Aeration Rate in Tertiary Treatment of Anaerobic Effluent from Soft Drink Industry by Co-Cultivation Between Penicillium gravinicasei and Microalgae

Abstract

The soft drink industry generates effluents with high organic loads and contaminants such as nitrogen and phosphorus, requiring sequential secondary and tertiary treatments to meet international discharge standards. Moving beyond traditional monocultures, this study developed a microbial consortium (forming microalga–fungus pellets), demonstrating a synergistic combination due to the resistance of the pellets, enhancing the treatment efficiency, and facilitating the recovery of the microbial sludge produced. Specifically, the treatment of anaerobic effluents (tertiary treatment) from the soft drink industry using consortia of the fungus Penicillium gravinicasei and the microalgae Tetradesmus obliquus and Chlorella sp. in aerated reactors was evaluated, analyzing the impact of aeration rates (0.5–3.5 vvm) on pollutant removal and microbial sludge production. The results showed that moderate aeration rates (1.5 vvm) optimized the removal of COD (up to 92.5%), total nitrogen (TN) (up to 79.3%), and total phosphorus (TP) (up to 83.4%) in just 2.5 h. Furthermore, excessive aeration reduced treatment efficiency due to microbial stress and difficulty in forming microalga–fungus pellets. The Chlorella sp. consortium showed greater stability, while T. obliquus was more sensitive to the aeration rate. Microbial sludge production was also optimized at around 1.5 vvm, consequence of the pollutant removal, with the formation of pellets that facilitated biomass harvesting.

1. Introduction

The advancement in the industrial sector dedicated to soft drink production corresponded to a 4% increase compared to the last global survey, quantifying annual output at approximately 754 billion liters, with higher consumption in countries such as the United States, China, and India. Consequently, the generation of wastewaters from manufacturing processes raises concerns regarding proper disposal, given the characterization of high levels of sugar, preservatives, mineral salts, and bicarbonates in their composition [1].

Given the wide variety of effluent origins in industries of this type, highlighting water from washing bottles, cans and tanks (syrup, sweeteners, concentrates and flavors), it is possible to detect a significant variation in the concentration of parameters such as BOD (86–47761 mg∙L⁻¹), oils, and greases (21–107 mg∙L⁻¹) [2,3]. Other parameters are chemical oxygen demand (COD) (8.600–33.6000 mg∙L⁻¹), volatile fatty acids (VFAs) (430–610 mg∙L⁻¹), ammoniacal nitrogen (500–2000 mg∙L⁻¹), phosphate (14–220 mg∙L⁻¹) and harmful recalcitrant compounds, including dye metabolites and heavy metals, complicating its management [4,5]. If released without adequate polishing, wastewaters pose a severe risk of eutrophication and pollution to receiving water bodies.

Furthermore, given the presence of a high organic load and dyes (used to improve the appearance of products), the application of anaerobic digestion as a secondary treatment stands out as an environmentally friendly option due to the reduction of organic compounds and conversion of the carbonaceous fraction into biogas, biofuel, and digestate, although some disadvantages have been demonstrated, such as conditions in long retention times and mineralization of nitrogen and phosphorus, present mostly in the fraction of microbial sludge produced but also in the liquid fraction. Consequently, this digestate often exhibits high concentrations of mineralized nutrients and residual organic matter, with typical values of chemical oxygen demand (COD), total nitrogen (TN), and total phosphorus (TP) ranging from 500 to 2000 mg/L, 50 to 200 mg/L, and 10 to 50 mg/L, respectively, often exceeding permissible discharge limits [6,7]. This anaerobic (secondary) effluent requires further treatment (tertiary or advanced treatment) to meet release standards and avoid pollution of soil and water bodies.

Therefore, in the context of wastewater treatment, the aeration process accounts for up to 50% of the total energy consumption in wastewater treatment plants (WWTPs) and plays a critical role in biological methods for degrading organic matter and sulfur-, phosphorus-, and nitrogen-containing compounds in conventional processes such as activated sludge or microalgal cultivation or co-cultivation with bacteria or fungi. For instance, the synergistic microalgae-microbe relationship, particularly during night-time, leads to reduced photosynthetic efficiency and, consequently, lower O₂ bioavailability. This affects respiratory activity due to competition between microalgae and bacteria/fungi, alongside other factors such as dissolved oxygen (DO) transfer and enhanced nutrient contact through cell suspension [8,9,10].

Even with the broad understanding of the study related to optimizations in energy consumption in treatment systems, there are still open gaps regarding the impact on extremely important parameters in the process of biofilm or pellet formation between microalgae and filamentous fungi, such as mass transfer dynamics, the effectiveness of the microbial community (co-cultivation) and consequently the applicability of the system [11,12]. The pelletization process occurs through electrostatic aggregation of microalgal cells onto fungal hyphae, driven by interactions between negatively charged microalgal surface groups (e.g., phosphate and hydroxyl) and positively charged fungal cell wall polysaccharides. This synergistic association enhances biomass production, improves contaminant removal efficiency, and increases resistance to shear stress and environmental challenges [13].

Among the biological treatment systems, some studies highlight the benefits of using the microalga–fungus consortium in wastewater treatment. This is due not only to the greater ease of collecting microalgae, but also to the bilateral contribution of these microorganisms, resulting in superior pollutant removal efficiency compared to a system composed of only one type of microorganism (monoculture). This mechanism operates through fungal-mediated conversion of organic matter into CO₂, which is subsequently assimilated by microalgae along with environmental nutrients (e.g., nitrogen and phosphorus) for photosynthetic processes and biosynthesis of proteins, phospholipids, and nucleic acids. Notably, genera such as Chlorella, Tetradesmus, Chlorococcum and Coelastrella (microalgae) as well as Aspergillus, Penicillium, Trichoderma and Cunninghamella (filamentous fungi) demonstrate contaminants removal efficiencies exceeding 80% under optimized cultivation conditions [14,15].

In addition, when bioremediation is carried out through a microalga–fungus consortium, higher COD removal rates are achieved when the system is maintained under conditions that allow heterotrophic or mixotrophic growth. Thus, filamentous fungi can degradate macromolecules or reacalcitrant compounds, producing extracellular enzymes, and facilitating their metabolization for microalgae during heterotrophic/mixotrophic growth [16,17].

The pursuit of sustainable waste management solutions has intensified research on biotechnological processes employing microbial consortia for digestate treatment. Digestates are characterized by organic loads and significant concentrations of nitrogen and phosphorus, requiring optimized operational conditions to enhance treatment efficiency and resource recovery [2,3].

Even though the benefits of the microalga–fungus consortia for wastewater treatment are known, the role of aeration in the formation of microalga–fungus pellets is not widespread and it is assumed that the aeration rate used for fungi development is the same for the consortium. The dynamic oxygen requirements of this symbiotic system create a complex balance. Crucially, the impact of aeration rate on the structural integrity of algal–fungal pellets, their synergistic metabolism, and the subsequent effect on nutrient removal efficiency in real high-strength industrial digestates remains poorly quantified. Most studies focus on synthetic media or standard municipal wastewater, leaving a significant gap in understanding how to optimize this energy-intensive parameter for complex industrial effluents to achieve simultaneous energy consumption reduction and improved treatment goals [5,8,9].

Due to the direct influence of aeration on crucial synergistic relationships—including fungal respiration, heterotrophic metabolism of organic pollutants, and photosynthesis and autotrophic nutrient assimilation by microalgae—this study aimed to investigate the impact of aeration rate on the tertiary treatment of anaerobic effluent from soft drink industry by evaluating the removal efficiencies of chemical oxygen demand (COD), total nitrogen (TN), and total phosphorus (TP). Thus, this study explored aeration rate optimization for co-cultivation between the fungus Penicillium gravinicasei and the microalgae Tetradesmus obliquus and Chlorella sp.

2. Materials and Methods

2.1. Wastewater Collection, Storage and Characteristics

The effluent was obtained from a company in the city of Maceió (−9°33′14.3″ S, 35°44′23.7″ W) in September 2024, resulting from the secondary treatment process under internal circulation in an anaerobic reactor with an inlet flow rate and pH of 70 m³ h⁻¹ and 6.5–7.5, respectively, and submitted for a residence time of between 16 and 24 h. It was taken to the laboratory, subjected to the simple filtration process on cellulose filter paper to remove coarse particles and stored frozen between −18 and −12 °C.

According to the methodologies detailed in Section 2.4, the digestate characterization showed chemical oxygen demand (COD), total nitrogen (TN) and total phosphorus (TP) of 172.86 ± 2.42 mg·L⁻¹, 14.51 ± 0.05 mg·L⁻¹ and 6.18 ± 0.02 mg·L⁻¹, respectively, in addition to a pH value equal to 8.60 ± 0.13, which demonstrated a good secondary treatment because the effluent before the anaerobic digestion exhibited the following characteristics for COD, TN, TP, and pH: 4200.43 ± 59.65 mg·L⁻¹, 13.59 ± 0.15 mg·L⁻¹, 6.80 ± 0.24 mg·L⁻¹ and 4.86 ± 0.09, respectively. However, the values obtained after the secondary treatment still exceed the limit for discharge and require an advanced treatment (tertiary treatment).

2.2. Species of Filamentous Fungus and Microalgae

The microalgal species Tetradesmus obliquus LCE-01 and Chlorella sp. were cultivated on solidified Nutrient Agar medium (Kasvi^®, Madrid, Spain) at 25 ± 2 °C under a 12:12 light/dark photoperiod (900 lumen LED lamps) in a growth chamber (SolidSteel^®, Piracicaba, Brazil) for 5 to 7 days. The filamentous fungus Penicillium gravinicasei LCE-06 was cultured on solidified Potato Dextrose Agar (PDA) medium (Kasvi^®, Madrid, Spain) at 25 ± 2 °C in a bacteriological incubator (Nova Instruments^®, Piracicaba, Brazil) in complete darkness for 7 days. All media were sterilized by autoclaving (FANEM^®, São Paulo, Brazil) at 121 °C and 1 atm for 15 min prior to use. Following the incubation period, fungal spores were harvested using a sterile 0.1% (v/v) Triton solution to generate a spore suspension. The spore concentration was subsequently quantified using a Neubauer counting chamber (Boeco^®, Hamburg, Germany) (Figure 1).

2.3. Treatment System

The experimental setup aimed to analyze the performance of the consortia formed by P. gravinicasei + T. obliquus and P. gravinicasei + Chlorella sp. using 500 mL Drechsel cylindrical glass flasks (60 mm external diameter), which simulate the behavior of an aerated tubular reactor in batch mode. The bioreactors received 350 mL of the effluent under study. The inoculum consisted of 8.5 × 10⁵ microalgae cells∙mL⁻¹ and a fungal spore concentration of 10⁴ spores∙mL⁻¹, imposing an initial pH of 7.5 [17,18] and a hydraulic retention time of 6 h, with intermittent sample collection. The microbial agents employed consisted of indigenous strains pre-acclimatized to wastewater treatment in prior studies.

Artificial lateral lighting (Taschibra^® LED 25 W 6500 K, Indaial, Brazil) of 100 µmol·m⁻²·s⁻¹ (measurement by HD 2302.0 radiometer, Delta Ohm^®, Caselle di Selvazzano, Italy) was provided. The reactors were aerated by an air supply system using a compressor (MOTOMIL^® 15/175 CMW, Naveantes, Brazil) at rates of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5 vvm. A 5 to 50 LPM air flow meter (maximum operating pressure: 0.6 MPa) (LZT M-6^®, Shanghai Shuangxu Electronics Co. Ltd., Shanghai, China) with a regulating valve function installed. Experiments with the monocultures were not carried out since previous works demonstrated lower treatment efficiencies than the consortia in all experiments [17,19,20].

2.4. Analyses

After collection, the samples were centrifuged at 3500 rpm for 30 min in a digital centrifuge (Tecnal^® NT-810, Piracicaba, Brazil) to separate the solid phase (microbial sludge) from the liquid phase, where contaminants were measured. Subsequently, mainly for COD determination, the samples passed through hydrophilic PES (polyethersulfone) and cellulose acetate filters of 0.45 and 0.22 µm (Chromastore^®, São Paulo, Brazil), respectively, to eliminate any solids that may not have been efficiently removed during centrifugation and that could affect the analyses.

2.4.1. Microbial Sludge Produced and pH Determination

To determine the microbial sludge produced (dry cell weight), the solids separated during the centrifugation step were used. They were transferred to pre-weighed capsules in an oven at 60 °C for 2 h, or until constant weight was reached. They were then cooled in a desiccator for 15 min and weighed. Cell dry weight was determined as the ratio of the dry residue mass to the sample volume. pH determination was performed using a digital pH meter R-TEC-7/2-MP (Tecnal^®, Piracicaba, Brazil) previously calibrated with 4.0 and 7.0 standards.

2.4.2. Chemical Oxygen Demand (COD) and Total Phosphorus (TP)

The analyses for chemical oxygen demand (COD) and total phosphorus (TP) were determined based on the recommendations of the Official Method of Analysis [21] using, respectively, the dichromate digestion method (absorbance reading obtained in a spectrophotometer at 600 nm) (Method 5220 B) and the ascorbic acid method (absorbance reading obtained in a spectrophotometer at 706 nm) (4500-P E).

2.4.3. Total Nitrogen (TN or TKN)

The procedure used was based on Standard Methods for the Examination of Water and Wastewater [22] structured in the digestion phases in a digestion block at 285 °C, distillation with nitrogen capture in boric acid solution and titration with 0.002 N sulfuric acid with calculation of the TKN concentration (Semi-Micro-Kjeldahl Method).

2.4.4. Contaminant Removal Rate, Sludge Production Rate and Conversion Factor

Contaminant removal rate, sludge production rate (biomass productivity) and conversion factor (contaminant consumed per biomass produced) were calculated by the following equations:

$$Contaminant Removal Rate \left({\displaystyle \frac{mg}{L·h}}\right)= {\displaystyle \frac{(S_{t=0}- S_{t_{cultivation}})}{t_{cultivation}}}$$

(1)

$$Sludge Production Rate \left({\displaystyle \frac{mg}{L·h}}\right)={\displaystyle \frac{(X_{t_{cultivation}}-X_{t=0})}{t_{cultivation}}}$$

(2)

$$Conversion Factor \left({\displaystyle \frac{{mg}_S}{{mg}_X}}\right)={\displaystyle \frac{Contaminant Removal Rate}{Sludge Production Rate}}={\displaystyle \frac{(S_{t=0}-S_{t_{cultivation}})}{(X_{t_{cultivation}}-X_{t=0})}}$$

(3)

where S is the contaminant concentration considered in mg·L⁻¹; X is the sludge (biomass) concentration in mg·L⁻¹; t is the cultivation time; t = 0 reflects the initial contaminant or sludge (biomass) concentration; and t_cultivation is the cultivation time considered in hours.

2.5. Experimental Design and Statistical Analysis

For each consortium (P. gravinicasei + T. obliquus and P. gravinicasei + Chlorella sp.) and each aeration rate (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 vvm), independent duplicates of the reactors were operated simultaneously. All analytical measurements (COD, TN, TP, and microbial sludge produced) were performed in duplicate for each replicate reactor. Data are presented as the mean ± standard deviation of the biological replicates. Statistical analysis was performed using analysis of variance (ANOVA) followed by Tukey’s Honestly Significant Difference (HSD) test to identify significant differences between group means at a 95% confidence level (p < 0.05).

3. Results and Discussion

3.1. Effect on Contaminants Removal

The effect of the microalga–fungus consortia resulted in significant reductions in anaerobic effluent when subjected to aeration rates around 1.5 vvm, with more pronounced variations under the effect of aeration for COD and TN removal (Figure 2). In parallel, for TP, similar removal efficiency was observed at aeration rates >1.0 vvm.

Furthermore, the maximum removal efficiency occurred in 2.5 h of hydraulic retention time (cultivation time), considering the set of three parameters, and the desirable discharge standard of 125 mg·L⁻¹, 10–15 mg·L⁻¹ and 1–2 mg·L⁻¹ for COD, TN and TP, respectively [23], which resulted in maximum removal percentages of 92.52 ± 1.01% (residual of 12.92 ± 1.62 mg·L⁻¹) for COD, 88.44 ± 0.05% (residual of 1.67 ± 0.02 mg·L⁻¹) for TN and 79.27 ± 0.03% (residual of 1.23 ± 0.02 mg·L⁻¹) for TP (Figure 3).

According to data described in the literature, aeration rates between approximately 0.5 and 1.5 L·min⁻¹ are commonly adopted in biological effluent treatments aiming at good mass and oxygen transfer during the process [24]. In the study of Yang et al. (2024) [18], an aeration rate of 0.3 vvm was applied in the remediation of secondary effluents in a membrane bioreactor using Chlorella proteinosa under a hydraulic retention time of 8 h, achieving removals of 65.6 ± 1.5% and 70.2 ± 0.7% for TN (initial of 10 mg·L⁻¹) and COD (initial of 50 mg·L⁻¹), respectively.

Similarly, Huang et al. (2023) [25] also found stability in the concentration of TP after the first moments of the treatment of synthetic municipal wastewater under aeration between 0.3, 0.6 and 0.9 vvm with an average removal efficiency of 97.23 ± 0.39% (initial of 7 mg·L⁻¹) using the microalga Chlorella vulgaris, in addition to better removal of COD and TN when higher aeration rate was applied.

Co-cultivating Chlorella vulgaris with aerobic sludge from a wastewater treatment plant, Radmehr et al. (2022) [26] found better TOC (total organic carbon) removals under aeration conditions of 0.8 and 1.2 vvm when compared to values of 0.2 and 0.4 vvm, as well as the absence of significantly different removals for PO₄-P. This study justifies better reductions not only in TOC but also of NH₄⁺ under conditions of greater aeration, directly relating the relation between the assimilation/degradation of these contaminants and the dissolved oxygen concentration.

This justification can be confirmed in studies of Huang et al. (2022) [27], who evaluated the influence of aeration on the bioremediation of wastewater by Tetradesmus dimorphus and obtained reductions in COD concentration (initial of 153 ± 17 mg·L⁻¹) of 92.10% and 99.40% for rates of 1.2 and 3.2 vvm, respectively, and an average of 89.95 ± 0.25% for TP (initial of 4.25 ± 0.75 mg·L⁻¹) in both aeration rates.

In another study, Kumkaew et al. (2023) [28] evaluated the performance of the filamentous fungus Phanerochaete chrysosporium (concentration 10⁷ spores·L⁻¹) in the treatment of anaerobic digestate with distillery residue or vinasse in a bubble column bioreactor under an operating volume of 4 L and varying the aeration rate at 0.5, 1.0 and 1.5 vvm. At the end of the seventh day of treatment, the best performance in COD removal was observed when subjected to 1.0 vvm, resulting in a removal of 46.33 ± 0.65% (initial COD = 29.45 ± 1.81 mg·L⁻¹). Still referring to the removal of organic matter by fungus, Pérez-Cadena et al. (2020) [29] analyzed the impact of aeration variation (1, 2 and 3 vvm) on the COD removal process (initial concentration 1600 mg·L⁻¹) in textile effluents by Trametes polyzona in an airlift reactor, observing removal levels of 95.2%, 95.4% and 96.2% for flows of 1, 2 and 3 vvm, respectively.

Applying a different genus of microalga, Liu et al. (2024) [30] used a photobioreactor and microalgae of the species Chlorococcum robustum AY122332.1 with a useful working volume of 15 L at a temperature of 28 ± 2 °C and 2 g·L⁻¹ of microalgae, as dry weight, for 30 days, divided into 4 feedback phases aiming at the remediation of NH₄⁺-N (initial concentration of 90 mg·L⁻¹), achieving removals in the order of 86% in each of the phases under aeration conditions of 1.0 vvm.

Investigating the use of higher aeration rates, Kusmayadi et al. (2022) [31] applied Chlorella sorokiniana to a gradual increase in the aeration rate from 1.0 to 1.5 and 2.0 vvm in the treatment of anaerobic digestate from the dairy industry, obtaining a directly proportional increase in the bioremediation potential in COD (4992 ± 96 mg·L⁻¹), TN (111 ± 4.8 mg·L⁻¹) and TP (30.4 ± 1.1 mg·L⁻¹) with removal efficiency percentages of 91.3 ± 3%, 96.7 ± 1% and 92.1 ± 2%, respectively.

It is important to note that in the type of reactor used, aeration not only provides oxygen to maintain fungal metabolic activity, and to a lesser extent, microalgae, or promotes better consortium interaction, but it also pneumatically agitates the system, promoting dispersion. If mechanical agitation were used, lower aeration rates could be achieved.

The aeration provides a constant flux of oxygen dissolved, but also interferes in the mixing inside the reactor and, consequently, the interaction between the gaseous phase and the cells, because dissolving oxygen is a physical–chemical process, and to absorb and metabolize it is a biological one; for this reason, its dynamics depends on the applied aeration rate and observed biological activity. Under lower aeration conditions of 0.5 and 1.0 volumes of air per volume of medium per minute (vvm), DO probably limited the mixing/metabolic activity of the heterotrophic fungus P. gravinicasei and reduced cooperative degradation of organic carbon. At 1.5 vvm, the combined effects of fungal respiration and photosynthetic oxygen production by microalgae with reasonable mixing was achieved, which enhanced nutrient uptake by the microbial consortium. At higher aeration rates, DO can be sufficient, but excessive shear stress disrupts microbial aggregation, impairs symbiotic interactions, and causes cellular damage [32].

This reinforces that, despite the benefits of aeration in terms of fluid and biomass homogeneity in the system, which are extremely important, especially in large-scale configurations, high rates result in increased shear stress, as they depend directly on the speed of the gas injected into the system, causing damage to the microorganisms in question, reducing growth and even cell lysis [33]. It is worth noting that the arrangement of agitation through air injection instead of mechanical methods minimizes the impacts of shear stress [34].

Regarding the conversion pathways present in the synergistic relationship, fungi perform heterotrophic metabolic activities by degradation and assimilating suspended solids through the release of extracellular polymeric substances (EPSs), composed of amino acids, nucleic acids, lipids, uronic acid, and various organic and inorganic compounds. This process facilitates the decomposition of complex and/or insoluble organic matter (such as proteins, carbohydrates, and fats) into smaller, soluble molecules that are readily absorbable by microalgae [33,34,35].

However, it is noteworthy that under conditions of high COD removal rates, the microalga–fungus consortium requires mixotrophic growth or the heterotrophic metabolism of both microalgae and fungi. This can involve the sequestration of free CO₂ and bicarbonate by carbonic anhydrase (in microalgae) [36]. Complementarily, to meet the consumption demands for cellular growth, microalgae assimilate soluble nutrients: nitrate through vacuolar sequestration, phosphate via ion exchange, ammonia through complexation, as well as heavy metal ions (Mg, Ca, Mn, Zn, Cu and Mo) by producing metallothioneins [36,37].

It is important to emphasize that aeration applied to the system directly influences key factors. It induces phosphorus precipitation in the medium (under high oxygen concentrations), consequently promoting its aggregation with microbial biomass and thereby enhancing the effective removal and recovery of phosphorus from the effluent. Furthermore, aeration provides the necessary agitation intensity to prevent an uneven distribution of flocculants during the pelletization process, an issue caused by the rapid dispersion of fungal spores under low agitation efficiency [38,39].

Finally, to evaluate the impact of aeration on pollutant removal, a statistical analysis was performed using analysis of variance (ANOVA), followed by the Tukey HSD (Honestly Significant Difference) test. ANOVA was applied to verify overall statistical differences between the results obtained after 2.5 h of treatment, while the Tukey HSD test allowed for pairwise comparison of means, identifying which aeration conditions presented significant differences in the removal of COD, TN, and TP (

Table 1. Tukey’s Honestly Significant Difference (HSD) test for COD, TN and TP under different aeration rates.

Aeration		P. gravinicasei + T. obliquus			P. gravinicasei + Chlorella sp.
Group 1	Group 2	COD	TN	TP	COD	TN	TP
0.5	1.0	YES	YES	YES	YES	YES	NO
0.5	1.5	YES	YES	YES	YES	YES	YES
0.5	2.0	YES	YES	YES	YES	YES	YES
0.5	2.5	YES	YES	YES	NO	YES	YES
0.5	3.0	YES	YES	YES	NO	YES	YES
0.5	3.5	YES	NO	YES	YES	YES	YES
1.0	1.5	YES	YES	YES	NO	NO	YES
1.0	2.0	YES	YES	YES	NO	NO	YES
1.0	2.5	NO	YES	YES	NO	YES	YES
1.0	3.0	YES	YES	YES	YES	NO	YES
1.0	3.5	YES	YES	YES	NO	YES	YES
1.5	2.0	NO	YES	NO	NO	NO	NO
1.5	2.5	YES	YES	NO	YES	YES	NO
1.5	3.0	YES	YES	NO	YES	NO	NO
1.5	3.5	YES	YES	YES	YES	YES	NO
2.0	2.5	YES	YES	NO	YES	YES	NO
2.0	3.0	YES	YES	NO	YES	NO	NO
2.0	3.5	YES	YES	YES	NO	YES	NO
2.5	3.0	YES	NO	NO	NO	YES	NO
2.5	3.5	YES	YES	YES	NO	YES	NO
3.0	3.5	NO	YES	NO	NO	YES	NO

).

It was possible to observe greater sensitivity to aeration of the bioremediation potential for the COD parameter, mainly in the P. gravinicasei + T. obliquus consortium, scoring the greatest average difference between the 0.5 and 1.5 vvm groups (value of 105.01), suggesting the achievement of better COD removals in the respective interval. This phenomenon also occurred for the P. gravinicasei + Chlorella sp. consortium, but with less intensity (value of 38.77). In contrast, for TP removal, there were no significant differences from 1.5 vvm onwards, suggesting that the phosphorus reduction is more pronounced in the first stages of increased aeration. This phenomenon is justified by microalgal consumption based on extracellular mechanisms using EPS with polyanionic properties promoting phosphorus adsorption in the cell wall in functional groups such as C=O, -OH and P=O [40] and, consequently, a sudden reduction in the first moments, followed by intracellular mechanisms (assimilation) for the synthesis of ATP, DNA, RNA and other key biomolecules [38,39,41]. Studies such as Luo et al. (2025) [42] found better treatment performance and phosphorus recovery for Chlorella sp. compared to species such as Scenedesmus sp. (FACHB-489), Selenastrum bibraianum (FACHB-271) and Chlamydomonas sp. (FACHB-359) with 1.59 times more extracellular than intracellular phosphorus at the end of the treatment.

3.2. Microbial Sludge Production and pH Variation

Analyzing the results shown in Figure 4, it is noted a higher biomass production up to 1.5 vvm, with values between 80 and 115 mg·L⁻¹ in 2.5 h. Despite this, in 6 h, there was continuous microbial sludge production, even with the reduction in effluent treatment efficiency, reaching values between 125 and 200 mg·L⁻¹. This behavior can be explained by the metabolism of microalgae, which, even under limiting nitrogen and phosphorus conditions, can capture carbon from the gas phase through autotrophy [28,43], and consequently, they can also end up providing nutrients to the filamentous fungus. The promotion of fungal growth and the consequent adhesion process of microalgae cells to their cell wall provides greater granulation/high-density flocculation, facilitating the sedimentation process and recovery of biomass for reuse, improving resistance to physical shocks and other environmental interferences (toxic and harmful substances), in addition to enhancing bioremediation efficiency [44,45].

Under conditions of low organic load (initial TOC of 170.11 ± 11.2 mg·L⁻¹) and aeration with 0.04% CO₂ supplementation, Daneshvar et al. (2019) [46] promoted the sequential cultivation of Scenedesmus quadricauda in wastewater, applying 90 μmol·m⁻²·s⁻¹ and an initial inoculation of 45 ± 5 mg·L⁻¹ with production of 430 mg·L⁻¹ and 360 mg·L⁻¹, respectively, in each 12-day incubation cycle and pH change from 6.3 ± 0.1 to 9.7 ± 0.3.

The application of aeration is indispensable in photobioreactor systems, serving critical functions in homogenizing culture media, facilitating gas–liquid mass transfer of CO₂ and O₂, and preventing biomass sedimentation. However, the present study reveals a critical operational threshold beyond which its benefits are negated by significant physiological impediments. Specifically, aeration rates exceeding 1.5 vvm induced pronounced hydrodynamic stress, which was detrimental to both symbiotic partners. This stress manifested as a substantial decrease in overall productivity and sludge (biomass) production. Furthermore, the elevated shear forces at these rates disrupted the morphological integrity of the fungi, critically impairing the formation of a compact mycelial network and thereby preventing the successful aggregation and maturation of stable pellets. The precise quantification of this upper tolerance limit constitutes a principal finding of this investigation, providing an essential scaling parameter for optimizing the energy efficiency and biomass yield of industrial-scale algal–fungal co-culture systems.

In accordance with the previous conditions, Chen et al. (2021) [47] cultivated less than 400 mg∙L⁻¹ of Chlorella vulgaris in the first 24 h in a reactor filled with a mixture of BG-11 and activated sludge extract characterized by an initial content of 352.4 ± 5.6 mg∙L⁻¹ of TOC.

The decrease in production and biomass with the increase in the aeration rate causes stress to both microalgae and fungi, and makes it difficult to form pellets due to high shear rates, especially for fungi in the formation of a compact mycelium.

According to Du et al. (2019) [48], the formation of biomass in the form of pellets can be explained by the absence of the smooth outer fraction of the microalgal cell wall, which is caused by a strong physical interaction. This process leads to the exposure of the fibrous surface and subsequent adhesion to the fungal hyphae (mycelium). As visualized in Figure 5, our results corroborate this mechanism, demonstrating clear cellular aggregation in both microbial consortia. It is plausible that, over a longer cultivation time and with the application of pre-pelletized fungal spores as an inoculum—as utilized by Du et al. (2019) [48]—this observed aggregation behavior could develop into the mature pelletization phenomenon.

There are also other factors with a high impact on aggregation/pelletization, such as the fungus–alga ratio (maximization to 1:1), in addition to pH values, light intensity, temperature, agitation, and exposure to phosphate and carboxylic groups, proven by FT-IR tests according to studies by Chu in the polarity charges [49].

The impacts of aeration on the characteristics of microbial biomass/sludge during pellets formation have been little investigated in the scientific community, as there is a dependence on the formation of pellets and their maintenance according to aeration, which can promote intrinsic characteristics, such as the formation of exopolysaccharides (EPS) that favor or do not favor the aggregation of species [50,51].

Regarding the pH behavior in the treatment systems (Figure 6), in both systems, the pH increases significantly during the first hour of treatment, starting from initial values close to 7.5 and stabilizing around 8.6 to 8.9 for all flow rates analyzed. According to Khalatbari et al. (2024) [52], pH increases can be justified by the growth of microalgae; however it is worth highlighting the adequacy of the pH levels discussed with the range required in the standards of the National Environmental Council of Brazil in resolution no. 430 of 2011 established between values of 5 and 9, thus not requiring adjustments to the effluent in the post-treatment discharge period.

Upon increasing the microalgal cell concentration and the consequent enhancement of the substrate-product effect in the photosynthesis process, the inorganic carbon system shifts predominantly towards the bicarbonate (HCO₃⁻) and carbonate (CO₃²⁻) species. Concurrently, the consumption of CO₂ in microalgal photosynthesis drives the equilibrium, causing bicarbonate to dissociate and release a proton (H⁺) [CO₂ + H₂O ⇌ H₂CO₃ ⇌ H⁺ + HCO₃⁻], which counteracts a further pH increase and establishes a stable alkaline equilibrium [53]. Such alkaline pH stability in the microalga–fungus consortium process is documented in the literature, for instance, in the studies by Mekpan et al. (2024) [54], who applied Scenedesmus sp. SPP and Aspergillus tubingensis TSIP9 for the bioremediation of palm oil mill effluent, maintaining the pH level at 9.5 from the second day until the end of the treatment.

In the study of Zhou et al. (2023) [55], the cultivation of the microalga Neochloris oleoabundans resulted in an increase in the pH of the medium from 7 to 9 within the first 6 hours of cultivation in an aerated photobioreactor at 1.0 vvm. In comparison, at high rates (1.2 and 3.2 vvm), Huang et al. (2022) [27] noticed an increase in pH in the Tetradesmus dimorphus cultivation system to approximately 8.0 (initial 6.5) in the first 24 h, highlighting a smaller change in this value since supplementation with CO₂ in the medium corroborates acidification through the formation of carbonic acid (H₂CO₃) (Yusuf et al., 2020) [56].

3.3. Contaminant Removal Rate and Sludge Production Rate

The consortium between P. gravinicasei and T. obliquus obtained better COD removal rates in relation to P. gravinicasei + Chlorella sp. with maximum values between 70 and 80 mg·L⁻¹·h⁻¹ while for TN and TP, values between 2 and 6 mg·L⁻¹·h⁻¹ were found in both consortia (Figure 7).

These values were higher than those found in the literature studies. For example, the performance of Chlorella was evaluated by Ran et al. (2023) [57] being subjected to anaerobic digestate treatment of swine wastewater and initial characterization equivalent to 1634.5 ± 24.5 mg·L⁻¹ of TN and 39.55 ± 2.45 mg·L⁻¹ of TP under condition of 0.2 vvm, 200 μmol·m⁻²·s⁻¹ and 25 ± 1 °C, resulting in removal rates equivalent to 1.09 and 0.125 mg·L⁻¹·h⁻¹, respectively. Similarly, and considering microbial consortia, Dong et al. (2022) [58] used anaerobic digestate from the pig farm to evaluate the bioremediation power of the consortium Chlorella vulgaris-Ganoderma lucidum (T1) and Scenedesmus obliquus-Pleurotus ostreatus (T2) in addition to the endophytic bacterial strain S395–2 in both systems, obtaining removal rates of 3.79 mg·L⁻¹·h⁻¹ for COD, 0.41 mg·L⁻¹·h⁻¹ for TN and 0.06 mg·L⁻¹·h⁻¹ for TP. Finally, Qian et al. (2022) [59] obtained values of 23.95 mg·L⁻¹·h⁻¹ for COD, 1.80 mg·L⁻¹·h⁻¹ for TN and 0.05 mg·L⁻¹·h⁻¹ for TP after symbiosis of Chlorella pyrenoidosa and Lichtheimia ornata.

Applying the study to the species Chlorella sorokiniana, Taghavijeloudar et al. (2021) [60] achieved removal rates equivalent to 0.66 mg·L⁻¹·h⁻¹ (initial COD of 211 ± 3 mg·L⁻¹), 0.07 mg·L⁻¹·h⁻¹ (initial NH₄⁺ of 34.1 ± 2.8 mg·L⁻¹) and 0.03 mg·L⁻¹·h⁻¹ (initial PO₄^3- of 6.1 ± 0.5 mg·L⁻¹) under 0.6 vvm. Under conditions of light intensity and temperature similar to the present study but with a higher contaminant load, Kusmayadi et al. (2022) [31] reached rates of 18.99 mg·L⁻¹·h⁻¹ (COD), 0.44 mg·L⁻¹·h⁻¹ (TN) and 0.12 mg·L⁻¹·h⁻¹ (TP) for 2.0 vvm.

For the microbial sludge production rate (Figure 7), maximum rates between 12.5 and 20 mg·L⁻¹·h⁻¹ were obtained at 1.5 vvm, which are high values compared to those found in the literature. For example, Yang et al. (2022) [61] verified the growth of the microalga Chlorella pyrenoidosa in a sequential photobioreactor with saline wastewater under aeration of 0.04 vvm and approximately 100 μmol·m⁻²·s⁻¹, recording a biomass productivity equal to 2.23 mg·L⁻¹·h⁻¹, very low in relation to that obtained. In another example, the cultivation of Chlorella minutissima was promoted by dos Santos et al. (2021) [62] in airlift bioreactors containing landfill leachate under conditions of light intensity of 125 μmol·m⁻² ·s⁻¹ and aeration flow of 0.45 vvm, resulting in a biomass productivity of 3.65 mg·L⁻¹·h⁻¹.

A study of the influence of aeration (0.3, 0.6 and 1.13 vvm) on the cultivation of Chlorella vulgaris was performed by Huang et al. (2023) [25], which was carried out in a photobioreactor containing synthetic municipal wastewater under 111 μmol·m⁻²·s⁻¹, increasing from 1.66 (lowest air flow) to 2.92 mg·L⁻¹·h⁻¹ (highest air flow) after initial inoculation of 0.36 g·L⁻¹.

In parallel, de Sá Filho et al. (2023) [63] conditioned T. obliquus in the treatment of groundwater contaminated with nitrate in a bubble column reactor, while aeration of 0.5 vvm and intensity of 100 µmol·m⁻²·s⁻¹ obtained a biomass production rate of approximately 2.10 mg·L⁻¹·h⁻¹. In another example, at an aeration rate of 1.0 vvm, Qin et al. (2016) [64] conditioned the microalgae Chlorella sp. and Scenedesmus sp. for growth consortium in dairy wastewater with a light intensity of 150 μmol·m⁻²·s⁻¹ and 5% CO₂ supplementation with final values of 31.32 mg·L⁻¹·h⁻¹.

Additionally, Kemel et al. (2025) [65] studied the biomass production of the microalga Botryococcus braunii in photobioreactors at 100 μmol·m⁻²·s⁻¹ with a final rate of 5.125 mg·L⁻¹·h⁻¹ (0.165 vvm). For the microalga Neochloris oleoabundans, Zhou et al. (2023) [55] used a horizontal aerated reactor at 1 vvm with 5% CO₂ supplementation and a productivity of 47.5 mg·L⁻¹·h⁻¹ (initial inoculation of 0.5 g·L⁻¹). For Nannochloropsis oculata, Sun et al. (2018) [66] obtained biomass productivity equal to 6.875 mg·L⁻¹·h⁻¹ after cultivation in an open channel pond (initial inoculation of 0.1 g·L⁻¹) with aeration of less than 0.1 vvm also supplemented with 5% CO₂.

Finally, Medeiros et al. (2025) [17], by treating dairy wastewater (COD, TN and TP concentrations of 223.28, 12.73 and 3.64 mg·L⁻¹, respectively) by the consortium between Tetradesmus obliquus and Cunninghamella echinulata (microalga–fungus) in photobioreactors operated during 7 days at 100 μmol·m⁻²·s⁻¹, obtained removal rates for COD, TN and TP of 1.125, 0.1125 and 0.05 mg·L⁻¹·h⁻¹, and sludge production rate of 1.64 mg·L⁻¹·h⁻¹, lower values than those found in this work.

It is also possible to point out that, after maximizing pollutant removal within 2.5 h, concentrations in the system gradually increased in both consortia, which can be justified by the reduction in growth rate with a proven impact on limiting nutrient bioavailability parameters and/or microbial stress through aeration rates and probable partial sludge death and cell disruption, as shown in Figure 7.

3.4. Conversion Factor of Contaminant Removed per Sludge Produced

The conversion factors were dependent on the aeration rate applied (Figure 8). Overall, for COD, the highest conversion factors were obtained at 1.5 vvm for the PN + TO consortium and reasonable values for the PN + CH consortium. However, the highest conversion factor values for the PN + CH consortium were between 3 and 3.5 vvm, but they were inversely negative for PN + TO. From this perspective, to cover both consortia, an aeration rate of 1.5 vvm met expectations well. It is important to emphasize that microalgae can capture atmospheric carbon, and for this reason, this conversion factor is particularly important.

The consumption of organic matter, quantified as COD, is primarily attributed to the metabolic activity of the fungal fraction of the consortium, but with the participation of microalgae during mixotrophy. The PN + TO and PN + CH consortia achieved conversion factor values of 1.63 and 1.67 mg_COD/mg_sludge, respectively, with PN + TO at 1.5 vvm, and PN + CH at higher aeration rates. It is important to emphasize that conversion factor values greater than 1 for COD in microbial sludge during wastewater treatment have already been observed [17,67,68,69].

Regarding the conversion factor of nitrogen in sludge, maximum values between 0.131 and 0.185 mg_TN/mg_sludge were achieved. Multiplying by 100 indicates an average nitrogen content in biomass of around 14–15%, achieving good nitrogen accumulation values in biomass at an aeration rate of 1.5 vvm, although not the highest values obtained, but in line with the findings for COD. Considering phosphorus, values between 0.050 and 0.069 mg_TP/mg_sludge reveal a great capacity of the sludge formed to accumulate phosphorus, showing a constancy of performance from 1.5 vvm even with 1.5 h of cultivation time. This behavior, already discussed previously, is characteristic of a removal mechanism by cellular assimilation to immediately meet the biochemical demand (synthesis of ATP and nucleic acids), which is more intense in the exponential growth phase of the biomass [42]. In the work of De Farias Silva and Sforza (2016) [43], under conditions of excess and limitation of nitrogen and phosphorus, cultivating Chlorella vulgaris at different residence times and light intensities led to nitrogen and phosphorus assimilation factors of at most 0.14 and 0.04 mg_{N or P}/mg_{dry biomass}.

4. Conclusions

Based on the results, it was possible to identify the relevance of the aeration rate as a critical factor in the performance of bioremediation systems using microalgae–fungal consortia. The T. obliquus + P. gravinicasei and Chlorella sp. + P. gravinicasei consortia showed significant efficiency in the removal of COD, TN, and TP, meeting the regulatory limits established for discharge. We showed that moderate aeration rates (such as 1.5 vvm) favor greater removal efficiency in terms of residual pollutant concentrations, while high rates demonstrate negative impacts due to biological stress and biomass dispersion, especially for the T. obliquus + P. gravinicasei consortium. In contrast, increasing the aeration rate up to 2.5 vvm in the consortium with Chlorella sp. showed higher performance for some parameters, although it can be operated with reasonable performance also at 1.5 vvm.

This study can be improved, and future perspectives include (i) the integration of real-time monitoring and control systems for dynamic adjustment of oxygenation conditions, ensuring pellet stability; and (ii) the technical–economic validation of the process on a pilot scale, increasing the feasibility of application under industrial conditions.