Efficient degradation of tetracycline by Mn(III)-microbial complexes mediated by mnOx@ACF in sequencing batch reactors: performance, mechanism, and effect on microbial community structure

Municipal wastewater treatment plants (WWTPs) are installed to remove pollutants from water, thus protecting the environment and human health. Biological treatment is the core component of WWTPs. With the continuous developments in nanotechnology, several engineered nanomaterials (ENMs) have been released into various environments, with their use being gradually increased in the WWTPs. More than 400,000 tons of ENMs are annually released into natural water bodies and WWTPs (Inshakova et al. 2020; Jin et al. 2024). The introduction of ENMs into WWTPs induces oxidative stress by generating reactive oxygen species, releasing toxic metal ions, and disrupting the cell membrane structure through physical interactions, thereby affecting the treatment efficiency and operational performance of the biological treatment (Wang & Chen 2016). ZnO nanoparticles have been shown to reduce the abundance of microorganisms, disrupt microbial diversity, and decrease the efficiency of biological treatment systems (Zhang et al. 2017; Cheng et al. 2019). Ag nanoparticles can inhibit the activity of nitrifying and denitrifying bacteria, which affects the nitrification and denitrification processes, respectively, in sequencing batch reactors (Chen et al. 2014). Hou et al. (2015) showed that the presence of copper oxide nanoparticles might affect the flocculation ability of activated sludge and its composition of extracellular polymeric substances. In addition, the introduction of carbon nanotubes in WWTPs can have a series of toxic effects on biological treatment processes, such as inhibiting nitrification, reducing organic matter removal, and disrupting sludge flocculation structure and granularity.

Manganese oxides have received widespread attention in environmental remediation because of their environmental friendliness, high natural abundance, low cost, and high activity. Mn(II) is essential for microbial metabolism, which facilitates sludge settlement and alters microbial activity in wastewater treatment. Jiang et al. (2020) found that the presence of Mn(II) prolonged the formation time of aerobic granular sludge and enhanced the secretion of extracellular polymers during the treatment of aniline wastewater. Li et al. (2018) successfully removed 93.95% of the ammonium from the water following the addition of Mn(II) to a sequencing batch reactor (SBR) with high nitrogen removal. He et al. (2019) found that Mn(II) can be oxidized by Mn-oxidizing bacteria or fungi to biogenic manganese oxides (bio-MnOx), and its combination with aerobic granules can be used to continuously oxidize and remove toxic pollutants from SBR.

In recent years, activated carbon fibers (ACFs) have been widely used as ideal support materials for nonhomogeneous catalysts because of their uniform microporous structure, large number of surface-active groups, excellent chemical stability, and inert structure. As ACFs can be processed into different textile structures (e.g., fibers, fabrics, and felts) (Huang et al. 2014; Sun et al. 2014; Wang et al. 2014; Zhou et al. 2015), they are easy to separate and recover from the reaction medium and thus have high practical applications in complex reaction system structures. Therefore, the use of ACFs as a carrier material loaded with manganese oxides can provide high activity and easy separation and recovery. Our group successfully prepared ACF-loaded manganese oxide nanomaterials (MnOx@ACF) and explored its catalytic ability in a previous study (Xiao et al. 2023). However, studies evaluating the environmental behavior and biotoxicity of MnOx@ACFs have been lacking, and exploring the stability, safety, and ecological benefits of MnOx@ACFs has become a priority.

In this study, tetracycline (TCH) was used as the target pollutant, and an SBR was used to simulate a biological treatment system for antibiotic wastewater to investigate the interference mechanism of MnOx@ACF composite catalysts in the biological treatment of antibiotic wastewater and the environmental response of activated sludge microbial flora. The DNA structures of all microorganisms in the samples were extracted from the simulated SBR reactor, and a macrogenomic library was constructed to study and analyze the genetic diversity and ecological information of microorganisms obtained from this environment using genomic research strategies to investigate the effects of the microbial community structure in the SBR reactor in the long-term presence of MnOx@ACF.

The activated sludge used in the experiments was obtained from a WWTP in Jiangxia District, Wuhan City, China, and was domesticated under TCH conditions at a concentration of 1 mg/L for 2 weeks before use. The composition and preparation methods of the synthesized wastewater are provided in Text S1 and Table S1, respectively.

The SBR reactors consisted of glass cylinders with an outer meridian of 136 mm, an overall height of 199 mm, and an effective volume of 2.0 L. An aeration device was mounted at the bottom of the reactor, which was connected to an air pump through a silicone tube to increase the dissolved oxygen level in the wastewater in the SBR. Domesticated sludge was inoculated into five SBR reactors (pH = 6.8–7.5) at a concentration of 4,000 ± 20 mg/L mixed liquor suspended solids. One SBR was set up as a no-MnOx@ACF control and named S0. The remaining SBR reactors were spiked with 0.05, 0.1, 0.2, and 0.4 g/L of MnOx@ACF and named S1, S2, S3, and S4, respectively. The five SBR reactors were operated continuously at 30 ± 2 °C for 30 cycles. Each operating cycle lasted for 12 h, including 10 min of filling, 11.5 h of aeration, 10 min of settling, and 10 min of draining. The hydraulic retention time was set to 20 h, and the exchange volume ratio was set to 60%. The airflow rate for aeration was set to 1.5 L/min.

The chemical oxygen demand (COD) and TCH concentrations in the influent and effluent were monitored during operation. The relevant analytical methods are presented in Text S2.

In this study, MnOx@ACF was prepared using a synthetic method previously reported by our group (Xiao et al. 2023). The preparation procedure is described in Text S3.

The scanning electron microscopy images and energy dispersive spectroscopy analysis of the ACF and MnOx@ACF are shown in Figure 1(b) and 1(c). The clean and unloaded ACF surfaces showed clear barred grooves, whereas in the MnOx@ACF-loaded with manganese oxides, the nanoparticles filled the ACF grooves. The change in the Mn content of the seeds also proved that the material was successfully prepared.

Figure 1(d) shows the adsorption of TCH by different systems. ACF alone could hardly adsorb TCH, while Mn₃O₄ and MnO₂ only adsorbed 15.5 and 18.0 mg/g of TCH, which was lower than 111.5 mg/g of MnOx@ACF. This indicates that the composites had better TCH adsorption capacity than the single materials, which may be attributed to the electrostatic interactions between the composites and TCH. Figure 1(e) demonstrates the variation spectra of Fourier transform infrared spectrometer (FTIR) before and after the reaction of MnOx@ACF. The small peak at 1,380 cm⁻¹ is usually attributed to the stretching vibration of carboxylates, which may be formed by the carboxyl groups on ACF with Mn ions during pyrolysis (Volkov et al. 2021). The stretching vibration of the peaks at 400 cm⁻¹⁻800 cm⁻¹ is attributed to the Mn–O bond and the Mn–O–Mn bond, and it can be found that the peaks before and after removal of TCH from MnOx@ACF hardly changed, which indicates the good mechanical stability and physicochemical properties of MnOx@ACF. When the amount of MnOx@ACF was adjusted (Figure 1(f)), the adsorption capacity decreased with an increase in the amount. The maximum adsorption capacity (176 mg/g) and the lowest removal rate of TCH (Figure S1) were observed when 0.05 g/L was used (39.1%). When the dosage was increased to 0.4 g/L, the TCH removal rate was 93.5%, but the adsorption capacity decreased since the increase in the dosage increased TCH adsorption sites by the material but also increased the adsorption resistance, eventually leading to a significant decrease in the adsorption capacity (Zhou et al. 2023).

Scanning electron microscopy was used to observe the morphological changes in the MnOx@ACF in the SBR biological treatment system. As shown in Figure 3(c) and 3(d), the MnOx@ACF originally dispersed on the surface of the ACF agglomerated after the SBR experiments. The obvious concave strip grooves on the surface of the MnOx@ACF after the SBR experiments were almost invisible, and the original, relatively dispersed MnOx@ACF particles almost exponentially agglomerated on the surface of the ACF after the SBR experiments. Elemental mapping analysis using energy dispersive spectroscopy of the localized regions showed that the mass percentages of Mn atoms were 1.02 and 5.24%, respectively. After the SBR experiments, the MnOx@ACF generated more peaks in the X-ray diffraction patterns (Figure S4), produced by the residual mineral components in the sludge of the nanomaterials (Pan et al. 2022).

To explore the valence changes in MnOx@ACF-Mn species in the SBR reactor, Figure 3(e) shows the Mn 2p spectra for 0, 10, and 30 cycles. Mn(IV), Mn(III), and Mn(II) correspond to 643.0–644.6 eV, 641.8–642.0 eV, and 641.2 eV, respectively. During the first 10 cycles, the Mn(III) content decreased from 65.95 to 28.90%, while the Mn (IV) content and Mn (II) content increased from 24.92 and 10.03% to 50.97 and 20.13%, respectively, which suggests that the microorganisms gradually adapted to the incorporation of the MnOx@ACF, at this stage, and the microorganisms continuously consumed Mn(III). As the system tended to stabilize, Mn(II) was almost completely converted into Mn(III) in the 10th–30th cycles. Thus, the stable removal of COD and TCH may be attributed to the mutual transformation of the Mn(III)-microbial complex and the Mn(II)-microbial complex, which was ultimately enriched in the prismatic grooves of the ACF. Indeed, previous studies have demonstrated that nanomaterials may influence the microbial biochemical reaction process, and Mn is involved in the transfer of extracellular electrons from bacteria to Mn minerals (Deng et al. 2022; Liu et al. 2023), thus facilitating the cycling of Mn(II) and Mn(III).

To investigate the effect of the long-term presence of MnOx@ACF on the microbial community structure in SBR reactors, mixed sludge samples were collected from five SBR (S0, S1, S2, S3, and S4) after 30 cycles of operation and subjected to microbial macrogenomic analysis on the Illumina NovaSeq sequencing platform.

The microbial community composition in the SBR reactors was analyzed at the phylum level for different MnOx@ACF concentrations (Figure 6(c)). Proteobacteria (including most nitrifying and denitrifying bacteria) play a key role in biological denitrification (Zhu et al. 2022) and had the highest relative abundance among the five SBRs. This indicates that Proteobacteria dominated in each reactor and that the addition of MnOx@ACF may have favored their enrichment in the environment. However, Actinobacteria, also an important denitrifying microorganism in SBR, showed opposite changes to Proteobacteria. Actinobacteria in the S1–S4 reactors all showed a significant decrease in relative abundance compared to the S0 reactor (17.57%), with relative abundances of 9.27, 6.86, 21.35, and 7.98%, respectively. These results indicate that different types of microbial populations undergo different degrees of population abundance changes when exposed to MnOx@ACFs for long periods. Bacteroidota are commonly found in wastewater treatment systems. Most of the Bacteroidota exist in anaerobic or low-oxygen environments, decompose proteins and amino acids, and effectively remove organic pollutants and nutrients. Their relative abundances were 3.97, 6.01, 4.63, 3.44, and 4.96% in S0–S4 reactors, respectively. In addition, the relative abundance of Chloroflexota, which is mainly a parthenogenetic anaerobic microorganism, did not change significantly in the S1–S4 reactors (1.8, 1.55, 1.64, 2.1, and 1.7%, respectively), suggesting that the presence of MnOx@ACFs provided a better growth environment for it.

First-order metabolic pathway annotations based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database revealed a functional profile of communities in the S0–S4 reactors, mainly including metabolism, genetic information processing, human diseases, environmental information processing, cellular processes, and organic systems. As shown in Figure 7(b), metabolism was the main function in all samples, which occupies about 70.0% of the functional composition in all S0–S4 samples, followed by genetic information processing and cellular processes. However, the functional composition relationship and the degree of contribution of each type of function in the different samples did not change significantly. The results indicated that the addition of MnOx@ACF did not significantly affect the major functional composition of the microbial communities in the SBR.

In addition, this was used as the abundance unit to map the functional abundance in the S0–S4 samples based on the KEGG database of second-class metabolic pathways, and the results are displayed in Figure S5. The left vertical coordinate shows the classification of the second-rank functional metabolic pathways based on the KEGG database, and the corresponding first-rank functional pathways are shown on the right. Annotation analysis was performed by function in each sample to elucidate the changes in vital metabolic processes caused by the presence of MnOx@ACF. As shown in the figure, the relative abundance of the overall microflora with metabolic levels of functionality increased to varying degrees as the amount of MnOx@ACF dosed in the SBR reactor increased. Among them, amino acid metabolism, carbohydrate metabolism, xenobiotic biodegradation and metabolism, and the metabolism of terpenoids and polyketides all showed significant increases in relative abundance. It has been suggested that the biotoxicity of TCs-like substances may stimulate the microbial community to activate protective mechanisms for degrading TCs, thus enhancing the overall metabolism (Ohore et al. 2021). Broad-spectrum polyketides are associated with the metabolic functions of terpenoids and polyketides (Abegaz & Kinfe 2019). Among all the primary functional pathways with increased relative abundance, nucleotide metabolism, replication and repair, glycan biosynthesis and metabolism, folding, sorting, and degradation, and cell growth and death subchannels showed the lowest increase in functional gene abundance, reflecting the biotoxicity of TCH by affecting cell replication and growth in epiphytic biofilm microbial communities (Xu et al. 2018; Ohore et al. 2021). In addition, although the overall functional abundance in the SBR was increased by the presence of TCH, different dosages of MnOx@ACF further influenced the increase in functional abundance. The results showed that either too high or too low MnOx@ACF affected the microbial functional profiles differently, and the S3 sample (0.2 g/L MnOx@ACF) influenced the strongest disturbance and stimulation of microbial functional abundance compared with the other samples, which may be related to the interactions among MnOx@ACF, TCH, and microbial populations.

This work was financially supported by the National Natural Science Foundation of China (Grant No. 51908432), the Natural Science Foundation of Hubei Province (2023AFB277), and the State Key Laboratory of Pollution Control and Resource Reuse Foundation (No. PCRRF22016). The authors would like to thank Technical Officer Zhengtao Gui of Shiyanjia Lab (www.shiyanjia.com) for the scanning electron microscopy and energy dispersive spectrometry elemental mapping analyses.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.