# Biodegradation of Sulfamethoxazole in Milkfish (_Chanos chanos_) Pond Sediments
## 3 Results and Discussion
### Isolation and Identification of Sulfamethoxazole-Degrading Bacteria  
To isolate SMX-degrading bacteria for further application, SMX was added to the sediment of milkfish ponds and incubated under aerobic or anaerobic conditions for three months. Aerobic and anaerobic microbial communities before and after SMX adaptation are shown in Figures 2 and 3, respectively. For SMX adaptation under aerobic conditions, Bacteroidetes increased from 10% to 30% (Figure 2A). Only one reported SMX-degrading bacterial genus (_Hyponomicobium_) was found before SMX adaptation [14]. Four reported SMX-degrading bacterial genera (_Brevundimonas_, _Escherichia/Stigella_, _Hyponomicobium_, and _Shewanella_) and one isolated in this study (_Vibrio_) were found after SMX adaptation [14; 15; 16; 17] (Figure 2B). For SMX adaptation under anaerobic conditions, archaea (Euryarchaeota, Pacearchaeota) decreased from 39% to 4% (Figure 3A). Proteobacteria increased from 38% to 56%. One reported SMX-degrading bacterial genus (_Escherichia/Stigella_) [16] and one isolated in this study (_Marinobacter_) were found before SMX adaptation. Three reported SMX-degrading bacterial genera (_Escherichia/Stigella_, _Flavobacterium_, and _Pseudomonas_) [15; 16; 18] and one isolated in this study (_Marinobacter_) were found after SMX adaptation (Figure 3B). More reported SMX-degrading bacterial  
Figure 1: Biodegradation of three sulfonamides in milkfish (_Chanos chanos_) pond sediments under aerobic (**A**) and anaerobic (**B**) conditions. SMX: sulfamethoxazole, SDM: sulfamethokine, SMZ: sulfamethazine. Data from three independent experiments are presented as the mean \(\pm\) SE. genera were observed after SMX adaptation under both aerobic and anaerobic conditions, indicating that SMX-degrading bacteria enrichment was achieved. Figure 2: Bacterial/archaeal community in milkfish (_Chanos chanos_) pond sediments under aerobic condition before and after sulfamethoxazole (SMX) adaptation. (**A**) Major bacterial/archaeal community (phylum level) in the sediments under aerobic conditions before and after SMX adaptation. (**B**) Major bacterial/archaeal community (genus level) in the sediments under aerobic conditions before and after SMX adaptation. Red “@”s indicate SMX-degrading bacteria that were isolated in this study. The isolation of SMX degrading bacteria was performed using marine agar plates containing 2 mg L\({}^{-1}\) SMX. Six bacterial strains (A1, A2, A12, A15, A16, and A20) and two bacterial strains (L and M) under aerobic and anaerobic conditions, respectively, were found to have the ability to use SMX as a carbon source and degrade SMX. SMX-degrading ability was tested under aerobic and anaerobic conditions. As shown in Figure 4A, SMX (2 mg L\({}^{-1}\)) was completely degraded by the bacterial strains A2 and A12 under aerobic conditions within 10 days. Under anaerobic conditions, SMX (2 mg L\({}^{-1}\)) was completely degraded by the bacterial strains L and M within 24 days (Figure 4B). The rate constants (\(k\)) and half-lives (\(t_{1/2}\)) of the SMX in the experiments shown in Figure 4 were estimated and listed in Table S3. Figure 3: Bacterial/archaeI community in milkfish (_Chanos chanos_) pond sediments under anaerobic condition before and after sulfamethoxazole (SMX) adaptation. (**A**) Major bacterial/archaeI community (phylum level) in the sediments under anaerobic conditions before and after SMX adaptation. (**B**) Major bacterial/archaeI community (genus level) in the sediments under anaerobic conditions before and after SMX adaptation. Red stars indicate SMX-degrading bacteria that have been reported. Red “@”s indicate SMX-degrading bacteria that were isolated in this study. Phylogenetic analysis using 16S rRNA sequences indicated that bacterial strains A2 and A12 are very close (99% sequence identity) to _Vibrio_ sp., and bacterial strains L and M are very close (99% sequence identity) to _Pseudomonas_ sp. and _Marinobacter_ sp., respectively (Figure 5 and Table S4).