A Comparative Study on Various Pretreatment Methods of Anaerobic Digestion Piggery Effluent for Microalgae Cultivation

Abstract

Anaerobic digestion piggery effluent (ADPE), dark brown with high turbidity and ammonium, inhibits algal growth and requires pretreatment for cultivation. This study compared various physical/biological pretreatment methods for microalgae cultivation. The results showed that the strategy of 10%ADPE fungal cultivation (10%AF) pretreatment and subsequent microalgae cultivation achieved maximum specific growth rate (0.094 d⁻¹) with productivity (0.014 g L⁻¹ d⁻¹) and significant nutrient removal: 100% ammonium nitrogen, 99% total nitrogen, 63% total phosphorus, 91% chemical oxygen demand. However, the pathogenic fungus used poses safety risks, requiring future screening of eco-friendly alternatives. This study demonstrated that the strategy could be a promising approach to algal biomass production and nutrient removal from ADPE.

1. Introduction

Piggery wastewater has become a serious environment issue in China [1]. To address this issue, the anaerobic digestion (AD) method for piggery manure, which could be the biogas resources has been widely used in China [2]. However, a large amount of AD piggery effluent (ADPE) would be produced during the AD process, which could cause secondary pollution issues [3]. Generally, the ADPE could be used for agricultural fertilization with high contents in nitrogen and phosphorus [4]. However, overuse of ADPE could also cause severe soil and water pollution. Therefore, harmlessly treatment for ADPE has aroused widespread concern [5]. Until now, many methods for ADPE treatment include physical (e.g., mechanical screening), chemical (e.g., flocculant precipitation), and biological (e.g., activated sludge) methods had been applied [6].

In recent years, the cultivation of microalgae to remove nutrients from ADPE has become a new promising biotechnology [7], since it has many advantages, e.g., higher efficiency, lower energy consumption, and no chemical addition, compared with other ADPE treatment methods [8]. Furthermore, microalgae biomass can also be processed into value-added products such as biofuels and animal feeds [9]. Theoretically, the treatment of wastewater by microalgae has great environmental and economic potential [10]. However, culturing microalgae with ordinary wastewater requires adding more nutrients, leading to the high treatment cost, which hinders the large-scale application of the microalgae-based wastewater treatment [10]. On the contrary, ADPE contains nutrient-rich nitrogen, phosphorus, and other trace elements, which are the main nutrients in traditional culture medium [2]. Therefore, cultivating microalgae in ADPE will reduce costs and simultaneously remove pollutants [11].

However, biogas fermentation substrates—typically mixtures of piggery wastewater and small amounts of agricultural waste (e.g., straw) from nearby swine farms of varying scales and feed types—result in ADPE with complex physical, chemical, and biological properties, hindering microalgae cultivation. High turbidity from suspended solids (SS) reduces light transmittance, weakening algal photosynthesis [12], while high concentrations of organic matter and ammonium inhibit microalgal growth but promote heterotrophic microbial proliferation [2], necessitating ADPE pretreatment. Although physical-chemical methods like ammonia stripping and cationic starch flocculation have been used [13], they incur high costs and secondary pollution risks due to energy/chemical requirements [14]. Thus, low-energy, chemical-free biological pretreatment—particularly fungal pretreatment—has emerged as a promising alternative [15], effectively removing nutrients and chromatic substances via biosorption and biodegradation [16]; fungi secrete extracellular enzymes (e.g., laccase, manganese peroxidase, lignin peroxidase) to break down complex recalcitrant organics into smaller molecules while reducing wastewater color intensity [17].

However, current biological pretreatment studies mostly focus on single-function optimization and lack systematic comparisons of different methods. This study compared the treatment effects of various physical/biological pretreatment methods on ADPE, with a focus on evaluating the application potential of the fungal pretreatment strategy in microalgae cultivation. It aims to clarify its enhancement effects on algal growth and nutrient removal, and provide a theoretical basis and technical reference for the efficient resource utilization of ADPE.

2. Materials and Methods

2.1. Preparation of ADPE

The ADPE used in this study was collected from an anaerobic digester in a biogas plant located in Xinyu City, Jiangxi Province, China, which could treat 4 million tons of piggery wastewater per year. Fresh piggery wastewater with 6–8% solid content was treated in a continuous stirred tank reactor (CSTR), simultaneously producing approximately 1000 m³ of ADPE per day. All the collected ADPEs were filtered through a solid-liquid centrifugal separator (LLW800, KAIDI, Suzhou, China) to remove large, suspended solids, autoclaved (121 °C, 40 min) to remove bacteria, and stored at 4 °C until use.

2.2. Microorganism and Culture Conditions

The algal strain for ADPE remediation was isolated from landfill leachate (Nanchang, China) and identified as Chlorella pyrenoidosa via gene sequencing [18]. Algal cells were cultured in 100 mL Tris-Acetate-Phosphorus (TAP) medium (

Table A1. Chemical composition of tris-acetate-phosphate (TAP) medium with 1 mL L⁻¹ of acetic acid.

Chemicals	Concentration (mg L−1)
Tris salt	2420
NH4Cl	375
CaCl2•2H2O	50
MgSO4•7H2O	100
K2HPO4	288
KH2PO4	144
Na2EDTA•2H2O	50
ZnSO4•7H2O	22
H3BO3	11.4
MnCl2•4H2O	5
FeSO4•7H2O	5
CoCl2•6H2O	1.6
CuSO4•5H2O	1.6
(NH4)6Mo7O24•4H2O	1.1

) within 250 mL flasks, incubated at 28 ± 2 °C under 100 μmol m⁻² s⁻¹ continuous cool-white fluorescent light with 120 rpm shaking.

The fungal strain Aspergillus fumigatus for ADPE pretreatment was isolated from biogas plant soil via streak plate method. Preliminary tests confirmed its growth in ADPE with 500 mg L⁻¹ NH₄⁺-N and tolerance to 2500 NTU turbidity. Fungal cultures were grown on potato dextrose agar (PDA) slants (

Table A2. Chemical composition of potato dextrose agar (PDA) medium.

Chemicals	Concentration (g L−1)
Potato powder	5
Glucose	15
Peptone	10
NaCl	50
Agar	15

) at 30 ± 1 °C for 72 h. Spore suspensions were prepared by vortexing agar slants with distilled water for 1 min. This suspension served as inoculum for 10%ADPE treatment, with fungal pellets removed via 100-mesh gauze prior to microalgae cultivation.

2.3. Different Pretreatment Processes Before Microalgae Cultivation

2.3.1. Turbidity Reduction

In this study, various pretreatment methods were used to reduce the turbidity of ADPE. (1) ADPE was first diluted with distilled water at three dilution levels (v/v): 5% ADPE, 7.5% ADPE, and 10%ADPE, representing 20, 13.3, and 10 dilution times of ADPE, respectively; (2) 10%ADPE was filtered by three types of filter paper, e.g., fast speed filter paper (pore size 80–120 μm, namely 10%F), medium speed filter paper (pore size 30–50 μm, 10%M), and slow speed filter paper (pore size 1–3 μm, 10%S), to remove SS; (3) 10%ADPE was naturally settled for 24 h (namely NS24 h) or 48 h (NS48 h), and the supernatant was taken out for subsequent experiments; (4) Using a heater (YGR-002, Yee, Shanghai, China) to maintain the temperature at 30 ± 1 °C, fungal cultivation was employed to reduce turbidity in 10%ADPE (referred to as 10%AF), with aeration filtered through a 0.45-µm pore-size syringe filter and controlled at 1 L min⁻¹ using an air pump.

2.3.2. Ammonia Stripping

NH₄⁺-N would be converted to NH₃ when air is aerated into the water under alkaline condition [19]. It has been successfully used to remove NH₄⁺-N from different wastewater since air stripping generates no secondary pollution and is simple to operate [20]. In this study, three factors were selected in the ammonia stripping experiment: temperature, pH, and air flow rate. The details of these experiments are shown in

Table 1. Experimental conditions in air stripping pretreatment process.

Experimental Conditions	Temperature Experiment	pH Experiment	Air Flow Rate Experiment
ADPE dilution ratio (%)	7.5	7.5	7.5
Initial pH of ADPE	9.5	8.0, 8.5, 9.0, 9.5 *	9.5
Temperature (°C)	25, 40	25	25
Air flow rate (L min−1)	1.5	1.5	0, 0.5, 1.0, 1.5, 2.0, 2.5
Stripping time (h)	6	6	6

* 9.5 included two pretreatment conditions of 7.5% anaerobic digestion piggery effluent (7.5% ADPE) pretreated at pH 9.5 via air stripping, with and without filtration using fast filter paper.

.

2.4. Microalgal Cultivation

C. pyrenoidosa was inoculated in the pretreated ADPE, which reduced the turbidity and NH₄⁺-N. The inoculation level was 1.0 × 10⁵ cells mL⁻¹. The optical density (OD), chlorophyll-a, and nutrient removal rates of all experimental groups were measured after 0, 1, 3, 5, 7, 9, 11, 13, and 15 days. During microalgae cultivation, these flasks were kept at 25 °C in a constant-temperature shaker at 120 rpm under a continuous cool-white fluorescent light illumination at 100 μmol m⁻² s⁻¹. In order to investigate the influence of turbidity, the concentration of NH₄⁺-N was adjusted to 300 mg L⁻¹ using NH₄Cl in the turbidity experiment.

2.5. Analytical Methods

2.5.1. Determination of Algal Growth

First, a 5-mL algal sample was centrifuged at 4000 rpm for 10 min to separate the biomass from the ADPE. Then, chlorophyll-a of C. pyrenoidosa was extracted by using 3–5 mL of methanol, which was shaken on the vortex mixer for 10 min in a dark place, and then the mixture was centrifuged at 4000 rpm for 20 min. Finally, the absorbances of the supernatant were measured at A_665nm and A_652nm, respectively [21].

The concentration of chlorophyll-a was calculated using Equation (1):

Chlorophyll-a (mg L⁻¹) = 16.72 × A_665nm − 9.16 × A_652nm

(1)

According to Equations (2) and (3), the dry cell weight (DW, g L⁻¹) of algae and the increase rate of DW (R, %) were calculated, respectively [22]:

DW = 0.2735 × OD_680nm + 0.0936 (R² = 0.98)

(2)

R = (DW_m − DW₀)/DW₀ × 100%

(3)

DW_m: the maximum DW of algae during growth; DW₀: the DW of algae on the initial day.

The specific growth rate (μ, d⁻¹) in an exponential phase of algal growth was calculated according to Equation (4) [11]:

μ = (lnDW₂ − lnDW₁)/(t₂ − t₁)

(4)

where DW₁ and DW₂ were algal DWs on day t₁ and day t₂. The biomass productivity (P, g L⁻¹ d⁻¹) was calculated according to Equation (5) [23]:

P = (DW₂ − DW₁)/(t₂ − t₁)

(5)

2.5.2. Nutrient Analyses

A DR6000 spectrophotometer (Hach, Loveland, CO, USA) was used to periodically determine algal cell concentration by OD measurement at a wavelength of 680 nm (Hach Co., Loveland, CO, USA). The pH was measured using a pH meter (PHSJ-4A, NESA Scientific Instrument Co., Ltd., Beijing, China). The methods for measuring NH₄⁺-N, chemical oxygen demand (COD), total nitrogen (TN), and total phosphorus (TP) are provided by the Ministry of Ecology and Environment of China (the HJ 535-2009, HJ 828-2018, HJ 636-2018, and GB 11893-1989 standards), and all calibrated with national environmental protection standard samples (error < ±5%) with detection limits of 0.025, 5, 0.05, and 0.01 mg L⁻¹, respectively. Turbidity (NTU) was measured using a nephelometer with calibration of R² > 0.999 and detection limits of 0.01 NTU (WZB-170 Turbidimeter, REX, Shanghai, China). All experiments were carried out in triplicate, and the average values were reported.

2.6. Statistical Analysis

All experiments were carried out in triplicate, and the experimental data were expressed as mean ± standard deviation, unless otherwise stated. Statistical analyses were performed using OriginPro 2021 (OriginLab Corporation, Northampton, MA, USA).

3. Results and Discussion

3.1. Characteristics of ADPE

The concentrations of NH₄⁺-N, TN, TP, COD, SS, turbidity, and pH were examined to be 4497.1 ± 16.3 mg L⁻¹, 5100.0 ± 101.4 mg L⁻¹, 202.0 ± 3.5 mg L⁻¹, 24,210.2 ± 224.0 mg L⁻¹, 20,300.0 ± 136.3 mg L⁻¹, 14,210.1 ± 182.5 NTU, and 8.34, respectively, in raw ADPE. The results indicated that the ADPE has highly variable characteristics, primarily due to the seasonal fluctuations in swine waste production and the co-substrates in digestion (

Table 2. Chemical composition of raw anaerobic digestion piggery effluent (ADPE) and various pretreated ADPEs.

	Raw ADPE	10%ADPE	10%F	10%M	10%S	NS24h	NS48h	10%AF
pH	8.34	9.25	9.24	9.22	9.21	9.18	9.16	6.10
NH4-N (mg L−1)	4497.1 ± 16.3	428.5 ± 1.2	401.3 ± 2.4	386.1 ± 2.1	360.4 ± 1.5	413.8 ± 0.6	406.5 ± 1.2	346.1 ± 0.8
TN (mg L−1)	5100.0 ± 101.4	449.0 ± 1.7	410.4 ± 2.7	391.2 ± 6.1	376.8 ± 3.2	432.6 ± 5.1	426.2 ± 3.5	349 ± 3.1
TP (mg L−1)	202.0 ± 3.5	24.4 ± 0.6	20.4 ± 0.1	19.6 ± 0.1	19.3 ± 0.3	20.2 ± 0.1	20.0 ± 0.2	17.6 ± 0.5
COD (mg L−1)	24,210.2 ± 224.0	2310.2 ± 23.0	1540.0 ± 18.0	1320.0 ± 64.1	1050 ± 66.3	2230 ± 42.2	2060 ± 34.3	672 ± 23.1
SS (mg L−1)	20,300.0 ± 136.3	2010.1 ± 3.1	1461.3 ± 0.8	1130.1 ± 1.6	982.6 ± 3.4	1966.1 ± 6.1	1814.6 ± 9.4	866.1 ± 10.1
Turbidity (NTU)	14,210.1 ± 182.5	1330.3 ± 2.3	765.1 ± 1.1	612.5 ± 0.6	466.3 ± 4.1	1210.2 ± 2.6	1102.8 ± 8.6	443.3 ± 5.1

).

According to the literature, all AD effluents (ADEs) are relatively alkaline due to temporary overloading of the digester, causing an incomplete digestion [21]. Compared with the concentrations of NH₄⁺-N (1220 mg L⁻¹) and SS (326 mg L⁻¹) reported by Scaglione et al. [24], the ADPE used in this study has much higher concentrations. The high SS of ADPE prevented the passage of sunlight through water bodies and thus greatly inhibited photosynthesis for algal growth [25], and the high level of NH₄⁺-N is potentially toxic to algal cells [26]. Thus, ADPE pretreatment was required to alleviate the light inhibition and ammonia toxicity for subsequent microalgae cultivation. Compared with the standard TAP medium, ADPE presented sufficient nutrients for algal growth.

3.2. Pretreatment of ADPE for Turbidity and Ammonia Removal

According to the previous studies of the cultivation of microalgae in ADPE, dilution would decrease the turbidity, which can avoid the limitation of light and the toxicity of ammonia [27]. Since the turbidity of the ADPE is caused by suspended matter or impurities that interfere with the penetration of light, the turbidity and nutrient concentrations of the ADPE would decrease after dilution. However, dilution pretreatment can also alleviate the color inhibition and ammonia toxicity in ADPE but increase water consumption [28]. For saving water resources, filtration, fungi cultivation, and air stripping were used to pretreat the ADPE in this study.

As shown in Table 2, COD concentrations in 10%ADPE decreased by filtration, e.g., 10%F, 10%M, and 10%S pretreatments from 2310 mg L⁻¹ to 1540 mg L⁻¹ (removal rate of 33.3%), 1320 mg/L (42.9%), and 1050 mg L⁻¹ (54.5%), respectively. The turbidities in 10%F, 10%M, and 10%S pretreatments also exhibited high removal rates of 42.5%, 54.0%, and 65.0%, respectively. In contrast, the filtration in all pretreatments had relatively low removal rates for NH₄⁺-N, TN, and TP. The characteristics of the ADPE remained almost unchanged for NS24h and NS48h pretreatments. The removal rates of COD, turbidity, NH₄⁺-N, TN, and TP by 10%AF pretreatment were 70.9%, 66.7%, 19.2%, 22.3%, and 27.9%, respectively. Therefore, the fungal pretreatment may be a more reliable procedure for turbidity and nutrient removal from ADPE. This is consistent with other studies, with the high removal rate of turbidity by fungi, and the removal mechanism was mainly biodegradation and biosorption [3]. However, the fungus used for fungal pretreatment and subsequent microalgae cultivation in this study was a pathogenic fungus, which would cause safety risks [29]. Therefore, it is necessary to screen out a non-pathogenic alternative and environmentally friendly fungus (e.g., Trametes versicolor or Phanerochaete chrysosporium) in future studies.

In the process of air stripping, NH₄⁺-N had high removal rates of 76.6%, 75.4%, and 72.8% for the temperatures, pH, and air flow rate, respectively. According to the change in removal rate for NH₄⁺-N, the stripping temperature, pH, and air flow rate with 40 °C, 9.5, and 1.5 L min⁻¹ were preferable, respectively (Figure 1). As pH increased, the removal rates of NH₄⁺-N also increased (Figure 1a). Kwon et al. reported that air stripping could reduce the concentration of TN and NH₄⁺-N, which is similar to our study [30]. Air stripping could effectively remove NH₄⁺-N and TN, but had no effect on COD and turbidity.

3.3. Algal Growth in ADPE

3.3.1. Algal Growth in Pretreated ADPE with Reduced Turbidity

C. pyrenoidosa could survive in 5%ADPE, 10%ADPE pretreated with filter paper and fungal cultivation. The maximum OD in 5%ADPE, 10%F, 10%M, 10%S, and 10%AF pretreatments were 0.87, 0.42, 0.46, 0.60, and 1.02, respectively (Figure 2a). C. pyrenoidosa did not grow in 7.5%ADPE, 10%ADPE, NS24h, and NS48h pretreatments. The ODs in 7.5%ADPE and 10%ADPE pretreatments dropped to 0 on the ninth day, while those in NS24h and NS48h pretreatments dropped to 0 on the seventh day. The specific growth rates of C. pyrenoidosa in 5%ADPE and 10%AF pretreatments were 0.079 and 0.094 d⁻¹, respectively (

Table 3. Algal growth parameters in various pretreated anaerobic digestion piggery effluent (ADPE).

Pretreatment	Specific Growth Rate (d−1)	Biomass Productivity (g L−1 d−1)
Dilution, filtration, and sedimentation
5%ADPE	0.079	0.011
10%F	0.024	0.002
10%M	0.035	0.003
10%S	0.044	0.005
10%AF	0.094	0.014
Air stripping at temperature (°C)
25	0.079	0.011
40	0.088	0.013
Air stripping at pH
8	0.060	0.007
8.5	0.080	0.011
9	0.085	0.011
9.5	0.087	0.013
10	0.072	0.009
Air stripping at air flow rate (L min−1)
0.5	0.052	0.006
1	0.077	0.011
1.5	0.082	0.013
2	0.076	0.012
2.5	0.070	0.013

). However, 10%F, 10%M, and 10%S pretreatments exhibited slow growth rates of 0.024, 0.035, and 0.044 d⁻¹, respectively. Wang et al. [22] showed that higher algal biomass was obtained at 5% diluted ADE from dairy manure rather than the other diluted ones during Chlorella sp. cultivation, which was consistent with our study. Likewise, the algal biomass productivity in 10%AF pretreatment was the highest in the turbidity experiment, reaching 0.014 g L⁻¹ d⁻¹. This was because 10%AF pretreatment removed a large amount of NH₄⁺-N and turbidity from ADPE, resulting in subsequent normal growth of microalgae [3].

After day 11, the algal growth decreased in 10%F, 10%M, and 10%S due to the exhaustion of nutrients, while they grew better in 10%AF pretreatment. Since fungi could produce some nutrients such as volatile fatty acids [31], and also degrade refractory organic matter to small molecule compounds, which can be more easily utilized for microalgae cultivation [32]. As shown in Figure 2b, the highest chlorophyll-a concentration was obtained at 12.55 mg L⁻¹ in 10%AF pretreatment. Correspondingly, the OD in 10%AF pretreatment increased faster than 5%ADPE, and the maximum biomass concentration reached 0.37 g L⁻¹ in the turbidity reduction experiment. The algal dry weight in 10%F, 10%M, and 10%S pretreatments increased by 41.7%, 30.7%, and 26.5%, respectively, compared to the initial dry weight. The results indicated that the algae might not grow when the turbidity exceeded 10%F pretreatment, i.e., ca. 800 NTU (Figure 2, Table 2). Different pretreatment methods had varying efficiencies in removing turbidity. Turbidity, which refers to the cloudiness or haziness of a fluid caused by suspended particles, could negatively impact the growth rate of microalgae by limiting light penetration. Fungal pretreatment offered a balanced approach by combining high turbidity removal efficiency with environmental sustainability.

3.3.2. Algal Growth in ADPE Pretreated by Air Stripping

As shown in Figure 3a, the maximum OD reached 1.20 in 40 °C pretreatment on day 11, while it reached 1.01 in 25 °C pretreatment on day 13. The maximum concentration of chlorophyll-a in 40 °C and 25 °C pretreatments reached 21.3 mg L⁻¹ and 18.8 mg L⁻¹ on day 11, respectively (Figure 3d). The specific growth rate in 40 °C and 25 °C pretreatments was 0.088 and 0.079 d⁻¹, respectively. The algal biomass productivity (0.013 g L⁻¹ d⁻¹) in 40 °C pretreatment was slightly higher than that (0.011 g L⁻¹ d⁻¹) in 25 °C pretreatment (Table 3). These results showed that the air stripping of ADPE at 40 °C was better for algae growth. Ammonia stripping pretreatment with high temperature significantly reduces the solubility of ammonia in water while enhancing molecular thermal motion, accelerating the diffusion of free ammonia (NH₃) from the liquid phase to the gas phase; after ammonia stripping via high-temperature pretreatment, cooling to 25 °C (see Section 2.4), which is suitable for algal growth, directly mitigates the photoinhibition of high NH₃ concentrations in ADPE on algal photosystem II (PS II) and prevents thylakoid membrane damage [33].

The OD and chlorophyll-a concentration in the air stripping experiment under different pH conditions are shown in Figure 3b,e. pH 9.5 pretreatment achieved the maximum OD with 1.02 on day 13, while pH 9.5F pretreatment (i.e., 7.5%ADPE pretreated by pH 9.5 with air stripping and filtered by fast filter paper) had the maximum OD value at 0.93 on day 9. Likewise, pH 9.5F pretreatment had the maximum biomass concentration (dry cell weight) in 0.34 g L⁻¹, which indicated that microalgae grow faster in the filtered ADPE with ammonia stripped. After day 11, the OD in pH 9.5F pretreatment began to decrease. As described above, the suspended matter removed by filtration reduced the concentration of available nutrients for algal growth. The algal specific growth rate and the biomass productivity in pH 9.5 pretreatment were the highest among all treatments, reaching 0.087 d⁻¹ and 0.013 g L⁻¹ d⁻¹, respectively (Table 3). As algal growth relies on a suitable intracellular weakly alkaline pH, the pH level after air stripping provides a weakly alkaline environment for subsequent algal growth, promoting carbonic anhydrase activity and CO₂ fixation [34]. To sum up, ADPE with air stripping at pH 9.5 is beneficial to the growth of microalgae.

The maximum OD reached 1.10 on day 13 with the stripping flow rate at 1.5 L min⁻¹, respectively. The removal rate of NH₄⁺-N increased with increasing flow rates (Figure 1b), so that the OD at 0 L min⁻¹ decreased to 0 on day 9. But for the growth of microalgae, the growth rate in 1.5 L min⁻¹ pretreatment (0.082 d⁻¹) was faster than that in 2.5 L min⁻¹ pretreatment (0.070 d⁻¹), while both pretreatments had the highest biomass productivity, reaching 0.013 g L⁻¹ d⁻¹ (Table 3). Microalgae can grow in the ADPE after air stripping, which proves the effectiveness of air stripping for the pretreatment of ADPE. Despite differing growth rates, the maintenance of biomass productivity parity between 1.5 and 2.5 L min⁻¹ flow rate pretreatment implied complex physiological adaptations in microalgae, possibly involving changes in cellular composition or density under different aeration regimes. Many physicochemical methods like air stripping and precipitation have been used for ADPE treatment [35]. The successful cultivation of microalgae in air-stripped ADPE validated the technical feasibility of this pretreatment method, highlighting its dual benefit of detoxifying wastewater while preserving essential nutrients for algal biomass production. This balance is crucial for developing sustainable wastewater treatment systems that integrate nutrient recovery with biomass production through algal cultivation.

3.4. Nutrient Removal by Microalgae in Pretreated ADPEs

Figure 4 shows the NH₄⁺-N, TN, TP, and COD removal by culturing C. pyrenoidosa in various pretreated ADPEs for 15 days. Different nutrient uptake of algal cells depended on various pretreatment methods (dilution, filter paper, natural sedimentation, and fungal cultivation). The algal cells did not grow in 7.5%ADPE, 10%ADPE, NS24h, and NS48h pretreatments, so the nutrient concentrations during the whole experimental period had low levels of removal rate. The results showed that during the 15-day growth period, the removal rates of NH₄⁺-N for algal growth, except for 7.5%ADPE, 10%ADPE, NS24h, and NS48 h pretreatment, were 93.6–99.8% in the other pretreatments (Figure 4a). The 10%AF pretreatment had the highest NH₄-N removal rate for algal growth among the treatments. The concentrations of TN were reduced by 62.9–91.7% with high removal rates of algal cells in 5%ADPE, 10%S, 10%M, 10%F, and 10%AF pretreatments (Figure 4b). Similar results were reported by Wang et al. [36]. They found that C. pyrenoidosa cultivated in 10-, 15-, 20-, and 40-fold diluted swine wastewater (34.7 mg L⁻¹–138.8 mg L⁻¹ of initial NH₄⁺-N concentration) showed high removal rates of 91.2–95.1% for NH₄⁺-N and 54.7–74.6% for TN [36]. Moreover, Kumar et al. reported that the maximum NH₄⁺-N reduction by Chlorella vulgaris was 61.8% in 50-fold diluted ADPE containing 20 mg L⁻¹ of initial NH₄⁺-N concentration [37]. TP reduced by 30.0–48.0% through algal uptake except for 7.5%ADPE, 10%ADPE, NS24h, and NS48h pretreatments (Figure 4c). A similar study also observed that TP was reduced by 34.7–62.5% in diluted ADPE [30]. The removal rate of COD in 5%ADPE, 10%S, 10%M, 10%F, and 10%AF pretreatments was 44.4–68.6% with the highest rates in 10%AF pretreatment during microalgae cultivation (Figure 4d). Therefore, it is obvious that microalgae exhibited the best performance in removing nutrients from ADPE pretreated by 10%AF. The 10%AF pretreatment likely played a crucial role in enhancing the bioavailability of nutrients. Acidification in fungal cultivation could break down complex organic matter, releasing nutrients in forms that were more accessible to algal cells. This 10%AF pretreatment step might reduce inhibitory substances; otherwise, it would hinder algal growth. The pretreatment might also remove certain toxic substances from algal cells in ADPE, thus improving the growth rate and nutrient removal efficiency of microalgae. During pretreatment, fungi can significantly degrade larger complex organic matters to smaller simple ones with short chains in water [32], which can be easily utilized by microalgae for algal growth [38]. Based on nutrient concentrations on the final day (Figure 4) and those in diluted 10%ADPE (Table 2), the high removal rates of NH₄⁺-N, TN, TP, and COD were achieved to 100%, 99%, 63%, and 91%, respectively, in the integrated treatment of 10%AF and subsequent microalgae cultivation. This provided the optimum strategy for microalgae-based nutrient removal among various pretreatments.

Li et al. found that the optimal consumed N/P ratio for freshwater microalgae cultivation is 5–10 [39]. The consumed N/P ratio of algal cells in various pretreated ADPE (except for 7.5%ADPE, 10%ADPE, NS24h, and NS48h pretreatments) was 38–65, which was much higher than the optimal consumed ratios of freshwater microalgae cultivation. Therefore, removing ammonia through air stripping or the extra addition of available phosphorus can enable a balanced N/P ratio [40]. Barbato et al. suggested that the optimized N/P ratio improves the nutrient removal capacity under high N and P concentrations [41]. This information can contribute to future research to improve microalgae cultivation in pretreated ADPE.

4. Conclusions and Outlook

This study demonstrated that 10%AF emerged as the most effective pretreatment for mitigating turbidity and nutrient load (particularly ammonium) in ADPE. The optimized 10%AF pretreatment facilitated robust microalgae cultivation, achieving a maximum specific growth rate of 0.094 d⁻¹ and biomass productivity of 0.014 g L⁻¹ d⁻¹, which surpassed outcomes from other pretreatment methods. Furthermore, the integrated strategy combining 10%AF pretreatment with microalgae cultivation exhibited high nutrient removal efficiency in 10%ADPE, with removal rates reaching 100%, 99%, 63%, and 91%for NH₄⁺-N, TN, TP, and COD, respectively. These results highlight the dual potential of this approach—simultaneously advancing algal biomass production and sustainable wastewater treatment—while offering a scalable solution for valorizing high-strength ADPE.

The pathogenic fungus (A. fumigatus) used in this study poses safety risks, and its potential pathogenicity may limit practical applications—this limitation requires significant attention. Based on this, future research should advance in three directions:

(i)

Screening of safe strains: prioritize the development of non-pathogenic fungi (e.g., T. versicolor or P. chrysosporium) to eliminate biosafety hazards while maintaining pretreatment efficiency;

(ii)

Scale-up challenges: explore scaled-up cultivation processes for fungal-microalgal coupling systems to address engineering issues such as difficulty in separating fungal mycelia and microalgal light limitation.

(iii)

Regulatory frameworks and standards: establish a safety assessment system for ADPE biological pretreatment, defining inactivation standards for pathogenic fungi and environmental release thresholds for product applications to ensure compliance with biosafety regulations. Overall, fungal pretreatment coupled with microalgal cultivation offers an innovative approach for ADPE treatment, but industrial application will require safety optimization and engineering breakthroughs.

(iv)

Life cycle assessment: Evaluate the environmental footprint of the entire process (e.g., fungal pretreatment, microalgae cultivation, biomass harvesting) compared to conventional ADPE treatment methods (e.g., physical-chemical pretreatment).

(v)

Economic analysis: Quantify operational costs (e.g., fungal strain maintenance, reactor construction) and potential revenue from biomass byproducts (e.g., biofuels, animal feed) to verify cost-effectiveness.”

(vi)

N/P balance: Adjusting N/P ratios in pretreated ADPE to align with microalgal stoichiometric demands (e.g., Redfield ratio or species-specific requirements) could further improve biomass productivity and nutrient assimilation efficiency.