Mutual impacts and interactions of antibiotic resistance genes, microcystin synthetase genes, graphene oxide, and Microcystis aeruginosa in synthetic wastewater

The physiological impacts and interactions of antibiotic resistance gene (ARG) abundance, microcystin synthetase gene expression, graphene oxide (GO), and Microcystis aeruginosa in synthetic wastewater were investigated. The results demonstrated that the absolute abundance of sul1, sul2, tetW, and tetM in synthetic wastewater dramatically increased to 365.2%, 427.1%, 375.2%, and 231.7%, respectively, when the GO concentration was 0.01 mg/L. Even more interesting is that the sum gene copy numbers of mcyA-J also increased to 243.2%. The appearance of GO made the significant correlation exist between ARGs abundance and mcyA-J expression. Furthermore, M. aeruginosa displayed better photosynthetic performance and more MCs production at 0.01 mg/L GO. There were 65 pairs of positive correlations between the intracellular differential metabolites of M. aeruginosa and the abundance of sul1, sul2, tetM, and tetW with various GO concentrations. The GO will impact the metabolites and metabolic pathway in M. aeruginosa. The metabolic changes impacted the ARGs, microcystin synthetase genes, and physiological characters in algal cells. Furthermore, there were complex correlations among sul1, sul2, tetM, tetW, mcyA-J, MCs, photosynthetic performance parameters, and ROS. The different concentration of GO will aggravate the hazards of M. aeruginosa by promoting the expression of mcyA-J, producing more MCs; simultaneously, it may cause the spread of ARGs.


Introduction
Antibiotic resistance gene (ARG) pollution has become a knotty problem that has attracted much attention around the world (Conley et al. 2009). The overuse of antibiotics in medicine, cultivation, and aquaculture field has caused the accumulation of ARGs in aquatic environment (Komijani et al. 2021). The ARGs have been detected in many water bodies in China, especially ARGs of sulfonamides and tetracyclines are ubiquitous and with a high concentration (Sun et al. 2017). The average level of ARGs in natural waters reached 1.2×10 8 gene copies/mL (Sun et al. 2017). There are not only large amounts of ARGs remaining in general water bodies, but also the problem of Microcystis aeruginosa (M. aeruginosa) should not be ignored. The overgrowth of M. aeruginosa not only caused harmful algae bloom but also produced very potent microcystins (MCs) which can stimulate oxidative stress of the cell, which would produce a large amount of reactive oxygen species (ROS) (Chen et al. 2016;McLellan and Manderville 2017). The ROS would promote the change of cell permeability, which accelerate the release of intracellular substances (Jiang et al. 2021). The increase in membrane permeability is one of the fundamental reasons for increasing the transfer efficiency of ARGs (Guo et al. 2021;Lu et al. 2020b;Sun et al. 2018). And then, to make matters worse, the graphene oxide (GO) would inevitably be released into the aquatic environment with its extensive application Zhu et al. 2019). The presence of GO might have a certain impact on the growth of M. aeruginosa, microcystin synthetase genes, and MC production . Aquatic environment is an important medium for the release and diffusion of MCs, ARGs, and GO. These pollutants can invade the human food chain by the water cycle, posing a serious threat to the aquatic ecological environment and human health (Avant et al. 2019;Jiang et al. 2020). It is known that the pollutants in natural water bodies are very complicated, and there are many kinds of pollutants such as ARGs, M. aeruginosa, and nano-pollutants (Rzymski et al. 2020). The GO might affect the microcystin synthetase gene expression and MC production when its concentration reaches a certain level (Wang et al. 2020a;Yin et al. 2020). People formerly believed that the abuse of antibiotics was the main reason for global accumulation and spread of ARGs (Sola 2020). However, more and more studies have shown that natural-occurring substances and some kinds of nanopollutants in the aquatic environment can promote the spread of ARGs (Sun et al. 2021). Studies have shown that extractive of M. aeruginosa and pure MCs and nanometer materials can cause the spread of ARGs in aquatic environment (Fan et al. 2021;Xu et al. 2021). Some researchers have inferred that MCs and nanometer materials might change the permeability and surface functional groups of microbial cells and accelerate the rate which ARG genetic material enters cells (Fan et al. 2021).
MCs are synthesized by the megazyme complex through non-ribosomal pathways (Yang et al. 2015). This type of complexus includes peptide synthase, polyketide synthase, and some other modified enzymes (Wei et al. 2020). By sequencing the gene cluster encoding synthase, it was found that the gene cluster contained a type of mixed non-ribosomal peptide synthetase genes including mcyA, mcyB, mcyC, mcyD, mcyE, mcyF, mcyG, mcyH, mcyI, and mcyJ (Lu et al. 2020a). Simultaneously, nano-pollutants in aquatic environment will also affect the production of MCs. In the presence of GO, the transcription levels of the synthetase genes mcyA, mcyB, and mcyD are significantly increased (Grasso et al. 2020). Therefore, what can be inferred is that although the production of MCs is determined by the genes in the microcystin-producing cells, the nano-pollutants such as GO in environment can also regulate their gene expression, thereby affecting the synthesis of MCs. The presence of MCs may increase the abundance of ARGs, and the GO in the aquatic environment might make the transcription of MCs synthase genes increase. While, there is still no result that can effectively verify this inference, this work is trying to prove this. What are the mutual impacts and interactions of ARGs, microcystin synthetase genes, MCs, GO, and M. aeruginosa? The researches on this aspect were currently rare.
The abundance changes of ARGs including sul1, sul2, tetW, tetM, and the gene copy numbers of MC microcystin synthetase genes including mcyA, mcyB, mcyC, mcyD, mcyE, mcyF, mcyG, mcyH, mcyI, mcyJ in GO-exposed M. aeruginosa with different concentrations were investigated in this study. Meanwhile, the correlativity between ARG abundance and mcyA-J expression quantity was evaluated. Moreover, the effect of GO with different concentrations on the ultrastructure, photosynthesis, metabonomics characters of M. aeruginosa was also studied. These results will reveal the mutual impacts and interactions of ARGs, microcystin synthetase genes, graphene oxide, and Microcystis aeruginosa in synthetic wastewater, which will provide some basics for the studies of multi-component pollutants in aquatic environment.

Experimental design
M. aeruginosa (FACHB-315) was purchased from the Institute of Wuhan Hydrobiology, Chinese Academy of Sciences and cultured in pH 7.0 BG11 medium (Table S1). The GO was purchased from the Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences. The GO sheet diameter was 0.1-10 μm in size with average thickness of 1.5 μm. The plasmids of ARGs including sul1, sul2, tetW, tetM were prepared by Genesis Biotechnology Co., Ltd. The gene sequences of sul1, sul2, tetW, tetM were listed in Table S2.
For the incubation experiment, photobioreactors as shown in Fig. 1 were used. The GO particles were dispersed in synthetic wastewater (Table S3) with initial concentrations of 0, 0.01, 0.1, 1, and 10 mg/L, and for the control group, the GO concentration was 0 mg/L. The GO concentration range was set close to that found in natural water (Zhao et al. 2020b). In these systems, GO particles had hydrodynamic diameters of 250-300 nm and zeta potential ranges from −30 to 50 mV, indicating that the GO particles had been stably dispersed in the synthetic wastewater at the four concentrations (Monteil et al. 2014). M. aeruginosa was inoculated at density of 1.0×10 5 cells/mL, and the reactors were placed in an illumination incubator (MGC-300A, China) at 28.0 ± 0.5°C and 75% humidity. At the beginning (0 h) and after 0, 24, 48, 96 h of incubation, samples were taken from each reactor and centrifuged at 8000×g for 10 min. The supernatants were used for analyses of ARG abundance, total nitrogen (TN), ammoniacal nitrogen (NH 3 -N), phosphate phosphorus (PO 4 3− -P), chemical oxygen demand (COD), and extracellular MCs (MC-LR and MC-RR). The precipitates were collected for intracellular MCs and reactive oxygen species (ROS) quantification, metabolic responses, mcyA-J gene expression analyses, and transmission electron microscopy (TEM) observation. Only samples taken at the end of the experiment (96 h) from the control, 0.01, and 10 mg/L GO were used for metabonomic analysis.

Measurement of algal photosynthetic response and growth rate
The phytoplankton classification fluorometer (Phyto-PAM, Germany, WALZ) was used to measure the various parameters of chlorophyll fluorescence. The specific steps are as follows: ①Start the Phyto Win software, place a certain amount of sample (the volume should be uniform each time) in a cuvette for 15 min. ② Start the instrument to determine the initial measurement fluorescence yield (F 0 ), measure the maximum fluorescence yield (F m ) after the saturation pulse at 4000 μmol/(m 2 ·s). ③ Calculate the maximum light energy conversion efficiency (F v /F m ). ④ Set the photochemistry intensity at 3000 μmol/(m 2 ·s) and irradiate for 1 min util the indicator light turns green, the initial fluorescence (F s ) and maximum fluorescence (F m ') were measured when the fluorescence value was stable. The measured chlorophyll fluorescence parameters are F 0 , F m , F 0 ′, F m ′, F s . The fluorescence parameters such as F v /F m , F v /F 0 , ETR max were calculated as follows (Poudyal et al. 2019): Maximum light conversion efficiency Eq. (1): Maximum photochemical quantum yield Eq. (2): Efficiency of light energy conversion Eq. (3): Quantum efficiency Eq. (4): Photosynthetic electron transport Eq. (5): The specific growth rate of algae is used to reflect the growth of M. aeruginosa. The formula is shown in Eq. (6): In Eq. (6): X n is for the cell density of M. aeruginosa at the end of the GO-exposure period (t n ), X n−1 is the cell density of M. aeruginosa at the GO-exposure period (t n−1 ) (Elser et al. 2007).

N, P nutrient removal determination
After the supernatants of samples were filtered through 0.44-μm filters, the concentration of nitrogen and phosphorus nutrients including TN, NH 3 -N, PO 4 3− -P, and COD was determined as described in their study (Ajayan et al. 2019).

MC quantification and microcystin synthetase gene expression measurement
The MCs in the supernatants were extracted with Oasis HLB and determined by liquid chromatography-mass spectrometry (LC-MS). Intracellular MC (MC-LR and MC-RR) extraction was performed as described in a previous study (Pinheiro et al. 2016). The sample was extracted with 75% methyl alcohol at 25°C for 20 min while stirring. The homogenate was centrifuged (10,000×g, 10 min) to remove the pellet. The MCs in the supernatant were eluted using 80% (v/v) methyl alcohol, concentrated at 35°C (Pinheiro et al. 2016), purified, and quantified using HPLC (Agilent 1200, USA). A reversed phase column equipped with a guard column at 45°C was used. For mcyA-J gene expression analysis, total RNA of M. aeruginosa was transcribed to cDNA for RT-qPCR analysis on a real-time PCR system (Thermo fisher, Step One Plus, USA). The qPCR amplification procedure was operated as Lee reported ).

TEM observation and ROS determination
The algal cells of M. aeruginosa were added to 2.5% glutaraldehyde with the final concentration of 2.5%, then fixed for 3 h. Centrifuged at 5000×g, the supernatant was removed, and 0.1 mol/L phosphate buffer was added to wash the samples for 3 times. Then, 4% osmic acid was added to fix the algal cell. The samples were centrifuged at 5000×g for 5 min after incubation overnight at 4°C, then the supernatant was removed. The acetone solutions of different concentrations of 10%, 30%, 50%, 70%, 90%, and 100% were used to dehydrate. Resin was used to embed, then the sample was sectioned (EMUC7, Lycra, Austria). The 3% uranyl acetate and 2% lead citrate were used to stain. Finally, the samples were observed by a transmission electron microscope (HT7700, Hitachi, Japan) (Soares et al. 2020). The ROS levels of samples were detected by a ROS kit (ML Elisa0255, R&D Systems, USA) according to its operating manual.

Metabonomic determination
Extraction, derivatization, and GC-MS detection process of metabolites were performed as the modified method of Weckwerth (Weckwerth et al. 2004). A certain amount of sample (grinded in liquid nitrogen) was added in a 1-mL pre-cooled extraction solution (volume ratio of methanol to water is 1:1) and 5 μL internal standard substance. Then, the mixture was vortexed for 3 min. After centrifugation (8000×g, 5 min), 500 μL supernatant was placed in liquid nitrogen for 30 min, then the sample was freeze-dried. The 50 μL methoxyammonium hydrochloride/pyridine solution (20 mg/ mL) was added, kept reacting at 40°C for 60 min. The 80 μL N-methyl-N-(trimethylsilane) trifluoroacetamide (MSTFA) was added, then reacted 80 min at 40°C. After centrifugation at 8000×g, for 10 min, the supinate was used for detection and analyzed by GC-MS.

Statistical analysis
The treatments and measurements were all performed in triplicate. Origin 8.5 was used for data processing for statistical analysis. The identification of metabolites was performed by the NIST database (2011). The metabolite data were normalized, then they were imported into SIMCA software (Version 11.5) for the PCA and PLS analysis. The HCE 3.5 software was used to perform hierarchical cluster analysis. The figures in this study were drawn by Graph pad Prism 7.0.

Analysis of mutual impacts between ARGs and microcystin synthetase gene expressions
The absolute abundance of the total ARGs including sul1, sul2, tetW, tetM of GO-exposed M. aeruginosa systems at a concentration of 0.01 mg/L was improved 4 times than that at a concentration of 0 mg/L, especially for sul1, sul2. The highest abundance of sul1 and sul2 in the GO-exposed M. aeruginosa system with a concentration of 0.01 mg/L reached 4.14×10 11 copies/L. The total gene copies of microcystin synthetase genes including mcyA-J reach up to 2.98×10 10 when the concentration of GO was 0.01 mg/L. In order to explain the impacts between ARGs (sul1, sul2, tetM, tetW) and microcystin synthetase genes (mcyA-J), the correlation analysis was performed, and the results were shown in Fig. 2.
The Pearson correlation analysis was performed between the expression of sul1, sul2, tetW, tetM, and the intracellular mcyA-J of M. aeruginosa in the synthetic wastewater when GO has a concentration of 0.01, 0.1, 1, 10 mg/L. The results demonstrated that there were 30 pairs, 30 pairs, 30 pairs, and 25 pairs of correlations (P<0.05) between ARGs and mcyA-J at 24 h, 48 h, 72 h, 96 h, respectively. It can be inferred that there was a positive correlation between the abundance of ARGs and the expression of mcyA-J when the GO was present. When the GO concentration is 0, there is no correlation between the abundance of sul1, sul2, tetM, tetW, and mcyA-J, which further demonstrated that the presence of GO made the abundance of ARGs closely related to the expression of mcyA-J. Interestingly, when the concentration of GO was 0.01 mg/L, the expression of mcyA-J was significantly increased (P<0.05), and the MC production was also significantly increased, and the expressions of sul1, sul2, tetM, tetW were also increased significantly (P<0.05). The presence of GO at the concentration of 0.01 mg/L made the positive correlation between ARG (sul1, sul2, tetM, tetW) abundance and mcyA-J expression further enhanced. What would be mentioned in the latter section was that the photosynthesis performance of M. aeruginosa and MC production was promoted when the GO concentration was 0.01 mg/L. It can be inferred that the presence of GO in aquatic environment will aggravate the overgrowth of M. aeruginosa, MC production, and spread of ARGs to a certain extent (Pan et al. 2015;Wu et al. 2020b).

Influence of GO on the N, P removal by M. aeruginosa
The nitrogen and phosphorus nutrients including total nitrogen (TN), ammoniacal nitrogen (NH 3 -N), phosphate (PO 4 3− -P), and chemical oxygen demand (COD) removal by M. aeruginosa are closely related to the growth rate (Ma et al. 2014). The removal rates of TN, NH 3 -N, PO 4 3− -P, and COD were 25%, 72%, 36.2%, and 42.9%, respectively, at 0.01 mg/L GO exposure (Fig. 3), indicating that 0.01 mg/L GO exposure can effectively stimulate and promote nutrient removal by M. aeruginosa from the growth environment (Aphale et al. 2015). Much smaller removals of TN, NH 3 -N, PO 4 3− -P, and COD were observed in the 10 mg/L GO treatment, which might be attributed to the negative effects of GO at a high concentration on the photosynthetic rates of algal cells [40], as evident by the low F v /F m and ETR max demonstrated in Fig. 1. Simultaneously, a high concentration of 10 mg/L GO inhibited nutrient removal by M. aeruginosa (Zhao et al. 2020a).

Cellular impacts of M. aeruginosa and GO
Significant effects (P<0.05) of GO on photosynthesis of M. aeruginosa were observed during the GO-exposure period at concentrations of 0.01, 0.1, 1, and 10 mg/L, respectively. The different concentration of GO exposure also affected the growth rate significantly (P<0.05). The results indicated that the intracellular production and extracellular release of MCs in GO-exposed groups were higher than that in the control (without GO exposure). As shown in Fig. 4A, the intracellular MC production in M. aeruginosa of the 0.01 mg/L GO-exposure group was the highest among all groups during the whole exposure period. Simultaneously, the number of gene copies   Fig. 5A showed obscure boundaries of the cytomembranes, indicating that severe peroxidation damage and plasmolysis of the algal cells occurred, and a large number of cells were ruptured in the high GO concentration treatments (the red circle in Fig. 5A). Thereupon, lots of intracellular MCs were released, and the percentage of extracellular MC release was increased significantly (P<0.05).
Furthermore, as shown in Fig. 3A,B, the value of F v /F m and ETR max of M. aeruginosa cells was the highest among all groups during the GO exposure, which was 0.01 mg/L. The F v /F m value reflects the potential maximum photosynthetic capacity of algal cells (Joonas et al. 2019), and the ETR max value reflects the maximum transmission rate of photons in photosynthesis of M. aeruginosa (Lee et al. 2019). The higher F v /F m and ETR max values would indicate the better photosynthetic performance (Cruces et al. 2021), and the F v /F m value of normal growth of algae is about 0.7-0.8 (Zheng et al. 2020). The highest F v /F m value is 1.1 in group of GO exposure at a concentration of 0.01 mg/L. Therefore, the photosynthetic performance was stimulated by GO exposure at a concentration of 0.01 mg/L. In contrast, the

Analysis of interactions from metabolomic aspects
The metabolic pattern of M. aeruginosa under GO exposures of 0.01 mg/L and 10 mg/L were compared with the control without GO exposure. The GO exposures at a concentration of 0.01 mg/L and 10 mg/L were close to the low and high contamination levels of GO in aquatic environments, respectively ). The metabolic profiling of M. aeruginosa in 0.01 and 10 mg/L GO-exposure groups is distinct (Fig. 7A), indicating that the metabolites in these two groups are significantly different (P<0.05). A total of 64 differential metabolites were screened (Fig. 7B), while the relative abundance of differential metabolites (Fig. 7C) and significantly different metabolic pathways (P<0.05) in the GOexposed groups were analyzed (Fig. 8).
After 96 h of exposure to 0.01 mg/L of GO, 56 metabolites were up-regulated while 8 metabolites were down-regulated ( Table 1). The identified metabolites were involved in 4 main physiological processes according to significant enriched pathways (P<0.05), including photosynthetic metabolism, glycometabolism, amino acid metabolism, and lipid metabolism. Much more metabolites were up-regulated instead of down-regulated, indicating that most physiological activities in M. aeruginosa were stimulated at the presence of 0.01 mg/L GO. In contrast, after 96 h of GO exposure at a concentration of 10 mg/L, 47 metabolites were up-regulated while 17 metabolites were down-regulated. More metabolites were down-regulated as compared with the 0.01 mg/L GO treatment, suggesting that the physiological activities were motivated to initiate the defensive mechanism against GO stress in the 10 mg/L GO treatment . The result of enrichment analysis of the KEGG pathway demonstrated that carbon fixation in the photosynthetic process, valine, leucine, and isoleucine biosynthesis, and galactose metabolism were significantly enriched (P<0.05) in M. aeruginosa exposed to GO at a concentration of 0.01 mg/L.
The metabolic network map reflects the important interactions between the altered metabolic pathways (Fig. 8).
Notably, in the 0.01 mg/L GO-exposure group, an increase in amino acid metabolism including increase in L-threonine, L-valine, L-alanine, and L-proline was observed. These findings are in accordance with previous studies where an increase in amino acid turnover in stimulated algal cells by a low concentration of GO was reported (Ouyang et al. 2020). The other important metabolic pathway found to be altered in M. aeruginosa in the 0.01 GO-exposure group was nucleotide metabolism, including increase of uracil and hypoxanthine. Proliferating algal cells of M. aeruginosa stimulated by GO often demand for nucleotides for the synthesis of cellular materials, which is fulfilled by purines and pyrimidines. Increases in nucleotides indicate that they are needed for cell proliferation ). Additionally, glycometabolism and fatty acid metabolism were indicated to be alerted in M. aeruginosa at GO exposure of 0.01 mg/ L. Specifically, increased levels of carbohydrates and numerous unsaturated fatty acids including D-glucose, galacturonate, linoleic acid, glutaric, and tetradecanoic acid were observed. The increase of glycometabolism indicates the vigorous growth of algal cell (Zhang et al. 2018), and unsaturated fatty acid will promote the photosynthetic performance of algal cells (Anto et al. 2020).
Moreover, the reticular correlativity between differential metabolites and other results including TN, NH 3 -N, PO 4 3− -P, COD, mcyA-J gene copies, MC production, ARGs (sul1, sul2, tetW, tetM), F v /F m , ETR max , and growth rate were calculated. Highly interconnected metabolites with high degrees play key roles in the interaction of M. aeruginosa and GO. T h e c o r r e l a t i o n be t w e e n m e t a b o l i t e s an d o t h e r factors (including sul1, sul2, tetM, tetW, mcyA-J, MC production, NH 3 -N, TN, PO 4 3− -P, COD, F v /F m , ETR max , growth rate, ROS, and 16S rRNA) were shown in Fig. 9. Moreover, it demonstrated that there were 23 pairs of positive correlations between the intracellular differential metabolites of M. aeruginosa and the abundances of sul1, sul2, tetM, and tetW with different GO concentrations. The metabolites that related ARG abundance were mainly amino acids. The metabolites that related mcyA-J expression were mainly amino acids and small molecule acids. There were 40 pairs of positive correlations between these metabolites and mcyA-J. Furthermore, there were 28 pairs of positive correlations between the abundance of sul1, sul2, tetM, tetW, and mcyA-J expression. The impacts and interactions were complicated of abundance of ARGs, mcyA-J expression, MC production, photosynthesis performance of M. aeruginosa, intracellular ROS levels, ultrastructure, and GO. Simultaneously, there is also a close correlation among various different metabolites in M aeruginosa (Kim et al. 2020).
Some chemical substances such as antibiotic contaminants and organic pollutant have been manifested to have toxic stimulant hormesis effects on algae at a certain concentration (Liu et al.  Particularly, the gene copies of microcystin synthetase (mcyA-J) increased. It was reasonable to conclude that the increased genetic expression of microcystin synthetase had resulted in the increased production of MCs. In contrast, the expression of mcyA-J was inhibited in the 10 mg/L GO-exposed group, and consequently, MC production decreased. The results suggested that the MC synthetic process is stimulated by low and inhibited by a high concentration of GO.
The ARGs and mcyA-J were significantly related with photosynthetic metabolites including phytol (an essential component of chlorophyll) and 3-6-anhydro-D-glucose (photosynthetic carbon fixes important metabolites) (Zhang et al. 2018). Moreover, some studies have demonstrated that microcystin synthetase gene (mcyA-J) and MC production was a kind of physiological response to environmental stressed factors . These results confirmed the role of MC production and synthesis in responsive process to GO exposure at environmental concentration .  The M. aeruginosa released more MCs in the 10 mg/L GOexposure group than in the control and the 0.01 mg/L GO treatment. The increased ROS level and membranolysis (Fig. 5) may facilitate the export of intracellular MCs . The cell rupturing induced by GO exposure might be an important explanation for the MC release by M. aeruginosa . With increased intracellular MC production and sul1, sul2, tetM, tetW abundance at a low concentration of GO and increased release of MCs at a high concentration of GO, the hazards of M. aeruginosa and ARGs would be exacerbated by GO in the aquatic environment (Bandara et al. 2019). It suggested that the harm of GO by regulating the ARG abundance, microcystin synthetase genes, and MC production has already become an ecological problem.
During the GO-exposure period of 96 h, impacts and interactions of ARGs, microcystin synthetase genes, MC production, photosynthesis were initiated. The relative abundance of carbohydrates related to the carbon fixation pathway in photosynthetic process in M. aeruginosa increased significantly (P<0.05) in GO exposure at a concentration of 0.01 mg/L. Simultaneously, the expression of mcyA-J in M. aeruginosa and sul1, sul2, tetM, tet W in synthetic wastewater increased significantly (P<0.05), resulting in the increase of intracellular MC production and ARG spread. The microcystin synthetase gene cluster of mcyA-J can regulate the ABC transporters (control the transportation and exchange of nutrients between extracellular and intracellular) Pearson et al. 2020). The result of KEGG pathway enrichment analysis suggested that the pathway of ABC transporters was significantly enriched, and the metabolites (valine, maltotriose, D-glucose, D-maltose, threonine, alanine, proline) which matched in the transporter pathway were up-regulated. It means that more extracellular nutrients (such as NH 3 -N) and ARG plasmid of sul1, sul2, tetM, tetW in synthetic wastewater will be transported into M. aeruginosa for cell growth, MC synthesis, and spread of ARGs (Yu et al. 2019). When the concentration of GO exposure increased to 10 mg/L, cytoderm rupture occurred, and large amounts of intracellular MCs were released. It indicated that the hormesis mechanism would be triggered in M. aeruginosa and ARG abundance when the GO is present. The presence of GO at a finite concentration in aquatic environment can aggravate the harm of M. aeruginosa and spread of ARGs (Duan et al. 2020;Huang et al. 2020).