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Article

Efficacy and Adaptation Mechanisms of Algal-Bacterial Granular Sludge Treatment for Phenolic Wastewater

by
Aoxue Yu
1,2,
Rui Ouyang
1,
Shulian Wang
1,*,
Bin Ji
3 and
Lu Cai
4
1
Key Laboratory of Health Intelligent Perception and Ecological Restoration of River and Lake, Ministry of Education, Hubei University of Technology, Wuhan 430068, China
2
College of Environmental Science and Engineering, Hunan University, Changsha 410082, China
3
Department of Water and Wastewater Engineering, School of Urban Construction, Wuhan University of Science and Technology, Wuhan 430065, China
4
Key Laboratory of Ecological Impacts of Hydraulic-Projects and Restoration of Aquatic Ecosystem, Institute of Hydroecology, Ministry of Water Resources and Chinese Academy of Sciences, Wuhan 430079, China
*
Author to whom correspondence should be addressed.
Water 2025, 17(1), 127; https://doi.org/10.3390/w17010127
Submission received: 15 December 2024 / Revised: 2 January 2025 / Accepted: 3 January 2025 / Published: 5 January 2025
(This article belongs to the Special Issue Application of Biotechnology in Water Treatment and Specific Treatments for Water Reuse)
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Abstract

:
The ubiquitous presence of phenolic compounds in effluents poses a risk to aquatic organisms and human health. This study investigates the responses of the emerging algal-bacterial granular sludge process in treating phenolic wastewater. The results showed that phenol at 1, 10, and 100 mg/L had little effect on ammonia-N, chemical oxygen demand (COD), and phosphate-P removal. At the highest phenol concentration of 100 mg/L, the average removal rates of ammonia-N, COD, and phosphate-P were 94.8%, 72.9%, and 83.7%, respectively. The presence of phenol led to a decline in chlorophyll content of the algal-bacterial granular sludge, concurrently resulting in an increase in the abundance of microbial diversity. Algal-bacterial granular sludge exhibited mechanisms such as elevated extracellular polymeric substances (EPSs), superoxide dismutase (SOD), and catalase (CAT) production, which may aid in coping with oxidative stress from phenols. This research underscores the algal-bacterial granular sludge’s potential for treating phenolic wastewater, thereby advancing knowledge in the field of phenol degradation with this innovative technology.

1. Introduction

In recent years, algal-bacterial granular sludge has received considerable attention in wastewater application [1,2]. This type of granular biomass, formed under natural light conditions, is composed of an outer layer rich in microalgae and an inner layer of non-photosynthetic bacteria [3]. A mutualistic symbiosis exists where microalgae supply oxygen through photosynthesis, supporting bacterial respiration that degrades organic matters, releasing carbon dioxide back to microalgae for further use in photosynthesis [4]. This process not only reduces greenhouse gas emissions like CO2 [5]; estimates suggest that algal-bacterial granular sludge treatment leads to a lower CO2 equivalent release (0.30 kg CO2 e/m3) compared to aerobic granular sludge (0.81 kg CO2 e/m3) [6,7]. In addition, algal-bacterial granular sludge holds considerable promise for concurrent wastewater treatment and the production of valuable by-products and biofuels [8]. Therefore, this technology aligns with sustainable environmental practices, aiming to concurrently address pollution removal, energy efficiency, carbon emission reduction, and resource recovery. Its potential for widespread application in wastewater management is anticipated [9,10]. However, it is crucial to recognize that the effectiveness of algal-bacterial granular sludge in wastewater treatment can be compromised by concurrently present contaminants [11], including heavy metals, antibiotics, microplastics, and persistent organic pollutants [12,13].
The contamination of soil, surface water, and groundwater by aromatic organic pollutants, including phenol and its derivatives, has attracted global concern. These pollutants are widely present in wastewater from a range of industrial activities, including petroleum refining, wood preservation, coking, and other processes. It is well documented that phenols are highly toxic to humans, aquatic life, and other organisms [14]. The United States Environmental Protection Agency stipulates limits, prohibiting phenol discharge in drinking water above 1 ppb and limiting its presence in water bodies to 1 ppm [15]. Biodegradation, considered eco-friendly and cost-effective, has become a crucial remediation strategy for phenol pollution [16]. Extensive research has focused on bacterial degradation of phenols, leading to the identification and characterization of numerous phenol-degrading bacteria, including Bacillus, Acinetobacter, Gliomas tix, and Pseudomonas [17]. Phenols can impair the function of key enzymes involved in nitrification and denitrification by bacteria, hindering nitrogen removal [18]. Fungi, too, play a pivotal role in aromatic compound cycling in the ecosystem. Studies have shown that various fungi are capable of mineralizing phenols through enzymatic processes, allowing for their degradation [16]. Microalgae, known for energy accumulation from toxic substances, can effectively degrade phenols by utilizing them for growth. When processing low-toxicity phenolic compounds, microalgae exhibit higher energy utilization to enhance biomass production [19]. For instance, Bucci et al. [20] employed a sequential batch reactor based on microalgal-bacterial particles for simultaneous removal of inorganic nitrogen and phenol, achieving complete phenol removal (100 mg/L). This was attributed to the synergistic interactions within the microalgal-bacterial partnership [21]. The capacity of bacteria and microalgae to remove phenolic compounds is contingent upon several factors, including microbial type, substrate concentration, and degradation time. It has been demonstrated that both bacteria and microalgae can utilize low concentrations of phenol as a carbon source. However, as the substrate content increases, the biotoxicity rises, and the growth and degradation abilities are progressively inhibited [22]. How does algal-bacterial granular sludge respond to different concentrations of phenols in terms of its reaction and treatment efficiency?
Research on algal-bacterial granular sludge predominantly focuses on municipal wastewater treatment, but the specific adaptation and effectiveness for dealing with phenolic effluents are yet to be fully investigated. This study seeks to fill that gap by examining the anaerobic degradation of synthetic phenolic wastewater by these granules, with the goal of establishing a theoretical framework and practical support for employing non-aerated algal-bacterial granules in the treatment of such waters. By closely monitoring the physicochemical changes in algal-bacterial granular sludge and evaluating the treatment results throughout the experiment, there exists a promising prospect for implementing these discoveries on a substantial scale in practical applications.

2. Materials and Methods

2.1. Synthetic Wastewater and Algal-Bacterial Granular Sludge

In the conducted experiment, a sequencing batch photobioreactor (equipped with an LED full-spectrum light source delivering a light intensity of 10,000 lux and maintaining a photoperiod of 12 h of light followed by 12 h of darkness) was employed to simulate domestic wastewater as a nutrient source. As an inoculation source, aerobic granular sludge was cultivated using activated sludge. The initial raw sludge was derived from activated sludge harvested from the aerobic tank of a standard wastewater treatment plant in Wuhan (Figure 1a). Following a 60-day period of incubation in continuous illumination and aeration, granular sludge was formed (Figure 1b), with the emergence of microalgae due to the photobiological conditions present. Subsequently, it was subjected to an additional 60-day maturation period to cultivate the fully developed algal-bacterial granules (Figure 1c). The mature granules exhibited a distinct elliptical shape with rounded boundaries, a sleek surface, a compact structure, and superior sedimentation capabilities (Figure 1d). Its average granular size was 4 mm, and the 5 min sludge volume index (SVI5) was 25.79 mL/g.
The simulated wastewater was composed of NaAc (400 mg/L), NH4Cl (30 mg/L), FeCl3 (10 mg/L), KH2PO4 (5 mg/L), CaCl2 10 (mg/L), MgSO4·7H2O (10 mg/L), and trace elements (1 mg/L). The following elements were present in the trace element stock solution: EDTA (10 g/L), MnSO4·H2O (100 mg/L), CuSO4·5H2O (30 mg/L), ZnSO4·7H2O (120 mg/L), H3BO3 (150 mg/L), Na2MnO4·2H2O (60 mg/L), KI (180 mg/L), and CoCl2·6H2O (150 mg/L).

2.2. Experimental Design

The experimental setup comprised five reactor groups, each initially containing model phenol wastewater with concentrations of 1, 10, and 100 mg/L. Incremental increments of 1-10-100 mg/L were introduced every 20 days in dedicated experimental groups for comparative analysis. A blank control group consisted of cultures without phenol. In a series of microreactors measuring 47 mm in diameter and 60 mm in height, 30 mL of simulated wastewater was mixed with 3 g of fresh algal-bacterial granular sludge in a batch process. These reactors were operated under 5 LED lights of 10,000 lux intensity, alternating in a 12 h cycle of light and darkness. The pH of the simulated wastewater was adjusted using 0.1 mol/L hydrochloric acid or sodium hydroxide solution and maintained at 7.0 ± 0.2. The reactor was operated at room temperature (20–25 °C) to mimic natural conditions. The initial volatile suspended solids (VSS) concentrations of algal-bacterial granular sludge was approximately 24.1 g/L. Water samples were collected daily at 9:00 AM and 9:00 PM, with a hydraulic retention time of 24 h and a volume exchange rate of 100%. Both influent and effluent samples were filtered through a 0.45-μm filter for analysis. The content of ammonia-N, chemical oxygen demand (COD), and phosphate-P were measured daily. The changes in chlorophyll, extracellular polymeric substances (EPSs), and antioxidant enzyme activity were measured every 20 days. At the end of the experiment, an adequate number of pellets were harvested for sequencing and analysis.

2.3. Analysis Methods

The content of ammonia-N, COD, and phosphate-P were determined according to standard methods [23]. For chlorophyll and carotenoid extraction from algal-bacterial granules, 5 mL of 95% ethanol was added to 0.2 g of fresh biomass in a 10 mL centrifuge tube. The sample was sealed with aluminum foil and stored at 4 °C overnight, after which it was centrifuged at 4000 rpm for 10 min.
The extraction of extracellular polymeric substances (EPSs) employed a modified heat treatment approach [24]. First, a 50 mL centrifuge tube containing 0.5× g of algal-bacterial granular sludge was rinsed thrice with deionized water, followed by the addition of 10 mL of 0.05% NaCl. The sample was then centrifuged at 1640× g for 10 min, and the supernatant was collected and designated as a soluble EPS (SB-EPS). Subsequently, the algal-bacterial granular sludge in the tube was resuspended in 10 mL of 0.05% NaCl, sonicated at 20 kHz for 2 min, shaken horizontally at 150 rpm for 10 min, sonicated again for 2 min, and then centrifuged at 8000× g for 30 min to separate the solids and the supernatant, which constituted a loose EPS (LB-EPS). The remaining granules were suspended in 10 mL of 0.05% NaCl, sonicated for 3 min, and subjected to heat extraction by incubation in a water bath at 60 °C for 30 min. Finally, the compact EPS (TB-EPS) was collected after centrifugation at 12,000 rpm for 30 min. The EPS-polysaccharide (EPS-PS) content was measured using the phenol–sulfuric acid method [25]. The EPS-protein (EPS-PN) content was measured using C504031 Lowry Protein Assay Kits (Sangon Biotech, Shanghai, China) according to the instructions [13].
The algal-bacterial granules were first frozen in liquid nitrogen and homogenized into a paste. To prepare the enzyme solution, the sludge was mixed at a 1:9 ratio with a 0.2 mol/L phosphate buffer (pH 7.8). This mixture was then subjected to centrifugation at 4000 rpm for 10 min at 4 °C using a refrigerated centrifuge (3H16RI, Hunan Herexi Instrument & Eqipment Co., Ltd., Changsha, China) to isolate the supernatant. The supernatant served as the enzyme source. The activities of malondialdehyde (MDA), catalase (CAT), and superoxide dismutase (SOD) were quantified employing specific assay kits procured from Nanjing Jincheng Bioengineering Institute (Nanjing, China).
At the end of the experiment, algal-bacterial granular sludge samples were extracted from the reactor, and DNA was extracted using the E.Z.N.A.® soil DNA kit (Omega Biotek Inc., Norcross, GA, USA). The quality of the DNA extracted was checked by 0.8% agarose gel electrophoresis, and the concentration and purity of the DNA were determined by Nanodrop ND-1000. The 16S rRNA and 18S rRNA genes were amplified using 515F/907R prokaryotic primers targeting the V4–V5 regions of the 16S rRNA gene and 528F/706R eukaryotic primers targeting the V4 region of the 18S rRNA gene, respectively [13,26]. Sequencing was performed on an Illumina MiSeq platform by PanRay Yunzhi Technology Co. (Zhengzhou, China). Concurrently, a scanning electron microscope (SEM) (SU8000, Hitachi, Tokyo, Japan) was employed to examine the microstructure of the algal-bacterial granular sludge.

3. Results and Discussion

3.1. Performance of Algal-Bacterial Granular Sludge on Phenolic Wastewater

Figure 2 shows the removal efficiency of algal-bacterial granular sludge on ammonia-N, COD, and phosphate-P. Ammonia-N removal by algal-bacterial granular sludge was high. The lowest average removal rate was 94.8% at a phenol concentration of 100 mg/L. After 60 days of operation, the concentration of ammonia-N in the effluent was only 1.23 mg/L (Table S1), which was lower than the Class A standard of 5 mg/L in the discharge standard of pollutants for municipal wastewater treatment plants (GB 18918-2002) [27]. Meanwhile, the increase in phenol concentration had a trace effect on the ammonia-N removal rate, which gradually decreased with the increased phenol concentration. For ammonia-N, on the one hand, bacteria consume ammonia-N during oxidative decomposition of COD, which accounts for 19% of ammonia-N removal [28]. On the other hand, microalgae can convert ammonia in wastewater into organic nitrogen through assimilation, which can be used to synthesize proteins, chlorophyll, energy transfer substances, and genetic material required for their own growth and metabolism, which accounts for 81% of ammonia-N removal [29]. Phenol has a strong toxic effect on denitrifying bacteria, and a very small amount of phenol can significantly inhibit bacterial reduction [18]. And in this study, no nitrifying or denitrifying bacteria were detected in algal-bacterial granular sludge. Therefore, the removal of ammonia-N relied mainly on assimilation by microalgae and bacteria rather than the biological deamination pathway. The slow reduction of ammonia-N may be due to the toxic effect of phenol on bacteria. Many studies have shown that phenol damages the membranes of algal cells, resulting in reduced photosynthesis [30]. Meanwhile, phenol and ammonia-N may compete for the same microbial pathways in the system, and phenol degradation is prioritized over ammonia-N conversion, resulting in lower ammonia removal [31].
However, for COD and phosphate-P (Figure 2b,c), the situation seems to be reversed, with a gradual increase in COD and phosphate-P removal with an increased phenol concentration compared to the control group, with an average removal rate of 72.9% and 83.7% at a phenol concentration of 100 mg/L, respectively. In the effluent, COD and phosphate-P were 77 mg/L and 0.12 mg/L (Table S1), which were better than the EU standard [32] of 125 mg/L and the discharge standard of pollutants for municipal wastewater treatment plants (GB 18918-2002) [27] of 1 mg/L, respectively. The concentration of phenol in the effluent was 5.4 mg/L when the influent contained 100 mg/L of phenol, whereas no phenol was detected in the remaining experimental groups (Table S1). Indeed, algae and bacteria can actually assimilate trace amounts of phenol into their cells, utilizing it as a carbon source for growth and metabolic processes, and subsequently detoxify phenol through a series of enzymatic degradation pathways [16]. Phosphate-P is also a key element in the energy metabolism of microalgae and bacteria, as inorganic phosphate is taken up by these microorganisms through active transport mechanisms within their cells.

3.2. Microbiological Analysis and Photosynthetic Pigment Analysis

As depicted in the data from observed_otus and Chao1 estimates (Table 1), after a 30-day incubation period, the microbial species richness showed a higher value in response to phenol concentrations of 1, 10, and 100 mg/L compared to the control, with a decreasing trend as the phenol concentration increased. This indicates that lower phenol levels promote a more diverse microbial community. In regard to Simpson’s diversity index, the experimental treatments with phenol addition demonstrated an improved community diversity when compared to the control (Figures S1 and S2).
Figure 3 shows the microbial community composition of prokaryotes at the phylum level and eukaryotes at the species level in algal-bacterial granular sludge. Most eukaryotes belonged to the phylum Chlorophyta, accounting for 98% of the total community (Figure 3a). The prokaryotes were Proteobacteria, Chloroflexi, Cyanobacteria, Firmicutes, Bacteroidota, Desulfobacterota, and Planctomycetota. Among them, Proteobacteria, Chloroflexi, Bacteroidota, Cyanobacteria, and Planctomycetota were the main species, accounting for 80% of the total community (Figure 3b and Figure S3). After 60 days of incubation, the relative abundance of Proteobacteria in the experimental group at phenol concentrations of 1-10-100, 1, 10, and 100 mg/L was 45.5%, 51.5%, 48.2%, and 32.6%, respectively, compared to 52.5% in the control. Cyanobacteria showed a relative abundance of 4.2%, 9.9%, 14.7%, and 20.9% in the experimental groups, compared to 3.7% in the control. Proteobacteria have been implicated in nitrogen and phosphorus removal and contaminant degradation processes [33]. Cyanobacteria, possessing chlorophyll a, can anchor various photosynthetic pigments in their cysts and conduct aerobic photosynthesis using low phenol concentrations as a carbon source for metabolism. This aligns with previously observed phenomena.
In summary, the presence of algae within the algal-bacterial granular sludge facilitated the system’s resilience to phenol exposure upon loading. Algal cells coalesced with bacteria, forming clusters, and numerous spherical and rod-shaped bacteria adhered to filamentous Cyanobacteria (Figure 1e,f). These filamentous cyanobacteria and bacteria interwove, resulting in a relatively stable spherical granule structure. Filamentous cyanobacteria served as cores or carriers, supporting the development of an initial framework for the algal-bacterial granules, thereby maintaining structural integrity.
Chlorophyll a, abundant in phototrophic microorganisms such as green algae, diatoms, and cyanobacteria, is distinct from chlorophyll b, which is exclusive to green algae. Assessments of photosynthetic pigments are often used to identify microalgal species [34]. Analysis revealed that on Day 20, the experimental group’s chlorophyll a content was lower than the control, while the chlorophyll b content was higher; the chlorophyll a/b ratio declined with an increasing phenol concentration (Figure 4). This suggested that phenol at higher concentrations (10 and 100 mg/L) initially boosted green algae growth and hampered cyanobacteria growth. However, from Day 40 to 60, chlorophyll a, total chlorophyll, and carotenoid levels started to decline, with chlorophyll b decreasing notably. The chlorophyll a/b ratio increased significantly, reflecting a greater inhibitory effect of phenol on green algae over time. This is evident in Figure 3b. Notably, a sudden drop in the chlorophyll a/chlorophyll b ratio occurred at a 1 mg/L phenol concentration, indicating that a lower phenol concentration of 1 mg/L had a lesser impact on algal particulate sludge when given extended incubation periods.

3.3. Defensive Responses of Algal-Bacterial Granular Sludge Under Phenols Stress

Exposure to phenol significantly impacts microbial dynamics and organization. Although extended exposure typically leads to decreased or complete impairment of specific growth rates, oxygen consumption rates, and enzymatic activities [35], microorganisms exhibit a remarkable ability to elicit various physiological adaptations, such as the copious secretion of extracellular polymers and enzyme-activating substances observed in these experiments, in an effort to enhance their tolerance to toxicity.

3.3.1. EPS Variations

EPSs are viscous metabolites secreted by microalgae and bacteria, mainly composed of polysaccharides, proteins, nucleic acids, and lipids, which help to maintain the stable structure of algal-bacterial granular sludge [36]. Polysaccharides can provide nutrients for microalgae and bacteria for their metabolism. EPS-PSs form a vesicle sheath around the algal-bacterial granular sludge, which helps it to resist the external adverse survival environment and enhances the adaptability of the algal particulate sludge to the changes in the complex environment [12,37]. In this study, the content of EPS-PSs and EPS-PNs in SB-EPSs and LB-EPSs was extremely low and negligible, and the content of EPS-PSs and EPS-PNs in TB-EPSs was mainly used to reflect the concentration change of the EPSs affected by phenol (Figure 5).
Figure 5a shows the variation of EPS-PN content. On Day 20, the EPS-PNs reached their maximum of 114.3 and 107.5 mg/g VSS, respectively, under the lower phenol concentrations (1 and 10 mg/L), nearly matching the control’s 71.1 mg/g VSS. As the phenol concentration escalated to 100 mg/L, the EPS-PNs dropped to a level similar to that of the control. On Day 40, the experimental group’s EPS-PNs were lower than the control and inversely proportional to the phenol concentration. By Day 60, the EPS-PNs in the experimental group gradually recovered to parity with the control. This indicated that the algal-bacterial granular sludge system produced a large number of EPSs to cope with the low concentration of phenol, but when the concentration of phenol is higher, it inhibited the activity of the cells to a certain extent, presenting a phenomenon that low concentrations promote and high concentrations inhibit. However, the system’s pollutant removal capability remained unaffected. Over time, as degradation progressed and the sludge adapted to the phenol environment, the EPS content stabilized, reflecting system maturation. Supporting this notion, Figure 5b reveals the dynamics of the EPS-PS content. It is noteworthy that between Days 40 and 60, the EPS-PS content in the experimental group was lower, suggesting phenol suppression of EPS-PS synthesis. This could also imply that the abundant EPS-PN production depleted a significant proportion of the energy required for EPS-PS synthesis. Generally, EPS-PSs aid in cell cohesion and adhesion, crucial for maintaining sludge structural integrity during granulation process [38]. The PN/PS ratio in the control group remained steady at around 14. However, at a 100 mg/L phenol concentration, it increased to 18, indicating heightened hydrophobicity and lower surface charge on the algal-bacterial granular sludge’s surface [39].

3.3.2. Antioxidant Enzyme Activity

Upon exposure to hostile conditions, cells produce substantial quantities of reactive oxygen species (ROS), such as superoxide radicals, hydrogen peroxide, and carboxyl radicals [40]. Chen et al.’s research [41] indicated that these reactive free radicals contribute to and worsen oxidative damage to cellular lipids and proteins. MDA, a small organic compound, serves as a common indicator of oxidative stress. The MDA content indeed escalated during the 20-day incubation period with the phenol concentrations ranging from 1 to 100 mg/L. The maximum value of 18.5 nmol/gprot was observed at 100 mg/L, nearly quadruple the control’s level, suggesting profound oxidative stress and membrane damage (Figure 6a). However, from 40 to 60 days, the MDA levels in the experimental group gradually declined and approached parity with the control, suggesting that the algal-bacterial granular sludge developed an adaptive mechanism to mitigate stress over time.
To counteract environmental-induced oxidative harm and preserve normal cellular metabolism, microalgae activate various enzyme activities, including SOD, CAT, and POD, forming an antioxidant defense system [37]. SOD, a key scavenger of oxygen radicals, facilitates the conversion of superoxide ions into water and oxygen, thereby reducing their harmful effects [42]. CAT, predominantly responsible for hydrogen peroxide scavenging, decomposes H2O2 into water and oxygen. These antioxidant enzymes effectively eliminate accumulated reactive radicals, maintaining cellular functionality [43]. As shown in Figure 6b, SOD activities were relatively high at Day 20, peaking at 115.88 U/gprot when the phenol concentration was 10 mg/L. This might be due to the initial exposure of algal cells to pollutants, leading to ROS production. The peak SOD activity at 10 mg/L rather than 100 mg/L could be attributed to high initial phenol concentrations affecting cellular enzyme protein secretion. Similarly, Figure 6c shows that CAT activity followed a similar rising pattern with increasing phenol concentrations, yet it exhibited a decline after 40 days, mirroring the SOD trend albeit with a delay, supporting the antioxidant enzyme mechanism.
The study demonstrates that as the incubation progresses, the removal rates of ammonia-N, phosphate-P, and COD by algal-bacterial granular sludge recovered, indicating that when phenol stress in the surrounding environment remained within the algal-bacterial granular sludge’s typical resilience, the system could effectively utilize antioxidant enzymes to neutralize ROS generated by oxidative stress, thus maintaining a balance in active oxygen metabolism. This, in turn, mitigated the detrimental impact of phenol on the overall performance of algal-bacterial granular sludge.

4. Conclusions

This study demonstrated the performance and adaptation strategy of algal-bacterial granular sludge technology in phenol-containing wastewater treatment. Over a 60-day period, varying phenol concentrations (1, 10, and 100 mg/L) were examined. Microorganisms were found to utilize low phenol levels (1–10 mg/L) as a carbon source for growth and metabolism. However, at higher concentrations (100 mg/L), bacterial growth in the algal-bacterial system was impaired, but the granular sludge produced increased EPSs, enhancing the system’s cohesion and mitigating the phenol’s direct toxic effects on microbes. To counteract oxidative damage from the phenol, the algae upregulated their production of SOD and CAT to neutralize excessive free radicals. Notably, the algal-bacterial granular sludge consistently maintained efficient COD, ammonia-N, and phosphate-P removal rates. This study provides insight into the algal-bacterial sludge’s response to environmental phenolic stress and proposes a promising and practical solution for treating phenolic wastewater.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/w17010127/s1; Table S1: Effluent quality after 90 days and typical discharge standards for WWTP effluent; Figure S1: Circos of prokaryotes in the phylum-level microbial community (R0: Control group; R1: Phenol content incremented from 1 to 100 mg/L at 20-day intervals; R2: Steady phenol concentration at 1 mg/L; R3: Steady phenol concentration at 10 mg/L; R4: Steady phenol concentration at 100 mg/L); Figure S2: Circos of prokaryotes in the genus-level microbial community; Figure S3: Prokaryotes in genus-level microbial communities.

Author Contributions

Conceptualization, A.Y. and S.W.; methodology, A.Y. and S.W.; validation, A.Y.; investigation, A.Y.; data curation, A.Y. and R.O.; writing—original draft preparation, A.Y.; writing—review and editing, S.W., B.J., and L.C. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the open project funding of Key Laboratory of Intelligent Health Perception and Ecological Restoration of Rivers and Lakes, Ministry of Education, Hubei University of Technology (HGKFYBP04).

Data Availability Statement

Data will be made available on request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Morphologies during different culture processes ((a): 0 d; (b): 60 d; (c): 120 d; (d): 150 d) and SEM images (e,f) of algal-bacterial granular sludge.
Figure 1. Morphologies during different culture processes ((a): 0 d; (b): 60 d; (c): 120 d; (d): 150 d) and SEM images (e,f) of algal-bacterial granular sludge.
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Figure 2. Removal profiles of ammonia-N (a), COD (b), and phosphate-P (c) across 60-day operation.
Figure 2. Removal profiles of ammonia-N (a), COD (b), and phosphate-P (c) across 60-day operation.
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Figure 3. Microbial community of prokaryotes at phylum level (a) and eukaryotes at species level (b).
Figure 3. Microbial community of prokaryotes at phylum level (a) and eukaryotes at species level (b).
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Figure 4. Changes in photosynthetic pigment content.
Figure 4. Changes in photosynthetic pigment content.
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Figure 5. Protein (a) and polysaccharide (b) content in EPSs.
Figure 5. Protein (a) and polysaccharide (b) content in EPSs.
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Figure 6. Activities of MDA (a), SOD (b), and CAT (c).
Figure 6. Activities of MDA (a), SOD (b), and CAT (c).
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Table 1. Microbial community diversity index.
Table 1. Microbial community diversity index.
SamplesObserved_otusChao1Simpson
Control480480.070.94
Phenol 1-10-100 mg/L383384.160.97
Phenol 1 mg/L657657.000.96
Phenol 10 mg/L565565.000.96
Phenol 100 mg/L525525.680.97
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Yu, A.; Ouyang, R.; Wang, S.; Ji, B.; Cai, L. Efficacy and Adaptation Mechanisms of Algal-Bacterial Granular Sludge Treatment for Phenolic Wastewater. Water 2025, 17, 127. https://doi.org/10.3390/w17010127

AMA Style

Yu A, Ouyang R, Wang S, Ji B, Cai L. Efficacy and Adaptation Mechanisms of Algal-Bacterial Granular Sludge Treatment for Phenolic Wastewater. Water. 2025; 17(1):127. https://doi.org/10.3390/w17010127

Chicago/Turabian Style

Yu, Aoxue, Rui Ouyang, Shulian Wang, Bin Ji, and Lu Cai. 2025. "Efficacy and Adaptation Mechanisms of Algal-Bacterial Granular Sludge Treatment for Phenolic Wastewater" Water 17, no. 1: 127. https://doi.org/10.3390/w17010127

APA Style

Yu, A., Ouyang, R., Wang, S., Ji, B., & Cai, L. (2025). Efficacy and Adaptation Mechanisms of Algal-Bacterial Granular Sludge Treatment for Phenolic Wastewater. Water, 17(1), 127. https://doi.org/10.3390/w17010127

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