The ecological effects of antibiotics in surface water have attracted increasing research attention. In this study, we investigated the combined ecotoxicity of erythromycin (ERY) and roxithromycin (ROX) on the microalgae, Chlorella pyrenoidosa, and the removal of ERY and ROX during the exposure. The calculated 96-h median effect concentration (EC₅₀) values of ERY, ROX, and their mixture (2:1 w/w) were 7.37, 3.54, and 7.91 mg∙L^-1, respectively. However, the predicted EC₅₀ values of ERY+ROX mixture were 5.42 and 1.51 mg∙L^-1, based on the concentration addition and independent action models, respectively. This demonstrated the combined toxicity of ERY+ ROX mixture showed an antagonistic effect on Chlorella pyrenoidosa. During the 14-d culture, low-concentration (EC₁₀) treatments with ERY, ROX, and their mixture caused the growth inhibition rate to decrease during the first 12 d and increase slightly at 14 d. In contrast, high-concentration (EC₅₀) treatments significantly inhibited microalgae growth (p < 0.05). Changes in the total chlorophyll contents, SOD and CAT activities, and MDA contents of microalgae suggested that individual treatments with ERY and ROX induced higher oxidative stress than combined treatments. After the 14-d culture time, residual Ery in low and high concentration Ery treatments were 17.75% and 74.43%, and the residual Rox were 76.54% and 87.99%, but the residuals were 8.03% and 73.53% in ERY+ ROX combined treatment. These indicated that antibiotic removal efficiency was higher in combined treatments than that in individual treatments, especially at low concentrations (EC₁₀). Correlation analysis suggested that there was a significant negative correlation between the antibiotic removal efficiency of C. pyrenoidosa and their SOD activity and MDA content, and the enhanced antibiotic removal ability of microalgae benefited from increased cell growth and chlorophyll content. Findings in this study contribute to predicting ecological risk of coexisting antibiotics in aquatic environment, and to improving biological treatment technology of antibiotics in wastewater.

Macrolide pollution has attracted a great deal of attention because of its ecotoxic effects on microalgae, but the role of phycospheric bacteria under antibiotic stress remains unclear. This study explored the toxic effects of erythromycin (ERY) on the growth and nitrogen metabolism of Auxenochlorella pyrenoidosa; then, it analyzed and predicted the effects of the composition and ecological function of phycospheric bacteria on microalgae under ERY stress. We found that 0.1, 1.0, and 10 mg/L ERY inhibited the growth and chlorophyll of microalgae, but the microalgae gradually showed enhanced growth abilities over the course of 21 days. As the exposure time progressed, the nitrate reductase activities of the microalgae gradually increased, but remained significantly lower than that of the control group at 21 d. NO₃⁻ concentrations in all treatment groups decreased gradually and were consistent with microalgae growth. NO₂⁻ concentrations in the three treatment groups were lower than those in the control group during ERY exposure over 21 d. ERY changed the community composition and diversity of phycospheric bacteria. The relative abundance of bacteria, such as unclassified-f-Rhizobiaceae, Mesorhizobium, Sphingopyxis, Aquimonas, and Blastomonas, varied to different degrees. Metabolic functions, such ABC transporters, the microbial metabolism in diverse environments, and the biosynthesis of amino acids, were significantly upregulated in the treatments of higher concentrations (1.0 and 10 mg/L). Higher concentrations of ERY significantly inhibited nitrate denitrification, nitrous oxide denitrification, nitrite denitrification, and nitrite and nitrate respiration. The findings of this study suggest that phycospheric bacteria alleviate antibiotic stress and restore the growth of microalgae by regulating nitrogen metabolism in the exposure system.

The ecotoxicity induced by macrolides has attracted widespread attention, but their impacts on the nitrogen metabolism and symbiotic environmental bacteria of microalgae remain unclear. This study examined the effects of roxithromycin (ROX) on the growth, chlorophyll levels, and nitrogen metabolism of Chlorella pyrenoidosa; investigated the changes in the composition and functions of environmental bacterial communities; and finally, analyzed the relationship between microalgae and environmental bacteria. The results indicated that all concentrations of ROX (0.1, 0.25, and 1 mg/L) inhibited microalgae growth, but the inhibition rates gradually decreased after a certain exposure period. For instance, the inhibition rate in the 1 mg/L treatment group reached the highest value of 43.43% at 7 d, which then decreased to 18.93% at 21 d. Although the total chlorophyll content was slightly inhibited by 1 mg/L ROX, the Chl-a/Chl-b value increased between 3 and 21 d. The nitrate reductase activities in the three treatments were inhibited at 3 d, but gradually returned to normal levels and even exceeded that of the control group at 21 d. Under ROX treatment, the consumption of NO₃⁻ by microalgae corresponded to the nitrate reductase activity, with slower consumption in the early stage and no obvious difference from the control group in the later stage. Overall, the diversity of environmental bacteria did not undergo significant changes, but the abundance of some specific bacteria increased, such as nitrogen-fixing bacteria (unclassified-f-Rhizobiaceae and Mesorhizobium) and organic contaminant-degrading bacteria (Limnobacter, Sphingopyxis, and Aquimonas). The 0.25 and 1 mg/L ROX treatments significantly enhanced the carbohydrate metabolism, cofactor and vitamin metabolism, amino acid metabolism, and energy metabolism of the environmental bacteria, but significantly downregulated nitrogen denitrification. This study provides new insights into the environmental bacteria-driven recovery mechanism of microalgae under antibiotic stress.