# Biodegradation of Sulfamethoxazole in Milkfish (_Chanos chanos_) Pond Sediments
## 3 Results and Discussion
### Test of Sulfamethoxazole-Degrading Ability of Isolated Bacteria  
To test the SMX-degrading ability of the four bacterial strains (A2, A12, M, and L) in milkfish pond sediments, batch experiments were performed. In the sterilized controls, the SMX residues were all above 98% after 8 and 24 days of incubation under aerobic and anaerobic conditions. Therefore, the decomposition of SMX should be mainly due to microbial processes. The results indicated that SMX (2 mg L\({}^{-1}\)) was completely degraded with bacterial strains A2 and A12 under aerobic conditions (Figure 7A), and completely degraded with bacterial strains L and M under anaerobic conditions (Figure 7B). The rate constants (\(k\)) and half-lives (\(t_{1/2}\)) of the SMX in the experiments shown in Figure 7 were estimated and listed in Table S5. These results indicate that the addition of isolated bacterial strains A2, A12, L, and M enhance SMX degradation in milkfish pond sediments. Based on the results of the SMX-degrading ability tests, bacterial strains A12 and L were used to perform aerobic and anaerobic continuous addition experiments, respectively. Figure 6: Photo pictures of isolated SMX-degrading bacteria by scan electron microscopy. (**A**) Bacteria strain A2. (**B**) Bacteria strain A12. (**C**) Bacteria strain L. (**D**) Bacteria strain M.  
SMX degradation in aerobic readdition experiments is shown in Figure 8A. The SMX in the readdition experiment with strain A12 was almost completely degraded on days 7, 21, 42, and 56. The SMX concentrations of the readdition experiment without strain A12 were 1.38, 2.61, 7.01, and 13.9 mg L\({}^{-1}\) on days 7, 21, 42, and 56, respectively. The rate constants (\(k\)) and half-lives (\(t_{1/2}\)) of the SMX after the third and fourth additions shown in Figure 8A were estimated and listed in Table S6. The results of SMX degradation in anaerobic readdition experiments are shown in Figure 8D. The SMX concentrations in the readdition experiment with strain L were 1.38, 1.71, 0.79, and 0 on days 7, 21, 42, and 81. The SMX concentrations in the readdition experiment without strain L were 1.75, 2.87, 10.88, and 8.01 mg L\({}^{-1}\) on days 7, 21, 42, and 81, respectively. The rate constants (\(k\)) and half-lives (\(t_{1/2}\)) of the SMX after the third and fourth additions shown in Figure 8D were estimated and are listed in Table S7. The results indicate that the ability of bacterial strains A12 and L to enhance SMX degradation in milkfish pond sediments could be increased by the readdition of SMX under both aerobic and anaerobic conditions. The degradation rates of the low SMX concentration (2 mg L\({}^{-1}\) for the first and second additions) for both aerobic and anaerobic continuous addition experiments were similar to the degradation rates in the batch experiments (Figure 7A,B). In the high SMX concentration (10 mg L\({}^{-1}\) for third and fourth additions) conditions, the degradation rate increased. It seems likely that SMX adaptation occurred in the readdition experiments, which led to SMX degradation at the late stages of the experiments without the addition of bacterial strains A12 and L. The degradation rate increased with SMX readdition (Tables S6 and S7). Microbes with SMX-degrading ability may increase in response to SMX readdition [11]. Figure 7: Biodegradation of sulfamethoxazole (SMX) by the four bacterial strains (A2, A12, L, and M) with fish pond sediments. (**A**) Aerobic SMX biodegradation by strains A2 and A12. (**B**) Anaerobic SMX biodegradation by strains L and M. Data from three independent experiments are presented as the mean \(\pm\) SE. Aerobic and anaerobic microbial communities of continuous addition experiments are shown in Figure S1. Two major stages (before day 40 and after day 40) were found in the results of the cluster analysis. The microbial community compositions before day 40 were largely distinct from the microbial community compositions after day 40.