The microalgal species used in this study was Chlorella sorokiniana (FACHB-26), which was supplied by the Freshwater Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Science (FACHB, Wuhan, China). Sterile Bold’s Basal Medium (BBM) (Zhang et al., 2021) supplemented with PAM was used in this study. The species was radiantly acclimatized on a shaker (25 ± 2°C, light intensity of 3,000 l×, 12:12 h light: dark cycle). The initial concentration of PAM was set at 100 mg/L, and the concentration was gradually increased to 400 mg/L during a 20 days acclimatization period. This enabled the species to gradually adapt to the presence of PAM for further experiments.

The samples used to isolate the filamentous fungi were collected from a revolving algal biofilm (RAB) reactor used to treat the wastewater that contained PAM (Zhang et al., 2021), which had been operating in the laboratory for 1 year. The algal biofilm was collected from the rotary belt of the RAB reactor by scraping, and it was then diluted to 10⁻⁵ using sterile water and coated on a solid medium with PAM as the sole source of nitrogen. Screened pure strains of fungi were picked for spore inoculation into liquid medium that contained PAM for shake flask domestication. The inoculum of fungal spores was 15%. The liquid medium was composed of 400 mg PAM, 1000 mg glucose, 100 mg CaCl₂, 500 mg MgSO₄, 500 mg KH₂PO₄, and 20 mL soil (unfertilized garden) suspension (per L) (Chang et al., 2020). The culture period (25 ± 2°C, 150 rpm) lasted for 7 days, and after the formation of complete mycelial pellets, the pellets were collected and transferred to new culture flasks for further acclimation and expansion.

The optimal culture conditions for the most stable structure and the highest biomass in the shortest period were obtained after preliminary experiments. The consortium was constructed using the as-prepared synthetic wastewater in a flask culture system with 24 h light exposure (25 ± 2°C, 3,000 l×) and continual shaking at 150 rpm. The preliminary experiments revealed that the stability and biomass of the spheres formed by the inoculation of mycelia were higher than those formed by the inoculation of mycelial spheres that had already formed, and therefore, mycelia were inoculated into the microalgae to form the consortium used in this study. The fungal mycelia can potentially combine with the microalgae through rotation, entanglement, and adsorption to ultimately form a sphere, which resulted in a significant decrease in the microalgal biomass. Therefore, to ensure that the consortium can bind enough microalgal cells, the microalgae were first cultured to achieve a high OD value (OD₆₆₀ = 0.8).

To establish whether the synergistic treatment effect of the microbial consortium on the wastewater that contained PAM was better than that of single fungal or microalgal systems, single system treatment experiments were conducted to compare the degradation by the three systems under the same culture conditions.

The pure strains were inoculated onto an inclined surface medium to detect the samples. After PCR amplification, the gene sequence that was isolated was identified as a species of Fusarium (Supplementary material). Fungi have mycelia, which use a process of winding and adsorption to form stable mycelial pellets in culture (Figure 1A). Compared with the use of single microbial strains to remove pollutants, the morphology and large specific surface area of the mycelial pellets enables greater contact and assimilation of the pollutants in wastewater (Li et al., 2023). More importantly, the formation of mycelial pellets is more conducive to the rapid separation of organisms and wastewater, which facilitates the reuse of microorganisms (Zhou et al., 2018). On this basis, the Fusarium mycelial pellet was used to package suspended algal cells, which consisted of the Fusarium mycelial pellet, and can effectively improve the ability to separate the algal cells and solution to achieve the goal of reusing them (Chen et al., 2020).

During isolation of the fungal strain, fungal growth was achieved on the plate when PAM was used as the sole nitrogen source, indicating that the isolated strain could effectively utilize PAM for its growth. The degradation of PAM was significantly affected by the addition of different co-metabolic nitrogen sources within 10 days of the completion of culture cycle (Figure 1B). The rate of degradation of PAM was the highest in the experimental group with added urea, which reached 53.76 ± 2.97%, and corresponded to a reduction in the concentration of PAM by approximately 200 mg/L. The rate of degradation of PAM reached 49.88 ± 3.2% in the experimental group with the addition of NH₄Cl and 46.69 ± 0.57% with the addition of yeast extract, while only 26.74 ± 1.46% of the PAM was degraded when PAM was used as the sole nitrogen source.

A previous study showed that the process of PAM biodegradation begins with deamination by amidase catalysis (Nyyssölä and Ahlgren, 2019). As a polymeric compound, PAM cannot enter the intracellular portion of the Fusarium sp. cells through biofilms (Zhao et al., 2020). Therefore, when PAM is used as the sole source of nitrogen, Fusarium sp. cannot secrete many extracellular enzymes for a short time, thus, the efficiency of PAM degradation was the lowest. Furthermore, the addition of co-metabolic nitrogen sources could ensure the rapid growth of biomass, which results in the secretion of more extracellular amidase, thereby effectively improving the degradation of PAM. Therefore, the addition of a co-metabolic nitrogen source can promote the secretion of extracellular amidase, thereby improving the rate of degradation of PAM to some extent. The addition of urea promotes the secretion of extracellular amidase enzymes by Fusarium sp. and improves the rate of degradation of PAM, which is consistent with the findings of a previous study (Bao et al., 2010).

During the culture period, the efficiency of TOC removal from the synthetic wastewater by Fusarium sp. exhibited significant differences when different co-metabolic nitrogen sources were added (Figure 1C). The concentration of TOC was significantly reduced in the experimental groups supplemented with yeast extract, which reached removal efficiencies of 58.77 ± 0.35%. The TOC removal efficiency in the other three groups was only approximately 10%. Compared with the other three nitrogen sources, yeast extract contains not only nitrogen but also many trace elements and growth factors, such as nucleosides and amino acids among others. It is hypothesized that these growth factors and trace elements are more conducive to the growth of fungi, which facilitates the synergistic degradation of PAM.

The change in algal biomass during the 6 days incubation period is shown in Figure 2A. Algal cultures exhibited significant growth when different co-metabolic nitrogen sources were added and reached a stable biomass until approximately 5 days before they started to decline.

The degradation of PAM by Chlorella sp. was also affected by the addition of a co-metabolic nitrogen source (Figure 2B). The rate of degradation of PAM reached 58.07 ± 3.1% when supplemented with NH₄Cl, 48.11 ± 1.05% when supplemented with yeast extract, 38.2 ± 0.27% when supplemented with urea and 40.28 ± 2.83% when PAM was used as the sole nitrogen source. To remove the organic nitrogen, algae first degraded it into ammonia using various enzymes, which enables it to then be absorbed by the algal cells (Simsek et al., 2016). These results indicate that the algae Chlorella sp. had a stronger ability to utilize inorganic nitrogen sources than those of organic nitrogen, although the degradation of PAM was effectively promoted by the addition of an external nitrogen source, regardless of whether it was organic or inorganic.

The ability of Chlorella sp. to remove TOC was significantly reduced during the 6 days culture cycle (Figure 2C). The rate of TOC removal reached 7.9 ± 1.28% when the system was supplemented with urea, −35.09 ± 2.72% when supplemented with NH₄Cl, 55.08 ± 0.14% when supplemented with yeast extract, and −24.5 ± 4.32% when PAM was used as the sole nitrogen source. This result shows that the microalgae were more effective at degrading TOC when adequate nutrients were available. However, as the experimental period extended, the organic matter generated by microalgal physiological activity accumulated, which led to a potential decrease in the efficiency of TOC removal and an increase in the levels of TOC.

During the culture period, Chlorella sp. was highly efficient at removing the IC from wastewater that contained PAM (Figure 2D). Approximately 90% of the IC was removed, which confirmed the suitability of the system for carbon sequestration. The efficiency of the IC removal was 42.97 ± 0.74% when the system was supplemented with urea, 85.28 ± 1.05% when supplemented with NH₄Cl, 79.14 ± 0.02% when supplemented with yeast extract and 82.62 ± 1.09% when PAM was used as the sole nitrogen source. This result also corresponds to a previous study that showed that microalgae are highly effective at fixing IC (Saravanan et al., 2022).

Chlorella can use both organic and IC sources (Nasir et al., 2015). A comparison of Figures 2C,D shows that when urea and yeast extract were used as co-metabolic nitrogen sources, the organic carbon in them could be utilized by Chlorella, thereby reducing the fixation of DIC in solution. Therefore, when urea and yeast extract were used as co-metabolic nitrogen sources, the TOC was removed when both nitrogen sources were used, but there was little removal of DIC. With ammonium chloride as a common metabolic nitrogen source and PAM as the only nitrogen source, there was little TOC in the system; Chlorella had to use DIC for growth, and during the process of growth, it secretes extracellular polymers, such as polysaccharides and proteins among others (Zhu et al., 2012). As a result, the two groups of TOC were negative, but the efficiency of the DIC fixed was positive.

A previous study has described the mechanism of flocculation of the actinomycete Streptomyces sp. hsn06 on Chlorella vulgaris (Li et al., 2017) and demonstrated the potential ability of both microalgae and fungi to flocculate. Microalgae are mostly adsorbed onto the surface of mycelial pellets, while only a small number of microalgal cells exist within the internal structure of the fungal-microalgal consortium pellets (Figure 3A). This was consistent with the findings of previous research (Leng et al., 2021). The ratio of microalgae to fungi was 2:1. The final consortium sphere that was formed adsorbed a large number of microalgal cells with a dense and stable structure that exhibited excellent settling performance (Figure 3A). This is advantageous for the subsequent harvesting of the algal biomass. The consortium was subjected to stability experiments in a gas column with an aeration rate of 135 L/min, and the consortium could remain stable without disintegrating for 48 h.

The results described above showed that both algae and fungi could degrade PAM separately, and the performance of the Fusarium-Chlorella consortium at degrading PAM was higher than the performance of single systems (Figure 3B). The rate of degradation of PAM reached 80.93 ± 0.7% when the system was supplemented with urea, 65.27 ± 1.32% when supplemented with NH₄Cl, 45.88 ± 3.48% when supplemented with yeast extract and 61.91 ± 0.6% when PAM was used as the sole nitrogen source. Overall, the concentration of PAM decreased significantly, and the rate of degradation reached 80%, which indicated that the Fusarium-Chlorella consortium had a significant effect on the degradation of PAM. Previous studies have shown that fungal-microalgal consortia have been widely used in wastewater treatment (Muradov et al., 2015), and the results of this study confirmed that the microbial consortium was highly adaptable to wastewater that contained PAM, which made it suitable for use in conditions where PAM is the sole nitrogen source available for growth.

The ability of the Fusarium-Chlorella consortium to remove TOC clearly differed when cultured with different co-metabolic nitrogen sources (Figure 3C). The rate of TOC removal reached 39.9 ± 0.41% when supplemented with urea, 3.56 ± 2.72% when supplemented with NH₄Cl, 51.58 ± 1.05% when supplemented with yeast extract and −4.4 ± 3.0% when PAM was used as the sole source of nitrogen. Therefore, the experimental groups supplemented with organic nitrogen sources provided another source of TOC in addition to PAM, which resulted in a significant decrease in the concentration of TOC. This may be due to the full utilization of additional sources of organic nitrogen. However, in the experimental groups where the inorganic nitrogen sources were supplemented or PAM was used as the sole nitrogen source, the concentration of TOC remained relatively stable with no significant changes observed. This may be attributed to the accumulation of some organic compounds that resulted from microbial metabolism during the cultivation period that were not utilized.

The degradation of IC by the Fusarium-Chlorella consortium over the 6 days culture period is shown in Figure 3D. The rate of IC removal reached 68.83 ± 1.32% in the group supplemented with urea, 93.58 ± 0.06% when supplemented with NH₄Cl, 93.48 ± 0.27% when supplemented with yeast extract and 88.97 ± 0.36% when PAM was used as the sole nitrogen source. Overall, the synergistic performance of the Fusarium-Chlorella consortium to treat IC was better than that of the single Chlorella sp. system with the ability to sequester carbon providing obvious advantages. These findings prove that the mechanisms of biodegradation by microalgae and fungi have synergistic effects, which results in their effective combination for wastewater treatment (Wang et al., 2022).

An analysis of the differences in the degradation efficiency of PAM among three different biological systems that did not include co-metabolic nitrogen sources was conducted. The results demonstrated a significant difference (p = 0.003) in the degradation capability among the three systems. This indicates that different biological systems exhibit significantly different abilities to degrade PAM, and the efficiency of the consortium to degrade PAM is significantly higher than that of a single system.

According to China’s Petrochemical Pollutant Discharge Standard (GB31570-2015, China), the concentrations of PAM and IC in effluent are not clearly required, while the relevant stipulated parameter is the chemical oxygen demand (COD). In the GB31570-2015, the COD discharge standard in oil flooding wastewater requires less than 100 mg/L. The COD values of the simulated flooding wastewater treated by mycelia were usually between 150 mg/L and 200 mg/L, which cannot directly reach the standard. However, in practical applications, oil flooding wastewater is treated by combining a variety of treatment processes, such as anaerobic baffled treatment process and aerobic biofilm treatment process among others (Sang et al., 2015; Song et al., 2018, 2019). In practice, the mycelial pellet can be combined with various processes to successfully process the DIC.

To investigate the structural changes to PAM molecules after sterilization and biodegradation, the FT-IR spectra of different PAM samples were analyzed (Figure 4). The peaks observed at 1115, 1616, 1,666 and 3,200 cm⁻¹ in the spectrum of PAM corresponded to the existence of C–N, N–H, C=O bonds and amide groups (Bao et al., 2010; Zhao et al., 2019).

A comparison of the FT-IR spectra for all three samples showed that the peaks that represented the C–N bonds, N–H bonds, C=O bonds and -NH₂ gradually disappeared, indicating that the side chains and amide groups of PAM were degraded. Furthermore, the peak that represented methylene at 1456 cm⁻¹ also gradually disappeared, indicating that the carbon backbone of PAM was also degraded. Furthermore, upon comparing the spectra of the three samples between 3,100 cm⁻¹ and 3,500 cm⁻¹, it was observed that the peaks had become wider. This indicated that the amide group had degraded into a carboxyl group (Bao et al., 2010). In comparison to the PAM degradation effects of high temperature sterilization and biodegradation, it was apparent that high temperature sterilization has some effect at degrading PAM, and the subsequent biodegradation fully degraded the PAM.

Based on the studies described above, three possible mechanisms for the removal of PAM, as well as that of the IC, by the biological systems were initially proposed.

When the experimental group without the use of additional nitrogen sources was used as an example, according to the metabolic pathway proposed in a previous study (Zhao et al., 2019) and the FT-IR results described above, the side chains of PAM were cleaved, which may be due to the role of extracellular amidases secreted by the microalgae and fungi to convert the amide group to carboxylate group; however, there was no carboxylate group that bound intramolecularly, which was slightly different from the findings of previous studies (Sang et al., 2015). Alternatively, it also proved that the microorganisms in this study could grow using PAM as the sole nitrogen source. In addition, the biomass still grew without the addition of an organic nitrogen source, and the FT-IR results showed that the main carbon chain of PAM was cleaved, which might be the result of the catalytic degradation of this carbon chain of PAM by a monooxygenase secreted by the microorganisms (Zhao et al., 2019). It also showed that PAM can be used as the sole source of carbon for microbial growth.

To remove the IC, we primarily rely on the biological fixation of IC by the microalgae in soluble carbonate compounds, such as NaHCO₃ (Molazadeh et al., 2019). Research has shown that microalgae primarily use CA to metabolize IC (Thanigaivel et al., 2022). In the Calvin cycle, microalgae absorb CO₂ and synthesize organic compounds to fix CO₂, and when the CO₂ dissolves, it converts the carbon into various forms of IC, thereby enabling the microalgae to remove the IC.