 33.5 ± 2.8 mg/L (Figure 2b). During this period, the differences between V1 and V3 were statistically significant (p ≤ 0.05), illustrating the influence of cultivation conditions on photosynthetic pigment synthesis. In the last phase of cultivation, on the tenth day, the highest chlorophyll a concentration was measured in variant V1 (AWW), which reached 67.6 ± 2.2 mg/L. By comparison, variant V2 (BM) reached 62.5 ± 1.8 mg/L, while V3 (AWW+M) reached a concentration of 59.2 ± 2.8 mg/L (Figure 2b).

The highest C/N ratio of 13.3 ± 0.5 was achieved in V1:AWW (p < 0.05), with the lowest TN concentration in the biomass (p < 0.05). The composition of lipids, saccharides, and proteins in the algal biomass depends on the nutrient composition of the growth medium [1]. In this study, the nitrogen and phosphorus content in AWW was lower than in the base medium, which could be responsible for the stress conditions of C. vulgaris. When C. vulgaris was grown on AWW, the concentration of lipids reached 83.2 ± 3.7 mg per 1 g biomass, but there was no differences (p > 0.05) at lipid, saccharides and protein concentrations in all experimental variants. In addition, the enrichment of AWW with microelements had no effect on the higher lipid content of the biomass of C. vulgaris (p > 0.05) (Table 2).

The decrease in biomass concentration and total chlorophyll content observed at the end of the study could be related to phosphate reduction in the growth medium, as Tossavainen et al. [52] reported inhibition of algae growth within a few days after nutrient depletion. Similar results were found by Hawrot-Paw et al. [37], who observed a significant reduction in the biomass growth of Chlorella minutissima after 10 days of cultivation in aquaculture wastewater as a culture medium. On the other hand, the decrease in total chlorophyll concentration in V2:BM was associated with the decrease in pH, since Chlorella sp. microalgae have an optimal growth rate at a pH between 6 and 8 and do not survive at a pH below 5 [47]. To summarise, nutrient concentrations in aquaculture wastewater are not sufficient to maintain the high productivity of the algal biomass. Therefore, a continuous supply of aquaculture wastewater should be provided to maintain the production of the microalgae biomass [53].

According to the literature, microalgae consume more nitrogen than phosphorus for the same productivity [9]. The ratio of N/P in the growth medium determines the growth potential of microalgae [35]. However, the availability of nitrogen in the growth medium affects the rapid cell growth and promotes the accumulation of chlorophyll in the microalgae cells [54,55]. The results of this study showed that the final growth of Chlorella did not depend on the form of nitrogen in the growth medium (Figure 1 and Figure 2, Table 2). In agreement with the results of this study, the experiments on the growth of C. vulgaris in different forms of nitrogen showed the highest biomass production at ammonia–nitrogen concentrations between 20 mg/L and 250 mg/L, and there were no differences in the biomass concentration achieved in commercial Bristol medium with nitrate as the nitrogen source [56]. According to Jakhwal et al. [57], phosphate concentration had no effect on the biomass production of marine microalgae in aquaculture wastewater, and even when phosphate was limited in the growth medium, the biomass accumulation was not hindered. However, sufficiently high nitrogen concentrations are required to ensure the effective phosphorus removal from wastewater [58].

The biochemical composition of the C. vulgaris biomass was evaluated. The contents of the analysed compounds at the end of cultivation time are summarised in

Table 3. Biomass concentration and biochemical composition in V1:AWW, V2:BM, and V3:AWW+M measured after 10 days of cultivation.

Parameters	Unit	V1:AWW	V2:BM	V3:AWW+M
Biomass concentration	g VS/L	2.4 ± 0.04	2.2 ± 0.08	2.1 ± 0.04
Chlorophyll a	mg/L	67.6 ± 1.8	62.5 ± 2.2	59.2 ± 2.8
Volatile solids (VS)	% TS	88.7 ± 1.3	86.9 ± 0.9	86.2 ± 1.4
Mineral solids (MS)	% TS	11.3 ± 1.1	13.1 ± 1.0	13.8 ± 1.1
Total carbon (TC)	mg/g TS	477 ± 14	471 ± 26	442 ± 31
Total organic carbon (TOC)	mg/g TS	431 ± 9	412 ± 21	409 ± 16
Total nitrogen (TN)	mg/g TS	32.5 ± 1.9	36.3 ± 1.3	37.3 ± 2.7
C/N		13.3 ± 0.5	11.3 ± 1.1	10.9 ± 0.9
Total phosphorus (TP)	mg/g TS	16.3 ± 2.1	16.0 ± 1.7	15.2 ± 1.8
pH	mg/g TS	7.81 ± 0.09	8.01 ± 0.12	7.94 ± 0.07
Protein	mg/g TS	203 ± 19	227 ± 14	233 ± 11
Saccharides	mg/g TS	212 ± 21	197 ± 13	216 ± 19
Lipids	mg/g TS	83.2 ± 3.7	81.4 ± 4.3	75.6 ± 6.7

.

After 10 days of cultivation, the highest biomass concentration was observed in the variant V1:AWW (2.4 ± 0.04 g TS/L), followed by V2:BM (2.2 ± 0.08 g TS/L) and V3:AWW+M (2.1 ± 0.04 g TS/L). Although the differences in the biomass yield between the variants were not statistically significant (p > 0.05), the slightly higher concentration in V1:AWW indicates that aquaculture wastewater alone can support the growth of microalgae comparable to synthetic media. This supports the findings of Hawrot-Paw et al. [37], who showed that Chlorella minutissima can effectively utilise nutrients from aquaculture effluents without the need for supplementation. The volatile solids (VS) content, an indicator of organic matter, ranged from 86.2 ± 1.4% TS in V3:AWW+M to 88.7 ± 1.3% TS in V1:AWW (Table 3). These values reflect a high organic content, which is typical of the Chlorella biomass grown under nutrient-rich conditions, and is consistent with previous studies [59,60].

Total carbon (TC) and total organic carbon (TOC) were highest in V1:AWW (477 ± 14 mg/g TS and 431 ± 9 mg/g TS, respectively) and decreased in V2:BM and V3:AWW+M, indicating a slightly higher efficiency of carbon assimilation in the non-supplemented AWW variant. The addition of microelements in V3:AWW+M did not increase the TOC content, suggesting that nutrient balance rather than quantity is more important for optimising carbon uptake, as also found by Zhai et al. [61]. Interestingly, the total nitrogen (TN) content was highest in V3:AWW+M (37.3 ± 2.7 mg/g TS), slightly exceeding that of V2:BM (36.3 ± 1.3 mg/g TS), while V1:AWW had the lowest nitrogen content (32.5 ± 1.9 mg/g TS) (Table 3). Consequently, the C/N ratio was highest in V1:AWW (13.3 ± 0.5), indicating a more carbon-rich biomass in untreated AWW, whereas the lower C/N ratios in V2:BM and V3:AWW+M reflect higher protein accumulation and nitrogen availability [62]. This was also confirmed by analysing the protein content, with V3:AWW+M (233 ± 11 mg/g TS) and V2:BM (227 ± 14 mg/g TS) having significantly higher protein contents than V1:AWW (203 ± 19 mg/g TS). The protein content correlated positively with the TN content, which is consistent with the known role of nitrogen in protein biosynthesis [63]. This indicates that supplementation with microelements slightly improved nitrogen utilisation, possibly by facilitating nitrogen assimilation pathways.

The saccharide content showed a less predictable trend, with the highest value in V3:AWW+M (216 ± 19 mg/g TS), comparable to V1:AWW (212 ± 21 mg/g TS), and the lowest in V2:BM (197 ± 13 mg/g TS) (Table 3). While saccharide synthesis can vary depending on environmental stressors and nutrient availability, no statistically significant differences were observed here. Lipid content ranged from 83.2 ± 3.7 mg/g TS in V1:AWW to 75.6 ± 6.7 mg/g TS in V3:AWW+M, indicating a moderate decrease in lipid biosynthesis with nutrient supplementation. Lipid accumulation is often inversely related to nitrogen availability, as nitrogen limitation causes a redistribution of carbon towards lipid storage [64]. This inverse relationship was also observed in our study.

Chlorophyll content, an indicator of photosynthetic activity, was highest in V1:AWW (67.6 ± 1.8 mg/g TS), followed by V2:BM (62.5 ± 2.2 mg/g TS) and V3:AWW+M (59.2 ± 2.8 mg/g TS). The decrease in chlorophyll levels in the supplemented variants could be due to microelement-induced oxidative stress or suboptimal nutrient balance, which can downregulate chlorophyll biosynthesis [65]. Of note, the pH values remained within the optimal range (6.8–8.0), with V2:BM having the highest pH of 8.01 ± 0.12, which could have affected pigment stability and enzymatic activity.

The microalgae biomass harvested after the AWW treatment can be used as aqua feed in aquaculture production [14]. A study by Gora et al. [66] showed that the addition of 1% chlorophyll extracted from microalgae improved the survival rate of fish larvae by 36.19%. An addition of 2.5% crude microalgae mixture (A. platensis and N. gaditana) was recommended for the rearing of juvenile European seabass to improve their growth [67]. According to Mueller et al. [15], an addition of 8% microalgae to the feed induces a local anti-inflammatory response in the gut, improves the response to oxidative stress, stimulates complementary and antibacterial responses in the liver and spleen, and also improves the transfer of important functional components of the microalgae (polyunsaturated fatty acids and pigments) to the fish muscle.

3.3. FAMEs Composition of the Algae Biomass

C. vulgaris can be used as a feedstock for biodiesel production [68]. Biodiesel is a mixture of fatty acid methyl esters synthesised by the transesterification process of an algal biomass rich in lipids or triacylglycerols. It is considered a renewable, biodegradable, and clean biofuel that replaces diesel from fossil fuels [3,69]. In order to valorise the resulting biomass, its composition in terms of FAMEs was also evaluated. The FAME profiles of the biomass samples cultivated under different conditions are shown in Figure 3. The dominant fatty acids in all variants were palmitic acid (C16:0) and oleic acid (C18:1) with concentrations of 21–24% and 19–22%, respectively. In V1:AWW, the highest proportion of C16:0 (24%) and C18:1 (22%) was observed, indicating increased lipid accumulation.

Linoleic acid (C18:2) also showed remarkable levels, especially in V1:AWW (19%) and V2:BM (18%), while linolenic acid (C18:3) ranged between 7 and 11%, with the highest value recorded in V2:BM (Figure 2). This variant also showed relatively high levels of stearic acid (C18:0, 7%) and pentadecanoic acid (C15:0, 3%), indicating a different pattern of lipid biosynthesis compared to the other cultivation media (Figure 3).

Saturated medium-chain fatty acids, such as C11:0 and C14:0, were present in smaller amounts (6–8% and 4–6%, respectively) (Figure 3). Minor differences were noted in the content of odd-chain and monounsaturated fatty acids, including C15:0, C16:1, and C17:1, which collectively contributed to 10–15% of total FAMEs depending on the variant (Figure 3).

In summary, the AWW medium promoted a higher accumulation of the main fatty acids relevant for biodiesel (C16:0 and C18:1), while the other growth media (BM and AWW+M) supported a higher diversification of FAMEs, possibly improving fuel properties through an improved compositional balance. Since fatty acid composition has a major impact on biodiesel quality, the results of the current study demonstrate that C. vulgaris strains can produce oil with a significant amount of saturated fatty acids consisting of stearic acid (C18:0) and palmitic acid (C16:0), which is consistent with previous results [70,71]. A high content of unsaturated acids impairs the oxidative stability of biodiesel and has a detrimental effect on storage stability [68]. According to Sadvakasova et al. [72], biodiesel with a large number of saturated fatty acids has a higher oxidative stability, cetane number, and viscosity. Therefore, C. vulgaris growing on AWW can be considered as a suitable candidate for biofuel production.

3.4. Biogas and Methane Potential of the Algae Biomass

Microalgae biomass cultivated on AWW can also be utilised in the production of biogas [3]. In the variant V1:AWW, the highest biogas yield of 358 ± 11 L/kg VS was obtained, which was significantly higher (p ≤ 0.05) than in the variants V2:BM and V3:AWW+M, where the yields were 319 ± 13 L/kg VS and 300 ± 12 L/kg VS, respectively (Figure 4,

Table 4. Biogas and methane production efficiency and the values of parameters characterising the process kinetics k—rate constant and r—production rate.

Parameter	Unit	Variant
		V0—Endogenous Production	V1:AWW	V2:BM	V3:AWW+M
Biogas	L/kgVS	83.5 ± 21	358 ± 11	319 ± 13	300 ± 12
k	1/day	0.16	0.16	0.13	0.13
r	mL/day	13.4	57.4	41.5	39.1
CH4	%	41.2 ± 0.5	60.2 ± 0.7	57.1 ± 0.9	57.4 ± 0.5
CH4	L/kgVS	34.0 ± 9	216 ± 7	182 ± 7	173 ± 6
k	1/day	0.15	0.15	0.14	0.15
r	mL/day	5.1	32.4	27.3	25.9

). A similar trend was observed for CH₄ production, with V1:AWW reaching 216 ± 7 L/kg VS, also statistically significantly higher (p ≤ 0.05) than the values obtained for V2:BM (182 ± 7 L/kg VS) and V3:AWW+M (173 ± 6 L/kg VS) (Figure 4, Table 4). The biogas production rate constant (k) in V1:AWW was significantly higher (p ≤ 0.05) compared to V2:BM and V3:AWW+M and was 0.16 1/day compared to 0.13 1/day in the latter variants. By contrast, no statistically significant differences were found in the k values for CH₄ production between the variants (Table 4). The biogas production rate (r) was highest in V1:AWW at 57.4 mL/day and showed significant differences (p ≤ 0.05) compared to V2:BM and V3:AWW+M, which produced 41.5 mL/day and 39.1 mL/day, respectively. The highest methane production rate was also found in V1:AWW at 32.4 mL/day, with statistically significant differences (p ≤ 0.05) compared to V2:BM (27.3 mL/day) and V3:AWW+M (25.9 mL/day) (Table 4).

The observed values are similar to the data reported in the literature. Prajapati et al. [73] found that biogas production ranged from 340 L/kg VS for C. minutissima to 464 L/kg VS for C. pyrenoidosa, with C. vulgaris at 369 L/kg VS. In addition, similar biogas yields (210–587 L/kg VS) were observed for other algal biomass, including the green algae Chlamydomonas reinhardtii, Dunaliella salina, Scenedesmus obliquus, C. kessleri, the euglenoid species Euglena gracilis, and the prokaryotic cyanobacterium Arthrospira platensis [74,75,76].

There are many factors that influence biogas production from algae biomass. These include the technological parameters of anaerobic digestion and the type of algae [77]. However, the chemical composition of the biomass has a strong influence on the biogas and methane yields [78]. The theoretical methane yields from lipids, proteins, and carbohydrates are 1014 L/kg VS, 851 L/kg VS, and 415 L/kg VS, respectively [79]. In this study, the higher biogas production in V1:AWW can be attributed to the slightly higher lipid enrichment in the biomass of C. vulgaris. Furthermore, the optimal C/N ratio for anaerobic digestion is 20–30 [80,81]. In this study, it was between 10.9 ± 0.9 in V3:AWW+M and 13.3 ± 0.5 in V1:AWW (Table 2), and thus below the optimal range for the anaerobic microflora. At a low C/N ratio, ammonia nitrogen is released during anaerobic digestion, that could inhibit methanogens if the concentration reaches a threshold of 1 g/L [82]. No inhibition by ammonia nitrogen was observed during the fermentation of the C. vulgaris biomass. This fact was confirmed by the significant and constant biogas production in all experimental variants (Figure 4). It was demonstrated that the composition and origin of the cultivation medium can significantly influence the biochemical composition and characteristics of the microalgae biomass, which directly affects the overall yield and kinetics of anaerobic digestion. This is one of the most important considerations when selecting biomass production strategies for microalgae intended for efficient methane fermentation and optimisation of biogas processes.

4. Microalgae in Wastewater Treatment—Potential and Challenges

The ability to utilise wastewater of different chemical composition and origin as a cultivation medium is one of the main advantages of the microalgae biomass production systems. It has been shown that such an environmentally friendly approach can significantly reduce the total cost of ownership of cultivation facilities and the unit cost of biomass production [83]. In addition to aquaculture applications, numerous attempts have been made to integrate microalgae photobioreactors into industrial [84], municipal and agricultural wastewater treatment processes [31], as well as into the neutralisation of wastewater from biogas plants [85,86] and anaerobic reactors [87]. In most reported cases, a high removal efficiency of organic pollutants as well as nitrogen and phosphorus compounds was achieved. Reports of photobioreactors effectively neutralising wastewater from industrial and domestic sources indicate the feasibility of commercial systems for the production of the microalgae biomass, including for the production of biofuels. There are also numerous concepts for siting photobioreactors near agricultural biogas plants, waste-to-biogas plants, wastewater treatment plants, district heating and cogeneration plants, landfills, and other industrial facilities that generate both CO₂ and nutrient-rich wastewater [86].

Promising research results have prompted institutions to incorporate microalgae technologies into environmental and energy strategies [88]. An example of this is the European Union’s framework for the development of the bioeconomy, in which microalgae occupy an important place as a raw material for environmental protection technologies, bioenergy production, and the extraction of high-quality nutrients for the food and feed industries [89]. The microalgae sector is expected to play an increasingly important role in the EU’s blue bioeconomy, especially in coastal areas [90]. The literature shows that microalgae cultivation technologies are among the most promising and sustainable solutions in the field of biotechnology [91,92]. Numerous studies have emphasised their competitiveness, cost-effectiveness, and potential for biofuel production [93,94,95]. Despite significant research progress and extensive experimental data, implementation on an industrial scale is still limited. It remains crucial to determine which technological aspects need to be optimised and which obstacles need to be removed in order to fully exploit the potential of this technology.

Microalgae systems based on the utilisation of wastewater and the subsequent conversion of the biomass into biofuel are complex processes that depend on numerous factors and parameters, which significantly limit their commercial applicability [96]. These technologies are much more complex compared to the conventional methods of wastewater treatment and waste-to-energy conversion. In activated sludge-based processes, for example, the design of large-scale plants is based on several key parameters, including the concentration of bacterial microflora in the bioreactor, the organic load of the sludge, the hydraulic load, and the oxygen content [97,98]. Anaerobic processes are even simpler—their design and operation are mainly based on the organic load index and hydraulic retention time [99]. The relative simplicity of these solutions is an important factor in their widespread application [100]. It should be emphasised that these systems compete to some extent with microalgae technologies, as they generate surplus sludge that can be used to produce fuels [101].

Most experiments on the use of microalgae in wastewater treatment are conducted at laboratory scale, which is a significant obstacle to industrial implementation [102]. While small-scale studies are invaluable for understanding biochemical mechanisms and testing different technological variants, they often do not provide the data required for economic, environmental, and technical assessments at the industrial level. Information on the energy balance, carbon footprint, material flow analyses, and long-term process stability, among other things, is lacking. In order to increase the technology readiness level (TRL), a gradual transition from laboratory to pilot scale to large-scale operation is required [103]. This phase is crucial for the precise definition of the type of raw materials, materials, equipment, and process parameters [104]. However, there are few reports in the scientific and technical literature describing the technical and operational aspects of large-scale microalgae plants [105].

In the context of environmental assessment, life cycle assessment (LCA) plays a crucial role. It enables a reliable assessment of potential waste streams (including wastewater, leachate, and waste gases) as elements to increase economic and environmental efficiency [106]. The LCA results are important both for the optimisation of the cultivation process and for the preparation of the environmental documentation required for investment processes [107]. The integration of LCA results into design decisions enables the minimisation of the environmental impact of the technology and the improvement of its efficiency [108].

A new research direction that can significantly impact the efficiency of wastewater treatment with microalgae is symbiotic systems that combine microalgae with bacteria, fungi, or yeasts [109]. Current work focuses on the optimisation of cultivation conditions, the analysis of interactions between autotrophic and heterotrophic organisms, and their effects on technological parameters [110]. In such systems, microalgae enhance the processes of nutrient degradation and produce oxygen, which supports the metabolism of aerobic microorganisms [111]. Heterotrophic microorganisms, in turn, break down organic pollutants into mineralised forms of elements and thus support the growth of microalgae [112]. Another significant advantage of symbiotic systems is the improved sedimentation properties of the biomass, which translates into lower costs for separation and dewatering, accounting for up to half of the total process costs [113]. In addition, such systems have been shown to have a positive impact on the quality and quantity of biofuels and other products with market value [114].

Despite ample scientific evidence confirming the potential of microalgae for bioenergy production, these technologies have been slow to materialise. At their current stage of development, they cannot compete with fossil fuels, and the volatility of their prices makes it even more difficult to predict the return on investment. Utilising wastewater as a nutrient source can improve the profitability of microalgae plants, but requires pre-treatment to eliminate pathogens, to remove impurities, and to reduce turbidity. High levels of organic matter and sulphur in the medium can promote the growth of competing microorganisms, limiting microalgae growth and process efficiency. The main obstacles to the development of microalgae technologies include the complexity of the cultivation process, the lack of data from pilot and large-scale plants that allow for a reliable assessment of the life cycle assessment, and insufficient regulatory and financial support. Possible areas for improvement include process optimisation, the use of genetic engineering and the development of advanced process monitoring and control systems.

5. Conclusions

C. vulgaris effectively removed nutrients from raw aquaculture wastewater (AWW) and achieved complete ammonia elimination (100%) within two days of cultivation and over 93% removal of orthophosphates. Nitrate removal reached 37.8%, emphasising the ability of the species to treat wastewater with complex nitrogen composition.

Raw AWW proved to be an efficient cultivation medium for C. vulgaris, providing the highest biomass yield (2.4 ± 0.04 g VS/L) after 10 days of continuous cultivation. The biomass productivity in AWW was comparable to or even exceeded that in the synthetic control medium. This indicates that AWW can serve as a cost-effective and nutrient-rich alternative for large-scale microalgae production. The increased chlorophyll a content (67.6 ± 1.8 mg/L) also confirmed the active photosynthesis and the favourable nitrogen and phosphorus conditions in the medium.

The biomass obtained from AWW exhibited a favourable fatty acid methyl ester (FAME) profile, in which saturated fatty acids—palmitic acid (C16:0, 24%) and stearic acid (C18:0, 22%) —dominated alongside a high proportion of monounsaturated oleic acid (C18:1, 22%). This composition improves the oxidative stability, the cetane number, and the combustion quality of the biodiesel, and makes C. vulgaris a suitable feedstock for the production of high-quality biofuels. The relatively low content of polyunsaturated fatty acids (C18:2–19%, C18:3–7%) minimises the susceptibility to oxidation and improves the shelf life of the biodiesel.

Supplementation of AWW with microelements (AWW+M) did not lead to a significant improvement in the biomass yield or nutrient removal efficiency. These results suggest that the inherent composition of raw AWW provides sufficient macro- and micronutrients to sustain algal growth and active photosynthesis, making additional supplementation unnecessary.

The biomass cultivated on raw AWW showed the highest biogas (358 ± 11 L/kg VS) and methane (216 ± 7 L/kg VS) yields, clearly outperforming the other experimental variants. The constant of the biogas production rate (k) was 0.16 day⁻¹, and the production rate (r) reached 57.4 mL/day—both statistically higher values. The methanogenesis process remained stable across all treatments, with no signs of ammonia inhibition, despite the relatively low C/N ratio (13.3), which remained within the acceptable threshold for anaerobic microbial activity.

The results obtained are characterised by a very low coefficient of variation, which does not exceed 10% for the most important parameters analysed, indicating a very high repeatability of the tests. Overall, the results clearly demonstrate that raw AWW can be effectively used as a stand-alone culture medium for C. vulgaris, allowing for simultaneous wastewater treatment and the production of an energy-rich biomass.

Treatment technologies for aquaculture wastewater based on microalgae are considered promising solutions with high application potential. However, there are barriers that significantly limit their rapid diffusion. The main limitations include the complex and technologically demanding processes of biomass cultivation and harvesting, as well as the insufficient amount of operational data from pilot and large-scale plants, which often prevents a reliable assessment of technological efficiency and cost-effectiveness. Given current technological advances, further performance improvements could be achieved through the application of optimisation techniques, genetic engineering, and improved process control and monitoring.